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Cation localization in growth hormone and prolactin cells of the rat pars distalis

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Cation localization in growth hormone and prolactin cells of the rat pars distalis

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Content CATION LOCALIZATION IN GROWTH HORMONE
AND PROLACTIN CELLS OF THE RAT PARS DISTALIS
by
Sara Barbara Yancey
A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(Anatomy)
February 1977
U N IVE R SITY O F S O U TH E R N C A LIFO R N IA
T H E G R A D U A T E S C H O O L
U N IV E R S IT Y P A R K
LO S A N G E L E S . C A L IF O R N IA 9 0 0 0 7
This dissertation, w ritte n by
Sara Barbara Yancey
under the direction of h.^x.... Dissertation C om
mittee, and approved by a ll its members, has
been presented to and accepted by The G raduate
School, in p a rtia l fu lfillm e n t of requirements of
the degree of
D O C T O R O F P H I L O S O P H Y
K
Dean
:OMMITTEE ERTAT
Chairman
This Dissertation is Dedicated
to
My Husband, Herb
11
ACKNOWLEDGEMENTS
I wish to express my gratitude to Dr. Joel Schechter,
my thesis advisor, who provided advice, support, criticism,
and encouragement throughout the years of my graduate train
ing. He smoothed the way and made every potentially trau
matic experience a joyous occasion. I am further indebted
to Dr. Barbara Monroe who gave generously of her time to
advise me and to Dr. Richard Weiner who provided advice in
the design of this study and the laboratory equipment and
staff for the radioimmunoassay. I also wish to thank Drs.
Bernard Slavin, Dwight Warren, and Wallace Frasher for
their unfailing support and encouragement and to express
my appreciation to Dr. Warren for the use of his laboratory
and equipment. The manuscript was read and critically exam
ined by Drs. Joel Schechter, Barbara Monroe, and Richard
Weiner. I am indebted to them for their interest, sugges
tions, and helpful comments. The manuscript was typed by
Mrs. Caroline Brown, whose expertise, concern, and good
humor place me greatly in her debt. I would also like to
thank Mrs. Sheila Odnert who always willingly and unfail
ingly provided technical assistance and aid in the execu
tion of the experiments.
Ill
TABLE OF CONTENTS
ABSTRACT .
FOREWORD .
CHAPTER I. EFFECTS OF VARIOUS MODIFICATIONS OF
THE POTASSIUM PYROANTIMONATE TECHNIQUE
ON CATION LOCALIZATION IN GROWTH
HORMONE AND PROLACTIN CELLS OF THE
RAT PARS DISTALIS ................
V
CHAPTER II
INTRODUCTION .
MATERIALS AND METHODS
RESULTS .............
DISCUSSION .
SUMMARY .............
A COMBINED ULTRASTRUCTURAL AND
POTASSIUM PYROANTIMONATE CYTOCHEMICAL
STUDY OF PROLACTIN CELLS AT VARIED
STAGES OF SECRETORY ACTIVITY .
INTRODUCTION .
MATERIALS AND METHODS
RESULTS .............
DISCUSSION .
SUMMARY .............
LITERATURE CITED ................
ABBREVIATIONS FOR FIGURES .
FIGURES .........................
TABLES .........................
5
45
53
64
77
79
93
99
109
119
121
149
150
202
IV
ABSTRACT
The cellular distribution of cation antimonate precipi
tate was examined in growth hormone (GH) and prolactin (PRL)
cells of the rat pars distalis by the use of several differ
ent modifications of the potassium pyroantimonate technique.
Tissues were either (1) fixed directly in an osmium tetrox-
ide-potassium pyroantimonate solution (OsO^/KSb) for periods
varying from two hours to overnight or (2) prefixed in a
glutaraldehyde-potassium pyroantimonate solution (Glu/KSb)
for periods varying from 15 minutes to two hours and post
fixed in OsO^/KSb.
(1) Direct fixation in OsO^/KSb for two to four hours
often yielded abundant precipitate at specific cellular
sites including nuclei, Golgi saccules, mitochondria, mem
branes of secretory granules, and multivesicular bodies.
However, use of this technique did not consistently produce
acceptable preservation of cellular fine structure.
(2) Prefixation in Glu/KSb consistently improved cellu
lar preservation and resulted in the deposition of coarser
grained precipitate at the periphery of some secretory gran
ules. However, increasing time of exposure of the tissue
to the prefixation solution resulted in a selective and pro
gressive decrease in cation precipitate in nuclei and Golgi
saccules. Results of prolonged exposure (two hours) were
V
uninterpretable due to striking variations in quantity and
distribution o£ precipitate. A very short prefixation time
(15 minutes) resulted in minimal changes in the distribution
of precipitate and still enhanced the preservation of cellu
lar structure.
The Glu/KSb (15 minute) technique was used to study the
cellular distribution of cation antimonate precipitate and
ultrastructural characteristics of PRL cells in varied
stages of secretory activity. Hemipituitaries were incu
bated in Krebs Ringer bicarbonate solution with glucose
(KRBG) (controls) or in KRBG with either 1 x 10 dopamine
- 3 6
or 5 X 10 M N -monobutyryl-cyclic adenosine monophosphate
(mbcAMP) (expérimentais), PRL release was measured by
radioimmunoassay. The concentration of antimonate deposits
associated with mitochondria, Golgi saccules, and secretory
granules was estimated by semiquantitative analysis of the
number of precipitate particles localized to these sites.
Dopamine inhibition of PRL release resulted in the accumula
tion of secretory granules in all stages of maturation
accompanied by a marked increase in mitochondrial antimonate
deposits and an increase in Golgi deposits as compared with
controls. The mbcAMP stimulation of PRL release resulted in
massive exocytotic activity at five and 15 minutes and a
decline in exocytotic activity after 30 minutes to one hour,
accompanied by a decrease in mitochondrial deposits at 30
VI
minutes and an increase in these deposits at one hour. The
concentration of antimonate deposits localized to secretory
granules was essentially the same in all experimental and
control groups. Plasma membranes and granule membranes
translocated to the plasma membrane during exocytosis were
not reactive. Exocytotic activity was accompanied by the
appearance of numerous coated pits, coated micropits at
sites of exocytosis, and the accumulation of microvesicles
at the margin of the Golgi apparatus.
Use of the potassium pyroantimonate technique in this
investigation has allowed the detection of intracellular
pools of cations that presumably consist predominantly of
calcium. Alterations in the concentration of mitochondrial
antimonate deposits following mbcAMP stimulation or dopa
mine inhibition suggest an active role for mitochondrial
calcium in stimulus - secretion coupling in PRL cells. Ultra-
structural changes in PRL cells during the secretory cycle
indicate that coated membrane structures and microvesicles
are involved in the process of membrane retrieval after exo
cytosis. Morphologic evidence of active synthesis and pack
aging of secretory product during dopamine inhibition indi
cates that the primary effect of this catecholamine is upon
the release of PRL, not its synthesis.
VI1
FOREWORD
The importance of calcium as a mediator of secretory
processes in endocrine and exocrine cells has been wel1
documented since the pioneering work of Douglas and associ
ates on secretory mechanisms in the adrenal medulla that
led to the formulation of the ’’stimulus-secretion coupling”
hypothesis (Douglas, 1968). This hypothesis has been
tested in physiological studies of all the cell types
present in the pars distalis of the rat anterior pituitary
gland. It is now generally accepted that calcium plays a
crucial role as an intermediary coupling the release of
pars distalis hormones to physiological stimuli (Rubin,
1974). Thus calcium serves as the intracellular link
between hypothalamic releasing hormone - induced cell mem
brane events and the release of hormones by exocytosis.
The means by which calcium effects hormone release
remains to be elucidated. Physiological evidence suggests
that the locus of a critical calcium fraction may be some
specific cytoplasmic site and that mobilization of this
calcium pool may be the crucial event in the release pro
cess (Milligan and Kraicer, 1974). Identification of cell
ular structures or organelles that possess the capacity to
sequester or release calcium according to the functional
state of Lhe cell could help clarify the mechanism of
action of calcium.
Calcium and other cations including sodium and magnes
ium can be visualized at the ultrastructural level by the
use of potassium pyroantimonate as a precipitating agent.
First developed by Komnick (1962) for the demonstration of
sodium, this technique has since been applied to the study
of a variety of tissues where it has been shown that
calcium is the predominant component of the intracellular
antimonate precipitate (Legato and Langer, 1969; Herman et
al., 1973; Yarom and Meiri, 1973; Clementi and Meldolesi,
1975; Stoeckel et al., 1975). General patterns of the
distribution of cation-antimonate precipitate have been
described in rat pars distalis cells by the use of two
different potassium pyroantimonate techniques. The first
account consisted of a brief description of precipitate
patterns in both the pars distalis and the pars intermedia
(Stoeckel et al., 1974). A more detailed description of
cation localization in pars distalis cells has recently
been published (Schechter, 1976).
The present investigation was initiated as a more
selective study to further characterize the distribution of
cation-antimonate precipitate in a specific pars distalis
cell under known conditions of stimulation or inhibition of
secretory activity as an approach to understanding the
mechanisms by which calcium regulates secretion. In addi
tion, since it has been clearly demonstrated in other
tissues that the subcellular distribution of antimonate
2
deposits varies depending upon specific factors in the fix
ation protocol (Simson and Spicer, 1975), it was essential,
first of all, to gain some insight into possible effects of
different fixation procedures on cation localization in
pituitary cells.
Therefore, the objectives of this investigation were
twofold :
(1). To determine those fixation parameters necessary
to yield optimal results in the precise cytochemical local
ization of cations in cells of the rat pars distalis by a
systematic, comparative study of the effects of several
modifications of Komnick’s potassium pyroantimonate tech
nique .
(2). To then characterize the morphological distribu
tion of cation antimonate precipitates in a specific pars
distalis cell type in varied stages of secretory activity
and to correlate the ultrastructural data with the measured
release of the hormone.
Growth hormone - secreting and prolactin-secreting cells
were selected as the focus for the first part of this study
since they may be reliably identified by morphological
criteria alone. For the second phase of the study,
prolactin-secreting cells were selected since they are
amenable to experimental manipulation, and i j i vitro secrete
high levels of prolactin autonomously (Meites et al., 1961).
Furthermore, they can be stimulated to secrete above
autonomous levels by the addition of cyclic adenosine
monophosphate derivatives to the incubation medium (Lemay
and Labrie, 1972) or inhibited significantly by the addi
tion of dopamine at micromolar levels (MacLeod and
Lehmeyer, 1974a). A further advantage which prolactin
cells offer as a model for the study of pituitary secretory
processes is the existence of a reliable radioimmunoassay
method for the measurement of prolactin.
CHAPTER I
EFFECTS OF VARIOUS MODIFICATIONS OF THE POTASSIUM PYRO
ANTIMONATE TECHNIQUE ON CATION LOCALIZATION IN GROWTH
HORMONE AND PROLACTIN CELLS OF THE RAT PARS DISTALIS
INTRODUCTION
Calcium: A Cation of Major Biological Importance
1. Cellular Calcium: Its Regulation and General
Effects on Cell Structure and Function
Calcium is a cation of profound biological signifi
cance, implicated both in the basic structural organization
of biological systems and in an amazingly broad spectrum of
physio logical processes. The classical experiments of
physiologists of the late nineteenth and early twentieth
centuries formed the foundation for myriad studies that
were to follow to investigate the role of calcium in
diverse cellular phenomena. Several among these are of
particular significance.
In a series of experiments. Ringer (1882, 1883a, 1883b,
1886) examined the effect of various salt solutions on per
fused hearts and skeletal muscle. These studies demon
strated a calcium requirement for the maintenance of muscle
contractility and formed the basis for further experiments
on the relationship between cell function and the external
electrolyte environment. Ringer (1890) also observed
dissociation of cells from one another in calcium-free
media, and concluded that calcium played an important role
in maintaining tissue integrity. A requirement for calcium
in neuromuscular transmission was demonstrated by Locke
(1894) , while Mines (I9l3) showed that omission of calcium
from the extracellular fluid caused a dissociation of the
electrical and mechanical responses of heart muscle.
Chambers and Reznikoff (1926) , investigating the role
of electrolytes in the maintenance of protoplasmic struc
ture and function, demonstrated that the cell membrane acts
as a primary barrier to the inward movement of calcium and
by injecting calcium into the cell (Amoeba proteus),
established the importance of a low intracellular calcium
concentration in normal protoplasmic function. The
importance of calcium in the regulation of cell membrane
permeability was shown by McCutcheon and Lucke (1928), who
noted that lack of calcium in the medium increased the rate
of swelling of sea urchin eggs in hypotonic solutions.
A direct effect of calcium deprivation on release
processes in cells was first described in the classical
experiments of Harvey and Macintosh (1940) . Measuring
directly the output of acetylcholine from preganglionic
fibers in the cat superior cervical ganglion, they found
that in the absence of extracellular calcium there was no
release of acetylcholine in response to electrical stimula
tion of the fibers. These experiments were of great
importance since they foreshadowed others which followed on
the role of calcium in secretory processes .
On the basis of a large amount of corroborative evi
dence from a variety of experiments, Heilbrunn (1945)
proposed a general theory of stimulation stating that any
stimulating agent causes a release of calcium from the
"outer region of the cell" and that the calcium that is
"released from the cortex enters the cell to affect the
interior".
More recent studies have contributed to a voluminous
literature on the actions of calcium and have further
defined its role both at the cell surface and within the
cell. Acting at the cell surface, calcium promotes cellu
lar adhesion and membrane structural integrity, influences
the permeability characteristics of the cell membrane,
decreases the charge density at the membrane surface, and
alters the electrical properties of the membrane by its
action as a membrane stabilizer (Manery, 1966; Rothstein,
1968; Baker, 1975). Many of these biological effects of
calcium have been attributed to its strong binding affinity
to membrane components, acidic phospholipids and proteins
(Manery, 1966 ; Bianchi, 1968). Dissociation of bound
calcium alters the physical properties of the membrane.
increasing its deformability and its permeability to water,
monovalent cations [i.e., sodium and potassium), calcium
itself, and other ions and solutes (Hodgkin and Keynes,
1957; Manery, 1966).
The relationship between calcium and biological mem
branes is an intricate one. While the binding or passage
of calcium influences the structure and function of the
membrane, the membrane in turn controls the passage of
calcium to the interior of the cell. Calcium is the major
divalent cation present in extracellular fluid. The extra
cellular calcium concentration [Ca^^Jp bathing most cells
is close to 10 ^ M (Baker, 1975; Berridge, 1975), much
higher than the intracellular concentration of calcium ions
2+ - 5 - 7
[Ca ]j, estimated to be in the range of 10 to 10 M
(Hodgkin and Keynes, 1957 ; Winegrad, 1969 ; Baker, 1975).
This large concentration gradient promotes entry of calcium
into the cell. If the concentration of ionized calcium
inside the cell were determined passively, the Donnan
equilibrium predicts that calcium ions would be accumulated
by most cells. However, the resting permeability of the
plasma membrane of most cells to calcium is low, limiting
its entry (Berridge, 1975). Nevertheless, if cells are
exposed to a solution of radio labeled calcium (^^Ca), the
isotope is taken up by the cells and lost. This indicates
that calcium exchange takes place across the membrane.
Calcium enters the cell by a passive process and is pumped
out by an energy dependent process (Rasmussen, 1970; Baker,
1975).
Although the exchange of calcium across the membrane
is important to cellular calcium homeostasis, it is not the
2 +
only mechanism operating to maintain [Ca ]j at a low con
centration. Free ionized calcium is a small fraction of
the total intracellular calcium (Baker, 1975). A number of
organelles possess the ability to accumulate calcium in
non-ionized form and act as intracellular buffers to
2 +
stabilize [Ca ]^. Of particular importance among these
organelles are the mitochondria and endoplasmic reticulum
(microsomal membrane). Mitochondria from a great variety
of tissues can sequester calcium against a very large con
centration gradient (Lehninger, 1964; Drahota et al., 1965 ;
Borle, 1973 ; Carafoli et al., 1975). Free calcium in the
mitochondrial compartment may exceed that in the cytosol
by a factor of 1000 (Borle, 19 73). Evidence from studies
of mitochondrial fractions from several cell systems
demonstrates that calcium may be released from mitochondria,
or that the mitochondrial uptake of calcium may be
inhibited, in response to alterations of the extramitochon-
drial medium (Dransfield et al., 1969; Carafoli et al.,
1974), and to a variety of agents including metabolic
inhibitors and low concentrations of cAMP (Borle, 1974).
The role of skeletal muscle sarcoplasmic reticulum in the
sequestration and release of calcium has been well defined
(Ebashi and Endo, 1968). Moreover, calcium sequestration
has been characterized in microsomal fractions derived from
sarcoplasmic reticulum of heart muscle (Dransfield et al.,
1969), and endoplasmic reticulum of brain (de Meis et al.,
1970), salivary gland (Selinger et al., 1970), blood plate
lets (Robblee et al., 1973), neurohypophysis (Thorn et al.,
1975), and liver (Moore et al., 1975). The microsomal
uptake also varies with changes in the ionic or chemical
environment (Rasmussen and Goodman, 19 75; Moore et al.,
1975; Dransfield et al., 1969). Plasma membranes are addi
tional potential storage sites for calcium (Schatzman and
Vincenzi, 1969 ; Sulakhe et al., 1973).
In summary, cellular calcium homeostasis is determined
by the interplay among three factors: (1) the rate of
calcium influx across the cell membrane, (2) the rate of
calcium efflux across the membrane, and (3) intracellular
buffering reactions. Calcium is distributed in a complex
compartmentalization that may be in part dependent upon the
morphological makeup of the cell, particularly its mito
chondrial content and development of endoplasmic reticulum.
In view of the large quantity of calcium that exists
in a bound state, small changes in binding could have
profound effects on [Ca^^Jj. It is this pool of calcium
10
which is significant in many cellular processes
in which calcium serves as the link coupling membrane
events to a cellular response. Among these processes where
calcium serves as a critical mediator are included the reg
ulation of cellular metabolism and proliferation
(Rasmussen, 1970; Rasmussen and Bikle, 1975; Rasmussen and
Goodman, 1975; Swierenga et al., 1976; Whitfield et al.,
1976), cell motility (Eckert, 1972; Gail, 1973; Hanson et
al., 1973; Hawkes and Holberton, 1973), muscular contrac
tion (Ebashi and Endo, 1968; Hurwitz and Suria, 1971;
Reuter, 1975), and exocytosis (Douglas, 1968). Although a
transmembrane influx of extracellular calcium may be
responsible for triggering changes in cells in some systems,
in others, calcium ions may be released into the cytoplasm
from intracellular reservoirs (Rubin, 1974). The essence
is that a translocation of calcium ions is a trigger
coupling an external stimulus (mechanical, electrical, or
hormonal) to some intracellular activity (enzymatic, con
tractile, secretory). Termination of the calcium action is
accomplished by extrusion of calcium to the exterior
through exchange or active transport or by sequestration
of calcium via intracellular binding (Rubin, 1974).
2. Calcium and Secretion
The concept that calcium serves as a general mediator
of secretory mechanisms was first formulated by Douglas
11
(Douglas and Rubin, 1961) who defined the concept of
"stimulus - secretion coupling." This was intended to em
brace all the events that followed a cell's exposure to a
stimulus which finally resulted in release of its secretory
product to the extracellular environment (Douglas, 1968).
The phrase was patterned after "excitation-concentration
coupling," coined by Sandow (1952) to describe the series
of events that occur in muscle stimulation: sarcolemmal
excitation, "inward acting link," and activation of con
traction. Sandow proposed that calcium served as the link
between excitation and contraction.
In a series of elegant experiments, Douglas and
colleagues studied physiological aspects of the secretory
process by using perfused adrenal glands and monitoring
the secretion of catecholamines. Douglas and Rubin (1961)
demonstrated that the excitant action of acetylcholine in
the adrenal medulla was dependent upon the presence of
calcium and suggested that acetylcholine evoked the secre
tion of catecholamines by causing calcium uptake from the
extracellular fluid into the medullary chromaffin cells.
Furthermore, the amount of catecholamines released by
acetylcholine was quantitatively related to the concentra
tion of calcium in the perfusion fluid over a wide range.
The response of the chromaffin cells to excess potassium
(assumed to depolarize the cells) was also dependent upon
calcium in the extracellular fluid.
12
Support for the concept that acetylcholine - evoked
secretion involved the movement of calcium into the cell
came from studies by Douglas and Poisner (1961) , which
demonstrated that uptake of ^^Ca into the chromaffin
cells was stimulated by acetylcholine. Not only was
calcium indispensable for the response to acetylcholine but
it was also sufficient for the response. Douglas and Rubin
(1963) showed that ions other than calcium, such as sodium,
potassium, or chloride, were unnecessary for the release
process; that calcium itself was a powerful secretogogue;
and that magnesium, known to compete with calcium in many
cellular reactions, had strong inhibitory effects in the
release of catecholamines. Other alkaline earth metals,
barium and strontium, were able to substitute for calcium
in the secretory process (Douglas and Rubin, 1964). Other
secretogogues, in addition to acetylcholine and elevated
potassium, were dependent upon calcium for their action
(Poisner and Douglas, 1966).
Direct evidence that acetylcholine acted on the plasma
membrane to depolarize the chromaffin cell Avas obtained by
intracellular recording of transmembrane potentials in
isolated chromaffin cells in culture before, during, and
after the application of acetylcholine (Douglas et al.,
1967a). Depolarization appeared to be due primarily to
inward sodium current; however, a portion of the
13
depolarizing action of acetylcholine seemed to be due to
the inward movement of calcium since acetylcholine retained
some depolarizing activity in the absence of sodium and
this remaining activity was proportional to the extracellu
lar calcium concentration (Douglas et al., 1967b). Never
theless, it appeared that the fall in potential across the
chromaffin cell membrane was of little direct significance
in stimulus - secretion coupling. While depolarization of
the chromaffin cell was an adequate stimulus for secretion
in the presence of calcium, it was not of itself a
sufficient stimulus if calcium was not present. Lack of
calcium did not prevent excess potassium from depolarizing
the membrane, yet it blocked secretion. Moreover, the
secretory response to acetylcholine was potentiated when
sodium was absent from the extracellular environment though
the membrane potential fell very little below the normal
resting value (Douglas and Rubin, 1961, 1963; Douglas et
al., 1967b). Depolarization seemed to be significant
primarily in that it represented a change in the molecular
arrangement of the membrane that resulted in altered trans
membrane calcium fluxes in response to the secretogogue
(Douglas, 1968).
It should be noted that prior to the work of Douglas
and associates, considerable work had already delineated
the pivotal role of calcium in the release of transmitter
14
from the squid giant axon (Hodgkin and Keynes, 1959) and
from cholinergic fibers both at the neuromuscular junction
(Locke, 1894 ; Eccles et al., 1941 ; del Castillio and Katz,
1954) and in autonomic ganglia (Harvey and Macintosh, 1940;
Hutter and Kostial, 1954). While these experiments were of
considerable importance, it was Douglas who recognized
basic similarities in the mechanisms by which cells that
store their product discharge it into the extracellular
fluid although cells differ widely in their secretory
products, morphology, electrical excitability, and physio
logical stimuli. It was Douglas who perceived that
calcium-activated exocytosis could be a general mechanism
of secretion. The initial research by Douglas and associ
ates into the mechanisms of release of catecholamines from
adrenal chromaffin cells was extended to studies of neuro
secretory cells of the posterior pituitary and secretory
cells in the submaxillary gland, and also prompted other
investigators to study the role of calcium in a variety of
secretory processes.
Noting the close developmental relationship of both
chromaffin cells and ordinary neurons with the neuro
secretory cells of the posterior pituitary, Douglas (1963)
began the first of a long series of experiments to deter
mine how electrical activity in the neurosecretory fibers
was translated into the secretion of vasopressin and to
15
explore the role of calcium in this process. The im
pressive array of experimental evidence for the unique and
critical role of calcium in neurosecretion has recently
been reviewed by Douglas (1974). In brief, the ionic re
quirements for neurosecretion evoked by depolarization
closely parallel those observed for the secretion of cat
echolamines from adrenal chromaffin cells and the release
of neurotransmitter from cholinergic neurons. Several
minor differences have been detailed by Rubin (1970) . In
each of these three cell types, secretion requires calcium,
varies with the extracellular concentration of calcium
over a wide range, and involves the influx of calcium.
Thorn et al. (1975a,b) and Dreifuss et al. (1975) have
presented more recent evidence in this regard.
During the past decade hosts of experiments have been
carried out on neural, endocrine, exocrine and other cells.
As seen in the extensive review by Rubin (1974), it has now
become clear that calcium has some general role in the
transduction sequence between membrane recognition of stim
ulant molecules and the release of hormone, neuro
transmitter or other cell product, from those cells whose
secretion involves the packaging of a product in membrane
bound vesicles or granules. This transduction is followed
by release of the vesicle or granule contents into the
extracellular fluid by the process of exocytosis. Thus,
16
for example, calcium has been shown to be a critical
element in the release of catecholamine from adrenergic
nerve terminals (Smith and Winkler, 1972), the release of
transmitter from presynaptic terminals in the squid stell
ate ganglion (Llinas and Nicholson, 1975) , the secretion of
insulin from beta cells (Malaisse et al., 1975; Heilman,
1975), histamine release from mast cells in response to the
antigen-antibody reaction (Forman and Mongar, 1975), the
secretion of enzymes by polymorphononuclear leukocytes
(Goldstein et al., 1975; Zabucchi et al., 1975), the
release of 5 - hydroxytryptamine from blood platelets in
response to thrombin (Sneddon and Williams, 1973), enzyme
secretion from the exocrine pancreas (Peterson and Ueda,
1975), the secretion of enzymes and fluid from salivary
glands (Selinger, 1975), and the secretion of hormones from
the anterior pituitary gland (Geschwind, 1971).
As originally formulated, the Douglas (1968) hypoth
esis of stimulus - secretion coupling stated that glandular
cells are caused to secrete by the entry of calcium which
is brought about by changes in the permeability properties
of the plasma membrane. However, there is evidence that
stimulation leading to release may not necessarily be
associated with transmembrane movement of calcium ions from
the extracellular fluid. Certain investigators have been
able to show that secretion from some cells can continue.
17
to some degree, under conditions of calcium deprivation.
For example, this appears to be the case for secretion of
enzymes from the rat parotid gland (Selinger, 1975) and
from the rat exocrine pancreas (Christophe, et al., 1974;
Peterson and Ueda, 1975). These findings suggest that the
critical calcium fraction may be localized to intracellular
stores. Moreover, in those instances where increased up
take of radiolabeled calcium has been demonstrated, experi
ments do not establish whether the increased ^^Ca in the
gland represents an absolute uptake of ^^Ca from the media
or a redistribution of cell calcium that causes a slowing
of calcium efflux.
3. Calcium and Secretion of Hormones of the Pars
Distalis of the Rat Anterior Pituitary Cland
In considering the role of calcium as a critical medi
ator in secretory mechanisms of cells of the pars distalis
of the rat anterior pituitary gland, one must take account
of the functional and structural complexity of the gland.
The pars distalis is a composite of at least five different
types of parenchymal cells that produce at least six major
hormones (Baker, 1974) whose release is controlled by hypo
thalamic factors or hormones (Porter et al., 1970). The
term hypothalamic hormone is used for chemically character
ized regulators, whereas the term hypothalamic factor is
given to those whose structure is unknown (Studer, 1976).
It is generally acknowledged that the adenylate cyclase -
3’,5’-cyclic adenosine monophosphate (cAMP) system plays a
role as a critical mediator of anterior pituitary hormone
secretion induced by hypothalamic releasing factors or
hormones (Wilber et al., 1964; Schofield, 1967; Fleischer
et al., 1969 ; Zor et al., 1969 , 1970; Jutisz and
de la Llosa, 1970 ; Ratner, 1970 ; MacLeod and Lehmeyer,
1970 ; Labrie et al., 1971 ; Borgeat et al., 1972 ; Lemay and
Labrie, 1972; Deery and Howell, 1973). Nevertheless, it
has been clearly demonstrated that the hypothalamus-
stimulated release of hormones is calcium-dependent. Cal
cium is required for releasing factor-induced secretion of
thyroid stimulating hormone (TSH) (Vale and Guillemin,
1967; Steiner et al., 1970), luteinizing hormone (LH)
(Samli and Geschwind, 1968; Wakabayashi et al., 1969),
follicle stimulating hormone (FSB) (Wakabayashi et al.,
1969 ; Jutisz and de la Llosa, 1970), adrenocorticotropic
hormone (ACTH) (Kraicer et al., 1969) and growth hormone
(GH) (Steiner et al., 1970). Thus both cAMP and calcium
have been implicated as second messengers mediating the
release of anterior pituitary hormones.
While both calcium and cAMP can serve as second
messengers, even the response to cAMP is calcium-dependent.
Steiner et al. (1970) observed a diminished release of TSH
in response to aminophy11ine in calcium-free medium.
19
Methyl xanthines such as aminophy11ine or theophylline act
by inhibiting a phosphodiesterase responsible for the break
down of cAMP, thereby increasing cellular levels of cAMP.
Calcium is also required for optimal release of : ACTH in
duced by dibytyryl cAMP or theophylline (Kraicer et al.,
1969; Zimmerman and Fleischer, 1970), FSH induced by cAMP
(Jutisz and de la Llosa, 1970), GH induced by aminophyl1ine
(Steiner et al., 1970), theophylline (Ewart and Taylor,
1971) or by -monobutyry1 cAMP (Lemay and Labrie, 1972)
and prolactin (PRL) induced by -monobutyryl cAMP (Lemay
and Labrie, 1972) or theophylline (Wakabayashi et al.,
1973).
The site of action of calcium appears to be distal to
adenylate eyelas e-activation of cAMP generation. Hypo
thalamic extract-stimulated increases in cAMP levels in the
pituitary gland are not diminished in the absence of cal
cium. This indicates that calcium is not required for
releasing factor activation of adenyl cyclase (Steiner et
al., 1970).
Cellular mechanisms in the release process of anterior
pituitary hormones appear to be similar for all the differ
ent cell types and in general resemble secretory processes
in adrenal chromaffin and other endocrine as well as exo
crine cells that prepackage, store, and release their
product in response to a specific stimulus.
20
Samli and Geschwind (1968) were the first to apply the
stimulus - secretion hypothesis to cells of the anterior
pituitary gland. The hypothesis has since been tested in
relation to mechanisms governing the release of all the
pituitary hormones. Potassium concentrations five to ten
times greater than the usual levels in incubation media
appear to mimic the action of releasing factors, stimu
lating the secretion of GH (Birge et al., 1969; MacLeod and
Fontham, 1970; Parsons, 1970), LH (Samli and Geschwind,
1968; Wakabayashi et al., 1969), ACTH (Kraicer et al.,
1969), TSH (Vale and Guillemin, 1967), and FSH (Wakabayashi
et al., 1969 ; Jutisz and de la Llosa, 1970). In contrast
to the stimulus-dependent secretion of these hormones, PRL
secretion occurs spontaneously dji vitro, and it is of
interest to note that elevated potassium does not signifi
cantly augment PRL secretion from anterior pituitaries of
female rats. This lack of effect of elevated potassium
suggests that PRL-secreting cells may depolarize spontane
ously i j i vitro (Parsons and Nicoll, 1969 ; MacLeod and
Fontham, 1970; Parsons, 1970). The effect of potassium has
an absolute requirement for calcium in the external medium
(Vale et al., 1967; Samli and Geschwind, 1968; Birge et
al., 1969 ; Kraicer et al., 1969 ; Wakabayashi et al., 1969 ;
Jutisz and de la Llosa, 1970). Moreover, the spontaneous
release of PRL is calcium dependent (Parsons, 1969, 1970;
21
MacLeod and Fontham, 1970). In addition, elevated
potassium has been reported to increase the "^^Ca space in
rat pituitaries iui vitro (Milligan and Kraicer, 1971;
Schofield and Stead, 1971). Martin et al. (1973) have
further demonstrated that elevated potassium causes a sig
nificant depolarization of anterior pituitary cells in
vitro .
High magnesium, a calcium competitor, inhibits the
action of both elevated potassium and releasing factors
(Birge et al., 1969; Wakabayashi et al., 1969; Parsons,
1970) and also the spontaneous release of PRL (Parsons and
Nicoll, 1969).
The physiological significance of the stimulatory in
fluence of depolarizing levels of potassium is unclear.
While the Douglas model of s timulus- secretion coupling
predicts a depolarization of the transmembrane potential
induced by releasing factor-plasma membrane interaction,
present evidence from limited i j i vivo studies has failed
to demonstrate any consistent pattern of response. Al
though Ashworth et al. (1968), using extracellular micro
electrodes, recorded a long-lasting depolarization in
response to hypothalamic extract, York et al. (1973), using
intracellular microelectrodes, recorded a range of
responses from hyperpolarization to depolarization, with no
change in the transmembrane potential as the most consistent
22
finding. Interpretation of results from these experiments
suffers from the inability to identify the cell types
penetrated by the microelectrode or recorded extracellu-
larly.
Evidence suggests that the mechanisms of action of
releasing factors and potassium differ. The effects of
high potassium and hypothalamic extract on LH release
(Samli and Geschwind, 1968) and of high potassium and
follicle stimulating hormone-releasing factor (FRF) on
FSH release (Wakabayashi et al., 1969) are additive. More
over, elevated potassium does not influence cAMP levels in
rat pituitaries (Zor et al., 1970). A possible explanation
for these varied results is that releasing factors may have
functions in addition to depolarization (Rubin, 1974). In
any case, depolarization itself is not sufficient for
hormone release. Depolarization was still significant
during stimulation with excess potassium in a calcium-free
medium, a condition known to inhibit hormone release
(Martin et al., 1973).
While an essential role for calcium in the cellular
mechanisms controlling secretion of pars distalis hormones
has been established, the exact source of the required cal
cium is not clear and may vary with the particular cell
type or stimulus. At present, increased uptake of ^^Ca in
response to a releasing factor has been demonstrated only
23
for a purified preparation of growth hormone releasing fac
tor (GRH) (Milligan et al., 1972). Crude extract of rat
hypothalamus did not increase the intracellular ^^Ca space
(Milligan and Kraicer, 1971). Moreover, there was no in
creased influx of ^^Ca in response to the administration of
either dibutyry1 cAMP or theophylline (Milligan and Kraicer,
1971). The action of releasing factors or of cAMP may re
quire a tightly bound intracellular pool of calcium. Mere
exclusion of calcium from the incubation medium does not
consistently result in significant inhibition of pituitary
hormone release in response to these agents. Several ex
periments have demonstrated a requirement either for a pro
longed preincubation in calcium-free medium or for the use
of the calcium-chelator EDTA in addition to a calcium lack
in order for the effect of calcium deprivation on releasing
factor-induced release to be fully manifested (Samli and
Geschwind, 1968; Jutisz and de la Llosa, 1970; Steiner et
al., 1970; Wakabayashi et al., 1969; Zimmerman and
Fleischer, 1970). In a series of experiments in which mild
versus rigorous procedures were utilized to remove calcium,
Milligan and Kraicer (1974) explored the possibility that
different calcium compartments might be involved in the
release of ACTH, depending upon the stimulus. A loosely
bound, easily accessible pool appeared to be essential for
the action of vasopressin and elevated potassium. The
24
calcium requirement for theophylline or hypothalamic ex
tract appeared to involve a more tightly bound, less
accessible pool. Citing evidence from their own and pre
vious studies, Milligan and Kraicer (1974) proposed that
calcium need not play a crucial role in the release of
hormones at the level of the plasma membrane but that cal
cium is critically important at some specific cytoplasmic
site involving translocation of calcium ions within the
cell.
The influx of ^^Ca associated with GRH may not be the
determinant factor in the release of GH but may reflect
changes in the calcium distribution within cellular com
partments (Milligan and Kraicer, 1974). There may not be
an absolute requirement for extracellular calcium in re
leasing factor- or cAMP-induced secretion of GH. Steiner
et al. (1970) observed that calcium exclusion inhibited the
GH response to both hypothalamic extract and aminophyl1ine,
but that the impairment in hormone response was significant
ly greater when EDTA was included in the calcium-free
medium. Ewart and Taylor (1971), utilizing EDTA and
calcium-free medium, observed that stimulation of GH secre
tion by theophylline was significantly inhibited. On the
other hand, simple incubation in calcium-free medium did
not significantly inhibit theophylline - induced release of
GH (Parsons and Nicoll, 1971). These results suggest that
25
a tightly bound intracellular pool of calcium may have been
removed by the use of EDTA and that this pool is essential
for optimal release of GH in response to its releasing fac
tor and for the release of GH induced by cAMP.
The control of PRL secretion is unique among the mech
anisms governing the release of hormones of the anterior
pituitary gland in that the primary input from the hypothal
amus is inhibitory, through the tonic release of prolactin-
inhibitory factor (PIE) (Neill, 1974). Incubation of
pituitary glands in the presence of acid extracts of hypo
thalamic tissue have shown the presence of PIP activity in
the extract (Pasteels, 1961b, 1962). Parsons and Nicoll
(1971) have examined the interactions of calcium and hypo
thalamic extract and theophylline on the release of PRL in
vitro. Results indicated that PIP did not interact direct
ly with calcium ions. Low calcium medium inhibited the
spontaneous release of PRL and the addition of PIP did not
further inhibit the release. Moreover, calcium at five to
ten times normal levels did not overcome the PIP action of
the extract. The inhibitory action of PIP was manifested
only when extracellular calcium was present to support the
spontaneous release. It appeared that PIP exerted a stabi
lizing effect on the plasma membrane that prevented calcium
entry. While a membrane stabilizing effect of PIP is
consistent with the data, an inhibitory action of PIP upon
26
the cAMP generating system is also a possibility. However
this appears to be a less likely alternative. Neither
potassium, which overcomes PIF action (Parsons and Nicoll,
1971), nor catecholamines, which mimic the action of PIF
(MacLeod, 1969; Birge et al., 1970) influence the levels of
cAMP in rat pituitary (Zor et al., 1970; Steiner et al.,
1970) .
A mobilizing action of theophylline on intracellular
calcium has been suggested by observations of Parsons and
Nicoll (1971). Theophylline restored the suppressed PRL
secretion in low calcium medium to control levels. A simi
lar effect of cAMP in calcium-free medium has been demon
strated by the data of Lemay and Labrie (1972). The fail
ure of hypothalamic extract to block the effect of theo
phylline in calcium-free medium indicated that PIF was
incapable of exerting any effect on intracellular calcium
(Parsons and Nicoll, 1971).
In summary, present evidence, though inconclusive,
suggests that the interaction of GRH with the plasma mem
brane of GH-secreting cells results in an alteration of the
membrane (depolarization), causing an influx of calcium
from the extracellular fluid (Milligan et al., 1972) and a
concomitant activation of adenyl cyclase, with the increased
production of cAMP (Zor et al., 1969 ; Labrie et al., 1971).
The increased cAMP in turn leads to the mobilization of an
27
intracellular pool of calcium (Parsons and Nicoll, 1969;
Steiner et al., 1970; Ewart and Taylor, 1971). PIF, on the
other hand, appears to stabilize the membrane of PRL-
secreting cells, preventing the uptake of calcium (Parsons
and Nicoll, 1971). Removed from the influence of PIF in
vitro, PRL cells may spontaneously depolarize (Parsons and
Nicoll, 1969 ; Parsons, 1970), allowing calcium to move into
the cell (Parsons, 1969, 1970). In addition, activation of
the adenylate cyclase -cAMP system in PRL cells appears to
stimulate the release of calcium from intracellular reser
voirs (Parsons and Nicoll, 1971).
Cation Localization Methodology
A cytochemical technique for the ultras truetural
detection of sodium was first demonstrated by Komnick
(1962) in a study of the sodium-rich cells of the salt
secreting gland of the Herring gull. Based on a long-known
chemical analytic method for the quantitative estimation of
sodium in biological fluids (Stieglitz, 1911 ; Kramer, 1920;
Kramer and Tisdall, 1921), this technique utilizes
potassium pyroantimonate as a precipitating agent. The
pyroantimonate anion is soluble in water as the potassium
salt, forms an insoluble product with sodium ions, and has
a relatively low molecular weight which makes it suitable
for introduction into tissues. Komnick’s original pro
cedure required fixation of tissue blocks at 1°C in an
28
unbuffered solution of 2% potassium pyroantimonate and 1 %
osmium tetroxide followed by a wash, then dehydration in
acetone. The rationale of the methodology developed by
Komnick is that potassium pyroantimonate will form an
electron-dense, insoluble precipitate consisting of sodium
pyroantimonate at the same time as the osmium tetroxide
fixes the tissues. The simultaneous occurrence of these
two events would presumably obviate diffusion artifact.
Komnick produced no direct evidence that the antimonate
precipitate in the cells of the salt gland was indeed
sodium, but utilized sections of gelatin impregnated with
known concentrations of sodium chloride to show the feasi
bility of the technique to localize sodium.
In subsequent studies by other investigators, Komnick's
technique and various modifications of the technique were
used to localize areas of presumed high sodium in sodium
transporting tissues (Kaye et al., 1965, 1966; Lee et al.,
1967; Ochi, 1968; Zadunaisky et al., 1968) frog muscle
(Tice and Engel, 1966 ; Zadunaisky, 1966), mammalian nervous
tissue (Siegesmund and Edelhauser, 1968 ; Spicer et al.,
1968) and lymphocytes (Spicer et al., 1968). In none of
these studies was precipitate specifically identified as
sodium pyroantimonate. Indeed Kramer and Tisdall (1921)
had observed that the potassium pyroantimonate reagent used
to precipitate serum sodium also precipitated ammonium and
29
calcium ions. Furthermore, Komnick and Komnick (1963)
stated that the solubility characteristics of calcium and
magnesium as well as other pyroant imonate salts were such
that they could be precipitated.
Both indirect and direct approaches have been used to
determine whether this subcellular precipitate in actuality
represents sodium ions. Alteration of tissue sodium prior
to fixation (Kaye et al., 1966 ; Zadunaisky et al., 1968;
Tani et al., 1969 ; Satir and Gilula, 1970) and autoradiog
raphy combined with potassium pyroantimonate fixation
(Amakawa et al., 1968 ; Tisher et al., 1969) provided
indirect evidence that precipitate contained sodium in those
tissues examined. Selected area electron diffraction
(Hartmann, 1966) and electron probe microanalysis (Lane and
Martin, 1969 ; Tandler et al., 1970; Kiers zenbaum et al.,
1971) gave direct evidence for the presence of sodium in
precipitate particles but did not allow the localization of
the sodium pyroantimonate deposits to specific cellular
sites. Lane and Martin’s (1969) study of the mouse vas def
erens demonstrated a distribution of sodium in extracellular
luminal and subepithelial deposits.
While the presence of sodium in some precipitate par
ticles was confirmed by these studies, substantial chemical
and electron microscopic evidence at the same time indicated
that the potassium pyroant imonate technique was not specific
30
for sodium (Bulger, 1969; Shiina et al., 1970; Tandler et
al., 1970 ; Torack and La Valle, 1970; Kiers zenbaum et al.,
1971). The reaction product was not exclusively represent
ative of metallic cations (Bulger, 1969; Clark and Ackerman,
1971b), and the reaction was influenced by such factors as
pH, fixative agent and method of fixation, type of buffer,
and extent of tissue rinsing before dehydration (Torack and
La Valle, 1970; Shiina et al., 1970; Clark and Ackerman,
1971a; Sumi, 19 71; Garfield et al., 19 7 2; Yarom et al.,
1974).
Calcium was clearly a component of the pyroantimonate
deposits, often a primary constituent of intracellular pre
cipitate. On the basis of results from pretreating control
tissues with calcium chelating agents, EGTA and EDTA, cal
cium has been identified as a predominant cation in intra
cellular antimonate deposits in nuclei of chick embryonic
myocardial cells (Yeh and Hoffman, 1967), mammalian cardiac
muscle (Legato and Langer, 1969) and frog skeletal muscle
(Yarom and Meiri, 1973). Elemental analysis of the antimo
nate precipitates in frog skeletal muscle and in dog and
pigeon cardiac muscle by electron microscopic X-ray micro -
probe analysis has allowed the specific identification of
intracellular deposits of calcium antimonate (Yarom and
Chandler, 197 2 ; Yarom et al., 1974 ; Saetersdal et al.,
1974) .
31
Klein et al. (1972) demonstrated in test tube experi
ments that the affinity of the pyroantimonate ion for cal
cium and magnesium was much greater than that for sodium.
Adding known concentrations of various cations to a 2%
potassium pyroantimonate solution, they observed an approx
imately linear rate of precipitate formation for calcium in
the range of 10 ^M, for magnesium in the range of 10 ^M,
_ 2
and for sodium at about 10 M. However, the specific in
vitro affinities of the antimonate anion for calcium,
sodium and magnesium have not been established. Shiina et
al. (1970) reported a tenfold greater affinity of potassium
pyroantimonate for sodium as opposed to calcium. Simson
and Spicer (1975) found that sodium was precipitated at a
_ 2
concentration of 10 M, while both calcium and magnesium
- 3 - 4
were precipitated at concentrations between 10 and 10 M.
In any case, it is apparent that physiological levels of
cellular calcium are in a range sufficient to allow precip
itation of calcium antimonate in tissues (Simson and Spicer,
1975) .
Moreover, studies of a variety of secretory tissues
employing potassium pyroantimonate fixation combined with
the use of chelators and X-ray microprobe analysis have
established the particular suitability of the pyroantimonate
technique for the localization of calcium. Calcium has been
identified as the primary constituent in antimonate
32
precipitate in beta cells of the endocrine pancreas (Herman
et al., 1973; Schafer and Kldppel, 1974; Ravazzola et al.,
1976), acinar cells of the exocrine pancreas (Clemente and
Meldolesi, 1975), cells of Lhe mouse anterior and posterior
pituitary (Stoeckel et al., 1975) and chromaffin cells of
the adrenal medulla (Ravazzola, 1976) .
Identification of the chemical nature of the antimonate
precipitate is only one factor among several that must be
considered in any interpretation of results from the use of
potassium pyroantimonate techniques. An additional factor
of considerable importance is the fixation protocol used in
any particular study. Variations of the parameters of the
fixation procedure may influence the subcellular distribu
tion of precipitate (Bulger, 1969 ; Clark and Ackerman,
1971a ; Sumi, 1971 ; Carfield et al., 1972 ; Herman et al.,
1973; Simson and Spicer, 1975). Since its introduction,
Komnick’s technique and many modifications of Komnick’s pro
cedure have been applied to the study of a variety of
tissues. Simson and Spicer (1975) have recently reviewed
the procedures and the reported results from many laborator
ies. Major differences in techniques will be briefly
summarized here.
In much of the research, Komnick’s technique has been
followed or only slightly modified (Kaye et al., 1965 ;
Hartmann, 1966 ; Kaye et al., 1966 ; Mizuhira and Amakawa,
1966 ; Tice and Engel, 1966 ; Lee et al., 1967; Amakawa et
al., 1968; Spicer et al., 1968; Bulger, 1969; Hardin et al.,
1969 ; Lane and Martin, 1969; Legato and Langer, 1969 ; Tani
et al., 1969; Hardin and Spicer, 1970a, 1970b; Klein et al.,
1970; Satir and Gilula, 1970; Shiina et al., 1970; Torack
and La Valle, 1970; Clark and Ackerman, 19 71a ; Garfield et
al., 1972; Klein et al., 1972 ; Spicer and Swanson, 1972;
Yarom and Meiri, 1972; McCallister and Hadek, 1973; Yarom
and Meiri, 1973; Simson and Spicer, 1974; Bogart, 1975;
Clemente and Meldolesi, 1975 ; Debbas et al., 1975 ; Haack et
al., 197 5 ; Sato et al., 1975; Schechter, 1976).
Of interest among the variant techniques is the use of
potassium pyroantimonate in the absence of a conventional
fixative, as both the primary fixative and the precipitant
of cations in animal and plant tissues (Tandler et al.,
1970 ; Kiers zenbaum et al., 1971; Davis et al., 1974).
Significant modifications of the basic technique,
utilized primarily in attempts to improve morphological
preservation, were incorporation of phosphate or collidine
buffer with the potassium pyroantimonate- osmium tetroxide
solution (Ochi, 1968 ; Bulger, 1969; Shiina et al., 1970;
Sumi, 1970; Sumi and Swanson, 1971) and exposure of the
tissue to an aldehyde solution at some time in the fixation
procedure. The aldehyde techniques can be grouped into
three major categories. First, buffered or unbuffered
34
glutaraldehyde is used in combination with potassium pyro
antimonate as the primary fixative, followed by postfixation
in an osmium tetroxide solution (Zadunaisky, 1966;
Zadunaisky et al., 1968 ; Bulger, 1969; Tani et al., 1969 ;
Sumi, 1971 ; Sumi and Swanson, 1971; Sato et al., 1975).
Second, tissues are prefixed in buffered glutaraldehyde and
postfixed in a solution containing potassium pyroantimonate
and osmium tetroxide (Clark and Ackerman, 1971a, 1971b;
Herman et al., 19 73; Hales et al., 1974; Clemente and
Meldolesi, 1975; Sato et al., 1975; Ravazzola, 1976).
Third, potassium pyroantimonate is added to solutions in all
steps of the fixation procedure exclusive of the final wash
before dehydration. Tissues are prefixed in buffered or
unbuffered glutaraldehyde combined with potassium pyroan
timonate, washed in a solution containing potassium pyro
antimonate, then postfixed in an unbuffered solution com
bining potassium pyroantimonate and osmium tetroxide
(Schafer and Kldppel, 1974 ; Stoeckel et al., 1974 , 1975).
While the cation distribution in cells of the rat
pituitary gland has been described by Stoeckel et al.,
(1974) and Schechter (1976), fixation procedures utilized
in these two laboratories differed. Stoeckel and colleagues
employed a procedure requiring prefixation of the tissues
in phosphate buffered glutaraldehyde-potassium pyroantimo
nate solution with postfixation in unbuffered osmium
35
tetroxide-potassium pyroantimonate solution. Schechter
fixed tissues directly in an unbuffered osmium tetroxide-
potassium pyroantimonate solution. No systematic compari
son of results from the use of different procedures has
been made to determine if there is any relationship between
the subcellular distribution of cations in anterior pitui
tary cells and the fixation protocol. However, Schechter
detected no marked differences in localization between
immersion and perfusion fixation. In fact, the 1974 paper
by Stoeckel et al. is very brief, relating only the general
characteristics of cation distribution in cells of both the
pars intermedia and pars distalis of male rat and mouse
pituitary tissue. In a more extensive account of cation
localization in the mouse anterior and posterior pituitary
(Stoeckel et al., 1975), the primary focus is upon cells of
the neural lobe. The portion of the paper related to the
anterior lobe is in essence a reprise of the brief descrip
tion provided in the previous publication. Included, how
ever, is evidence from the use of EGTA and EDTA controls
and electron microprobe analysis, indicating that the
intracellular precipitates throughout the hypophysis con
sisted essentially of calcium pyroantimonate.
The demonstrated ability of potassium pyroantimonate
techniques to localize intracellular cations has led to
their utilization in showing that shifts in intracellular
36
cation pools may occur in response to alterations in the
functional state of the cell. For example, cation shifts
related to functional state have been demonstrated in skel
etal muscle (Yarom and Meiri, 1972; McCall is ter and Hadek,
1973; Davis et al., 1974) and in arterial smooth muscle
(Haack, 1975). In addition, a few reports describe cation
fluxes in response to stimulation of secretory cells. Beta
cells of mouse pancreatic islets have been studied both in
vitro and iji vivo. An alteration of antimonate precipita
tion patterns was observed in beta cells in response to
high glucose medium as compared to patterns seen in the
cells in low glucose medium iui vitro (Herman et al., 1973;
Ravazzola et al., 1976). Varying precipitate patterns were
also seen in beta cells of normo-, hypo -, and hyperglycemic
mice iui vivo (Schafer and Kldppel, 1974). Alterations in
the distribution of cation precipitate have also been ob
served after isoproterenol stimulation of secretory activ
ity in rat parotid gland (Simson and Spicer, 1974) and sub
mandibular gland (Bogart, 1975).
Ultrastructure of Growth Hormone and Prolactin Cells
Electron microscopic studies of the pars distalis of
the rat anterior pituitary gland have revealed a multi
plicity of cell types that can be distinguished by the
characteristics of their secretory granules and the struc
ture of other cellular organelles (Costoff, 1973; Herlant,
37
1975). It is now generally agreed that there are at least
five distinct secretory cell types in the pars distalis
(Nakane, 1975) in addition to non-secretory elements con
sisting of follicular cells and vascular and connective
tissue cells (Farquhar, 1961b). The secretory cells are
known to produce six hormones: GH, PRL, TSH, ACTH, FSH and
LH. Available evidence indicates that there are separate
cell types for the elaboration of each hormone with the
possible exception of FSH and LH which may be produced by
the same cell (Nakane, 1975). According to presently pre
ferred nomenclature, a particular cell type is named accord
ing to the hormone it secretes (Baker, 1974).
GH and PRL cells have several characteristics in
common. Both secrete protein hormones consisting of single
polypeptide chains closely related to each other in chemical
structure (Pantic, 1975). PRL and GH are each stored in
large secretory granules, the largest present in the rat
pars distalis. The secretory granules of both cell types
are acidophilic if stained by classical methods (Pasteels,
1972).
The cells that produce GH were first described at the
ultras truetural level by Rinehart and Farquhar (1953) but
were at that time identified only as the ultras truetural
correlates of light microscopic acidophils and were not
directly associated with their hormonal product nor
38
distinguished from PRL cells. Later, Farquhar and Rinehart
(1954a) recognized two ultrastrueturally distinct types of
acidophils, both of which contained large secretory gran
ules : (1) an abundant class of cells containing spherical-
shaped granules about 350 nm in diameter and (2) a less
abundant class of cells with elliptical-shaped 600 nm gran
ules. Earlier light microscopic studies of experimentally
altered pituitaries had allowed the differentiation of two
types of acidophilic cells based upon the response to
thyroid hormone deficiency or castration and estrogen admin
istration (Purves and Griesbach, 1952). On the basis of
data from these earlier experiments, Farquhar and Rinehart
(1954a) proposed that the more numerous acidophils, identi
fied by 350 nm spherical granules, were producing GH, while
those with 600 nm elliptical granules could be producing
PRL. In an ultrastruetural investigation of pituitaries of
thyroideetomized rats, Farquhar and Rinehart (1954b) ob
served alterations in many of the acidophils, indicating
the suppression of activity consequent to thyroidectomy.
When correlated with physiological data showing the inhib
itory effects of thyroidectomy on growth (Evans et al.,
1939) and the secretion of growth hormone (Eartly and
Leblend, 1954), and data showing the absence of an effect
of thyroxine on the growth of hypophysertomized animals
(Ray et al., 1950), these observations provided evidence
39
that ultrastructurally identifiable cells corresponding to
light microscopic acidophils were the site of production of
GH. On the other hand, it appeared that cells containing
the 600 nm granules produced, stored, and secreted PRL.
Dynamic changes occurred in these cells during lactation
(Hedinger and Farquhar, 1957; Hymer et al., 1961) or upon
withdrawal of the suckling stimulus (Smith and Farquhar,
1966) or upon estrogen administration (Hymer et al., 1961).
Cell fractionation studies have supported these observa
tions. GH and PRL secretory granules have been isolated
and assayed for their hormonal content. The mean diameters
and morphological characteristics of the granules from sep
arated fractions have been correlated with those same
features of secretory granules in intact cells (Costoff,
1973; Zanini and Giannattasio, 1974). In addition, direct
confirmation for the production of GH and PRL by distinct
classes of cells has been obtained by the use of immuno-
cytochemical techniques (Nakane, 1970; Moriarity, 1973).
The unique characteristics of both GH and PRL cells reveal
ed by these and other investigations allow their identifi
cation by morphologic criteria. The single most useful
criterion is secretory granule size and shape.
GH cells are the most prevalent cell type in the rat
pars distalis. In male rats, they comprise 50% to 60% of
all cells ; in the female, they comprise about 40% (Costoff,
40
1973). These medium-si zed ovoid or pyramidal - shaped cells,
often situated along capillaries (Baker, 1974), are distrib
uted throughout the gland except for those areas immediate
ly adjacent to the pars intermedia and the anterior ventral
portion of the gland (Nakane, 1975). Typically, the cell
nucleus is round or ovoid in shape and central or slightly
eccentric in position. Spherical, mature secretory gran
ules, 300 to 350 nm in diameter, are distributed in abun
dance throughout the cytoplasm (Costoff, 19 73; Nakane,
1975). The granules are very osmiophi1ic, homogeneously
electron dense, and have a closely applied limiting mem
brane. Rough endoplasmic reticulum may vary from minimal
to extensive stacks of elongated cisternae (Costoff, 1973;
Baker, 1974; Farquhar et al., 1975; Nakane, 1975). The
Golgi apparatus generally consists of vesicles and vacuoles
with a few stacks of flattened saccules (Baker, 1974).
Mitochondria occur in clusters near the nucleus and
occasionally among the secretion granules (Nakane, 1975).
Multivesicular bodies and lysosomes are present to a vari
able degree in and around the Golgi region (Costoff, 1973;
Farquhar et al., 1975).
PRL cells comprise about 10% of pars distalis cells in
the male rat and about 40% in the female (Costoff, 1973).
Variable in shape, they may appear polygonal or may be
cupped around gonadotrophs. They may be situated along a
41
capillary or located more centrally in a cell cord, extend
ing long projections between cells to reach a capillary
(Baker, 1974; Nakane, 1975). The cells are distributed
primarily in areas near the pars intermedia and are sparse
in the anterior ventral portion of the gland (Nakane, 1975).
The oval nucleus is centrally placed or slightly eccentric.
Mature secretory granules are the largest of any pars dis-
talis cell type, ranging from 600 to 900 nm, and show con
siderable pleomorphism. Within the same cell they vary
considerably in size and shape (Baker, 1974; Farquhar et
al., 1975; Nakane, 1975). Granule content is homogeneously
electron-dense and often appears slightly separated from
the granule membrane by an electron-lucent space (Baker,
1974; Farquhar et al., 1975; Giannattasio et al., 1975).
In contrast to GH cells, PRL cells rarely become filled
with secretory granules. PRL granules are generally dis
tributed toward the periphery of the cell (Baker, 1974).
Rough endoplasmic reticulum is characteristically well-
developed, arranged in parallel lamellae at one pole of the
cell (Costoff, 1973; Nakane, 1975). The Golgi area gener
ally is extensive, consisting of stacks of flattened
saccules, vesicles, and vacuoles, and containing immature
secretory granules (Costoff, 1963; Baker, 1974). Mitochon
dria are numerous and distributed among the granules.
Lysosomes and multivesicular bodies are present to a
42
variable degree (Costoff, 1973; Smith and Farquhar, 1966).
Morphological aspects of the secretory process (synthe
sis, storage, and release) are essentially similar in both
GH and PRL cell types and analogous to those described in
cells of the exocrine pancreas (Caro and Palade, 1964;
Jamison and Palade, 1967a,b). The ultrastructural compo
nents of the secretory pathway have been defined by elec
tron microscope studies. The kinetics of the process have
been determined by autoradiography adapted to electron
microscopy (Racadot et al., 1965). The hormone is synthe
sized on the polysomes attached to the membrane of the
rough endoplasmic reticulum and concomitantly segregated in
the cisternal space. The hormone is then transferred,
probably via intermediary vesicles, to the cisternae of the
Golgi apparatus where it is concentrated into secretory
granules (Farquhar and Wellings, 1957; Farquhar, 1961a,
1971; Howell and Whitfield, 1972). From the Golgi system,
mature secretory granules move toward the periphery of the
cell (Pasteels, 1972; Farquhar, 1961a). Upon stimulus to
secretion, granules discharge their content into the peri
vascular space by the process termed exocytosis (Farquhar,
1961a; Pelletier et al., 1971). During this process the
granule membrane fuses with the plasma membrane. Reorgani
zation of the membrane leads to fission of the fused mem
branes and establishes continuity between the granule
43
membrane and the plasma membrane around an orifice through
which the granule contents are released (Palade, 1975).
Fusion of one granule membrane to an adjacent granule mem
brane may lead to discharge of two or more secretory gran
ules in series (Farquhar, 1971). The core material of the
granule maintains its structural integrity for a brief time
after release allowing ready identification of sites of exo
cytosis (Farquhar, 1961a). The final result of exocytosis
is a discharge of the hormone and a relocation of the gran
ule membrane to the plasma membrane. Subsequent internali
zation of the membrane appears to occur by the process of
endocytosis with recovery of the membrane in the form of
vesicles (Pelletier, 1973; Farquhar et al., 1975).
When hormone release is inhibited or unstimulated,
secretory granules accumulate in the cytoplasm. Lysosomes
incorporate and degrade the excess hormone in undischarged
granules (Smith and Farquhar, 1966; Pasteels, 1972;
Farquhar, 19 71).
44
MATERIALS AND METHODS
Collection of Tissues
Adult Sprague-Dawley female rats weighing 180 to 200
gm., purchased from the Holtzman Laboratories, were util
ized in this investigation. A minimum of two animals was
used in each experiment. For those experiments designed to
determine effects of differing immersion fixation proce
dures, rats were decapitated without anesthetic. The cal-
varium was rapidly removed and the brain reflected to allow
transection of the optic nerves in order to expose the
pituitary gland. Fixative was immediately flooded into the
floor of the cranial cavity, the dura encasing the gland
was removed with fine forceps, and the entire pituitary was
carefully elevated from the sella turcica on the flat sur
face of a small, round, scalpel blade and quickly placed in
fresh fixative in an acid-cleaned container. Care was taken
to keep the pituitary moistened by dripping fixative onto
the surface of the gland during the removal process. For
those experiments on the effects of perfusion fixation, rats
were anesthetized with nembutal. The thoracic cavity was
opened and fixative solution was perfused through the left
ventricle of the heart at a perfusion pressure of 100 mm of
Hg for four to five minutes and a total volume of 150 to 200
ml. of fixative. The pituitary was then excised and placed
in fresh fixative.
45
Basic Fixation Procedures
1. Basic fixation protocols
Two different basic fixation protocols were used, each
utilizing KSb as a precipitating agent:
a) Method 1 : Direct fixation in unbuffered 1%
osmium tetroxide-2% potassium pyroantimonate
solution.
b) Method 2 : Prefixation in 5% glutaraldehyde-
2% potassium pyroantimonate in 0.1 M potassium
phosphate buffer
Two brief rinses in the 2% potassium pyroantim-
onate-0.1 M potassium phosphate wash
Postfixation in the unbuffered 1 % osmium
tetroxide-2% potassium pyroantimonate
Prior to use, fixatives were cleared of any suspended
or precipitated pyroantimonate particles by centrifuging
at 10,000 RPM for 10 minutes in a Sorvall refrigerated cen
trifuge using a Type 30 rotor. The pH of the solutions was
always checked and readjusted if necessary just before use.
Fixation with the OsO^/KSb solution was always carried out
in the cold (4°C) in order to minimize tissue damage.
Tissues were always fixed in large volumes of the solu
tions. After fixation and prior to dehydration, tissues
were thoroughly rinsed three times in 2% collidine buffer
or a 7% sucrose solution for a total rinse time of 10 min
utes in order to prevent any nonspecific deposition of
46
potassium pyroantimonate precipitated in the alcohol used
for dehydration.
2. Preparation of solutions
Reagent grade chemicals, double glass distilled, de
ionized water, and acid cleaned polyethylene plasticware or
inert glassware were used for the preparation of all solu
tions. Potassium pyroantimonate, Lot #157-9, was obtained
from Polysciences, Inc.
a) Unbuffered 1% osmium tetroxide-2% potassium
pyroantimonate solution (OsOy j /KSb) :
50 ml. of 0.01 M acetic acid were adjusted to a pH
within the range of 7.4 to 7.8 by the dropwise addition of
0.1 M KOH. This solution was stirred and heated at 80°C to
90°C for 30 minutes, 1.5 gm. of potassium pyroantimonate
(KSb) were added, and the solution was heated and stirred
for at least an additional 30 minutes in order to complete
ly dissolve the KSb. To compensate for any losses during
heating, the volume was readjusted to 50 ml. by the addi
tion of H^O. The solution was cooled to room temperature
and then combined with sufficient crystalline osmium tetrox-
ide (OsO^) or 4% aqueous OsO^ to yield a final concentra
tion of 1% OsO^. The pH was adjusted to 7.4 to 7.8 by addi
tion of dilute acetic acid.
47
b) 5% glutaraldehyde-2% potassium pyroantimonate
in 0.1 M potassium phosphate buffer (Glu/KSb):
Stock solutions of 0.1 M K2HP0^ and 0.1 M KH2 PO^ were
prepared and stored in the refrigerator for several months.
For preparation of the fixative, 65 ml. of 0.1 M potassium
phosphate buffer were made up by the addition of sufficient
K2HP0 ^ stock solution to the KH2P0 ^ stock solution to yield
a pH between 7.5 and 7.6. This buffer solution was stirred
and heated at 80°C to 90°C for 30 minutes, 1.5 gm. of KSb
were added, and heating and stirring continued for 30 min
utes to dissolve the KSb. After cooling to room tempera
ture, the 65 ml. of total solution were divided into two
lots consisting of 45 ml. and 20 ml. each. To the 45 ml.
were added 5 ml. of 50% glutaraldehyde to yield a final
concentration of 5% glutaraldehyde. The pH of this solu
tion was adjusted with dilute HCl to 7.5 or 7.6.
c) 2 % potassium pyroantimonate in 0.1 M potassium
phosphate buffer (KSb/Phosphate wash):
The 20 ml. of phosphate buffer and dissolved KSb re
maining from the preparation of the Glu/KSb fixative were
reserved as a wash solution.
Variations in Fixation Protocols
1. Method 1 : Direct fixation in unbuffered OsO^/KSb
solution :
a) Variations in method and total time of immer-
sion fixation:
Pituitary glands were fixed for periods of time vary
ing from two hours to overnight. In some experiments,
vials containing the fixative and immersed tissue were
placed in an ice bath on a rotary shaker at slow speed for
a short (10-15 minute) period at the beginning of fixation
in order to aid penetration of the fixative. Tissues were
then placed in the refrigerator for the remainder of the
fixation time.
b) Addition of an osmoregulator :
In several experiments, 0.15 M sucrose was added to
the basic OsO^/KSb solution.
c) Storage of fixative solution:
The basic fixative with the addition of 0.15 M sucrose
was utilized either fresh or after refrigerated storage for
periods varying from several days to two months.
d) Handling of tissue :
1) Tissues were minced in the fixative, or 2) cuts
were made into, but not through, the gland, or 3) whole,
unsectioned glands were immersed in the fixative. In the
latter two procedures, pituitaries were cut into approxi
mately 1 mm.^ blocks during dehydration in 7 5% ethyl alcohol.
e) Vascular Perfusion:
Pituitaries were fixed with OsO^/KSb solution contain
ing 0.15 M sucrose by vascular perfusion, then immersed in
the same fixative for approximately two hours.
49
2. Method 2 : Prefixation in the Glu/KSb solution,
postfixation in OsO./KSb for two to four hours:
a) Variation in total prefixation time:
Pituitaries were prefixed by immersion for 15 minutes,
30 minutes, one hour, and two hours. Immediately after ex
cision, pituitaries from each rat were quickly sectioned in
the fixative into approximately eight blocks of tissue of
similar size, then placed in fresh fixative for the remain
der of the prefixation time.
b) Vascular perfusion :
Pituitaries were perfused, as described above, with
Glu/KSb solution, then immersed in the same fixative and
sectioned into small blocks of tissue and prefixed for an
additional 30 minutes.
Dehydration and Embedment
In all experiments, tissues were rapidly dehydrated in
ascending concentrations of ethyl alcohol (50%, 60%, 75%,
95%, and 100%), placed in propylene oxide and infiltrated
and embedded. Several different Epon 812 and Araldite 502
or 6005 epoxy resin mixtures or Spurr’s low viscosity medium
were utilized in order to determine those media with super
ior sectioning qualities when used for support of tissues
containing antimonate deposits. The following mixture
proved superior and therefore was used throughout the major
portion of the study : Epon 812, 25 ml.; DDSA, 55 ml.,
50
Araldite 50 2 , 15 ml., DMP-30, 5% by volume.
Sectioning and Staining
Tissue blocks were sectioned on a Porter Blum MT-2
ultramicrotome. Several blocks from each rat were sectioned
and sampled at several different levels from the surface of
the tissue, beginning with an area very near the surface
exposed directly to the fixative. Preliminary observations
showed that precipitates were often absent from the periph
ery of the block after Glu/KSb prefixation. Therefore,
sections taken for a comparison of results of different ex
periments were always taken from comparable areas well re
moved from the surface of each block of tissue.
Thin sections, with interference colors varying from
silver to pale gold, were collected on copper grids, viewed
unstained or lightly stained with lead citrate or with
uranyl acetate and lead citrate, and photographed with an
Hitachi 12A transmission electron microscope.
Analytical Procedure
The calcium chelator ethylene - glycol-bis (B-aminoethy1
ether) tetraacetic acid (EGTA) was used to test for the
presence of calcium within the electron-dense deposits in
the tissue. Adjacent serial sections were collected on
copper grids. Unstained control sections on one grid were
photographed. Adjacent sections on other grids were
immersed at 50° C for 20 minutes in either an aqueous
51
solution of 20 mM EGTA at pH 7.5 or in distilled water, and
then photographed.
52
RESULTS
Method 1 : Direct Fixation in Unbuffered Osmium Tetroxide
Potassium Pyroantimonate Solution
1. Results of Variations in Fixation Protocol:
a) Variations in total time of immersion fixation
in OsO/ | /KSb solution :
Optimal preservation of cellular structure with precise
localization of antimonate precipitates in cellular sites
was achieved with a total fixation time of two to four
hours and with an initial 10 to 15 minute gentle agitation
of the immersed tissues (Fig. 1). Prominent sites of depo
sition of the abundant, fine grained precipitates were
heterochromatin areas of the nucleus, Golgi saccules, and
mitochondria. No significant differences in cellular pre
servation or distribution of precipitates were noted within
the two to four hour interval. Overnight fixation, however,
resulted in considerable destruction of all membranous ele
ments (Fig. 2). While cellular and organellar spatial re
lationships were generally retained, along with some degree
of structural integrity of the contents of nuclei, nucleoli,
secretory granules, and mitochondria, there was an almost
complete loss of membranes delimiting the cells and the
cellular compartments. Antimonate precipitates were abun
dant but appeared to be specifically localized only to
heterochromatin areas of the nucleus and were diffusely
53
distributed throughout the remainder of the cell (Fig. 3).
b) Addition of an osmoregulator:
Increasing the tonicity of the OsO^/KSb fixative solu
tion by the addition of 0.15 M sucrose was beneficial in
the reduction of cellular swelling which occurred in cells
fixed in the basic solution alone.
c) Storage of the fixative:
Attempts to use OsO^/KSb fixative that had been stored
in the refrigerator for periods longer than a few days
often resulted in poor morphological preservation and in
consistent antimonate precipitate formation. In some sec
tions nuclear deposits were abolished and precipitate
grains were randomly deposited in the cytoplasm. Other
sections showed abundant, nuclear and cytoplasmic deposits
but with loss of structural detail, similar to that illus
trated in Figs. 2 and 3.
d) Handling of the tissues :
Procedures involving as little disturbance of the
tissue as possible resulted in the most successful fixation.
Mincing of the tissues resulted in considerable destruction
of cells at and beneath the cut surface. Pituitaries that
were fixed whole by immersion in the fixative and cut into
small pieces after hardening during dehydration showed over
all better retention of cellular and organellar integrity.
54
e) Fixation by vascular perfusion:
Vascular perfusion with freshly prepared OsO^/KSb solu
tion with 0.15 M sucrose (Fig. 4) generally resulted in
good preservation of cellular morphology and the same
pattern of distribution of antimonate precipitates as that
observed after immersion fixation of tissues in the same
fixative for two to four hours. However, some areas of
tissue showed the deposition of a heavy, diffuse background
precipitate.
f) Summary of results of variation in protocol:
Optimal results from the use of the OsO^/KSb fixative
were obtained by the use of the following protocol : Intact
pituitary glands were fixed by immersion in cold (4°C),
freshly prepared, unbuffered OsO^/KSb solution, pH 7.6 to
7.8 containing 0.15 M sucrose for a total fixation period
of two to four hours including an initial 10 to 15 minute
gentle agitation of the immersed tissue.
This procedure resulted in a consistent, reproducible
pattern of distribution of pyroantimonate precipitate at
discrete cellular sites in those areas of tissue that were
well preserved (Fig. 1). However, acceptable preservation
of cellular morphology was not consistently achieved. Some
blocks of tissue from each gland showed evidence of struc
tural damage. Cellular membranes of all types were suscep
tible to fragmentation. Limiting membranes of secretory
55
granules in particular exhibited discontinuities. (For ex
ample , see Fig. 7).
2. Cation Localization in GH and PRL Cells after
Direct Fixation:
Figures 5, 6, 7, and 8 are representative examples of
CH and PRL cells fixed directly in the OsO^/KSb solution.
Fine, granular, electron-dense precipitate was most clearly
visualized in unstained sections (Figs. 5, 6). However,
no differences in distribution or noticeable loss of pre
cipitate occurred after sections were contrasted either with
lead citrate or uranyl acetate and lead citrate (Figs 7, 8).
At low magnification, both CH and PRL cells exhibited abun
dant precipitate in nuclei and within Colgi saccules and
mitochondria with no significant variations between the two
cell types (Figs. 5, 6, 7, 8). Nuclei had a heavy deposi
tion of precipitate that was confined predominantly within
the areas of heterochromatin. Relatively little precipitate
was dispersed throughout the euchromatin areas and in the
perinuclear space of the nuclear envelope and was absent
from areas immediately adjacent to nuclear pores (Figs. 5,
9). In the nucleolus (Fig. 9) precipitate was absent from
the condensed components and confined to the nucleolar-
associated heterochromatin or to euchromatin channels within
the nucleolus.
Variable sized aggregates of fine precipitate were
56
present within dilated Golgi saccules (Figs. 5, 6, 7, 8,
10) while smooth and coated Golgi-associated vesicles often
contained a single, small antimonate particle (Fig. 10).
Immature secretory granules in the Golgi region were marked
by the presence of precipitate grains in the area between
the dense core of the forming granule and its enclosing
membrane (Fig. 10). Very fine grains were associated with
the limiting membranes of some mature GH (Fig. 11) and PRL
secretory granules but were never observed deposited within
the core material. Mitochondrial deposits, variable in
quantity from one mitochondrion to another, were usually
dispersed within the matrix with occasional depositions be
tween the cristal membranes (Fig. 10). Multivesicular
bodies consistently contained small grains of precipitate
within their internal small vesicles and a variable quantity
of precipitate within the matrix (Figs. 12, 13). Some
lysosomes exhibited no deposition while others showed moder
ate to dense accumulations of precipitate that was often
associated with membranous material (Fig. 5). A small
quantity of diffuse fine-grained precipitate was generally
present in the cytoplasmic matrix and in some cisternae of
rough endoplasmic reticulum (Fig. 14). No deposits of pre
cipitate were specifically localized to the cytoplasmic face
of the plasmalemma; however, clusters of precipitate some
times occurred in close approximation to its external sur
face (Fig. 14) .
57
Method 2 : Prefixation in Glutaraldehyde-Potassium Pyro
antimonate Solution
1. General Effects of Prefixation
Prefixation in the Glu/KSb solution followed by post
fixation in the OsO^/KSb solution consistently resulted in
improved cellular detail due to better preservation of
cytomembranes. However, distinct alterations in the
pattern of distribution of antimonate precipitate were
noted when these tissues were compared with those directly
fixed in the OsO^/KSb solution. These changes were most
evident in the nucleus, in the Golgi region, and in precip
itate localized to secretory granules. A systematic, com
parative study of cation localization in GH and PRL cells
prefixed for periods of time varying from 15 minutes to two
hours revealed that some of these alterations were time
dependent.
2. Effects of Varied Prefixation Times and Methods
on Cation Localization in GH and PRL Cells:
a) 15-minute prefixation:
Prefixation for 15 minutes resulted in the deposition
of fine-grained precipitate particles approximately the
same size as observed after direct fixation in OsO^/KSb
solution. However, use of the prefixation step produced an
ablation of much of the nuclear deposits, an alteration in
pattern of distribution of nuclear precipitate, and the
localization of coarser-grained and more abundant precipi
tate to the periphery of many GH and PRL secretory granules,
Precipitate localized to other organelles did not appear
significantly altered (Figs. 15, 16).
In the nucleus of both PRL and GH cells, precipitate
was dispersed almost exclusively in areas of euchromatin
(Figs. 15, 16), as opposed to the abundant precipitate in
heterochromatin areas observed after direct fixation. The
size of the aggregates varied from section to section in
dependent of cell type. Precipitate was also absent from
the nucleolar-associated chromatin and within the nucleolus
was deposited only in areas of euchromatin. The peri
nuclear space frequently contained small clusters of pre
cipitate (Figs. 15, 16).
Fine precipitate was deposited in Golgi saccules in
both GH (Fig. 15) and PRL cells (Fig. 16) in a pattern
closely resembling that observed after direct fixation in
the OsO^/KSb solution. Deposits localized to Golgi-
associated vesicles (Fig. 15), multivesicular bodies (Fig.
17), lysosomes and mitochondria (Figs. 15, 16) were also
essentially identical to those observed after direct fixa
tion. In addition, precipitate was clearly evident within
clusters of micro-vesicular and tubular structures near the
Golgi apparatus or near the plasma membrane (Fig. 18).
These deposits were also observed in cells fixed directly
59
with OsO ./KSb, but were not clearly displayed, perhaps be
cause of the generally less satisfactory preservation of
cellular membranes.
Some of the immature and mature PRL secretory granules
(Fig. 19) were ringed by precipitate that was 1arge-grained
when compared with that present after direct fixation in
OsO^/KSb. Many mature GH secretory granules were also en
circled by a discontinuous line of precipitate (Fig. 20).
Precipitate generally appeared to be associated with the
innermost aspect of the limiting membrane of both GH and
PRL granules; however, some deposits extended beyond the
contours of the granule and its membrane and could not be
strictly localized to either the inner or outer surface
(Figs. 19, 20). On rare occasions precipitate appeared in
creased in areas of close approximation of granules and the
plasma membranes (Fig. 20). At times, small vesicles,
occasionally containing precipitate, were observed in close
proximity to secretory granules or appeared to be in the
process of budding from or fusing with the granule membrane
(Fig. 21).
Cytoplasmic matrix deposits were variable from one
section to another but most commonly were minimal or absent.
Clusters of precipitate were present in some dilated
cisternae of rough endoplasmic reticulum but were generally
absent from flattened parallel lamellae (Fig. 19). As
60
after direct fixation, no precipitate was found associated
with the cytoplasmic surface of plasma membranes (Fig. 15,
20). A variable quantity of relatively coarse precipitate
was present in the extracellular space but never strictly
localized to the external face of the plasma membrane (Fig.
15) .
b) 30-minute to one-hour prefixation :
During a 30-minute to one-hour prefixation in the
Glu/KSb solution, there was further loss of nuclear deposits
and a considerable decrease in precipitate within Golgi
saccules. These shifts in distribution were accompanied by
an artifactual, random deposition of rounded masses of pre
cipitate (Fig. 22). Rather large clumps of precipitates
were widely dispersed, largely in the euchromatin of the
nucleus (Fig. 22) in place of the fine nuclear precipitate
observed after 15 minutes prefixation.
Golgi saccules in most GH and PRL cells contained no
precipitate (Figs. 22, 23). However, on rare occasions
coarse clumps of precipitate were localized in some saccules
(Fig. 24). In all other aspects, the pattern of distribu
tion of precipitate, including that of the nucleolus (Fig.
22), secretory granules (Figs. 22, 23, 24, 25), mitochondria
(Figs. 22, 23), rough endoplasmic reticulum (Fig. 23), lyso
somes and multivesicular bodies (Fig. 23) was similar to
that observed after 15 minutes prefixation. A fortuitous
61
tissue section from this group of animals showed a small
vesicle containing precipitate apparently either budding
from, or fusing with, the limiting membrane of a secretory
granule (Fig. 26).
c) Two-hour prefixation:
Results from prolonged exposure of the tissue to the
Glu/KSb solution (two hours) were uninterpretable due to
striking variations in the quantity and distribution of
precipitate from section to section and from cell to cell.
Some cells showed an almost complete absence of all cellu
lar precipitate, while adjacent cells were covered by a
diffuse, heavy precipitate (Fig. 27).
d) Vascular perfusion of the Glu/KSb solution :
Results of vascular perfusion of the Glu/KSb solution
were similar to those seen after prolonged prefixation in
the same solution. This method was generally unsatisfac
tory for obtaining specific localization of antimonate pre
cipitates .
3. Analysis of Antimonate Precipitates by Chelation
Exposure of thin sections of OsO^/KSb fixed tissue to
20 mM EGTA solution for 20 minutes resulted in an almost
complete disappearance of the electron-dense precipitates
(Figs. 28, 29). Exposure of thin sections of Glu/KSb pre
fixed tissues to the chelator at the same concentration for
the same period of time resulted in the extraction of most
62
of the antimonate deposits. However, a few precipitates
were resistant to the chelator. These rare, resistant pre
cipitates were localized to dilated cisternae of rough endo
plasmic reticulum and to mitochondria (Figs. 29, 30).
63
DISCUSSION
Results of this investigation demonstrate that the
pattern of distribution of pyroantimonate precipitate in GH
and PRL cells of the rat pars distalis varies depending
upon the primary fixative and the fixation protocol. No
significant differences were noted when tissues were fixed
directly in an unbuffered osmium tetroxide-potassium pyro
antimonate solution either by immersion for two to four
hours or by perfusion. However the size and location of
pyroantimonate deposits were profoundly influenced by pre
fixation in a glutaraldehyde-potassium pyroantimonate solu
tion. Moreover, alterations induced by the prefixation
procedure occurred as a function of time in the glutaralde
hyde -pot ass ium pyroantimonate solution and consisted pri
marily of a selective and progressive loss of pyroantimo
nate precipitates from areas of the nucleus and from Golgi
saccules.
In the selection of a potassium pyroantimonate tech
nique for cation localization, direct fixation in an un
buffered osmium tetroxide-potassium pyroantimonate solution
would a priori appear to be the preferred procedure. In
order to preserve the i j i vivo distribution of cations, the
pyroantimonate anion should penetrate the tissue rapidly to
immobilize cations at the same time that fixation occurs.
64
This is, of course, the rationale on which the original
Komnick methodology was based. Analysis of results from
many laboratories utilizing various modifications of
Komnick’s technique has conclusively demonstrated that the
presence of osmium tetroxide is essential as a minimum re
quirement for adequate penetration of the pyroantimonate
anion concomitant with adequate preservation of cellular
s trueture.
The presence of osmium tetroxide is apparently not
absolutely essential for the entry of the pyroantimonate
anion into cells since aqueous potassium pyroantimonate
used as both the primary fixative and the precipitating
agent prior to exposure of the tissue to glutaraldehyde,
can result in deposition of voluminous intracellular pre
cipitates (Tandler et al., 1970 ; Kiers zenbaum et al., 1971 ;
Tisher et al., 1972). However, cells treated with this
’’antimonate technique” exhibit obvious signs of damage.
Morphologic preservation is clearly inadequate for precise
localization of antimonate deposits. On the other hand,
glutaraldehyde as the primary fixative either limits the
entry of the anion or prevents its interaction with intra
cellular cations, so that tissues fixed in solutions con
taining glutaraldehyde and potassium pyroantimonate in the
absence of any osmium postfixation exhibit predominantly
extracellular precipitates with minimal or no intracellular
65
deposition of precipitate ( Zadunaislcy, 1966; Bulger, 1969 ;
Legato and Langer, 1969 ; Satir and Gilula, 1970 ; Sumi, 1971;
Garfield et al., 1972; Simson and Spicer, 1975; Stoeckel et
al., 1975). Osmium tetroxide apparently alters the perme
ability of the membrane allowing the rapid entry of the
pyroantimonate anion and under optimal conditions provides
good morphology and the localization of precipitate in dis
crete cellular sites. However, poor preservation has evi
dently been a frequent problem with procedures involving
fixation in osmium tetroxide-potassium pyroantimonate solu
tions . Difficulties similar to those described in the pres
ent study have been described by others (Clark and Ackerman,
1971a; Sumi, 1971 ; Herman et al., 1973 ; Sato et al., 1975)
and are suggested by the poor preservation of cytoplasmic
detail displayed in many of the electron micrographs of
Spicer and associates (Spicer et al., 1968; Spicer and
Swanson, 19 7 2).
Use of the OsO^/KSb procedure for cation localization
in GH and PRL cells in the present investigation resulted
in a consistent, differential and reproducible subcellular
distribution of pyroantimonate precipitate and produced the
same pattern of localization whether tissues were fixed by
immersion or perfusion. These results are in substantial
agreement with those of Schechter (1976) who used similar
procedures. Optimal results from this procedure therefore
66
serve as a standard for comparison with results of the
Glu/KSb prefixation procedures.
Alteration in the distribution of precipitate due to
initial prefixation in a glutaraldehyde solution is not an
unexpected result since similar observations have been re
ported from studies comparing effects of direct fixation
and prefixation techniques on cation localization (Clark
and Ackerman, 1971a,b; Herman et al., 1973; Simson and
Spicer, 1975). Not previously reported, however, is the
significant time - dependence of the alterations that occur
in response to the Glu/KSb prefixation observed in the
present investigation.
The solutions employed in the Glu/KSb prefixation pro
cedures closely match those utilized by Stoeckel and associ
ates (1974, 1975). However, the time sequence of their
fixation protocol was not reported. While their descrip
tions of cation localization in cells of both the posterior
and anterior pituitaries of the male rat and mouse do not
include descriptions of the distribution of precipitate in
GH and PRL cells specifically, their brief, general descrip
tion of cation localization in cells throughout the pars
dis tails and pars intermedia is consistent in most regards
with the present observations in GH and PRL cells after a
relatively brief prefixation. However no mention is made of any
localization of precipitate at the periphery of PRL
67
secretory granules. In the present study, the deposition
of relatively large - grained precipitate was often observed
at this site. At the same time a frequent association of
precipitate with GH secretory granules was noted by
Stoeckel and associates. The basis for this discrepancy
is unknown. Since their study was of pituitaries of male
animals and PRL cells were not specifically discussed, it
is impossible to say whether PRL granules in male rats do
not exhibit pyroantimonate reactivity or whether deposits
were present and simply overlooked by these investigators.
It is of interest to note that Stoeckel and coworkers
reported that precipitate within Golgi saccules varied from
none or minimal to abundant. Golgi-associated vesicles and
multivesicular bodies were consistently reactive. These
results support the observations of a time-dependent alter
ation in Golgi saccule localization during the Glu/KSb pre
fixation. Cations sequestered within Golgi saccules may
represent a more labile pool than those present in other
cellular compartments. It is also possible, however, that
glutaraldehyde interaction with Golgi saccule membranes in
well-fixed tissues immobilizes cations but prohibits their
interaction with the pyroantimonate in the OsO^/KSb solu
tion.
The distribution of pyroantimonate deposits primarily
in euchromatin areas of the nucleus after prefixation in the
68
Glu/KSb solution appears to be a generally observed con
sequence of prefixation procedures utilizing glutaraldehyde
(Clark and Ackerman, 1971a,b; Garfield et al., 1972; Herman
et al., 1973; Schafer and Kloppel, 1974; Stoeckel et al.,
1974, 1975 ; Simson and Spicer, 1975 ; Ravazzoli, 1976). It
appears to have gone unnoticed, or at least unmentioned in
the literature, that this pattern of distribution of pre
cipitate closely resembles that seen after application of
the "antimonate" techniques that utilize potassium pyro
antimonate as both the precipitating agent and the primary
fixative (Tandler et al., 1970; Kierszenbaum et al., 1971;
Tisher et al., 1972). This may indicate that the distribu
tion of precipitate in areas of heterochromât in seen after
fixation in osmium tetroxide-potass ium pyroantimonate is
not attributable to a direct interaction of the pyroantimo
nate anion with cations located in these areas but may be
secondary to some interaction requiring the presence of
osmium tetroxide at the time precipitation occurs.
The biochemical basis of these differing nuclear
patterns of distribution has not been completely defined.
Clark and Ackerman (1971b) , using a number of digestive and
blocking procedures as well as electron microprobe analysis,
have attributed the heterochromatin reactivity with osmium
tetroxide-potass ium pyroantimonate fixation to calcium
bound to nucleic acids as well as to reactive amino groups
69
on his tones. Glutaraldehyde appears to dissociate the cat
ions from the nucleic acid binding sites and to block the
reactivity of the ionized amino groups (Clark and Ackerman,
1971b) .
The greater reactivity of GH and PRL secretory gran
ules after glutaraldehyde prefixation may be attributable
to better preservation of the granule membranes. Others
have commented upon a noticeable fragility of membranes of
secretory granules of cells of the anterior pituitary gland
fixed in osmium tetroxide solutions and have noted that
these membranes are better preserved after glutaraldehyde
fixation (Siperstein and Miller, 1970; Costoff, 1973).
If a translocation of cations from original sites of
sequestration were to occur to a significant degree, one
would expect a random deposition of reaction product. The
random deposition of rounded clumps of precipitate after
prefixation for periods of 30 minutes or longer and the non
specific pattern of distribution of precipitate after two
hours prefixation would argue that such artifactual trans
location does occur during prolonged prefixation in glutar
aldehyde. In addition, glutaraldehyde apparently has
effects on cellular membranes so that after two hours pre
fixation it either prevents pyroantimonate interactions in
well-fixed cells or excludes the pyroantimonate from the
well-fixed cells exposed to the potassium pyroantimonate in
70
the osmium tetroxide post-fixâtion step. Results similar
to those of this investigation after two hours prefixation
in the Glu/KSb solution have been described by Simson and
Spicer (1975) in rat parotid gland after a 30 minute pre
fixation with buffered glutaraldehyde but no exposure to
potassium pyroantimonate until the osmium tetroxide post
fixation step. While this has not been tested in the pres
ent investigation by the utilization of glutaraldehyde
alone during prefixation, a comparison of the results of
this study with those of Simson and Spicer (1975) implies
that the presence of potassium pyroantimonate in the glutar
aldehyde is of some benefit.
Results of EGTA chelation indicate that divalent cat
ions are major components of the pyroantimonate deposits
seen after either direct fixation in the OsO^/KSb solution
or after the Glu/KSb prefixation procedure. The relatively
high specificity of EGTA for calcium (Ebashi and Endo, 1968;
Ogawa, 1968) would seem to indicate that the precipitate is
primarily calcium pyroantimonate; however, EGTA in the
presence of a large excess of tissue calcium will also
chelate magnesium (Bjerrum et al., 1957), and on the basis
of this evidence alone, it is impossible to state that the
observed removal of precipitate is due only to chelation of
calcium. However, the possibility that sodium is localized
to areas where precipitates are removed can be excluded with
71
more confidence. vitro, EGTA chelates calcium from cal
cium antimonate precipitates formed when ions are added to
a pH controlled potassium pyroantimonate solution but does
not chelate sodium from sodium antimonate precipitates
(Saetersdal et al., 1974). It can be concluded that sodium
was not precipitated in the GH and PRL cells of this study.
Failure to precipitate significant amounts of sodium antim-
onate in GH and PRL cells by either of the fixation methods
is somewhat surprising but may be related to a weak or un
stable interaction of sodium with the potassium pyroantimo
nate so that much of the sodium is not preserved during
tissue processing (Hartmann, 1966; Garfield et al., 1972;
Tisher et al., 1972; Chandler and Battersby, 1976).
Evidence indicates that much of the cellular calcium
is retained in tissues fixed in the presence of potassium
pyroantimonate. This has been demonstrated by analysis of
tissues and fixative solutions by atomic absorption spectro
photometry (Garfield et al., 1972) and comparative X-ray
fluorescence analysis of fixed and unfixed tissues (Stoeckel
et al., 1975). Furthermore, Stoeckel et al. (1975), using
X-ray microprobe analysis, found that calcium was the major
component of intracellular precipitates in anterior pitui
tary cells reacted with potassium pyroantimonate solutions.
Localization of calcium deposits to the Golgi region was
confirmed by Schechter (1976) with microprobe analysis.
72
There is very little evidence from biochemical measure
ments of calcium in isolated subcellular fractions from
pituitary cells to corroborate the results obtained from
the use of potassium pyroantimonate techniques in the same
tissue. However, the distribution of pyroantimonate
deposits in GH and PRL cells observed in the present in
vestigation is consistent with intracellular sites of cal
cium sequestration known to exist from application of bio
chemical techniques to other cell systems. The results are
in general agreement with the known characteristics of cal
cium accumulation by mitochondria and endoplasmic reticulum
(microsomal fractions) from a variety of mammalian tissues
and with the known large ratio of membrane-bound or seques
tered calcium to free cytoplasmic calcium. The only direct
biochemical evidence of calcium binding sites in either GH
or PRL cells is contained in a single study demonstrating
that isolated PRL secretory granules contain a small but
measurable amount of calcium (Zanini and Giannattasio,
1974). In a separate study, magnesium was not found to be
present at detectable levels in isolated granule fractions
that contain both GH and PRL secretory granules (Hymer and
McShan, 1963) . Data from these two studies combined with
the present results from EGTA analytical procedures indicate
that precipitate localized to secretory granules of PRL and
probably GH cells most likely consists of calcium antimonate.
73
An association of calcium with secretory granules of
both endocrine and exocrine cells does not appear to be an
uncommon phenomenon. Use of the pyroantimonate technique
coupled with X-ray microprobe analysis has allowed the
identification of heavy deposits of calcium antimonate
within the halos of secretory granules of pancreatic islet
cells (Herman et al., 1973) and also the identification of
calcium antimonate associated with the membranes of zymogen
granules of the exocrine pancreas and of the parotid gland
(Clementi and Meldolesi, 1975). In addition, both norepin
ephrine- and epinephrine-storing granules of adrenal
chromaffin cells are major intracellular storage sites for
calcium as demonstrated by the use of the potassium pyro
antimonate technique (Ravazzola, 1976) and cell fractiona
tion studies (Borowitz et al., 1967). Secretory granules
in subcellular fractions from the posterior pituitary gland
also have a high calcium concentrât ion (Thorn et al., 1975).
There appears to be remarkably good correspondence of
results when a variety of techniques for the demonstration
of cellular calcium is applied to identical tissues or
similar tissues from closely related species. In these
cases it has been possible to compare patterns of distribu
tion of antimonate precipitate with calcium distribution in
subcellular fractions or in freeze-dried sections of unfixed
tissue. For example, Clementi and Meldolesi (1975) in their
74
study of the rat exocrine pancreas combined potassium pyro
antimonate cytochemical techniques and analysis of ^^Ca up
take in subcellular fractions and reported substantial
agreement in results from both procedures. In addition,
observations of Howell and associates (19 75) from X-ray
microprobe analysis of frozen sections of unfixed rat endo
crine pancreas and analysis of ^^Ca uptake in isolated sub
cellular fractions are in good agreement with the subcellu
lar localization of pyroantimonate precipitate in mouse
endocrine pancreas (Herman et al., 1973; Schafer and
Kloppel, 19 74).
In conclusion, potassium pyroantimonate techniques can
be useful for the identification of intracellular struc
tures or organelles that possess the ability to sequester
calcium. Several organelles in GH and PRL cells possess
the capacity to bind the pyroantimonate reaction product,
probably consisting primarily of calcium antimonate. Prom
inent among these cytoplasmic structures are Golgi saccules
and vesicles, mitochondria, secretory granules, and multi-
vesicular bodies and other lysosomal elements. The fact
that the pattern of localization of pyroantimonate precipi
tate to cytoplasmic sites observed after 15 minutes prefix
ation in the Glu/KSb solution closely resembles that seen
after direct fixation in the OsO^/KSb solution supports the
validity of the localization obtained by this former method.
75
In addition, the 15 minute prefixation procedure has the
obvious advantage of enhanced preservation of cellular
structure. Use of either of these fixation procedures may
be a valuable tool in the study of the role of calcium in
secretory mechanisms as long as comparable fixation con
ditions are strictly maintained for all experimental manip
ulations. Longer periods of prefixation in the Glu/KSb
solution leads to a loss of reaction product or pyroantimo
nate reactivity and a random deposition of precipitates
and should be avoided.
76
SUMMARY
The subcellular distribution of cation precipitates
was examined in GH and PRL cells of the rat pars distalis
through the use of different fixation procedures employing
potassium pyroantimonate as the precipitating agent.
Tissues were either (1) fixed directly in osmium tetroxide-
potassium pyroantimonate solutions (OsO^/KSb) for periods
varying from two hours to overnight or (2) prefixed in a
glutaraldehyde-potassium pyroantimonate solution (Glu/KSb)
for periods varying from 15 minutes to two hours and post
fixed in OsO^/KSb solution.
(1) Direct fixation in OsO^/KSb for two to four hours
often yielded abundant precipitate at specific cellular
sites (notably in nuclei, Golgi saccules, mitochondrial
matrix, multivesicular bodies, and membranes of secretory
granules) but did not consistently produce acceptable pre
servation of cellular details.
(2) The use of glutaraldehyde (Glu/KSb) consistently
improved cellular preservation and revealed precipitate
associated with secretory granule membranes more clearly
than did the use of OsO^/KSb. However, increasing the
times of exposure of the tissue to the glutaraldehyde solu
tion resulted in a selective and progressive loss of cation
précipita Les from nuclei and Golgi saccules despite the
presence of KSb in the fixative. Results of prolonged
77
exposure (two hours) were uninterpretable due to striking
variations in quantity and distribution of precipitates.
Precipitates were almost totally lacking in some cells
while adjacent cells contained diffuse heavy precipitate.
Results of these experiments suggest that a very short pre
fixation time (15 minutes or less) in Glu/KSb can enhance
the preservation of cell features and still keep to a mini
mum the loss of cation precipitate from previously estab
lished sites of localization.
78
CHAPTER II
A COMBINED ULTRASTRUCTURAL AND POTASSIUM PYROANTIMONATE
CYTOCHEMICAL STUDY OF PROLACTIN CELLS AT VARIED STAGES
OF SECRETORY ACTIVITY
INTRODUCTION
Hypothalamic Control of Prolactin Secretion
The major influence of the hypothalamus upon the
secretion of PRL in mammalian species appears to be inhibi
tory in nature. Rat anterior pituitary glands isolated
from neurovascular linkage to the hypothalamus possess the
capacity for autonomous secretion of PRL both i j i vivo and
in vitro. Transplantation of the gland to extracranial
sites (Everett, 1954, 1956 ; Nikitovitch-Winer and Everett,
1958; Chen et al., 1970), transection of the pituitary
stalk (Nikitovitch-Winer, 1965), and lesions of the median
eminence (Chen et al., 1970; Bishop et al., 1971), result
in increased PRL secretion. Organ culture and short-term
incubation experiments have demonstrated that autonomous
secretion of PRL occurs in substantial amounts iji vitro
(Meites et al., 1961 ; Pasteels , 1961a). The presence of a
substance in the hypothalamus inhibitory to PRL secretion
has been demonstrated by exposure of the pituitary to
79
hypothalamic tissue or extracts by various means : by
transplantation of the gland to the sella turcica of a
hypophysectomized rat (Nikitovitch-Winer and Everett, 1958),
by injection of rat hypothalamic extract into lactating
(Grosvenor et al., 1965; Kuhn et al., 1974), proestrous
(Amenomori and Meites, 1970), or normal male rats (Watson
et al., 1971), by the infusion of rat hypothalamic extract
into pituitary portal vessels (Kamberi et al., 1971b), and
by the addition of hypothalamic tissue fragments or extract
to the cultured or incubated gland (Pasteels, 1961b, 1962;
Talwalker et al., 1963; Gala and Reece, 1964). These ob
servations and numerous others (Meites et al., 1972; Meites
and Clemens, 1972; Neill, 1974) indicate that the hypothal
amus-median eminence region produces a substance which
reaches the anterior pituitary gland by the hypothalamic-
pituitary portal veins and inhibits PRL secretion. This
agent has been termed "prolactin-inhibiting factor" (PIP)
(Talwalker et al., 1963). Its exact chemical nature is
still unknown although there is a growing body of evidence
implicating the catecholamine, dopamine, as a possible
physiological PIP.
Alterations in hypothalamic PIP activity occur in
response to a variety of physiological stimuli as well as
pharmacological agents that alter the hypothalamic content
of catecholamines. Considerable experimental evidence has
80
demonstrated a reciprocal relationship between hypothalamic
catecholamine content and PIF activity of the hypothalamus
and serum PRL levels. Among those agents that have been
shown to depress PRL secretion and to increase catechol
amines and PIF activity are PRL itself (Chen et al., 1967;
MacLeod and Lehmeyer, 1974b), ergot alkaloids (Wuttke et
al., 1971; Meites et al., 1972), the catecholamine precur
sor L-Dopa, and monoamine oxidase inhibitors (Donoso et al.,
1971; Lu and Meites, 1971, 1972). Agents demonstrated to
increase PRL release and to decrease catecholamine and PIF
activity include the suckling stimulus (Ratner and Meites,
1964; Meites et al., 1972), stress (Nicoll et al., 1960;
Meites, 1970; Meites et al., 1972), estrogens (Ratner and
Meites, 1964; Meites et al., 1972), reserpine (Ratner et
al., 1965; Lu et al., 1970), and catecholamine synthesis
inhibitors a-methyl para-tyrosine (aMPT) and methyldopa
(Lu et al., 1970; Donoso et al., 1971; Lu and Meites, 1971;
Meites, 1970) .
Coppola and associates (1965) proposed that hypothal
amic catecholamines, specifically norepinephrine, con
trolled the synthesis of PIF. However, since that time,
substantial evidence has accrued that PRL secretion is reg
ulated through an inhibitory dopaminergic pathway. The
median eminence is rich in dopaminergic nerve terminals of
hypothalamic tubero-infundibular neurons (Fuxe and Hdkfelt,
1966) whose activity is influenced by such states of en
hanced endocrine activity as pregnancy and lactation (Fuxe
et al., 1969). Implantation of reserpine in the basal hypo
thalamus caused depletion of monoamines in this region and
increased the secretion of PRL as indicated by the induc
tion of pseudo-pregnancy (van Maanen and Smelick, 1968).
Complete deafferentation of the medial basal hypothalamus
caused depletion of norepinephrine in the isolated island
while dopamine content was unaltered (Weiner et al., 1972).
These dopaminergic neurons appeared to be those involved in
the tonic inhibition of PRL secretion since the PRL
response of completely deafferented rats to aMPT (Weiner,
1973), reserpine (Krulich et al., 1975), or L-Dopa (Krulich
et al., 1975) was unaltered from that in control rats.
Kamberi and associates (1971a,c) reported that direct per
fusion of the pituitary through a portal vessel with large
doses of dopamine, norepinephrine or epinephrine was
without effect on serum PRL. However, infusion of dopamine
into the third ventricle of rats evoked an increase in PIF
activity in portal blood and a decrease in serum PRL.
Dopamine was much more potent than either norepinephrine or
epinephrine. Moreover, Donoso and associates (1971) used
drugs that specifically augmented or inhibited dopamine
synthesis to demonstrate that alterations in dopamine and
not in norepinephrine or epinephrine were associated with a
82
converse change in PRL release.
These observations supported the concept advanced by
Lu and Meites (1972) and Kamberi and coworkers (1971a,b,c)
that dopamine acts as a neurotransmitter to increase the
activity of PIF in the hypothalamic-pituitary portal blood.
van Maanen and Smelick (1968) were the first to sug
gest that dopamine itself could be PIF, citing morphologi
cal evidence that the transmitter substance of the tubero-
infundibular dopaminergic system could be released directly
into the portal circulation (Sano et al., 1967). By radio-
enzymatic assay, dopamine has been detected in the portal
blood of female rats at levels sufficient to indicate a
role for dopamine in the regulation of anterior pituitary
function; moreover, dopamine was the only catecholamine
found in the portal blood (Ben-Jonathan et al., 1976).
Convincing evidence has established that dopamine is able
to exert a direct effect on pituitary secretion of PRL
both iui vitro and iui vivo. Reports of Birge and associates
(1970) and MacLeod and associates (1970) demonstrated that
dopamine in a concentration of 10 ^ M (1 yg-5 pg/ml) caused
a profound inhibition of the release of PRL from pituitary
glands iji vitro, accompanied by an increase in PRL re
tained within the gland. Norepinephrine (Birge et al.,
1970; MacLeod et al., 1970) and epinephrine (Birge et al.,
1970) were also inhibitory in vitro. These reports were
83
confirmed by Koch and colleagues (1970) ; however the latter
investigators observed that smaller doses of norepinephrine
or epinephrine (10-20 ng/ml), which are within the concen
tration range of catecholamines present in the hypothalamus,
stimulated PRL release, while similar concentrations of dop
amine had no effect. These reported effects of smaller
doses have not been confirmed by other investigators. In
deed Shaar and associates (Shaar et al., 1973; Shaar and
Clemens, 1974) demonstrated that very small quantities of
dopamine and norepinephrine at a concentration at, or below,
their hypothalamic concentration showed a marked inhibitory
effect iui vitro. Doses of dopamine from 1 to 100 ng. per
- Q - 7
ml. (10 to 10 M range) significantly inhibited PRL re
lease in a dose-related response. Dopamine was a more po
tent inhibitor than either norepinephrine or epinephrine.
_ 7
MacLeod and Lehmeyer (1974a) reported that dopamine at 10
M (76 ng/ml) always produced a large decrease in PRL secre
tion. MacLeod (1976) has suggested that lower doses were
less effective because of pituitary monoamine oxidase and
also because of active oxidation of dopamine by atmospheric
oxygen. Contrary to the earlier observations of Kamberi et
al. (1971a), recent reports of Takahara and colleagues
(1974a,b) have demonstrated a direct effect of dopamine when
perfused into a pituitary portal vessel in a solution con
taining glucose to protect the catecholamine from oxidation.
Further evidence for a direct effect of dopamine iai
84
vivo has been presented in a series of experiments by
Donosa and associates. L-Dopa was able to decrease plasma
PRL in rats with median eminence lesions that interrupted
the central nervous system (CNS) control of PRL release.
L-DOPA was still effective although its conversion to nor
epinephrine was blocked. This suggests that L-Dopa convert
ed to dopamine acted directly at the pituitary level to in
hibit PRL release (Donoso et al., 19 73). Additional exper
iments were performed with rats bearing pituitary grafts
under the kidney capsule (Donoso et al., 1974). Administra
tion of L-Dopa produced the expected decrease in plasma PRL.
Pre-treatment with L-a-methyldopa hydrazide, which specific
ally blocks the peripheral conversion of L-Dopa to dopamine
but allows dopamine to accumulate in the CNS, prevented the
expected decrease in PRL in response to L-Dopa. This indi
cates that dopamine was not acting at the hypothalamus to
release PIF, but was acting at the level of the grafts to
directly inhibit the release of PRL.
The concept that hypothalamic dopamine inhibits PRL
secretion by a direct action in the pituitary has been
further strengthened by the demonstration that drugs that
affect dopaminergic receptors have effects on the anterior
pituitary gland. This argues for the presence of pituitary
dopamine receptors. Thus, the dopamine receptor stimulator,
apomorphine, inhibited PRL secretion both in vivo (Smalstig
et al., 197 4) and dji vitro (MacLeod and Lehmeyer, 1973,
1947a; Smalstig et al., 1974); haloperidol, a catecholamine
receptor blocker, was able to overcome the dopamine inhib
ition of PRL release in vitro (Quij ada et al., 19 73) ;
pimozide, a specific dopamine receptor blocker, was able to
reduce the inhibitory action of apomorphine iji vitro
(Smalstig et al., 19 74); and pimozide implants in the an
terior pituitary as well as in the median eminence - arcuate
region evoked an increase in plasma PRL iji vivo (Ojeda et
al., 1974).
Although catecholamines, particularly dopamine, can
inhibit PRL release by a direct action on the pituitary,
they may not represent the only PIF activity in the hypo
thalamus. Schally and associates (1976a) recently reported
the purification from porcine hypothalami of a catechol-
amine-rich fraction which powerfully inhibited the release
of PRL. However, other types of fractions with very
different physio-chemical characteristics also inhibited
PRL release (Schally et al., 1974), including a fraction
later identified as gamma-aminobutyric acid (Schally et al. ,
1976b) . Fractions with PIF-activity separate from cate
cholamine-containing fractions have been isolated from
porcine (Griebrokk et al., 1974), ovine (Dhariwal et al.,
1968) and beef hypothalami (Dular et al., 1974). Further
more it appears that the rat hypothalamus liberates a
86
substance with PIF activity in response to dopamine in
vitro. Quij ada and colleagues (1973) used the dopamine
blocker, haloperidol, to demonstrate that this activity was
not due to dopamine itself. Therefore, while it has been
well-documented that dopamine is a possible physiological
PIF, the inhibitory control of PRL release is a complex
mechanism involving a number of factors whose interrelation
ships are not yet completely understood.
Moreover, in recent years it has become clear that the
release of PRL is governed by both inhibitory and stimula
tory factors from the hypothalamus and that the circulating
level of PRL i j i vivo is a resultant of both PIF and PRF
(prolactin-releasing factor) activity. Meites and his
associates (1960) reported that injection of crude extracts
of rat hypothalamic tissue could evoke mammary secretion in
estrogen-primed rats. This report was later confirmed by
Mishkinsky and coworkers (1968) who further noted that
hypothalamic extracts from lactating rats had a greater
effect than those from non-lactating females. This was
interpreted as an indication of the presence of PRF in the
extracts. Everett (1964) and Grosvenor and colleagues
(1967) proposed the existence of PRF to explain the very
rapid discharge of PRL occurring in response to the suck
ling stimulus.
A stimulatory effect of rat hypothalamic extracts on
87
PRL release i j i vitro was first reported by Nicoll and asso
ciates (1970) who observed an increase in PRL release sub
sequent to an initial four-hour inhibition of release from
pituitaries incubated in the presence of the extracts. The
presence of PRF activity in the median eminence area has
been demonstrated by assay of sections of rat hypothalami
(Krulich et al., 1971). Moreover, Milmore and Reece (1975)
have reported that the same hypothalamic extract could
either stimulate or inhibit PRL release depending upon the
experimental conditions (i.e., the physiological state of
the rat).
Although not yet completely substantiated, evidence
supports the concept that thyroid stimulating hormone-
releasing hormone (TRH) represents a functional PRF (Bowers
et al., 1973). It has been convincingly demonstrated that
TRH evokes the release of both TSH and PRL from pituitaries
of several species, including man (Jacobs et al., 1971;
Bowers et al., 1973). Elucidation of the effect of TRH on
PRL secretion in rats has been more difficult. No, or min
imal, effects of TRH have been demonstrated on PRL secre
tion i j i vitro from normal pituitaries (Lu et al., 1972;
Vale et al., 1973). This is perhaps related to an inabil
ity of TRH to further stimulate a release that is already
proceeding at a high rate (Meites et al., 1961; Pasteels,
- 9
1961a). However, TRH, at a concentration as low as 10 M,
stimulated the secretion of PRL from rat pituitary tumor
cells (Tashjian et al., 1971) or pituitaries of hypothyroid
rats (Vale et al., 1973) iii vitro. In addition, intravenous
administration of TRH caused a significant rise in serum PRL
levels in normal male rats (Mueller et al., 1973), estrogen
or estrogen-progesterone treated male rats (Mueller et al.,
1973 ; Rivier and Vale, 1974), and proes trous (Mueller et al. ,
1973; Blake, 1974) or lactating rats (Blake, 1974).
Rather large doses of TRH (100-1000 ng) infused into
the portal vessels raised serum PRL levels ; however, the
response was not dose - related and smaller doses were in
effective. These experiments, nevertheless, provided evi
dence for a direct effect of TRH on the rat pituitary in
vivo (Takahara et al., 1974c). Furthermore, it appears
that PRL cells have functional TRH receptors (Rivier and
Vale, 1974) whose concentration can be modulated by estro
gens or thyroid hormones (DeLean et al., 1974). Chen and
Meites (1975) have recently presented results of experi
ments suggesting involvement of a serotoninergic pathway in
the TRH-mediated release of PRL and TSH.
An ability of TRH and dopamine to interact on the
release of PRL has been demonstrated both iui vivo and dji
vitro. Pretreatment of male rats with L-Dopa prevented
the TRH-evoked rise in serum PRL but not the elevation of
serum TSH. This indicated that L-Dopa interfered with the
89
ability o£ TRH to stimulate the release of PRL but did not
influence TRH effects on TSH cells (Chen and Meites, 1975).
Conversely, addition of TRH to the incubation medium re
sulted in blockade of the dopamine inhibition of PRL re
lease iji vitro (Hill-Samli and MacLeod, 1974), an interac
tion analogous to that between haloperidol and dopamine
(MacLeod and Lehmeyer, 1974a). TSH also exhibited an abil
ity to block the inhibition of PRL release in response to
the dopamine receptor stimulators apomorphine or ergocryp-
tine iji vitro (Hill-Samli and MacLeod, 1975).
As the exact relationship between dopamine and PIF
remains undefined, so the relationship between TRH and PRF
is unclear. Evidence for the existence of a PRF distinct
from TRH has come from several different laboratories. Ex
tracts of porcine (Valverde-R. et al., 1972; Szabo and
Frohman, 1976), rat (Valverde-R. et al., 1972; Rivier et
al., 1976), and beef hypothalami (Dular et al., 1974), con
tain PRL-releasing factors distinct from TRH and other
recognized hormones of the hypothalamus. Moreover, in
several situations, including the proes trous surge of PRL
and the response to either stress or the suckling stimulus,
acute alterations in PRL secretion are dissociated from
changes in TSH secretion (Blake, 1974). Though there is
ample evidence for the existence of PRF and though it has
been convincingly demonstrated that TRH causes release of
90
PRL, the significance of TRH in the physiological control
of PRL secretion has not been fully clarified.
TRH has been shown to stimulate cAMP accumulation in
pituitary tissue (Bowers, 1971; Labrie et al., 1975) and in
clonal strains of pituitary cells that secrete GH and PRL
(Dannies et al., 1976), and it appears likely that the
action of TRH involves binding of the peptide to a plasma
membrane receptor and the subsequent activation of the
adenylate cyclase-cAMP system (Labrie et al., 1975).
Cyclic AMP derivatives, dibutyryl cAMP (Nagasawa and Yanai,
1972; Wakabayashi et al., 1973; Sundberg et al., 1976) and
N ^-monobutyryl cAMP and other N^- and C^- derivatives
(Lemay and Labrie, 1972; Pelletier et al., 1972) markedly
enhance the release of prolactin iai vitro. These effects
of cAMP derivatives could be duplicated by the addition of
the phosphodiesterase inhibitor, theophylline, to the
medium (Parsons and Nicoll, 1971; Wakabayashi et al., 1973;
Sundberg et al., 1976). Administration of the N^-mono-
butyryl derivative (5mM) stimulated the release of PRL
within five minutes as revealed by the measurement of the
hormonal content of the medium and ultras truetural evidence
of increased exocytotic activity (Pelletier et al., 1972).
Studies of the interactions of hypothalamic extract
and cAMP or theophylline demonstrated that cAMP was able to
reverse the inhibition of PRL release induced by the
91
extract (Parsons and Nicoll, 1971; Sundberg et al., 1976).
However, cAMP appeared to influence PRL secretion via a
different mechanism from that of the PIF activity of the
extract, since the inhibitory activity of the extract was
still expressed in the presence of cAMP. Tlius pituitary
explants incubated in medium containing extract and
theophylline or cAMP secreted PRL at a rate similar to con
trol rates, not at the elevated levels evoked by the
theophylline or cAMP (Parsons and Nicoll, 1971).
These observations support the concept that the adenyl
ate cyclase-cAMP system is involved in the control of the
activity of prolactin cells, and that cAMP may act as the
intracellular mediator of the PRF stimulation of PRL
release.
92
MATERIALS AND METHODS
General
Adult female Sprague-Dawley rats weighing 180 to 200
gm., purchased from the Holtzman Laboratories, were used
throughout these experiments. Rats were housed in group
cages at 25°C with access to laboratory chow and water ad
1ibitum and with a controlled 14 hour, 10 hour light
darkness cycle for at least two weeks before use. In order
to eliminate an effect of diurnal variation, experiments
were always begun between 8:00 and 9:00 A.M.
Incubation of Hemipituitaries
Rats were decapitated without anesthetic, and pitui-
taries were rapidly excised and placed in small Petri
dishes containing incubation medium equilibrated with
9 5% 0^ and 5% CO2 at room temperature. Posterior pitui-
taries were removed and each anterior pituitary was care
fully sectioned into two identical halves. One half then
served as a control and the corresponding half served as
experimental. Each hemipituitary was placed in a 10 ml.
siliconized beaker containing 1 ml. of incubation medium
equilibrated with 95% O 2 and 5% CO2 at room temperature.
Three pituitaries were similarly prepared for each experi
mental situation. Two populations of hemipitui Laries
93
(controls and matched experimentals) were thus obtained,
matched in origin. Tissues were incubated in a Dubnoff
metabolic shaker at low speed, at 37°C, and in a water
saturated atmosphere of 95% O2 and 5% CO^. A preincubation
for 30 to 60 minutes was routinely done in order to allow a
period of time for adjustment and for release of hormone
from injured cells. Incubation media was then decanted and
replaced with 1 ml. of fresh media (controls) or with 1 ml.
of fresh media containing either 5 mM -monobutyryl cAMP
or 10 ^ M dopamine (expérimentais). Reservoirs containing
the fresh media for exchange were always kept in the
Dubnoff metabolic shaker. The duration of the incubation
period varied within the series of experiments and will be
indicated for each experiment.
Incubation Medium
The incubation medium utilized in all experiments was
Krebs - Ringer bicarbonate solution with 200 mg% glucose
(KRBG) as described by Umbreit et al. (1972). This medium
contains: 116 mM NaCl; 4.7 mM KCl, 2.5 mM CaCl2 ; 1.2 mM
KIl2P0^ ; 1.1 mM MgSO^ ; and 2.5 mM NaHCO^- Fresh medium was
prepared from stock solutions of the salts. The solution
was gassed with 9 5% O2 and 5% CO2 for 30 minutes at room
temperature and the pH adjusted to 7.35 just before use.
Viability of Pituitary Cells in Vitro and
Selection of Fixation Procedure
Preliminary experiments were run to assess the
94
viability of pituitary cells incubated in KRBG for periods
of time varying from five minutes to six hours, and to
select a potassium pyroantimonate fixation technique suit
able for incubated tissues. In order to assess the viabil
ity of cells, hemipituitaries were fixed in 2% glutaralde-
hyde, 2% paraformaldehyde, in 0.1 M cacodylate buffer with
0.02% CaCl2 , pH 7.3 for two hours, then postfixed in 1%
OsO^ in 0.1 M cacodylate with 0.02% CaCl2 , pH 7.3. Tissues
were then dehydrated in a graded ethanol series, followed
by propylene oxide rinses and embedded. Tissue blocks were
sectioned and checked for quality of morphologic preserva
tion by light and electron microscopy. In order to select
a potassium pyroantimonate technique, samples of tissues
incubated for varying periods of time were fixed by either
the direct OsO^/KSb procedure or prefixed in Glu/KSb for 15
minutes and postfixed in OsO^/KSb. Results from these pre
liminary experiments indicated that reasonably good cellu
lar morphology and integrity was maintained when tissues
were incubated for periods of three hours. Longer incuba
tions resulted in extensive necrosis of the tissue. The
direct OsO^/KSb fixation procedure was unsatisfactory for
incubated tissues due to considerable damage to cellular
membranes even at shorter incubation times. Prefixation in
Glu/KSb considerably improved cellular preservation.
95
Cyclic AMP Stimulation of Prolactin Secretion
Hemipituitaries were incubated for five minutes, 15
minutes, 30 minutes, and one hour, in 1 ml. of freshly
prepared KRBG with 5 mM -monobutyry1 cyclic adenosine
monophosphate (mbcAMP) (Sigma Co.). Control halves were
incubated for the same intervals of time in 1 ml. of KRBG.
At the end of the incubation period the medium was withdrawn
and placed in test tubes and immediately frozen for later
radioimmunoassay of PRL. Upon removal of the medium, pitui
taries were quickly immersed in the Glu/KSb solution,
minced, and prefixed for 15 minutes, rinsed, then postfixed
in the OsO^/KSb solution, dehydrated and embedded as
described in Chapter I.
Dopamine Inhibition of Prolactin Secretion
Hemipituitaries were incubated in 1 ml. of KRBG con
taining 10 ^M dopamine hydrochloride (Sigma Co.) with
•7
10 M dithiothreitol as an anti- oxidant for periods of
one, two, and three hours. Matched halves were incubated
in 1 ml. of KRBG for the same time periods. Media was with
drawn at the end of each incubation period and rapidly
frozen for later radioimmunoassay. Pituitaries were fixed
in Glu/KSb, postfixed in OsO^/KSb, dehydrated and embedded
as described above.
Radio immuneas say of Prolactin
PRL activity in individual media samples was measured
96
by radioimmunoassay in collaboration with Dr. Richard Weiner,
Department of Obstetrics and Gynecology, University of
California, San Francisco. Each sample was assayed at two
or three dilutions, averaged, and expressed in terms of the
purified PRL reference standard (NIAMD-Rat Prolactin-RP-1)
obtained from the National Institutes of Health, Rat Pitui
tary Hormone Distribution Program.
Semiquantitative Estimation of the Concentration of
Antimonate Deposits
A semiquantitative estimation of the concentration of
antimonate deposits in mitochondria, Golgi saccules and
associated with PRL secretory granules, was made by deter
mining the number of precipitate particles at these sites.
A minimum of six blocks of tissue representing each experi
mental and control stage were thin sectioned. Sections of
areas of blocks in which cation localization was not
successfully demonstrated were omitted from the sample
(i.e., superficial sections of blocks as described in
Chapter I, page 51). In those areas showing acceptable
cation localization, PRL cells were photographed at two
magnifications (X 15,000 and X 37,500). Ten micrographs at
each of the magnifications were chosen at random from those
representing each experimental manipulation. Micrographs
were coded and intermixed so that the secretory state of
97
the cells was not immediately obvious during the count.
Counts were made over the total area of the PRL cell
present in the micrograph. Precipitate particles at each
of the designated cellular sites were counted and averaged.
In addition, the proportion of secretory granules exhibit
ing antimonate reactivity was calculated. Those counts in
the median range were designated as moderate. Counts at
either extreme were designated minimal or heavy, respec
tively. The number of precipitate particles present in
aggregates was estimated from the size of individual par
ticles present in the field. This procedure is based on
the method of Klein et al. (1970).
98
RESULTS
Effects of Dopamine and -monobutyry1 cAMP
on Rat Pituitary Prolactin Release In Vitro
As indicated in Table 1, dopamine at a concentration
of 1 X 10 ^ M greatly reduced PRL release from hemipitui-
taries during a one, two, or three hour incubation, as com
pared with the release from untreated control hemipituitar-
ies. Inhibition was consistently greater than 80%. In
contrast, the administration of 5 x 10 ^ M mbcAMP had a
dramatic effect in stimulating the release of PRL from in
cubated hemipituitaries as compared with the release from
untreated controls (Table 1). This effect was maximal
within five minutes, with levels of PRL reaching 800% of
control levels. The percent of increase then gradually
decreased with increasing time of incubation so that total
PRL released from mbcAMP treated hemipituitaries during a
one hour incubation was approximately 130% of the total PRL
released from control hemipituitaries during a one hour
incubation.
Autonomous Release of Prolactin In Vitro
1. Early Stages of the Response to Incubation
(Five Minutes to 30 Minutes)
a) Ultrastructural features
After five to 30 minutes incubation in KRBG, PRL cells
99
showed variation in the number and distribution of secre
tory granules and in the features of other organelles,
primarily the Golgi apparatus. Some cells were well granu
lated (Fig. 32), with secretory granules distributed pre
dominantly in peripheral areas of the cytoplasm at the pole
of the cell opposite that containing the nucleus. Other
cells possessed moderate amounts of secretory granules that
were distributed throughout most areas of the cytoplasm
(Fig. 33).
The Golgi apparatus varied in appearance from cell to
cell. Golgi regions in some cells appeared to be relative
ly inactive, i.e., consisting primarily of flattened, par
allel saccules with few or no immature secretory granules
(Fig. 33). Other cells exhibited larger Golgi regions con
taining areas of dilated saccules and immature secretory
granules in various stages of formation. Smooth and coated
vesicles were abundant (Fig. 34).
PRL release iji vitro was reflected in exocytotic pro
files that usually were limited to the extrusion of the
contents of a single secretory granule. One or a few sites
of exocytosis were generally present along the cell mem
brane (Fig. 35). Some cells showed no evidence of exocyto
sis. Release of the contents of more than one secretory
granule at a single site was a seldom observed phenomenon.
By 30 minutes of incubation, coated pits were found
100
frequently at irregular intervals along the membrane
(Fig. 36) and also associated with, or close to, membranes
of exocytotic depressions or pits (Figs. 37, 38). In some
instances, the entire membrane forming exocytotic pits
appeared to be coated (Fig. 38). Both coated and smooth
surfaced vesicles were present in the cytoplasm near the
plasma membrane, and occasionally the membrane of a coated
vesicle appeared to be in continuity with that of a smooth
surfaced vesicle (Fig. 39).
b) Cytoplasmic distribution of antimonate deposits
The distribution of antimonate deposits in PRL cells
incubated in KRBG was essentially identical to that previ
ously described (Chapter 1) for PRL cells fixed immediately
following sacrifice. However, semiquantitative analysis of
cation precipitates in PRL cells after five to 30 minutes
incubation indicated that there had been some loss of antim
onate reactive cations during the 30 minute incubation
(Table 2). These changes were not easily detectable in a
cursory examination of electron micrographs. After five
minutes incubation (Figs. 32, 33, 35), mitochondria gener
ally contained a moderate amount of precipitate that was
only slightly reduced at 30 minutes (Fig. 34). After five
minutes, most PRL cells contained moderate amounts of pre
cipitate in Golgi saccules (Fig. 3 3) and a few cells showed
a relatively large amount. After 30 minutes incubation.
101
Golgi saccules containing large amounts of precipitate were
no longer evident. In contrast to these alterations in
mitochondrial and Golgi saccule precipitate, no changes
were observed either in the amount of precipitate localized
to secretory granules or the proportion of secretory gran
ules exhibiting antimonate reactivity (Table 2).
Although antimonate deposits were always localized to
the limiting membranes of some mature secretory granules,
no deposits appeared to be specifically bound to the secre
tory granule membrane that had fused with the plasma mem
brane at sites of exocytosis (Figs. 35, 40, 41, 42). Secre
tory granules with coarse precipitate were commonly seen in
very close proximity to the plasma membrane or to exocy-
totic pits (Figs. 40, 41, 42).
No antimonate reactivity was detected on coated mem
branes at sites of exocytosis, and only rarely were antimo
nate particles present in coated vesicles found adjacent
to the plasma membrane (Figs. 36, 37, 38). However, coated
vesicles in the Golgi region often contained antimonate
particles (Fig. 34).
2. Later Stages of the Response to Incubation
(One Hour to Three Hours)
a) Ultrastructural features
After one to three hours of incubation, PRL cells dis
played no striking differences in ultrastructural features
102
as compared with cells after five or 30 minutes. The pri
mary change discernible with prolonged incubation was a
trend toward depletion of mature secretory granules in
some cells. Frequent profiles of secretory granule release
and abundant coated vesicles and pits were observed through
out the three hour period (Fig. 44). By three hours, accu
mulations of microvesicles were frequently observed at the
margins of the Golgi facing toward the plasma membrane
(Figs. 43, 45).
b) Cytoplasmic distribution of antimonate deposits
As can be seen in Table 2, PRL cells at one, two, and
three hours incubation showed no further alterations in the
concentration of antimonate deposits associated with mito
chondria, Golgi saccules, or secretory granules as compared
with concentrations measured at 30 minutes of incubation.
Mitochondria and Golgi saccules still contained moderate
amounts of antimonate precipitate at three hours. Some
secretory granules were reactive; others were not. Many of
the microvesicles (noted primarily at three hours) contain
ed small grains of antimonate precipitate (Fig. 45).
Dopamine Inhibition of Prolactin Release In Vitro
1. Ultrastructural Features
After one hour of dopamine inhibition, PRL cells gen
erally contained moderate numbers of secretory granules
(Fig. 46). Profiles of exocytosis and coated pits were
103
rarely observed. Golgi regions showed variable degrees of
development but often appeared to be active as indicated by
the formation of immature secretory granules (Fig. 46).
By the end of two hours of exposure to dopamine, PRL
cells showed an increase in the average number of secretory
granules per cell. Large mature secretory granules were
distributed throughout the cytoplasm and considerable num
bers of immature secretory granules were accumulated within
the Golgi region in which the saccules were often quite
dilated (Figs. 47, 48).
After three hours of inhibition, many PRL cells had
accumulated large numbers of secretory granules, most of
which were situated in more central regions of the cell. In
some cells, granules were concentrated toward one pole of
the cell, and although granules were often present at the
plasma membrane adjacent to the pericapillary space, there
was little evidence of exocytosis (Fig. 49).
2. Cytoplasmic Distribution of Antimonate Precipitate
Semiquantitative analysis of antimonate precipitate in
dopamine-treated PRL cells (Table 3) revealed no alterations
in antimonate deposits associated with Golgi saccules or
secretory granules after one hour inhibition of PRL release
as compared with antimonate concentrations at these sites in
one hour controls. However, in mitochondria, there
appeared to be a trend toward the deposition of a greater
104
amount of precipitate after one hour of dopamine treatment.
After two to three hours of inhibition of PRL release,
the amount of precipitate within mitochondria was distinct
ly increased over that in controls (Table 3 and Fig. 50) .
The concentration of mitochondrial deposits was even great
er after three hours exposure of the cells to dopamine than
the concentration measured after two hours. The concentra
tion in Golgi saccules, though unchanged after one hour,
was increased after two hours, primarily due to the pres
ence of large aggregates of precipitate in dilated saccules
(Table 3 and Figs. 48, 51). However, at no time were there
detectable alterations in the proportion of secretory gran
ules exhibiting antimonate reactivity; some granules were
reactive while others were not (Table 3 and Fig. 50).
Cyclic AMP Stimulation of PRL Release
1. Early Stages of the Response to cAMP
(Five to 15 Minutes)
a) Lfltrastruetural features
PRL cells exposed to mbcAMP for five to 15 minutes re
vealed marked exocytosis, with many cells showing numerous
sites of granule extrusion (Fig. 52). Many of the mature
secretory granules were situated in the peripheral cyto
plasm (Figs. 52, 53), and often adjacent to the perivascu
lar space (Fig. 53). Complex profiles of fusion of secre
tory granules were extensive and often occurred at some
105
distance from the cell membrane (Fig. 53). Coated pits and
more extensive areas of coated membrane were commonly ob
served at sites of granule to granule fusion, and coated
vesicles sometimes appeared in very close proximity to
these sites (Fig. 54), Golgi regions in some PRL cells at
this stage exhibited areas of smooth membrane vésiculation
and collections of relatively large diameter vesicles
(Fig. 55).
b) Cytoplasmic distribution of antimonate deposits
Semiquantitative analysis of antimonate deposits in
PRL cells exposed to mbcAMP for five to 15 minutes revealed
no significant alterations in the concentration of antimo
nate deposits at any of the cytoplasmic sites analyzed as
compared with the concentration in controls (Table 4). A
moderate quantity of antimonate precipitate was present in
most Golgi saccules, in large diameter vesicles seen at
this stage, and mitochondria (Fig. 55). The proportion of
secretory granules showing antimonate reactivity was similar
to the proportion observed in controls (Table 3 and Fig.
56). As in controls there was no evidence of reactivity of
granule membranes translocated to the plasma membrane dur
ing exocytosis, although on one occasion small precipitate
particles were evident at sites of membrane fusion (Fig.56).
2. Intermediate Stage of the Response to cAMP
(30 Minutes)
a) Ultrastructural features
106
After 30 minutes of incubation of PRL cells with
mbcAMP, there appeared to be a trend toward degranulation
and a decline in exocytotic activity. Some PRL cells dis
played fewer secretory granules and fewer profiles of exo
cytosis when compared with cells at the earlier stages (Fig.
57). In other PRL cells, considerable numbers of mature
secretory granules and multiple sites of exocytosis were
still present (Figs. 58, 59).
The most notable ultrastructural feature emerging at
this stage was an amassing of microvesicles in the cyto
plasm between the Golgi apparatus and the cell membrane.
These large collections of closely packed microvesicles
were often encircled by lamellar arrays of rough endoplasmic
reticulum. The membrane at the periphery of these arrays
was generally studded with ribosomes while closely abutted
membranes nearer the interior of these arrays lacked identi
fiable ribosomes (Figs. 60, 61). Rough endoplasmic retic
ulum in general was characteristically well developed in
PRL cells at this stage (Figs. 57, 58).
b) Cytoplasmic distribution of antimonate deposits
Semiquantitative analysis of the concentrations of
antimonate precipitate after 30 minutes incubation with
mbcAMP demonstrated a distinct decrease in the average num
ber of precipitate grains localized to mitochondria in com
parison with controls and cells at earlier stages in the
107
response to mbcAMP (Table 4). In many sections of PRL
cells, a large proportion of the mitochondria contained min
imal deposits (Fig. 62). Precipitate localized to secre
tory granules and Golgi saccules was unchanged from that in
controls (Table 4). Golgi saccules usually contained moder
ate amounts of precipitate (Fig. 62).
3. Later Stages of the Response to cAMP
(One Hour)
a) Ultrastructural features
After one hour exposure to mbcAMP, some PRL cells con
tained only a few, scattered mature secretory granules, al
though profiles of exocytosis could still be found. Other
PRL cells still contained moderate numbers of secretory
granules. Coated pits were readily observed as at earlier
stages (Fig. 63). Characteristics of the Golgi apparatus
and the rough endoplasmic reticulum were similar to those
seen in cells after 30 minutes exposure to mbcAMP.
b) Cytoplasmic distribution of antimonate deposits
After one hour incubation with mbcAMP, mitochondria of
PRL cells contained increased amounts of antimonate precip
itate compared to decreased, even minimal amounts seen
after 30 minutes and in comparison with amounts in controls
(Table 4 and Fig. 63). However, in the case of granules
and the Golgi, there was no significant change in amounts of
precipitate as compared with those in controls or expérimen
tais exposed for shorter times to mbcAMP (Table 4).
108
DISCUSSION
Results of the present study indicate that alterations
in the concentration of antimonate precipitate at specific
subcellular sites may be correlated with changes in the
secretory activity of PRL cells. At one extreme, inhibition
of PRL release in response to dopamine was associated with
an increase in mitochondrial antimonate deposits as com
pared with the deposits in mitochondria in cells of con
trols . This increase was detectable at the earliest time
interval sampled (one hour) and continued throughout a
three - hour incubation with dopamine. Also, an increase in
precipitate localized to Golgi saccules was noted after
both two and three hours inhibition of PRL release. At the
other extreme, the marked stimulation of PRL release in
duced by mbcAMP was associated with a decrease in mitochon
drial antimonate deposits at 30 minutes and an increase in
these deposits at one hour. No changes were detected in
the concentration of precipitate localized to Golgi saccules
in cAMP stimulated cells.
Limitations inherent in the potassium pyroantimonate
technique do not allow an unequivocable statement that
mitochondrial antimonate precipitate represents only mito
chondrial calcium. However, a preponderance of evidence
(discussed in Chapter I) supports the interpretation that
109
calcium is a primary component of the precipitates. More
over, the results are in concordance with biochemical data
demonstrating a direct effect of cAMP on mitochondrial cal
cium. Borle (1974) showed that cAMP could promote the
efflux of calcium from mitochondria isolated from liver,
kidney, and heart. Subsequent cell fractionation studies
by Howell and associates (1975) demonstrated that cAMP
diminishes the net uptake of calcium in mitochondria iso
lated from 3 cells of the rat endocrine pancreas. The
present results are also in agreement with observations of
Schafer and Kloppel (1974) on the distribution of antimo
nate deposits in 3 cells of normo-, hypo -, and hyperglycemic
mice. Mitochondrial antimonate deposits were decreased in
actively secreting cells of hyperglycemic mice and in
creased in inactivated cells of hypoglycemic mice. Further
more there is some evidence that cAMP induces an efflux of
calcium from anterior pituitary cells. Milligan and
Kraicer (1971) observed a net decrease in ^^Ca in rat hemi-
pituitaries after 30 minutes exposure to dibutyryl cAMP.
In the present study, the mbcAMP stimulated release of
PRL had markedly declined by one hour. Total PRL released
into the media during a one hour incubation with mbcAMP was
only slightly elevated as compared with the total PRL re
leased from one hour controls. The rebound increase in the
concentration of antimonate deposits observed in
110
mitochondria of PRL cells at this stage in the response to
mbcAMP suggests the existence of a feedback mechanism to in
hibit the mobilizing action of mbcAMP on mitochondrial cal
cium stores in order to prevent excessive secretion. Evi
dence for the existence of such feedback mechanisms in a
variety of cells has been reviewed by Berridge (1975).
The progressive accumulation of mitochondrial antimo
nate deposits observed during the time course of the re
sponse of PRL cells to dopamine presumably reflects mito
chondrial sequestration of calcium. Results are again con
sistent with biochemical data demonstrating the remarkable
ability of mitochondria from a variety of tissues to seques
ter calcium against a large concentration gradient (Borle,
1973; Carafoli et al., 1975). Combined with this evidence
from previous studies the present results suggest that mito
chondria play an important role in the regulation of intra
cellular calcium levels in PRL cells.
The significance of the increased concentration of an
timonate deposits in dilated Golgi saccules in dopamine in
hibited cells after two hours inhibition is unknown. These
organelles may play some secondary role in the regulation of
intracellular calcium. Active accumulation of calcium by
microsomal membrane fractions from a variety of tissues has
been demonstrated (Robblee et al., 1973; Moore et al., 1975;
Thorn et al., 1975b). However, microsomal membrane fractions
111
include both rough and smooth membrane and some plasma mem
brane fragments. Therefore, it is impossible to make any
direct comparison between the present results and those of
biochemical studies. The time course of the increase in
deposits in Golgi saccules suggests that these changes re
flect some alteration in the synthetic - secretory pathway
secondary to inhibition of PRL release.
The potassium pyroantimonate technique does not allow
the detection of small shifts in intracellular cations. In
the present investigation, variations in the quantity of
antimonate deposits in samples of cells from rats in the
same treatment group tended to obscure differences between
the experimental groups. Semiquantitative estimation of
the concentration of antimonate precipitate was useful to
reveal trends leading to a marked increase or decrease in
precipitate but did not provide definitive evidence for
cation shifts in PRL cells. Thus the maximal stimulation
of PRL release observed within five minutes after the addi
tion of mbcAMP may have been associated with the mobiliza
tion of a relatively small fraction of the total calcium
present in mitochondria. The relative insensitivity of the
potassium pyroantimonate technique would not allow the de
tection of subtle changes in cation concentration. Further
more, the cytoplasmic concentration of free ionized calcium
[Ca^^lj is at or below the limits of the binding affinity
112
of the pyroantimonate anion for calcium. Thus one can make
no statement with regard to this calcium pool that has been
implicated as the "activator” calcium in the induction of
the secretory process (Rubin, 1974). The present results,
however, do implicate mitochondria as a possible source of
at least a portion of this "activator" calcium in cAMP stim
ulated cells and as a site for sequestration of calcium to
2 +
reestablish a low [Ca ]j after cAMP stimulation or to in
sure low levels of [Ca^^jj during prolonged inhibition of
PRL release induced by dopamine.
The site of action of the "activator" calcium remains
unknown. Secretory granules would appear to be one possible
site of action. However, it was not possible to detect any
consistent pattern of distribution of antimonate precipi
tate localized to secretory granules related either to the
cellular location of the granule, or to its state of matur
ation, or to the state of secretory activity of the PRL
cell. In any experimental situation, roughly the same pro
portion of secretory granules exhibited antimonate reaction
product. The fact that some granules exhibited reactivity
and others did not may be due to a failure of the antimo
nate anion to penetrate the membranes of some of the gran
ules. This possibility is suggested by a comparison of the
results with those of Clementi and Me1dolesi (1975). In
their study of the rat exocrine pancreas, isolated membranes
113
of zymogen granules always exhibited heavy antimonate reac
tion product; however, zymogen granules in the intact cell
often exhibited no reactivity.
In the present study, no antimonate precipitate was lo
calized specifically to the plasma membrane of PRL cells
or to the secretory granule membrane translocated to the
plasma membrane during exocytosis. This suggests that an
timonate reactive cations bound to the granule membranes are
released, or somehow masked, at the time of membrane fusion
during exocytosis. This loss of antimonate reactivity may
reflect some alteration in the molecular structure of the
granule membrane concomitant to membrane fusion. There was
no indication that antimonate reactive cations were associ
ated with the secretory material enclosed by the granule
membrane or extruded during exocytosis.
The addition of mbcAMP induced a marked extrusion of
secretory material from PRL cells within five to 15 minutes.
Simultaneously there was a greatly elevated level of PRL in
the incubation medium. After 30 minutes to one hour, the
exocytotic activity had subsided and the comparative in
crease in PRL in the media had declined. The magnitude and
time course of this response differ from those reported by
Pelletier and associates (19 72) in their study of the ultra-
structural correlates of mbcAMP stimulation of GH, PRL, and
ACTH release. Pelletier reported a lower, more sustained
114
release of PRL in response to mbcAMP used at the same con
centration as in the present investigation. In the
Pelletier et al. study, PRL release (measured by polyacrila-
mide gel elecLrophoresisj was maximal within 10 minutes and
remained linearly elevated for one hour. The present find
ings also differ from morphological observations of
Pelletier et al. in several respects. Pelletier made no
reference to coated pits or micropits at sites of exocytosis
as were readily observed in this study. Whereas Pelletier
described hypertrophy of the Golgi apparatus in PRL cells
by one hour incubation in mbcAMP with the accumulation of
smooth vesicles and immature secretory granules, no such
hypertrophy of the Golgi was apparent in the present study.
In fact, a significant proportion of PRL cells incubated
for even a short time in KRBG alone showed active Golgi re
gions as judged by the presence of numerous smooth vesicles
and immature secretory granules. In the present study, the
most distinctive feature of the Golgi region after the add
ition of mbcAMP was the amassing of numerous microvesicles
at the periphery of the Golgi apparatus after 30 minutes.
These collections of closely packed microvesides closely
resemble microvesicles found in terminals of the posterior
pituitary after a secretory stimulus (Douglas, 1974) and
may be related to a process of membrane retrieval that
occurs subsequent to exocytosis. Considerable experimental
115
evidence indicates that in many secretory cells the translo
cation of granule membrane to cell membrane during exocyto
sis is followed by a process of endocytosis or membrane
vésiculation that results in the removal of redundant cell
membrane and may provide a mechanism for its reuti1ization
(Abrahams and Holtzman, 1973; Heuser and Reese, 1973;
Douglas, 1974; Geuze and Kramer, 1974).
Douglas (1974) has proposed that coated vesicles form
as a result of rounding up and pinching off of excess cell
membrane in cells that have received a strong secretory
stimulus. According to the Douglas (1974) "exocytosis-
vesiculation" hypothesis, coated vesicles give rise to
microvesicles that move centripetally. In the present study
membranes of exocytotic pits often appeared to be coated,
and coated vesicles appeared to pinch off from the trans
located granule membrane at sites of exocytosis or were
present in peripheral areas of the cell near sites of exocy
tosis. Masses of microvesicles were seen to accumulate in
the cytoplasm near the plasma membrane and at the margin of
the Golgi subsequent to the strong secretory stimulus of
mbcAMP. All these observations are consistent with the
schema proposed by Douglas. However, convincing evidence
is lacking for the transformation of coated vesicles into
microvesicles in PRL cells. In the present study and lliat
of the Pelletier et al. (19 7 2) study, smooth vesicles of
116
various sizes were seen in close proximity to sites of exo-
cytosis. Pelletier (1973) demonstrated that these smooth
membrane vesicles were involved in the uptake of peroxidase
in mbcAMP-stimulated PRL cells. It is possible that a pro
cess of membrane retrieval may be accomplished by mechanisms
that involve the direct formation of both smooth and coated
vesicles as suggested by Nordman and associates (1974) in
their study of the posterior pituitary.
Coated vesicles in PRL cells seldom contained antimo-
nate reaction product whereas smooth vesicles more often
contained antimonate precipitate. This may be related to a
relatively poor penetration of the antimonate anion into
coated vesicles. Small smooth vesicles and microvesicles
commonly contained antimonate particles. Nordman and asso
ciates (1974) believe that membrane retrieval is analogous
to micropinocytosis since they have demonstrated that an up
take of extracellular fluid occurs secondarily to secretion
in the posterior pituitary. They have further proposed
that this may provide a route for the uptake and transport
of calcium to various intracellular sites. The presence of
antimonate particles in smooth vesicles and microvesicles
in PRL cells lends some support to this concept.
The ultrastruetural correlates of dopamine inhibition
of PRL release have not previously been described. The
morphological findings of very little exocytotic activity
117
and an accumulation of secretory granules in all stages of
maturation in dopamine treated cells closely resemble the
results observed in PRL cells after treatment of rat pitui-
taries with ergocornine (a probable dopamine agonist) both
in vivo (Meites and Clemens, 1972) and iji vitro (Ectors et
al., 1972). As in ergocornine treated PRL cells, there was
no morphological evidence of an impairment in PRL synthesis
in dopamine treated PRL cells. Rough endoplasmic reticulum
was well-developed and immature secretory granules were
present even after three hours of inhibition with dopamine.
These observations are in concordance with physiological
data indicating that the primary effect of dopamine is on
release of PRL, not its synthesis (MacLeod, 1976).
118
SUMMARY
The potassium pyroantimonate technique (Glu/KSb)
described in Chapter I was used to study the cellular dis
tribution of cation antimonate precipitate and ultrastruc-
tural characteristics of PRL cells in varied stages of
secretory activity. Hemipituitaries were incubated in KRBG
alone, in KRBG with 1 x 10 ^M dopamine, or in KRBG with
5 X 10 ^M mbcAMP.PRL release was measured by radioimmuno
assay. The concentration of antimonate deposits associated
with mitochondria, Golgi saccules and secretory granules
was estimated by semiquantitative analysis of the number
and size of precipitate particles localized to these sites.
Dopamine inhibition of PRL release resulted in the accumula
tion of secretory granules in all stages of maturation
accompanied by a marked increase in mitochondrial antimonate
deposits and an increase in Golgi deposits. The mbcAMP
stimulation of PRL release resulted in massive exocytotic
activity at five and 15 minutes and a decline in exocytotic
activity after 30 minutes to one hour. Exocytotic activity
was followed by the appearance of numerous coated pits,
coated micropits at sites of exocytosis, and the accumula
tion of microvesicles at the margin of the Golgi apparatus.
These changes in the stimulated cells were accompanied by a
decrease in the concentration of mitochondrial antimonate
119
deposits at 30 minutes and an increase in these deposits at
one hour. The concentration of antimonate deposits local
ized to secretory granules was the same in either inhibited
or stimulated cells. Plasma membranes and granule mem
branes translocated to the plasma membrane during exocyto
sis were not reactive.
120
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148
LIST OF ABBREVIATIONS FOR ELECTRON MICROGRAPHS
bl = basal lamina
cap = capillary
chr = nucleolar-associated
chromatin
cv = coated vesicles
eu = euchromat in
G = Golgi apparatus,
Golgi saccules
h = heterochromât in
ig = immature secretory
granules
ly = lysosome
m = mitochondria
mv = microvesicles
mvb = multivesicular
body
N = nucleus
nu = nucleolus
pm = plasma membrane
ps = pericapillary
space
rer = rough endoplasmic
reticulum
sg = mature secretory
granules
sv = smooth vesicles
V = vesicles
149
Figures 1 through 4 are electron micrographs of stained
sections of normal rat pars distalis fixed by a variety of
procedures, all of which involve direct fixation in the
OsO^/KSb solution.
Figure la. Pars distalis cells; direct fixation in
OsOz^KSb with 0.15 M sucrose; four hour immersion;
uranyl acetate -lead citrate counterstain. This
electron micrograph is an example of the best results
achieved by the use of any direct fixation procedure.
Cellular details are well preserved and antimonate
precipitate is localized to specific cellular sites.
Deposits are evident within Golgi saccules (G), hetero
chromatin (h) areas of the nucleus (N), and mitochon
dria (m). (X 18,000).
Figure lb. At higher magnification, details of the nuclear
deposition of antimonate precipitate are more clearly
displayed. Abundant fine-grained precipitate overlies
the heterochromâtin (h). (X 33,600).
150
WHÆfeî-
ismmm
^6;
Figure 2. Pars distalis cells; overnight immersion in
OsO^/KSb; uranyl acetate -lead citrate counterstain.
Note the absence of all cytomembranes. This result
of prolonged fixation in OsO^/KSb was consistently
observed. A semblance of normal tissue and cellular
architecture persists. While plasma membranes appear
to be absent, outlines of individual cells are
vaguely discernible and cell to capillary (cap) and
organellar spatial relationships are maintained to
some degree. Nuclei (N), mitochondria (m), and
secretory granules (sg) are recognizable. Cytoplasmic
deposition of antimonate precipitate reflects the
pattern of lamellar rough endoplasmic reticulum
(arrow) although membranous elements are not discern
ible. (X 9,000).
152
Wi
y
m
s
• V /Tk'
% .
153
Figure 3. Pars distalis cell, overnight immersion in
OsO^/KSb; uranyl acetate-lead citrate counterstain.
Again note the poor preservation of cytomembranes
in cells subjected to prolonged fixation as seen in
Figure 2. In this higher magnification view, abun
dant precipitate appears throughout the cytoplasm
but with no specific pattern of distribution.
Nuclear deposits are localized primarily to hetero
chromatin (h). Remnants of mitochondria (m),
secretory granules (sg). (X 25,000).
154
K
1 3 M
mmm
m m %
"' W il
155
Figure 4. Pars distalis cells; vascular perfusion with
OsO/^/KSb ; uranyl acetate- lead citrate counters tain.
This electron micrograph is illustrative of the best
results achieved with vascular perfusion of the
OsO^/KSb solution. Structure is well-preserved and
antimonate deposits are precisely localized in the
same pattern of distribution as observed after
immersion fixation for two to four hours (compare
with Figure 1)• Antimonate deposits in hetero
chromatin (h) , Golgi saccules (G) , mitochondria (m).
(X 15,000).
156
m m
mm
mmm
H
m
mim
< 3 . * . . ..r* er-njK*. u ^ ^ » ' w ' « .<
m m rnm m
&mrn
•hi’ ’
©
157
Figures 5 through 14 are electron micrographs of both
stained and unstained sections of normal rat pars distalis
illustrating cation localization in GH and PRL cells after
direct fixation by immersion in the OsO /KSb solution with
Ü.15 M sucrose for two to four hours. In both GH and PRL
cells in stained and unstained sections, precipitate is
localized to heterochromatin areas of the nucleus, to Golgi
saccules and Golgi-associated vesicles, within mitochondria,
in multivesicular bodies, in some cisternae of rough endo
plasmic reticulum and is associated to a variable degree
with the membranes of secretory granules.
Figure 5. GH cell; identifiable by the presence of abun
dant, large, round secretory granules (sg); direct
fixation in OsO^/KSb; unstained. At low magnifica
tion, antimonate deposits are clearly displayed as
clusters of precipitate in Golgi saccules (G) and
mitochondria (m) and as accumulations of particles
overlying the heterochromâtin (h). Little precipi
tate is present in euchromatin areas (eu) and deposits
are absent from areas of nuclear pores (arrows).
Lysosomes (ly) contain a variable quantity of precip
itate. (X 22,200).
Figure 6. PRL cell, identifiable by the presence of large
secretory granules (sg) showing wide variation in the
size and shape ; direct fixation in OsO/|^/KSb; unstained.
At low magnification in unstained sections, the dis
tribution of antimonate deposits in PRL cells is
similar to that observed in GH cells. (Compare with
Figure 5). Abundant precipitate is localized in the
heterochromâtin (h), Golgi saccules (G) and mito
chondria (m). (X 22,500).
158
M
A # ' . . 'à
ly
sg--'
k-m.
- ‘ w ■
/?
®
j3 • + n ' y
.*
-a
f-
-sg.
J .
& % # %
. r
■ h
■t'^« * » -
JTl
1 1
159
Figure 7. GH cell; direct fixation in OsO^/KSb; uranyl
acetate-lead citrate counterstain. Section staining
does not cause any apparent alteration in the pattern
of distribution or quantity of antimonate deposits
(compare with Figure 5). Precipitate is evident in
the heterochromatin (h), in Golgi saccules (G), and
in mitochondria (m). Fragmented membranes of some
secretory granules (arrows). (X 22,400).
160
•-■ T 'i ,
161
Figure 8. PRL cell; direct fixation in OsO^/KSb; uranyl
acetate-lead citrate counterstain. Precipitate is
the same in distribution and quantity as in unstained
section of Figure 6; predominantly localized to the
heterochromâtin (h), Golgi saccules (G), and mito
chondria (m). Fine grains of precipitate appear be
tween the dense core material of an immature secre
tory granule and the enclosing membrane (arrow).
(X 20,400).
162
m m m
K m # #
Figure 9. Nucleus of a PRL cell; direct fixation in
OsO^/KSb; uranyl acetate -lead citrate counters tain.
In this higher magnification view, details of the
nuclear and nucleolar distribution of antimonate
deposits may be observed. Heterochromatin (h) areas
contain massive accumulation of fine-grained precipi
tate while euchromatin (eu) areas contain widely dis
persed, fine deposits. Nuclear pore areas appear
free of precipitate (arrows). Precipitate overlies
the nucleolar-associated chromatin (chr) but is not
evident in the condensed components of the nucleolus.
(X 23,400).
Figure 10. Golgi region of a GH cell; direct fixation in
OsO^/KSB ; uranyl acetate-lead citrate counters tain.
Clusters of fine precipitate lie within Golgi saccules
(G); single antimonate particles are confined within
smooth and coated Golgi vesicles (short arrows).
Immature secretory granules (ig) have deposits be
tween the dense core and the membrane (long arrows).
Precipitate is distributed throughout the mitochondria
(m). (X 52 , 500) .
164
r ^
.•v^«
165
Figure 11. Secretory granules in a GH cell; direct fixa
tion in OsO/^/KSb ; unstained. Fine antimonate grains
form a discontinuous line along the limiting membranes
of some mature secretory granules (arrows). (X 41,400).
Figure 12. Multivesicular body (mvb) in a PRL cell; direct
fixation in OsO/^/KSb ; uranyl acetate- lead citrate
counterstain. Single grains of precipitate are
deposited within the small vesicles. (X 40,000).
Figure 13. Multivesicular body (mvb) containing secretory
material in a PRL cell ; direct fixation in OsO/^/KSb ;
uranyl acetate-lead citrate counterstain. Precipitate
is seen within internal vesicles and in the matrix.
(X 40,500).
Figure 14. Plasma membrane and adjacent cytoplasm of a
PRL cell ; direct fixation in OsO^/KSb; unstained.
Notice that no precipitate appears to be specifically
localized to the cytoplasmic surface of the plasma
membrane (pm). Fine precipitate is distributed as
single grains or small clusters along the external
surface of the plasma membrane where it abuts on the
perivascular space, but the particles do not appear
to be strictly localized to the external surface.
Intracellularly, small deposits are diffusely dis
tributed within the cisternae of rough endoplasmic
reticulum (long arrows) and overlie the cytoplasmic
matrix (short arrows). (X 52,500).
16 6
"A -
* f . > . .
/
It
w t
' V
.v' \ ^ P r
wm
. r , • ... *.
V ;,pm
, ' ' ' \
167
Figures 15 through 21 are electron micrographs of stained
and unstained sections of normal rat pars distalis showing
GH and PRL cells after prefixation in Glu/KSb for 15 min
utes, followed by postfixation in OsO^/KSb. In both GH and
and PRL cells, the pattern and quantiLy of nuclear antim
onate precipitate are altered as compared with patterns
seen after direct fixation in the OsO^/KSb solution. Pre
cipitate localized to secretory granules is generally
larger grained. Deposits localized to other cellular
organelles are essentially unchanged.
Figure 15. GH cell; 15 minute prefixation in Glu/KSb; lead
citrate counters tain. Observe the distinct altera
tions in the pattern and quantity of nuclear precipi
tate after 15 minute prefixation (compare with Figures
5 and 7). Small aggregates of fine antimonate par
ticles overlie the euchromatin (eu) and generally are
not evident in the heterochromatin (h). Deposition
at other cellular sites within this micrograph does
not appear significantly altered. Scattered grains
are enclosed within the perinuclear space (long
arrows). In the cytoplasm, clusters of precipitate
are localized to Golgi saccules (G) and single antim-
onate grains are enclosed within Golgi vesicles (short
arrows). Mitochondria (m) contain deposits, sometimes
in large quantities. No precipitate is specifically
localized to the cytoplasmic surface of the plasma
membrane (pm). Deposits are present in the extra
cellular space (upper left). (X 37,500).
Figure 16. PRL cell; 15 minute prefixation in Glu/KSb;
unstained. Alterations in nuclear deposits were also
observed in PRL cells as compared with patterns seen
after direct fixation (compare with Figures 6 and 8).
Nuclear precipitate is reduced in quantity and local
ized to euchromatin areas (eu). As in GH cells,
deposits at other cellular sites appear to be
unchanged: antimonate deposits are present in the
perinuclear cistern (arrows), in Golgi saccules (G)
and mitochondria (m). (X 19,800).
168
smrnmi
a.
v - t
-«• f
: i
#
G
m
V t l
169
Figure 17. Multivesicular bodies (mvb) in a GH cell; 15
minute prefixation in Glu/KSb; lead citrate counter
stain. As after direct fixation in OsO^/KSb, precipi
tate grains are precisely localized within the internal
vesicles and a small quantity is occasionally present
in the matrix. (X 37,500).
Figure 18. Microvesicles (mv) in a PRL cell; 15 minute pre
fixation in Glu/KSb; lead citrate counterstain. Single
precipitate grains are localized within some of these
very small vesicles (arrows). Membranes of these
structures are difficult to visualize since they seldom
appear in precise transverse section. (X 51,000).
Figure 19. Secretory granules, Golgi saccules, and rough
endoplasmic reticulum in a PRL cell; 15 minute prefix
ation in Glu/KSb; unstained. In place of the fine
grains of precipitate localized to PRL secretory gran
ules after direct fixation in OsO^/KSb, relatively
coarse grains encircle the periphery of immature (ig)
and more mature (sg) secretory granules and overlie
the contents of the granules that appear to be cut in
tangential section (long arrow). Aggregates of fine
precipitate grains lie within the Golgi saccules (G),
are localized to dilated cisternae of rough endo
plasmic reticulum (short arrow), and are absent from
most of the flattened lamellae (*)» (X 31,000).
Figure 20. Secretory granules in a GH cell; 15 minute pre
fixation in Glu/KSh; uranyl acetate -lead citrate
counterstain. Antimonate grains associated with the
limiting membranes of secretory granules (sg) in GH
cells also are increased in quantity after prefixation
(compare with Figure 11) and overlie the contents of
granules that appear to he cut in tangential section
(arrows). (X 46,500).
Figure 21. Secretory granules in a PRL cell; 15 minute
prefixation in Glu/KSb; lead citrate counterstain.
This detailed view shows a budding or fusion process
that occasionally appeared to be occurring between
small antimonate-containing vesicles (arrow) and the
membranes of secretory granules (sg). (X 60,000).
170
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171
Figures 22 through 25 are electron micrographs of stained
and unstained sections of normal rat pars distalis showing
examples of GH and PRL cells prefixed in Glu/KSb for 30
minutes to one hour, then postfixed in OsO/|^/KSb. These
micrographs demonstrate that lengthening the time of pro
fixation results in a further loss of nuclear deposits
and a notable decrease in precipitates localized to Golgi
saccules accompanied by the artifactual deposition of
rounded clumps of precipitate. Antimonate deposits local
ized to other cellular sites appeared to be unaltered
despite longer prefixation.
Figure 22. GH cell; 30 minute prefixation in Glu/KSb;
unstained. This electron micrograph illustrates
the selective alterations that occur in GH cells
during a 30 minute prefixation. Relatively coarse
precipitate is widely dispersed in the nucleus.
Note the absence of precipitate in Golgi saccules
(G) and its presence in Golgi vesicles (short arrows).
Some secretory granules (sg) show precipitate at their
periphery and deposits are present within mitochondria
(m) and dilated rough endoplasmic reticulum (rer).
Observe the artifactual deposition of rounded masses
of precipitate (long arrows). (X 29,000).
Figure 23. PRL cell ; one hour prefixation in Glu/KSb;
uranyl acetate-lead citrate counterstain. This
micrograph is representative of the effects of
one hour prefixation on cytoplasmic deposition
in PRL cells. No precipitate is localized to Golgi
saccules (G). However, deposits are present in
mitochondria (m), within some vesicular elements of
rough endoplasmic reticulum (arrows) and associated
with membranes of immature (ig) and mature (sg)
granules. Vesicles within a multivesicular body
(mvb) contain precipitate. (X 25,200).
172
;
173
Figure 24. Golgi region of a GH cell; 30 minute prefix
ation in Glu/KSb; uranyl acetate -lead citrate counter
stain. In this rare instance, a few coarse, rounded
precipitates lie within Golgi saccules (G). Golgi
vesicles contain deposits (arrows) as do immature
secretory granules (ig). (X 36,000).
Figure 25. Secretory granules in a GH cell; one hour
prefixation in Glu/KSb, uranyl acetate-lead citrate
counters tain. Antimonate deposits ring the periphery
or overlie the contents of the granules (sg) as seen
after 15 minute prefixation (compare with Figure 20).
(X 37,200).
Figure 26. Secretory granules in a PRL cell ; one hour
prefixation in Glu/KSb; uranyl acetate-lead citrate
counterstain. This electron micrograph more clearly
illustrates the process of budding or fusion (arrow)
that was occasionally observed between antimonate-
containing vesicles (arrow) and secretory granules
(sg) of both GH and PRL cells (compare with Figure 21).
(X 46,500).
174
»
u
Figure 27 is an electron micrograph of an unstained section
of pars distalis representative of the effects of prolonged
(two-hour) prefixation in Glu/KSb. Results from this pro
cedure are uninterpretable due to striking variations in
both the quantity and distribution of precipitate.
Figure 27. Pars distalis cells; two-hour prefixation
in Glu/KSb; unstained. Note the almost complete
absence of precipitate from some cells while
adjacent cells contain a heavy, diffuse precipitate.
(X 15 , 840) .
176
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- T
j W r •.
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SfaSi
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• ' - ' r-â'j .
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177
Figures 28 through 31 are results of EGTA analytical pro
cedures and indicate that precipitates localized by either
direct fixation or prefixation techniques probably consist
primarily of calcium antimonate.
Figure 28. GH cell ; direct fixation in OsO^/KSb; unstained.
In the absence of the chelating agent, antimonate
precipitates are evident in the heterochromatin (h),
mitochondria (m), and Golgi saccules (G). (XI3,800).
Figure 29. Adjacent section to that of Figure 28; direct
fixation in OsO^/KSb; unstained; EGTA chelation.
Observe that the chelation results in almost complete
extraction of antimonate precipitates from all areas
including the heterochromatin (h), mitochondria (m),
and Golgi saccules (G) (compare with Figure 28).
(X 13,800).
Figure 30. PRL cell ; 15 minute prefixation in Glu/KSb;
unstained. In the absence of the chelating agent,
antimonate deposits are evident in euchromatin (eu),
Golgi saccules (G), dilated rough endoplasmic retic
ulum (rer), mitochondria (m), and around some secre
tory granules (arrow). (X 13,800).
Figure 31. Adjacent section to that of Figure 30; 15 min
ute prefixation in Glu/KSb; unstained; EGTA chelation.
Notice that chelation results in removal of most of
the intracellular precipitates . Antimonate deposits
remain only as a few isolated grains in the rough
endoplasmic reticulum (rer) and mitochondria (m).
Golgi (G), euchromatin (eu), and secretory granule
(arrow) precipitates are extracted (compare with
Figure 30). (X 13,800).
178
28
m
f eu : y
@
• >
• t.
m
' ' &r'
h
- % -
' M k v
3 7 ' . # / % .
% / . %; -% .
®
: ÎT-' a.s.f
eu
• - : , ■ ^ -r^' • ■ ■ '• .
t
y ••• ':■ ' W ' F ’ . rer . i
m
- " S
w sm.
' y %.
; v
0
179
Figures 32 through 45 are electron micrographs of sections
of pars distalis showing PRL cells incubated in KRBG for
periods of time varying from five minutes to three hours
(control hemipituitaries). All tissues were prefixed by
immersion in Glu/KSb for 15 minutes, then postfixed in
OsO^/KSb.
Figure 32. PRL cell; five minute incubation in KRBG;
uranyl acetate-lead citrate counters tain. In this
well granulated cell, secretory granules (sg) are
distributed predominantly in the peripheral areas
of the cytoplasm at one pole of the cell. Mito
chondria (m) contain moderate amounts of antimonate
deposits. (X 12,600).
Figure 33. PRL cell ; five minute incubation in KRBG;
uranyl acetate -lead citrate counterstain. In this
PRL cell, moderate numbers of granules (sg) are
scattered throughout the cytoplasm. (Compare with
Figure 32). The Golgi apparatus (G) contains
relatively few immature granules (ig). Golgi saccules
(G) and mitochondria contain moderate amounts of
antimonate precipitates (X 16,200).
180
yy'ti.
%',
181
Figure 34. Golgi region in a PRL cell; 30 minute incuba
tion in KRBG; lead citrate counters tain. The Golgi
apparatus is well developed and includes areas of
dilated saccules (G), containing moderate amounts
of antimonate deposits. Immature secretory granules
(ig) in various stages of formation are evident.
Abundant smooth and coated vesicles (v), some con
taining antimonate grains, are distributed throughout
the Golgi region. Mitochondria (m) contain moderate
amounts of antimonate precipitate. (X 40,500).
Figure 35. PRL secretory granules ; five minute incubation
in KRBG; uranyl acetate-lead citrate counters tain.
Secretory material, apparently of a single PRL gran
ule, is being extruded at two sites along the cell
membrane (arrows). Some of the mature secretory
granules (sg) exhibit coarse antimonate deposits
associated with their limiting membranes. Character
istically, no antimonate deposits are localized to
the granule membrane that has been translocated to
the plasma membrane during exocytosis. Mitochondria
(m) contain a moderate amount of precipitate.
(X 30 ,000) .
182
& #
.:#->
Xlr.rs
r --
^ 'v.
/•
183
Figure 36. Plasma membrane (pm) and peripheral cytoplasm
of a PRL cell; 30 minute incubation in KRBG; uranyl
acetate - lead citrate counters tain. Coated pits (arrows)
are seen at intervals along the cell membrane.(X 24 ,750).
Figure 37. Plasma membrane (pm) and cytoplasm in an area
adjacent to that of Figure 36; 30 minute incubation in
KRBG ; uranyl acetate -lead citrate counters tain. A coated
micropit or caveola (arrow) projects from the membrane
forming an exocytotic pit containing secretory material .
(X 64,800).
Figure 38. Two adjacent exocytotic pits in a PRL cell; 30
minute incubation in KRBG; uranyl acetate-lead citrate
counterstain. The membrane of these exocytotic pits ap
pears to be coated (arrows). A coated vesicle (cv) is
closely associated with the adjacent cell membrane.
(X 60 , 000) .
Figure 39. Portion of a PRL cell; 30 minute incubation in
KRBG ; uranyl acetate -lead citrate counters tain. Coated
vesicles (cv) and smooth vesicles (sv) are present in
the cytoplasm near the cell surface. The membranes of a
coated and smooth vesicle appear to be in continuity
with one another (*) in this section. Single antimonate
particles are enclosed in some of the vesicles,(X 60 ,000).
Figure 40. Exocytosis in a PRL cell; five minute incubation
in KRBG; uranyl acetate - lead citrate counterstain. Al
though coarse antimonate grains are localized to the mem
brane of a secretory granule (sg) near the plasma mem
brane (pm) and adjacent to the site of extrusion of the
contents of another granule, the granule membrane (arrow)
that has been translocated to the cell membrane exhibits
no reactivity to the antimonate anion. (X 36,000).
Figure 41. Exocytosis in a PRL cell; five minute incubation
in KRBG; uranyl acetate -lead citrate counterstain. In a
similar view to that of Figure 40, a secretory granule
(sg) with abundant coarse antimonate deposits appears
very close to the membrane of another granule (arrow)
that has already fused with the cell membrane. Note that
the translocated membrane and the plasma membrane (pm)
are not reactive to the antimonate anion. (X 36,000).
Figure 42. A site of multiple exocytosis in a PRL cell ;
30 minute incubation in KRBG; lead citrate counters tain.
Secretory material from two or possibly three granules
is being released in series (arrows). Note the antimo
nate reactive secretory granule (sg) in close proximity
to the site of exocytosis. Cell membrane (pm) and
translocated granule membrane exhibit no reactivity.
(X 45,000).
184
y m ' y .
mm
1 ■ '
■ ' A . - z ^
'V
i
m m m m m
' 34t^:*'
185
Figure 43. PRL cell; three hour incubation in KRBG; lead
citrate counters tain. After three hours in incubation,
this cell contains relatively few mature secretory
granules. A few granules (sg) remain at the periphery
of the cell. This trend toward depletion of granules
was the primary change observed in PRL cells subjected
to prolonged incubation. Note the collection of micro
vesicles (mv) at the margin of the Golgi (G).
(X 18 ,000) .
Figure 44. Exocytosis in a PRL cell ; one hour incubation
in KRBG; uranyl acetate -lead citrate counters tain. A
portion of the membrane (arrow) at the exocytotic de
pression seems to be pinching off from the remainder
of the membrane and possibly forming a coated vesicle.
(X 45 , 000) .
Figure 45. A portion of a PRL cell near the cell surface ;
three hours incubation in KRBG; lead citrate counter
stain. As noted at all previous incubation times,
some secretory granules (sg) exhibit antimonate pre
cipitate while others do not (compare with Figure 35).
Microvesicles (mv) accumulating at the margin of the
Golgi apparatus (G) contain antimonate particles.
These were most conspicuous after three hours of incu
bation. (X 39,000).
186
187
Figures 46 through 51 arc electron micrographs of sections
of pars distalis showing PRL cells incubated in KRBG with
dopamine (1 x 10"^M) for periods of one, two, or three
hours (experimental hemipituitaries). All tissues were
prefixed by immersion in Glu/KSb for 15 minutes, then post
fixed in OsO^/KSb.
Figure 46. PRL cell; one hour incubation in KRBG with
dopamine ; uranyl acetate -lead citrate counters tain.
PRL cell after one hour’s inhibition of PRL release
contains moderate numbers of mature secretory gran
ules (sg). The Golgi region (G) appears to be active
as indicated by the formation of immature secretory
granules (ig). Some mitochondria (m) show the deposi
tion of relatively large amounts of antimonate pre
cipitate. Cells at this stage were generally similar
to controls except for a marked decrease in exocytotic
activity and an increase in mitochondrial antimonate
deposits in some cells. (X 15,000).
Figure 47. PRL cell ; two hour incubation in KRBG with
dopamine ; lead citrate counterstain. Large secretory
granules (sg) exhibiting considerable pleomorphism are
distributed throughout the cytoplasm. At this stage,
PRL cells generally contained more secretory granules
than in controls or after one hour exposure to dopa
mine. Immature granules (ig), apparently in all
stages of formation, are evident in the Golgi region.
Antimonate deposits are present in some Golgi saccules
(G) . (X 10 ,800) .
■ .r- •
fasiif
189
Figure 48. Golgi region in a PRL cell; two hours incuba
tion in KRBG with dopamine; lead citrate counterstain.
After two hours inhibition, Golgi saccules (G) are
dilated and contain relatively large clusters of
antimonate precipitate. Many of the mitochondria (m)
also show large aggregates of precipitate. Antimonate
deposits both in mitochondria and Golgi saccules were
increased above those in controls. (X 35,250).
Figure 49. PRL cell ; three hours incubation in KRBG with
dopamine ; uranyl acetate-lead citrate counterstain.
The cytoplasm at the pole of the cell opposite the
nucleus is almost entirely filled with large mature
secretory granules (sg). In this cell, granules line
the plasma membrane adjacent to the pericapillary
space as indicated by the basal lamina (bl) but there
is no evidence of exocytosis. (X 18,600).
190
##;####
W ÿ m m
Ê&tà$SÊBÊSSM
\zy' .'Æ-y-ÿ
; a # K .
îmmmyyû
. f j
m a # 6
«
V.
:-yv
.<
r .
' /
191
Figure 50. PRL secretory granules; Lliree hours incubation
in KRBG with dopamine; lead citrate counterstain.
Precipitate is associated with the membranes of some
secretory granules (sg) in a similar pattern to that
seen in controls. Mitochondria (m) now contain dis
tinctly greater amounts of precipitate in comparison
to mitochondria of controls [compare with Figure 34).
[X 37,500).
Figure 51. Golgi region in a PRL cell; three hours incu
bation in KRBG with dopamine; lead citrate counter
stain. Some of the Golgi saccules (G) are greatly
dilated and contain large aggregates of antimonate
particles. Immature secretory granules [ig), varying
in degree of maturation, are evident. [X 31,500).
Figures 52 through 63 are electron micrographs of sections
of pars distalis showing PRL cells incubated in KRBG with
5 X 10"^M mbcAMP [experimental hemipituitaries). All
tissues were prefixed by immersion in Glu/KSb for 15 min
utes, then postfixed in OsO^/KSb.
Figure 52. PRL cell; five minute incubation in KRBG with
mbcAMP ; uranyl acetate - lead citrate counterstain.
Numerous sites of exocytosis [arrows) are evident,
correlating with the rapid response to mbcAMP.
[X 15,900).
Figure 53. PRL cell; 15 minute incubation in KRBG with
mbcAMP ; uranyl acetate-lead citrate counterstain.
PRL secretory granules [sg) have accumulated in the
cytoplasm adjacent to the pericapillary space [ps).
Complex profiles of granule fusion [arrows) appear
at sites relatively distant from the cell membrane.
[X 17 , 500) .
192
( g )
\
M
r-
ps f
%9ê
# #
193
Figure 54. Plasma membrane and peripheral cytoplasm of a
PRL cell; 15 minute incubation in KRBG with mb cAMP ;
uranyl acetate - lead citrate counterstain. Sites of
multiple granule extrusion are evident. Extensive
areas of coated membrane (arrows) are evident at one
site of exocytosis. A coated micropit appears to be
forming at the adjacent site (*). A coated vesicle
(cv) containing antimonate precipitate, is closely
associated with the membrane at a site of granule
fusion. (X 48,000).
Figure 55. Golgi region of a PRL cell; five minute incu
bation in KRBG with mbcAMP ; uranyl acetate -lead
citrate counterstain. Collections of large diameter
smooth-surfaced vesicles (arrows), containing antimo
nate deposits, are located in the Golgi region. Moder
ate quantities of precipitate are localized to Golgi
saccules (G) and mitochondria (m). (X 37,500).
Figure 56. Plasma membrane and peripheral cytoplasm of a
PRL cell; five minute incubation in KRBG with mbcAMP;
lead citrate counterstain. Many of the secretory
granules (sg) exhibit antimonate precipitate associ
ated with their membranes. No deposits are associated
with the membranes of exocytotic pits (*) except in
one area where small grains of antimonate are evident
at the site of membrane fusion (arrows). (X 37,500).
194
' V V ■
# ;
* : "
:^r
' # 6'
: a '
#
Â / % -r
L. * ✓ ^ T,
r - . ^ T jc. < ' 1 % /
195
Figure 57. PRL cell; 30 minute incubation in KRBG with
mbcAMP ; lead citrate counterstain. This cell exhibits
few secretory granules (sg) and no evidence of exocy
tosis, in accordance with a trend toward degranulation
and reduced exocytosis observed as the intermediate
stage of the response to cAMP. Rough endoplasmic
reticulum (rer) appears well developed, forming con
centric whorls at one pole of the cell. (X 13,300).
Figure 58. PRL cell; 30 minute incubation in KRBG with
mbcAMP ; uranyl acetate -lead citrate counterstain.
Considerable variation in the number and distribution
of secretory granules was evident after 30 minute
incubation with mbcAMP (compare this figure with
Figure 57). In this PRL cell numerous secretory gran
ules (sg) and multiple sites of exocytosis (long
arrows) are evident. Coated pits (short arrows)
appear at intervals along the cell membrane.
(X 14, 700) .
196
197
Figure 59. Exocytosis in a PRL cell; 30 minute incubation
with mbcAMP ; uranyl acetate -lead citrate counters tain
A coated micropit or caveola (arrow) projects at the
base of an exocytotic pit. A coated vesicle (cv)
appears to have just pinched off from the membrane at
the same site of exocytosis. (X 37,500).
Figure 60. Peripheral cytoplasm of a PRL cell; 30 minute
incubation in KRBG with mbcAMP ; uranyl acetate -lead
citrate counters tain. A large collection of closely
packed microvesicles (mv) is evident in an area of
the cell between the plasma membrane and the Golgi
apparatus. This was a characteristic response to
cAMP stimulation at this stage. Lamellar rough and
smooth endoplasmic reticulum (arrow) partially en
circles the microvesicles. (X 21,000).
Figure 61. Peripheral cytoplasm of a PRL cell ; 30 minute
incubation in KRBG with mbcAMP ; uranyl acetate - lead
citrate counters tain. In a view similar to that of
Figure 60, a collection of microvesicles (mv) is
encircled by endoplasmic reticulum, part of which
forms a lamellar array (arrow). (X 27,000).
Figure 62. Golgi region of a PRL cell; 30 minute incuba
tion in KRBG with mbcAMP ; uranyl acetate - lead citrate
counters tain. At this stage, mitochondria (m) show
a decrease in antimonate precipitate as compared with
controls (compare with Figure 34) and mbcAMP treated
cells at earlier stages (compare with Figure 55).
Some Golgi saccules (G) contain a moderate amount of
precipitate, which remained unchanged during the cAMP
exposure. (X 37,500).
198
'-"-.M
•} ws
; : W
199
Figure 63. PRL cell; one hour incubation in KRBG with
mbcAMP ; uranyl acetate-lead citrate counterstain.
Secretory granules (sg) are few and scattered.
Coated pits (arrows) are evident at several sites
along the cell membrane. Mitochondria (m) contain
moderate amounts of precipitate. (X 15,600).
200
mi
201
Table 1. Effects of dopamine (1 x 10 ^ M) or mbcAMP
(5 X 10"^ M) on rat pituitary prolactin
release in vitro (NIAMD rat prolactin assay)
Addition
to KRBG®'
Incubation
Time
Number
of Rats
% of Control
(av)^
dopamine 1 hr. 5 10.8 (P<0.033
dopamine 2 hr. 5 9.3 (P<0.03)
dopamine 3 hr. 3 15.2 (P<0.125)
mb cAMP 5 min. 6 803.5 (P<0.043
mb cAMP 15 min. 3 371.9 (P<0.125)
mb cAMP 30 min. 6 204.8 CP<0.015)
mb cAMP 1 hr. 5 134.3 (P<0.03)
Half of each anterior pituitary was exposed to these
treatments while each corresponding control hemi-
pituitary received only incubation medium (KRBG).
Significance of differences in PRL released between
treated and control hemipituitaries computed using the
Wilcoxin Paired-Comparison Test (Hodges and Lehman,
1970) .
202
Table 2. Concentrations o£ antimonate deposits in PRL
cells after incubation in KRBG for periods
of 5 minutes to 3 hours.
Time of
Incubation
Organelle
Mitochondria
Golgi
saccules
secretory
granules
5 min. moderate moderate to heavy moderate
30 min. moderate^ moderate moderate
1 hr. moderate^ moderate moderate
2 hrs . moderate^ moderate moderate
3 hrs. moderate^ moderate moderate
Slightly decreased from concentrations measured at
5 minutes.
203
Table 3. Alterations in the concentration of antimonate
deposits in PRL cells after KRBG-dopamine
(1 X 10 ^ M) incubation compared with the
concentration in controls (KRBG alone)
Organelles
Time of
Incubation Mitochondria
Golgi
saccules
secretory
granules
1 hr. unchanged or
increased
unchanged unchanged
2 hr. increased increased unchanged
3 hr.
,a
increased increased unchanged
; The amount of precipitate was greater at 3 hours than
at 2 hours.
204
Table 4. Alterations in the concentration of antimonate
deposits in PRL cells after KRBG-mbcAMP
(5 X 10 ^ M) incubation as compared with the
concentration in controls (KRBG alone)
Time of
Incubation
Organelles
Mitochondria
Golgi
saccules
secretory
granules
5 min. unchanged unchanged unchanged
15 min. unchanged unchanged unchanged
30 min. decreased unchanged unchanged
1 hr. increased unchanged unchanged
205
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Asset Metadata

Creator Yancey, Sara Barbara (author)

Core Title Cation localization in growth hormone and prolactin cells of the rat pars distalis

School Graduate School

Degree Doctor of Philosophy

Degree Program Anatomy

Degree Conferral Date 1977-02

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag anatomy,biology,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c30-204451

Unique identifier UC11228454

Identifier DP23584.pdf (filename),usctheses-c30-204451 (legacy record id)

Legacy Identifier DP23584.pdf

Dmrecord 204451

Document Type Dissertation

Rights Yancey, Sara Barbara

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus, Los Angeles, California 90089, USA