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Dynamic regulation of vasopressin, a mnemonic neuropeptide, induction of calcium signaling and V1a vasopressin receptors in the rat cerebral cortex

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Dynamic regulation of vasopressin, a mnemonic neuropeptide, induction of calcium signaling and V1a vasopressin receptors in the rat cerebral cortex

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Content DYNAMIC REGULATION OF VASOPRESSIN, A MNEMONIC
NEUROPEPTIDE, INDUCTION OF CALCIUM SIGNALING AND V IA
VASOPRESSIN RECEPTORS IN THE RAT CEREBRAL CORTEX
by
Michael Chang Son
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
(MOLECULAR PHARMACOLOGY AND TOXICOLOGY)
August 2002
Copyright 2002 Michael Chang Son
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UMI Number: 3094417
UMI
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UNIVERSITY OF SOUTHERN CALIFORNIA
THE GRADUATE SCHOOL
UNIVERSITY PARK
LOS ANGELES. CALIFORNIA 90007
This dissertation, written by
.............
under the direction of h .i s . Dissertation
Committee, and approved by all its members,
has been presented to and accepted by The
Graduate School in partial fulfillment of re
quirements for the degree of
DOCTOR OF PHILOSOPHY
Dean of Graduate Studies
Date ........ .
DISSERTATION COMMITTEE
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TABLE OF CONTENTS
List o f Figures............................................................................................................. iii
Abstract....................................................................................................................... v
Chapter One
Introduction..................................................................................................... 1
Chapter Two
Vasopressin-Induced Calcium Signaling in Cultured Cortical
Neurons........................................................................................................... 25
Chapter Three
Regulation and Mechanism of L-Type Calcium Channel Activation
via V 1 a Vasopressin Receptor Activation in Cultured Cortical
Neurons........................................................................................................... 58
Chapter Four
Mechanism of Vasopressin-Induced Phosphatidylinositol Signal
Desensitization and V ia Vasopressin mRNA Regulation by
Protein Kinase C Activation in Cultured Cortical Neurons...................... 8 6
Chapter Five
Overview and Summary.............................................................................. 110
Bibliography............................................................................................................... 125
ii
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LIST OF FIGURES
Figure 1 - Concentration-response analysis of Vi agonist-stimulated
[3H]IPi accumulation in cortical neurons..............................................35
Figure 2 - Time course for 250 nM Vi agonist-stimulated [3H]EPi
accumulation in cultured cortical neurons............................................36
Figure 3 - Effects ofbisindolylmaleimide I and U-73122 on V]
agonist-induced [3H]IPi accumulation..................................................37
Figure 4 - Antagonism of Vi agonist-stimulated [3H]IPi accumulation
by a Via antagonist in cortical neurons................................................. 38
Figure 5 - Vi agonist-induced rise in intracellular Ca2 + concentration
in cultured cortical neurons.................................................................... 40
Figure 6 - Vi agonist induction of [ H]EPi accumulation in the
presence and absence of extracellular calcium ....................................41
Figure 7 - Concentration-response analysis of Vi agonist-stimulated
45Ca2 + uptake in cultured cortical neurons........................................... 42
Figure 8 - Time course analysis of Vi agonist-stimulated 45Ca2 +
uptake in cultured cortical neurons........................................................43
Figure 9 - Antagonism of Vi agonist-induced calcium influx by a
Via antagonist in cortical neurons..........................................................44
Figure 10 - The L-type calcium channel antagonist, nifedipine,
blocked Vi agonist-induced uptake of extracellular Ca2 +
in cortical neurons....................................................................................69
Figure 11 - Blockade of Vi agonist-induced rise in intracellular
calcium concentration by L-type calcium channel antagonist,
nifedipine, in cultured cortical neurons................................................ 70
Figure 12 - Inhibitory effects of bisindolylmaleimide I (PKC
inhibitor) and U-73122 (PLC inhibitor) on Vi agonist-induced
calcium channel activation..................................................................... 71
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Figure 13 - Effects of bisindolylmaleimide I (PKC inhibitor) and
U-73122 (phospholipase C inhibitor) on V) agonist-induced
[3H]IPi accumulation.............................................................................72
Figure 1 4 - V) agonist induction of protein kinase C activation................... 73
Figure 15 - Inhibition of V) agonist-induced intracellular calcium
increase by protein kinase C inhibition in cultured cortical
neurons......................................................................................................74
Figure 16 - Time course for 250 nM V) agonist-induced [3H]IPi
accumulation desensitization in cultured cortical neurons...............96
Figure 17 - Desensitization of V) agonist-induced increase in
intracellular calcium concentration.....................................................97
Figure 18 - Time course for 250 nM Vi agonist-induced [ H]IPi
accumulation desensitization/recovery in cultured cortical
neurons...................................................................................................... 98
Figure 19 - Southern blot analysis of regulation of V) agonist-
induced V ia vasopressin receptor mRNA expression levels
in cortical neurons by R T-PC R ..............................................................99
Figure 20 - Quantitative RT-PCR analysis of regulation of V ia
vasopressin receptor mRNA expression by protein kinase C ............100
iv
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ABSTRACT
Vasopressin (AVP) is a nine amino acid neuropeptide found to have
memory-enhancing properties. Our earlier studies have demonstrated broad
expression of V ia vasopressin receptor (VlaR) mRNA throughout the cerebral
cortex. In addition, our studies have demonstrated neurotrophic effects of A VP
in cortical neurons. However, the effector mechanism of A VP in the cerebral
cortex has yet been determined.
We have studied the effector mechanism of A VP action and the
characterization of the A VP-induced signaling pathway in the cerebral cortex to
determine the functionality and the regulation of VlaRs. As a result, our
investigation of the mechanism of A VP enhancement of cognitive function in
the cerebral cortex via V laRs has led to the following novel findings: (1) In
cerebral cortical neurons, A VP induces calcium influx and phosphatidylinositol
hydrolysis that lead to a rise in intracellular calcium concentration via V laR
activation. (2) A VP-induced calcium signaling follows “calcium-induced
calcium release” model where initial calcium influx through L-type calcium
channel is required for the increase in intracellular calcium concentration. (3)
A VP-induced L-type calcium channel activation is mediated by protein kinase C
(PKC). And (4) desensitization of A VP-induced signaling and V laRs are
mediated via PKC regulation in cortical neurons.
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Understanding the mechanism of A VP function at the biochemical and
molecular 1 evels m ay p rovide i nsights i n d eveloping t herapeutic s trategies f or
various cognitive disorders. In addition, present findings provide insights in
understanding the mechanism of signal desensitization at an early and later
phase that could be essential in providing strategic approaches in treatment of
drug tolerance development at different stages.
Results of our study provide the first documentation of the effector
mechanism of V ia vasopressin receptor and its regulation in the cerebral cortex.
Furthermore, these data demonstrate that V ia vasopressin receptors in the
cerebral cortex are functional entities and open the intriguing possibility that
vasopressin can influence the complex functions of the cerebral cortex.
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CHAPTER ONE
Introduction
How does learning and memory work? Research into human and animal
memory has expanded at an enormous rate in recent decades, spanning fields
from epidemiology and psychology to single-cell neurophysiology and
molecular biology. During the past several decades, the mnemonic effects of
arginine vasopressin on memory consolidation and retrieval have been
demonstrated in a variety of behavioral paradigms not only in rodents, but also
in primates, including humans (DeWied et al, 1988 and 1993; Jolles, 1987).
However, its mnemonic effects at the cellular and molecular levels remain to be
elucidated. Therefore, understanding the mechanisms of learning and memory
function can provide us with fundamental insights in development of therapeutic
strategies for treating memory and other cognitive disorders.
Vasopressin, a neuropeptide
Arginine vasopressin, the endogenous ligand for the V ia vasopressin
receptor, is a neuropeptide that is synthesized in a number of sites in the brain
including the paraventricular, supraoptic and suprachiastmatic nuclei of the
hypothalamus, the bed nucleus of the stria terminalis and the medial amygdala
(Buijs, 1987; Caffe et al., 1987; Sofroniew et al., 1985). Within the nervous
system, vasopressin impacts a broad spectrum o f functions including those
1
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regulating homeostasis (DeWied et al., 1988 and 1993; Kasting et al., 1982), the
stress response (DeWied et al., 1988 and 1993; Jezova et al., 1995; McEwen et
al., 1987), and learning and memory (Brinton et al., 1998; DeWied et al., 1988
and 1993; Jolles, 1987; Kovacs et al., 1994; Strupp et al., 1985).
Vasopressin and memory
The pioneering work of De Wied has clearly demonstrated the
importance of pituitary peptides in the acquisition and maintenance o f learned
behavior (De Wied, 1964 and 1969). The belief that vasopressin influences
learning and memory originated in De W ied’s early demonstration that the
removal of the neurohypophysis resulted in disruption of avoidance behavior
which was restored after treatment with subcutaneous infections of pitressin
(vasopressin and oxytocin), an extract of the posterior pituitary (De Wied, 1965
and 1 966). In accordance w ith these findings, avoidance 1 atencies in various
passive avoidance paradigms are increased upon vasopressin challenge (Bohus
et al., 1972, 1978a and 1978b). Treatment of volunteers with lysine8 -
vasopressin improves memory, as measured by visual retention, recognition and
recall. Immediate memory and learning are also improved by vasopressin
(Legros et al., 1978). Thus, De Wied et al. (1976) concluded that vasopressin
facilitates memory consolidation (the input stage of memory) as well as the
retrieval of stored information (the output stage of memory).
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Vasopressin challenge also improves memory in amnestic patients
(Oliveros et al., 1978). Studies of retrograde amnesia also support the memory
hypothesis where vasopressin and behaviorally-active peptide fragments protect
against CO2- and electroconvulsive shock-induced amnesia (Rigter et al., 1974,
1978), against pentylenetetrazol-induced amnesia in rats (Bookin et al., 1977),
and against puromycin-induced amnesia in mice (Lande et al., 1972; Walter et
al., 1975). De Wied et al. (1976) further suggested that vasopressin might be
involved in the normal, physiological regulation of learning and memory
processes. This conclusion was based on experiments involving Brattleboro rats
with hereditary hypothalamic diabetes insipidus (Valtin et al., 1964) as well as
the effects of centrally administered anti-vasopressin serum. Both normal
animals, following neutralization of centrally available vasopressin by specific
antiserum (Wimersma-Greidanus et al., 1975), and homozygous Brattleboro rats
with diabetes insipidus (De Wied et al., 1975; Bohus et al., 1975 and 1998)
exhibit a memory deficit whereas the inferior learning and memory is r eadily
restored by the administration of vasopressin and vasopressin fragments (De
Wied et al., 1975). The therapeutic value o f vasopressin in amnestic patients
(Oliveros et al., 1978) and its correlation with alterations o f mood (Gold et al.,
1978; Raskind et al., 1979) have also provided evidence that our understanding
of behavioral and biochemical processes leading to central nervous system
effects o f posterior pituitary peptides is o f significance.
3
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Cerebral cortex
Various regions of the brain are uniquely evolved to serve specific
functions such as memory storage and process. One o f the hallmark
characteristics of the cerebral cortex is its integrative capacity. The cerebral
cortex mediates complex integrative functions such as abstract reasoning,
planning, language, sensory perception, and is generally held to be the site of
long-term memory storage (Goldman-Rakic, 1988; Posner et al., 1988; Ojeman,
1991; Chapman, 1959). Information derived from the external environment;
from homeostatic systems and from internal conscious and unconscious events
all converge onto the cerebral cortex for processing and storage (Chapman,
1959). Results from many laboratories, utilizing both animal and human
experimental paradigms, have shown that the cerebral cortex operates as a
highly integrated unit. Within the array of integrating networks are distributed
functional domains that operate as parallel systems (Bressler, 1995; Posner et
al., 1988) that are capable of performing perceptual and mnemonic operations
(Goldman-Rakic, 1988). Based on these data, the cerebral cortex can be
conceptualized as a constellation o f autonomous functional domains that are
highly interactive throughout the cerebral cortex thereby achieving integration
of information processing and storage. A critical, as yet unanswered question
concerns the mechanism(s) by which integration of information occurs in the
cerebral cortex.
4
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Behavioral studies, using paradigms in rodents that ranged from aversive
(De Wied et al., 1988; Koob et a l, 1982) to positively motivated tasks (Bohus,
1977; Messing et al., 1985), have demonstrated that vasopressin can enhance
memory function. In addition, Messing and colleagues have found that the
memory enhancing effects of vasopressin are positively correlated with the
complexity the task (Messing et al., 1985). The greater the task complexity, the
greater the memory enhancement by vasopressin where the behavioral tasks of
increased complexity can be associated with cerebral cortex function (Goldman-
Rakic, 1988 and 1996). Later, experiments by Greenough and his colleagues
showed that memory formation is accompanied by structural changes in the
rodent brain, including changes in number, distribution, and size of synapses on
neurons (Black et al., 1990; Weiler et al., 1995). In our previous studies have
demonstrated that vasopressin induce neurotrophic effects in cultured cortical
neurons (Chen et al., 1996).
Vasopressin distribution in cerebral cortex
A feature that one might expect of an integration system would be a
broad distribution of elements that has the capacity to both receive and transmit
information. Neuropeptides and neurotransmitters are integral constituents of
the central nervous system. Certain neuropeptides (e.g. vasopressin, oxytocin,
ACTH, MSH) affect the central nervous system process underlying memory
(Kovacs et al., 1994). In addition, a broadly distributed receptor system is
5
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dependent on a ligand delivery system that can simultaneously activate receptors
at d istant 1 ocations. T here are se veral w ays s uch a d elivery s ystem c ould b e
constructed: a broadly and densely distributed fiber innervations from a central
synthetic source; paracrine secretion from multiple time linked sources or
diffusion from a reservoir with access to a large portion of the cerebral cortex.
Vasopressin-like immunoreactive fibers are widely present in the central
nervous system (Buijs, 1987 and 1980). In addition to the strong vasopressin
immunoreactivity found in the hypothalamic paraventricular, supraoptic, and
suprachiasmatic nuclei, vasopressin immunoreactive cell bodies and axons were
also found in the amygdala and hippocampus (Dorsa et al., 1983; Caffe et al.,
1987). Immunoreactive activity of vasopressin peptides was detected in the
extract of rat cerebral cortex tissue samples at the low concentrations
(Hashimoto et al., 1985). However, a dense collection of vasopressin
immunoreactive fibers was seen in the parietal cortex and a moderate amount of
immunoreactive fibers were observed throughout the cortex in the adult
Brazilian opossum (Iqbal et al., 1995). Vasopressin mRNAs have been
demonstrated to express in the amygdala by in situ hybridization (Urban, 1990)
and in the cerebral cortex and hippocampus by Northern blot analysis (Grant et
al., 1993) in normal rats. In transgenic mice, vasopressin is expressed under its
own promoter in the neocortex and hippocampus (Grant et al., 1993; Ang et al.,
1993).
6
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Because the vasopressin immunoreactive fibers detected thus far in the
cerebral cortex are extremely sparse, the most accessible known source for
vasopressin in cortex is the cerebrospinal fluid. Importantly, pharmacokinetic
and behavioral evidence accumulated over the past several decades have
documented that vasopressin can traverse the cerebrospinal fluid-brain barrier
(DeWied et al., 1988; Strupp et al., 1985; Koob et al., 1982; Pardridge, 1983,
1986; Palkovits, 1982). Work from De Wied’s group has indicated that
vasopressin is present in the cerebrospinal fluid where it can readily cross and
enter the brain to influence behavior (DeWied et al., 1988) and in the cells of
choroid plexus within the lateral ventricles (Szmydynger-Chodobska et al.,
1995). In addition, a number of new sites for vasopressin have been suggested
including the hippocampus, dentate gyrus, the diagonal band of Broca, and the
choroid plexus (Chodobski et al., 1997; Hallbeck et al., 1999; Planas et al.,
1995).
However, one might still question the significance of the low
cerebrospinal fluid levels o f vasopressin relative to the concentrations required
to induce biochemical and cellular responses. It is very interesting to note that
vasopressin levels in cerebrospinal fluid are elevated in various physiological
and pathological situations (Kasting et al., 1983; Szczepanska-Sadowsks et al.,
1983; Simon-Oppermann et al., 1983; Sorensen et al., 1984 and 1985; Seckl et
al., 1988). Specifically, vasopressin levels in cerebrospinal fluid have been
7
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shown to increase dramatically during behavioral tests of memory (Laczi et al.,
1984). DeWied and colleagues showed more than a decade ago that while the
basal levels of vasopressin in cerebrospinal fluid are low, the level of
vasopressin increases dramatically following a single passive avoidance trial
(Laczi et al., 1984). Moreover, the levels of vasopressin in cerebrospinal fluid is
most likely from synthesis by the choroid plexus (Szmydynger-Chodobska et al.,
1995) and by transport from the median eminence where levels of vasopressin
are quite high (Palkovits, 1982; Dogterom et al., 1977; Brinton et al., 1986).
Interestingly, we found that under conditions associated with a decrease in
memory function, vasopressin content in the median eminence decreased
significantly which would also influence the content of vasopressin in
cerebrospinal fluid (Brinton et al., 1986; Trembleau et al., 1995). Although the
amount of release is small in magnocellular neurons of the supraoptic nuclei and
paraventricular nuclei compared with the amount released from the
neurohypophysis, the concentration of vasopressin in the extracellular fluid of
the supraoptic nuclei resulting from the somato-dendritic release has been
calculated to be 1 0 0 to 1 0 0 0 fold higher than the basal plasma concentration
(Landgraf et al., 1991a). Thus, while the exact source of vasopressin in the
cerebral cortex remains to be determined, the most promising source, the
cerebrospinal fluid, shows remarkable changes in vasopressin content under
cognitively relevant conditions.
8
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V ia vasopressin receptor characterization
The neurohypophysial hormone vasopressin whose actions are mediated
by stimulation of specific G protein-coupled receptors is classified into Vi-
vascular (V ia), V2-renal (V2), and V3-pituitary (V lb) vasopressin receptors
(Michell et a l, 1979; Jard, 1983; Thibonnier, 1992, 1993a,b). Recently, several
groups cloned various members o f the family of human and animal vasopressin
receptors (Bimbaumer et al., 1992; Lolait et al., 1992, 1995; Morel et al., 1992;
De Keyser et al., 1994; Thibonnier et al., 1994; Hutchins et al., 1995). V ia
vasopressin receptor gene was first isolated from a rat liver cDNA library using
a Xenopus oocyte in vitro expression system (Morel et al., 1992 and 1993).
Another expression cloning strategy was to use mouse Ltk-cell system (Barberis
et al., 1993). Since then, V ia vasopressin receptor gene has been isolated from
different species including human, rat, and sheep (Morel et al., 1992 and 1993;
Thibonnier et al., 1994 and 1995; Hutchins et al., 1995). Thus, stable expression
o f these cloned receptors in heterologous mammalian cells allowed the detailed
characterization of the receptor and signal transduction pathways coupled to V ia
vasopressin receptor stimulation, without the possible interference from other
receptor subtypes and endogenously bound hormone.
The G proteins coupled to the V ia vasopressin receptor are mainly
members of the G qm family, but also o f the Gj family, as some of the signals
activated by V ia vasopressin receptors stimulation are reduced by pertussis
9
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toxin pretreatment (Thibonnier et al., 1995). Binding of vasopressin to V ia
vasopressin receptor leads to the activation of phospholipases C, D, and A2, the
production o f inositol 1,4,5-triphosphate and diacylglycerol, the activation of
protein kinase C, the mobilization of intracellular calcium, the influx of
extracellular calcium via receptor-operated calcium channels, and the activation
o f the Na+ -H+ exchanger (Thibonnier, 1992; Briley et al., 1994). Stimulation of
V ia vasopressin receptors also leads to the activation of the MAP kinase
pathway that is pertussis toxin-insensitive, via protein kinase C-dependent and -
independent pathways (Granot et al., 1993; Nishioka et al., 1995). The
secondary nuclear signal mechanisms triggered by activation of V ia vasopressin
receptors include induction of immediate-early response gene expression and
protein synthesis. In addition, there are 12 and 8 threonine-serine residues in the
third cytoplasmic loop and the C-terminal region of the Via receptor,
respectively. These residues could be sites for regulatory phosphorylation,
suggesting that receptor function is regulated by protein kinase.
V ia vasopressin receptor expression
More than a decade ago, our laboratory detected vasopressin recognition
sites in the cerebral cortex o f the mammalian brain (Brinton et al., 1984).
Autoradiographic studies demonstrated vasopressin recognition sites throughout
the cortex, from the frontal lobe to the occipital lobe, which appeared to be
distributed throughout the cortical layers (IV and VI). We have consistently
10
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observed these receptor sites using both agonist and antagonist receptor ligands
(Brinton et al., 1984; Chen et al., 1993). Recent in situ hybridization
localization of mRNA for the V ia vasopressin receptor in adult rat brain
demonstrated V ia vasopressin receptor mRNA expression in cerebral cortex of
adult male and female rats (Szot et al., 1994; Ostrowski et al., 1992). The
presence of V ia vasopressin receptor mRNA in these brain regions supports our
findings that vasopressin receptors continue to exist in the cortex of adult
animals. In addition, results from our studies on V ia vasopressin receptor
mRNA expression using reverse transcriptase-polymerase chain reaction
showed consistent result which demonstrated V ia vasopressin receptor mRNA
expression in three different cell types derived from four major regions of the
cerebral cortex (Yamazaki et al., 1997). Therefore, we have determined the
functionality of V ia vasopressin receptors in the cerebral cortex by investigating
the calcium and phosphatidylinositol signaling mechanisms induced by V ia
vasopressin receptor activation in cultured cortical neurons.
V ia vasopressin receptor desensitization
Desensitization is a key characteristic o f second-messenger pathways
and is manifested by diminished responsiveness to persistent agonist stimulation
where agonist stimulation causes receptor desensitization. Like most G-protein-
coupled receptors already studied, V ia vasopressin receptor undergoes
desensitization when stimulated by its agonist which results in
11
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phosphatidylinositol and calcium signal desensitization in native tissues and in
cell lines expressing the receptor (Cantau et al., 1988; Collins et al., 1984;
Fishman et al., 1985; Grier et al., 1989; Lutz et al., 1990 and 1991; Nathanson et
al., 1994; Plevin et al., 1992). In addition, work from several investigators
suggests that receptor internalization may also contribute to V ia vasopressin
receptor signal desensitization (Cantau et al., 1988; Fishman et al., 1985; Lutz et
al., 1991). On the other hand, the mechanism of down-regulation of V ia
vasopressin receptor, which may last up to hours, has not been extensively
studied. Thus, we have investigated the regulation of V ia vasopressin receptor
mRNA expression to persistent activation o f V ia vasopressin receptors in
cultured cortical neurons.
Most frequently invoked mechanism for rapid desensitization refers to
phosphorylation of threonine and serine residues by protein kinase A or C and/or
by G receptor kinase (Ancellin et al., 1997; Lohse, 1993). The V ia vasopressin
receptor has a number of threonine and serine residues which are putative sites
for phosphorylation and may be involved in desensitization. Results from
Morel’s group suggest that desensitization o f the V ia vasopressin receptor is
mediated by protein kinase C activation in Xenopus oocytes (Ancellin et al.,
1997). Based on these findings, we have investigated the mechanism of the
induction of signal desensitization via V ia vasopressin receptor activation and
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the possible role of protein kinase C activation in vasopressin-induced signal
desensitization in cultured cortical neurons.
Because long-term memory formation and behavioral tasks of increased
complexity are associated with cerebral cortex function, the behavioral effects of
vasopressin suggested to us that the V ia vasopressin receptors we detected in
the cerebral cortex may be of functional importance and that elucidation of the
cellular, biochemical and genomic events underlying integration and long-term
storage of information may provide us with the understanding of the mechanism
of cognitive functions in the cerebral cortex. Therefore, we have pursued the
following studies to determine the functional significance of V ia vasopressin
receptors in the cerebral cortex and its possible mechanism of memory storage,
( 1) to investigate the vasopressin induction of calcium signaling, (2 ) to
determine the regulatory mechanism of vasopressin-induced calcium influx, and
(3) to determ ine the mechanism of V ia vasopressin receptor desensitization via
protein kinase C activation in cultured cortical neurons.
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CHAPTER TWO
Vasopressin-Induced Calcium Signaling in Cultured Cortical Neurons
Abstract
Earlier autoradiographic studies from our laboratory detected
vasopressin recognition sites in the mammalian cerebral cortex (Brinton et al.,
1984; Chen et al., 1993). More recently, we have detected mRNA for the V ia
vasopressin receptors in cultured cortical neurons (Yamazaki et al., 1997). To
determine whether these recognition sites are functional receptors, we have
pursued the signal transduction mechanism associated with the V ia vasopressin
receptor in enriched cultures of cortical neurons. Results of these studies
demonstrate that exposure o f cortical neurons to the selective V 1 vasopressin
9 R
receptor agonist, [Phe ,Om ]-vasotocin, (Vi agonist) induced a significant
accumulation of [3H]inositol-1 -phosphate ([3H]IPi). Vi agonist-induced
accumulation of [3H]IPi was concentration dependent and exhibited a linear
dose response curve. Time course analysis of Vi agonist-induced accumulation
of [3H]IPi revealed a significant increase by 20 min which then decreased
gradually over the remaining 60 min observation period. Vi agonist-induced
accumulation o f [ HJIPi w as b locked b y a s elective V la vasopressin r eceptor
antagonist, (Phenylac1 , D-Tyr(Me)2, Arg6’ 8 , Lys-NH29)-vasopressin. Results of
calcium fluorometry studies indicated that V] agonist exposure induced a
25
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marked and sustained rise in intracellular calcium concentration that was
abolished in the absence of extracellular calcium. The loss of the rise in
intracellular calcium concentration was not due to a failure to induce PIP2
hydrolysis since activation of the phosphatidylinositol pathway occurred in the
absence of extracellular calcium. Vi agonist-induced 45Ca2 + uptake was
concentration dependent with a biphasic time course at 250 nM. These results
indicate that in cultured cortical neurons, V ia vasopressin receptor activation
leads to induction of the phosphatidylinositol signaling pathway, influx of
extracellular calcium, and a rise in intracellular calcium concentration which is
dependent on V ia vasopressin receptor activated influx of extracellular calcium.
These data are the first to demonstrate an effector mechanism for the V ia
vasopressin receptor in the cerebral cortex and provide a potential biochemical
mechanism that may underlie vasopressin enhancement of memory function.
Introduction
Vasopressin, the endogenous ligand for the V ia vasopressin receptor, is
a neural peptide that is synthesized in a number of sites in the brain including
the paraventricular, supraoptic and suprachiastmatic nuclei o f the hypothalamus,
the bed nucleus o f the stria terminalis and the medial amygdala (Buijs, 1987;
Caffe et al., 1987; Sofroniew, 1985). In addition, vasopressin is present in the
cerebral spinal fluid where it can readily cross and enter the brain to influence
26
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behavior (DeWied et al., 1988). Within the nervous system, vasopressin
impacts a broad spectrum of functions including those r egulating homeostasis
(DeWied et al., 1988 and 1993; Kasting et al., 1992), the stress response
(DeWied et al., 1988 and 1993; Jezova et al., 1995; McEwen et al., 1987), and
learning and memory (Brinton, 1988; DeWied et al., 1988 and 1993; Jolles,
1987; Kovacs et al., 1994; Stmpp et al., 1985). Behavioral studies, using
paradigms in rodents that ranged from aversive (DeWied et al., 1988; Koob et
al., 1982) to positively motivated tasks (Bohus, 1977; Messing et al., 1985),
have demonstrated that vasopressin can enhance memory function. In addition,
Messing and colleagues have found that the memory enhancing effects of
vasopressin are positively correlated with the complexity the task (Messing et
al., 1985). The greater the task complexity, the greater the memory
enhancement by vasopressin. Behavioral tasks o f increased complexity can be
associated with cerebral cortex function (Goldman-Rakic, 1988 and 1996).
Furthermore, in cortical neurons, our preliminary evidence indicates that
vasopressin induces neurotrophic effects (Chen et al., 1996), a model for
learning and memory.
More than a decade ago, our laboratory detected vasopressin recognition
sites in the cerebral cortex of the mammalian brain (Brinton et al., 1984; Chen et
al., 1993). The recognition sites for vasopressin were observed by
autoradiography to be uniformly distributed throughout the cortical regions and
27
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were present using both agonist and antagonist receptor ligands (Brinton et al.,
1984; Chen et al., 1 993). M ore r ecently, in situ hybridization 1 ocalization o f
mRNA for the V ia vasopressin receptor in adult rat brain revealed abundant
V ia vasopressin receptor mRNA expression in cerebral cortex of adult male and
female rats (Szot et al., 1994; Ostrowski et al., 1992). The presence of mRNA
for the V ia vasopressin receptor in cerebral cortex supports earlier findings that
vasopressin receptors continue to exist in the cortex during adulthood
(Ostrowski et al., 1994). Our recent studies to determine the cellular
localization of V ia vasopressin receptor mRNA has demonstrated expression in
cultured c ortical n eurons f rom e ach o f the f our r egions o f the c erebral c ortex
(Yamazaki et al., 1997).
Three reasons governed the selection of investigating V ia vasopressin
receptor a ctivation o f calcium s ignaling i n c ortical n eurons. First, c alcium i s
critical to the development of the cellular and structural features of the nervous
system (Gu et al., 1997; Komuro et al., 1996). Our findings of Vi agonist-
induced neurotrophism in cultured cortical (Chen et al., 1996) and hippocampal
neurons (Brinton et al., 1 994) are consistent with calcium regulation of n erve
cell growth and is consistent with the actions and signal transduction
mechanisms of many growth factors that increase intracellular calcium by the
rapid turnover of inositol phospholipids (Berridge, 1993 and 1998; Majerus et
al., 1990). Second, our previous work has demonstrated that V ia vasopressin
28
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receptor a ctivation i n c ultured h ippocampal n eurons 1 eads to a ctivation of t he
phosphoinositide signaling pathway, uptake of calcium from the extracellular
medium, and induction o f complex intracellular calcium signaling (Brinton et
al., 1994). Lastly, calcium has been found to be a critical biochemical signal in
electrophysiological models of learning and memory (Brinton et al., 1998).
Thus, we have investigated vasopressin regulation of phosphoinositide
signaling, calcium influx, and intracellular calcium levels in cultured cortical
neurons.
Materials and Methods
Cell culture preparation
Cultures of cortical neurons were prepared following the method
described by Brinton et al. (Son et al., 1998). Cortices were dissected from the
brains of embryonic day 18 (E18: with EO as breeding day) Sprague-Dawley rat
fetuses. The tissue was treated with 0.05% trypsin in Hank’s Balanced salt
solution (50 mM KC1, 3 mM KH2P 0 4, 80 mM NaCl, 0.9 mM NaH2P 0 4-7H20 ,
10 mM Dextrose, 0.3 M HEPES) for 5 minutes at 37 °C. Following incubation,
trypsin was inactivated with cold phenol red free Dulbeccos Modifies Eagle
Medium (DMEM; Gibco) supplemented with 10 mM NaHC03, 10% fetal
bovine serum, 5 U/ml penicillin and 5 fig/ml streptomycin, and 10% F12
nutrient medium for 3 minutes. Tissue was then washed with Hank’s Balanced
29
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salt solution (2 x) and dissociated by repeated passage through a series of fire
polished constricted Pasteur pipettes. Cells were plated at a concentration of 1 x
106 cells/ml onto polyethylenimine (PEI) coated (10 pg/ml; Sigma. St. Louis) 35
mm Petri dishes. Cells were grown in 2 ml of phenol red containing Neurobasal
Medium (which does not promote glial cell proliferation; Gibco #320-1103),
B27 medium supplement (Gibco #680-7504), 25 pM glutamate, 0.5 mM
glutamine, 5 U/ml penicillin, and 5 pg/ml streptomycin and maintained in a 37
°C 5% CO2 incubator.
Assessment o f 1 3 H lIP i accumulation
After 2 days of incubation, 1 ml of the media was aspirated off and
replaced with 0.5 ml of media containing 4 pCi/ml of [3H]myo-inositol (specific
activity = 23.45 Ci/mmol). Preliminary studies indicated that 24 hours of
incubation with [3H]myo-inositol was optimal for incorporation into the cell
lipids. Cells were rinsed twice with 1 ml of Krebs Ringer bicarbonate (KRB)
buffer (124 mM NaCl, 5 mM KC1, 1.3 mM MgCl2- 6 HaO, 1.2 mM KH2PO4, 26
mM NaHCCh, 10 mM dextrose, 1 mM C aC k) then pre-incubated in 1 ml of
KRB for 2 0 m in at 3 7 °C. F ollowing thepreincubationperiod, solution was
exchanged for KRB + 10 mM LiCl (inositol phosphatase inhibitor) + test
peptides at 20 min at 37 °C for the dose response analysis and at varying time
points for the time course analysis. Peptides were dissolved in KRB solution
(without LiCl) immediately prior to use.
30
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The reaction was terminated by the addition of 750 pi o f ice cold
methanol, cells were scraped from the petri dishes with a cell scraper, and
transferred to test tubes containing 1 ml of chloroform and 0.5 ml of deionized
distilled water. An additional 750 pi of ice-cold methanol was added to the petri
dishes then transferred to the same test tubes. Chloroform samples were
vortexed then centrifuged (5 min, 2000 rpm). The aqueous phase was transferred
to test tubes containing 4 ml of deionized distilled water, vortexed, and
centrifuged (5 min, 2000 rpm). Five ml of the sample was filtered through 1 ml
Dowex columns (Bio-rad, MO), which had been generated using 1 ml of 1 M
ammonium formate/0.1 M formic acid. Columns were washed with 5 ml of
distilled deionized water (2x), which was discarded, followed by 2.5 ml of 1 M
ammonium formate/0.1 M formic acid. This eluate containing the inositol
phosphates was collected and 1 ml of the 2.5 ml eluate was counted by
scintillation in 5 ml of scintillation fluid. In order to present comparable data
cross experiments, [3H]IPi accumulation data were analyzed by determining the
ratio of aqueous CPM/organic CPM and expressed as a percent of basal
accumulation.
Calcium fluorometry
Cortical neurons were cultured onto PEI-coated coverslips (Hitachi,
ANO-2103, Danbury, CT) that are sized to fit within the fluorometry cuvette
and allowed to grow for 2 days in phenol red free DMEM supplemented with 10
31
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mM NaHC03, 10% fetal bovine serum, 5 U/ml penicillin and 5 pg/ml
streptomycin, and 10% F I2 nutrient medium. Media was then removed and the
cultures rinsed 2x in 37 °C Krebs buffer (137 mM NaCl, 5.3 mM KC1, 1.0 mM
MgCl2-6 HaO, 1.2 mM KH2PO4, 10 mM HEPES, 25 mM Dextrose, and 1.5 mM
CaCF) for 10 min at 37 °C. Fluo-3 acetoxymethyl ester (5 mM) was added to
neurons for 30 min in Krebs buffer at 37 °C (same as above) followed by 3
buffer washes and allowed to equilibrate for 15 min in Krebs buffer without
fluo-3 as described in (Kao et al., 1989). Fluorescent signals were detected
using a Flitachi f-2000 spectrometer. Fluo-3 was excited at 488 nm and
emission signals above 515 nm were collected. Data are expressed as relative
absorbance across time. Various controls were performed prior to the actual
experiments. Ten pM glutamate +2 mM glycine was used as a positive control
to increase intracellular calcium concentration, 200 nM o f the calcium
ionophore A23187 was used to detect the maximal intracellular calcium rise,
and 4 mM MgCh was used as negative control to quench the fluo-3.
Calcium imaging
Cortical neurons to be used in calcium imaging studies were cultured at a
density o f lxlO 6 cell/ml onto (+)poly-L-lysine coated coverslips then placed on
to coverslip clamp chamber MS-502S (ALA Scientific Instruments; NY).
Unless stated otherwise, neurons were used 7 days following seeding. Neurons
were briefly washed with Krebs buffer (137 mM NaCl, 5.3 mM KC1, 1.0 mM
32
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MgCl2-6H20 , 1.2 mM KH2PO4, 10 mM HEPES, 25 mM Dextrose, and 1.5 mM
CaCl2 ) then loaded with fura-2 acetoxymethyl ester (5 pmol / L; Molecular
Probes, Inc., OR) by incubating for 45 minutes at 37 °C. Excess fura-2 dye was
removed by washing with Krebs buffer and incubated for 30 minutes at 37 °C
for neurons to equilibrate. Fluorescence measurements of intracellular calcium
concentration were performed using InCyt2™, Fluorescence Imaging System
(Intracellular Imaging, Inc., OH). Neurons were placed on a stage of an inverted
microscope (MT-2, Olympus) equipped with epifluorescence optics (20X,
Nikon). Fluorescence was excited at wavelengths of 340 and 380 nm
alternatively using rotating 4-wheel filter changer. To minimize the background
noise of the fura-2 signal, successive values (16 sample images, 8 background
images), were averaged (~13 images / min).
45Ca2 + uvtake
Cultures were similar in age and cellular density to those used for
[3H]BPi accumulation. Cultures were washed in Krebs buffer for 20 min at 37
°C. Following this wash, V) agonist + 1.0 pCi 45Ca2 + per 2 ml were
simultaneously added to the cultures (specific activity of 45Ca2 + = 30.7 mC/mg).
Following the incubation period with V) agonist, the treatment solution was
decanted and cultures were washed two times with 2 ml of Krebs buffer. After
decanting the last Krebs wash, 45Ca2 + uptake was terminated by the addition of 1
ml 7 % ice cold trichloroacetic acid to each culture dish and incubated for 45
33
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min at 4 °C. Trichloroacetic acid extracts were removed and placed into
scintillation vials for counting and 1 ml of NaOH added to the cultures to
solubilize protein for analysis of protein content by the Lowry method (Lowry et
al., 1951).
Chemicals
[3H]Myo~inositol ( spec. act. 2 3.4 Ci/mmol) and 45Ca2 + (spec. act. 3 0.7
mC/mg) were purchased from Dupont New England Nuclear. V) agonist ([Phe2,
Om8]-vasotocin), V) antagonists ([ 1 -(P-Mercapto-(3, (3-cyclopentamethylene
propionic acid), 2-(0-methyl)tyrosine] -Arg -vasopressin (Pennisula
2 4 8
Laboratories, Inc.); [d(CH2 )5, D-Ile , lie , Arg ]-vasopressin (Peninsula
1 0 C i 8
Laboratories, Inc); Des-Gly-[Phaa, D-Tyr(Et) , Lys , Arg ]-vasopressin
2 8
(Pennisula Laboratories, Inc.); ([d(CH2)5, Tyr(Me) , Arg ]-vasopressin
(Manning compound, Bachem); and linear Via receptor antagonist, [Phenylac1 ,
D-Tyr(Me)2, Arg6’ 8 , Lys-NH2 9 ]-vasopressin (Bachem). Dowex, ammonium
formate, and formic acid for the ion exchange columns were obtained from
Sigma Chemical. Fluo-3 acetoxymethyl ester was purchased from Molecular
Probes (Portland, Oregon).
Data analysis
[3H]IPi accumulation data are presented as mean percent of basal ±
S.E.M., as determined by the ratio of aqueous cpm/organic cpm. 45Ca2 + uptake
data are presented as mean percent o f basal ± S.E.M. Statistical analysis was
34
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performed by a Student’s f-test or by a one-way analysis of variance (ANOVA)
followed by Newman-Keuls post hoc analysis.
Results
To determine the relationship between the concentration of Vi receptor
agonist (Vi agonist) and its response, cultured cortical neurons were exposed to
varying concentrations of V) agonist. Results of this analysis showed a linear
increase of [3H]EPi accumulation as the concentration of V) agonist increased.
At 250 nM V) agonist, a significant increase in [ HJIPi accumulation was first
observed (144.8% + 16.3, /><0.01; Fig. 1) which progressed with 1000 nM Vi
agonist producing an 89.5% increase over control (189.5% ± 16.3, /K0.001;
Figure 1).
220
200
o
5 ?
180
e
o 160
3
S
3
O
tj
<
140
120
6 "
b- 1 0 0
Basal 10 100 250 500 1000
V, Agonist (nM)
Figure 1. Concentration-response analysis of Vi agonist-stimulated [3H]IPi
accumulation in cortical neurons. Cultures were exposed to varying concentrations of
Vi agonist for 20 min. Values are from one experiment and are representative of three
separate experiments, with 5-6 cultures per condition per experiment. Data points
represent mean + S.E.M, ***/><0.001, **/K0.01.
35
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
O Basal
® V, Agonist (250nM)
125 -
& T 100 -
15 20 30 60
Time (min)
Figure 2. Time course for 250 nM V! agonist-stimulated [3H]IPi accumulation in
cultured cortical neurons. Cultures were exposed to 250 nM Vi agonist at different time
periods. Values are from one experiment and are representative of three separate
experiments, with 6-8 cultures per condition per experiment. Data points represent
mean± S.E.M, **/K0.01.
Time course analysis demonstrated a significant accumulation of [ H]IPi
at 20 minutes of exposure to 250 nM Vi agonist (143.3% ± 8.9,/><0.01; Figure
2) which began to decline in magnitude by 30 minutes, but which was still
significantly greater than control. To account for the gradual decrease in Vi
agonist-induced [3H]IPi accumulation, protein kinase C inhibitor,
bisindolylmaleimide (BIS I), was applied to block the possible activation of
negative feedback mechanism. U-73122 is a phospholipase C inhibitor that
blocks the production of inositol phosphates whereas BIS I is a protein kinase C
inhibitor which allows continuous production o f inositol phosphates by
inhibiting the negative feedback mechanism of the phosphoinositide signal
36
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
pathway. The results of the experiments to confirm the efficiency of U-73122
and BIS I showed that 5 pM U-73122 effectively blocked the accumulation of
inositol phosphates (83.7% ± 4.5; Figure 3) whereas 1 pM BIS I and 250 nM Vi
agonist allowed greater accumulation o f inositol phosphates (206.94% ± 13.91,
/K0.001; Figure 3) compared to 250 nM Vi agonist alone (176.30% ± 1 5.07,
pO .001; Figure 3).
240
1 ^
S Q 200
C m
0 1 8 0
■ 'S
U i®
§ 1 4 0
•-S
120
1 100
3 8 0
( J
< 60
& 40
E 2 0
0
Figure 3. Effects of bisindolylmaleimide I and U-73122 on V] agonist-induced [3H]IPt
accumulation. One set of cultures was pre-incubated with 1 pM BIS I for 20 min, then
incubated with 250 nM Vi agonist and 1 pM BIS I for 60 min. Another set of cultures
was treated the same except with 5 pM U-73122. Values are mean ± S.E.M. from one
experiment and are representative of data obtained in two separate analyses with 7-8
cultures per condition per experiment, *** p<0 .0 0 1 .
To confirm that Vi agonist induction of [3H]IPi accumulation was
mediated by the V ia vasopressin receptor, a linear V ia receptor antagonist,
37
***
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Des-Gly-jThaa1 , D-Tyr(Et)2, Lys6, Arg8]-vasopressin, was tested for its ability to
block Vi agonist-induced [3H]IPi accumulation. The V ia antagonist alone at
100 nM concentration had no significant effect upon [3H]IPi accumulation
(108.1 ± 5.0, p<0.65; Figure 4) whereas, 100 nM V ia antagonist was able to
block Vi agonist induction of [3H]IPi accumulation (108.1% ± 5.0, p<0.65;
Figure 4).
■H p-eiraiatiminthe presence
■ I (flCOnMVj attagprist
lit
Peptide;
Figure 4. Antagonism of Vi agonist-stimulated [3H]rPj accumulation by a Vla
antagonist in cortical neurons. One set of cultures was exposed to the 250 nM Vi
agonist and 100 nM Via antagonist alone for 60 min. A second set of cultures was pre
exposed to 100 nM Via antagonist for 20 min then exposed to 250 nM Vi agonist for 60
min. Values are mean ± S.E.M. from one experiment and are representative of data
obtained in three separate analyses with 7-8 cultures per condition per experiment, **
/KO.Ol.
In addition, we have used various other Vi antagonists to study the
receptor specificity of V i agonist-induced calcium signaling in cultured cortical
38
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
neurons that demonstrated agonist and inverse agonist effects. Ten nanomolar
o f the Vi antagonists, [d(CH2)5, D-Ile2, lie4, Arg8 ]-vasopressin and [l-((3-
Mercapto-P, P-cyclopentamethylene propionic acid), 2-(0-methyl)tyrosine]-
Arg8 -vasopressin (Bachem), showed agonist properties resulting in [3H]IPi
accumulation which were 64.6% and 105.5% over control, respectively. In
contrast, 10 nM Vi antagonist, ([d(CH2)51 , Tyr(Me)2, Arg8 ]-vasopressin
(Peninsula Laboratories, Inc.), demonstrated an inverse agonist effect to produce
25% inhibition of [3H]IPt accumulation compared to control.
The Vi agonist-induced accumulation of inositol phosphates suggested a
regulation of intracellular calcium. To determine whether V ia vasopressin
receptor activation resulted in an increase in intracellular calcium concentration,
calcium imaging was used. Our results indicated that Vi agonist induced
intracellular calcium concentration increase (Figure 5A and 5B), which
interestingly, was inhibited upon removal o f extracellular calcium (Figure 5C
and 5D). In addition, we used the calcium sensitive dye, fluo-3, to determine
quantitative changes and to characterize the rise in intracellular calcium
concentration induced by Vi agonist in cultured cortical neurons. At 1.0 mM
extracellular calcium, 250 nM Vi agonist induced a sustained increase in
intracellular c alcium c oncentration for the d uration o f the e xposure (< 7 min;
Figure 5E). Removal o f extracellular calcium resulted in a loss in V] agonist-
induced rise o f intracellular calcium concentration (Figure 5E).
39
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
(3
1.0 m M G ?
O.OmMCa"" j j i
0.30 -
W
y
o
C O
0.25
250nMjV| aonist
250nMV, agonist
i ! i i , 1
100 200 300
TDvECsoc)
100 200 300
TIME (sec)
Figure 5. Vi agonist-induced rise in intracellular Ca2 + concentration in cultured cortical
neurons. Cortical neurons were loaded with fura-2 (A and C), then exposed to 250 nM
Vi agonist in the presence of 1.0 mM extracellular Ca2 + (B) and in the absence of
extracellular Ca2 + (D). Quantitation and characterization of Vi agonist-induced rise in
intracellular Ca2 + concentration demonstrate a pronounced and sustained increase in
intracellular Ca2 + concentration (E). However, not only the sustained intracellular Ca'
2+
rise was abolished but also Ca2 + increase was blocked in the absence of extracellular
Ca (F). Note that while the absolute numbers for absorbance for E and F are different,
the axis (total range of absorbance and time) are equal. Observations in both calcium
containing and calcium free buffer have been replicated at least 3 times from cultured
cortical neurons derived from different culture dates.
40
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
To further investigate the extracellular calcium dependence of Vi agonist
induction of calcium signaling, we used calcium-free media to detect changes in
[3H]IPi accumulation in cultured cortical neurons. Removal of extracellular
calcium, Vi agonist-induced [3H]IPi accumulation was not significantly affected
(156.3 ± 15.7; /K0.01; Figure 6 ) compared with 1.0 mM extracellular calcium
(158.9 ± 17.1,/><0.05; Figure 6 ).
2 0 0
73
§ 180
C Q
160
o
g , 140
.2 1 2 0
| to o
| 80
o
% ®
o 7 40
^ 20
0
Figure 6 . V x agonist induction of [3H]IP 1 accumulation in the presence and absence of
extracellular calcium. In Ca2 + -free KRB buffer, additional Mg2 + was added. Cultures
of cortical neurons were exposed to 250 Vi agonist in 1.0 mM and 0.0 mM Ca2 +
containing KRB buffer for 60 min. Values represent mean ± S.E.M. from one
experiment and are representative of two separate analyses with 7-8 cultures per
condition per experiment, * p<0.05, **/K0.01.
Because removal of extracellular calcium blocked the rise in intracellular
calcium concentration but not IPi accumulation, it was clear that the increase in
41
LOrnMCd
OOmM Cd
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
intracellular calcium concentration detected in the calcium fluorometry
experiments was generated from another source. We therefore pursued whether
V laR a ctivation r egulated c alcium i nflux from the extracellular c ompartment.
Vi agonist (250 nM) significantly increased 45Ca2 + influx in a concentration
dependent manner (Figure 7). The significant increase in 45Ca2 + influx was
observed at a concentration as low as 1 nM (131.2% ± 5.9, p<0.05) and as high
as 1000 nM (227.4% ± 6.8,/X0.001).
250 -
5 sec
S ' 225
S 1 2 5 -
+
7* ioo -
75 -
Basal 1 10 100 250 500 1000
V, A g o n ist (nM)
Figure 7. Concentration-response analysis of V i agonist-stimulated 45Ca2 + uptake in
cultured cortical neurons. 45Ca2 + was added simultaneously with varying concentrations
Vi agonist or control buffer and cells incubated for 5 s at room temperature. Values are
from one experiment and are representative of three separate experiments, with 5
cultures per condition per experiment. Data points represent mean ± S.E.M, ***
/K0.001, **/?<0.01, *p<0.05.
A time course analysis indicated that Vi agonist-induced 45Ca2 + uptake
was biphasic over time (Figure 8 ). Within 5 seconds of Vi agonist (250 nM)
exposure, significant 45Ca2 + influx occurred (125.9% ± 7.2, p<0.05). After 5
42
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
seconds, the concentration of 45Ca2 + influx decreased over time then increased
rapidly again at 30 seconds (136.3% ± 10.0, /K0.05).
o Basal
9 Vj agonist (250 nM)
C 3
e a
©
+
100 -
15 30 1 5 10 60
Time (sec)
Figure 8 . Time course analysis of Vi agonist-stimulated 45Ca2 + uptake in cultured
cortical neurons. 45Ca2 + was added simultaneously with 250 nM Vj agonist or control
buffer at different time periods. Values are from one experiment and are representative
of four separate experiments, with 5 cultures per condition per experiment. Data points
represent mean ± S.E.M, ***/?<0.001, *p<0.05.
A linear V ia antagonist was used to determine the specificity of Vj
agonist-induced calcium influx via V laR activation. Results of our analysis
showed that 100 nM VI a antagonist alone did not induce calcium influx (95.4 ±
5.0, Figure 9). Whereas the same concentration o f V ia antagonist effectively
blocked the Vi agonist (250 nM)-induced calcium influx in cultured cortical
neurons (101.9 ± 9.4, Figure 9).
43
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
14 0 |
pmnculn&on in the presence
"3 120 - m m o // CDnMVja artagorist
e 3
P Q
100
o
N O
8 0
o
3
£ *
o
20
Peptides
Figure 9. Antagonism of Vi agonist-induced calcium influx by a Via antagonist in
cortical neurons. One set of cultures was exposed to the 250 nM Vi agonist and 100
nM V]a antagonist alone for 5 sec. Second set of cultures was pre-exposed to 100 nM
Via antagonist for 20 min then exposed to 250 nM Vt agonist for 5 sec. Values are
mean ± S.E.M. from one experiment and are representative of data obtained in three
separate analyses with 7-8 cultures per condition per experiment, **p<0.01.
Discussion
The purpose of this study was to investigate whether the V ia vasopressin
receptor in the cerebral cortex was functional and the signal transduction
mechanism associated with this receptor. Results o f this study demonstrate that
the neural peptide vasopressin induces calcium signaling via activation of V ia
vasopressin receptors in enriched cultures o f cortical neurons. Using a selective
agonist for Vi vasopressin receptor, the data show that V ia vasopressin receptor
activation leads to phosphoinositide hydrolysis and uptake of calcium from the
44
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extracellular compartment which ultimately leads to an increase in intracellular
calcium in cultured cortical neurons.
Vasopressin induction of [3H]IPi accumulation and the pharmacological
linkage of this effect to the V] receptor are consistent with Vi receptor action in
a host of other tissues including neural preparations (Bone et al., 1984; Hatton et
ah, 1992; Shewey et ah, 1988). The Vi agonist induced a concentration
dependent increase in [3 H]IPi accumulation with maximal stimulation, 89%,
increase over basal level, occurring at 1000 nM V] agonist. The linear
concentration response curve o f Vi agonist-induced [ H]IPi accumulation m
cultured cortical neurons was distinct from the inverted U-shaped curve
exhibiting the [3H ]ffi accumulation in cultured hippocampal neurons (Ozawa et
ah, 1989).
The time course analysis revealed a complex relationship between time
and IPi accumulation. The time course required for Vi agonist-induced [ H]IPi
accumulation to reach statistical significance is consistent with other reports in
neural tissue (Bone et ah, 1984; Horn et ah, 1987). At 20 min of exposure to
- 3
250 nM Vi agonist, significant [ H]IPi accumulation was induced. Following
20 min, [3H]IPi accumulation declined over the period of time studied (60 min)
although a significant increase still was apparent at 30 min. Since 10 mM LiCl
was used to block polyphosphoinositide metabolism in brain and to prevent
inositol recycling by an uncompetitive inhibition o f inositol monophosphatase
45
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(Mota de reitas et al., 1994; Nahorski et al., 1991), the accumulation of BPi was
expected to sustain or increase. A potential explanation for the decline in
accumulation over time is negative regulation of phosphatidylinositol / calcium
signaling pathway by protein kinase C (Bird et al., 1993). Activation of
phospholipase C catalyses the breakdown of phosphatidylinositol-4,5-
bisphosphate into the two intracellular messengers inositol-1,4,5-trisphosphate
and diacylglycerol to release calcium from intracellular stores and to activate
protein kinase C, respectively (Berridge, 1993). Phospholipase C-induced
protein kinase C activation can lead to phospholipase C inhibition thus
preventing further production of inositol phosphates (Mota de reitas et al., 1994;
Nahorski et al., 1991). Thus, prolonged exposure with V) agonist may activate a
negative feedback mechanism to prevent excess production of inositol
phosphates in cultured cortical neurons. Alternatively, the effect of lithium may
not be permanent and the intracellular concentration of LiCl may have been
decreased by being incorporated into the membranes or by e scaping from the
neurons via transports such as sodium-lithium countertransport demonstrated in
erythrocytes (Canessa et al., 1994). Another possibility is the presence of other
substrates with higher affinity to lithium in the cell in addition to inositol
monophosphatase or the presence of other mono- or divalent cations (such as
Na+ and Mg2 + ) which could interfere with the effect of lithium (Johansson et al.,
1992; Mota de reitas et al., 1994; Nahorski, et al., 1991; Reader et al., 1990).
46
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During the course of these studies, we used several Vi antagonists to
study the receptor specificity o f Vf agonist-induced calcium signaling in
cultured cortical neurons. These experiments using various V] antagonists
demonstrated agonist and inverse agonist effects of these antagonists in cultured
cortical neurons. Ten nanomolar of the Vf antagonists, [d(CH2)s, D-Ile2, lie4,
Arg8 ]-vasopressin and [l-((3-Mercapto-(3, P-cyclopentamethylene propionic
acid), 2-((9-methyl)tyro sine] - Arg8 -vasopressin (Bachem), showed agonist
properties resulting in [3H]EPi accumulation which were 64.6% and 105.5% over
control, respectively. In contrast, 10 nM Vi antagonist, ([d(CH2)s1 , Tyr(Me)2,
Arg8 ]-vasopressin (Peninsula Laboratories, Inc.), demonstrated an inverse
agonist effect to produce 25% inhibition of [ H]IPi accumulation compared to
control. These results may be due to different antagonists having different Kj
properties for V ia vasopressin receptors in cortical neurons. Alternatively,
these antagonist’s inverse actions o f Vi receptor antagonists may be the result of
different receptor characteristics throughout development which is not
uncommon and is increasingly found to be a function of receptor subunit
composition or developmentally specific isoforms of the receptor (Minakami et
al., 1995; Tribollet et al., 1991) which may explain such opposing actions of the
antagonists. One such example relevant to the vasopressin receptor system was
reported in Szot and colleagues (Szot et al., 1989). In this study, the Vi
antagonist was a potent competitor for the adult rat cingulate gyrus A VP
47
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
receptor whereas in the rat pup it was the least potent of the Vi compounds
studied. The change in labeling pattern observed during development may
reflect changes in the pharmacology and specificity of A VP binding sites in the
brain (Tribollet et al., 1991) and their role in maturation of the central nervous
system. In these studies, we have used the linear V ia antagonist (Phenylac1 , D-
Tyr(Me)2, Arg6’ 8, Lys-NH29)-vasopressin; Bachem) which effectively
antagonized the 250 nM V i agonist-induced [ 3H]IPi accumulation in cultured
cortical neurons. This linear V ia antagonist demonstrated to be a ‘true’
antagonist such that it was devoid of any effect alone but inhibited the action of
the agonist.
Vi agonist induced an influx of extracellular calcium in a time and
concentration dependent manner. The calcium fluorometry data demonstrated
that the influx of extracellular calcium is required for the increase in intracellular
calcium concentration. In the absence of extracellular calcium, no rise in
intracellular calcium concentration was observed, whereas, Vi agonist-induced
[3H]IPi accumulation was unaffected. Two postulates emerge from our findings
that the rise in intracellular calcium concentration was dependent upon
extracellular calcium. First, these data indicate that the influx of calcium is an
obligatory step for the rise o f intracellular calcium concentration. The
requirement of extracellular calcium influx is consistent with a calcium
dependent release o f intracellular calcium from the endoplasmic reticulum and is
48
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
consistent with a calcium-induced calcium release model (Berridge, 1993). In
this system, the JP3 receptor displays a bell-shaped response to calcium which
functions as a coagonist with IP3 to release stored calcium (Berridge, 1993;
Finch et al., 1991; lino, 1990; Bezprozvanny et al., 1991). In the absence of
calcium, IP3 has little effect but becomes increasingly active as the concentration
of calcium rises. The synergistic interaction between IP3 and calcium on
calcium release is followed by inhibition when high intracellular calcium levels
are attained (Berridge, 1993). The influx of extracellular calcium induced by V)
agonist plays a key role in triggering the release of calcium from the
intracellular stores in cultured cortical neurons. Therefore, our data
demonstrating that Vi agonist-induced rise in intracellular calcium concentration
is dependent upon the presence of extracellular calcium are consistent with the
“calcium-induced calcium release” model (Berridge, 1993).
The purpose o f this study was to investigate the signal transduction
mechanism associated with the V ia vasopressin receptor in the cerebral cortex.
Results of our study, along with the results of V) agonist-induced neurotrophism
have demonstrated the functionality o f V ia vasopressin receptors in the cerebral
cortex. Results o f this investigation indicate that Vi agonist activates
phosphatidylinositol diphosphate hydrolysis leading to an accumulation of
[3H]IPi that is independent of extracellular calcium. Moreover, V ia vasopressin
receptor activation induced a rapid and significant influx of extracellular
49
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
calcium which resulted in an immediate and sustained increase in intracellular
calcium concentration. Future studies will further investigate the source and the
mechanism o f Vi agonist-induced Ca2 + influx, and the downstream signals
induced by the increase in intracellular calcium concentration in cultured
cortical neurons.
Results of this study provide the first documentation of the biochemical
signaling pathway induced by vasopressin in the cerebral cortex. Furthermore,
these data demonstrate that V ia vasopressin receptors in the cerebral cortex are
functional entities and open the intriguing possibility that vasopressin can
influence the complex functions of the cerebral cortex.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
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CHAPTER THREE
Regulation and Mechanism of L-type Calcium Channel Activation via V ia
Vasopressin Receptor Activation in Cultured Cortical Neurons.
Abstract
Our previous studies demonstrated that in cultured cortical neurons, V ia
vasopressin receptor (VI aR) activation resulted in a sustained rise in
intracellular calcium concentration that was dependent on calcium influx (Son et
al., 1998). To investigate the mechanism underlying the dependency on calcium
influx, we pursued the identification of calcium channel subtypes that were
regulated by V laR activation. Results of these analyses demonstrated that the
L-type calcium channel blocker, nifedipine, blocked VI vasopressin receptor
agonist (Vi agonist)-induced calcium influx. Intracellular calcium imaging
analyses using fura-2AM demonstrated that blockade o f L-type calcium
channels prevented Vi agonist-induced rise in intracellular calcium
concentrations. To determine the mechanism of V laR activation of L-type
calcium channels, intermediate signaling components of the phosphatidylinositol
signal pathway were investigated. Results o f these analyses demonstrated that
Vi agonist-induced calcium influx was blocked by both the phospholipase C
inhibitor (U-73122) and protein kinase C inhibitor (bisindolylmaleimide I).
Analysis o fV laR activation o fp ro tein k in ase C ( PKC) demonstrated that V i
agonist induced PKC activity within 1-minute exposure in cultured cortical
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neurons. These results indicate that in cultured cortical neurons, V laR
activation regulates influx of extracellular calcium via L-type calcium channel
activation through a protein kinase C dependent mechanism.
Introduction
Vasopressin, the endogenous 1 igand for V la v asopressin receptor, is a
neuropeptide that is synthesized in a number of sites in the brain including
paraventricular, supraoptic and suprachiastmatic nuclei of the hypothalamus, the
bed nucleus of the stria terminalis and the medial amygdala (Buijs, 1987; Caffe
et al., 1987; Sofroniew et al., 1985). Within the nervous system, vasopressin
impacts a broad spectrum of functions including those r egulating homeostasis
(DeWied et al., 1988; Kasting et al., 1982), the stress response (DeWied et al.,
1988; Jezova et al., 1995; McEwen et al., 1987), and learning and memory
(Brinton, 1998; DeWied et al., 1988; Jolles, 1987).
Our previous studies have demonstrated that at the biochemical level,
V laR activation leads to phosphatidylinositol signaling which results in
regulation of intracellular calcium concentrations in cultured cortical neurons
(Son et al., 1998). Calcium is critical to the development o f the cellular and
structural features of the nervous system (Berridge, 1993; Nishizuka, 1992).
One major factor in determining the nature of a neuronal calcium signal is the
opening o f permeability pathways for calcium in the cell membrane to allow
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influx o f calcium from the extracellular space. Typically, these have been
grouped into voltage-sensitive calcium channels and receptor-operated calcium
channels. Our previous findings indicate such channels may be involved in Vi
agonist-induced calcium influx in cultured cortical neurons (Son et al., 1998).
There are a growing number of different types of calcium channels
detected in neurons such as L-, N-, P-, Q-, R-, and T-type calcium channels
(Ishibashi et al., 1997; Randall et al., 1995; Sabatier et al., 1997; Zhang et al.,
1993). Calcium channels play a critical role in many aspects of neuronal
function such as neurotransmitter release, neurite outgrowth, gene expression
and modulation of learning and memory (Bliss et al., 1993; Ginty et al., 1992;
Goelet et al., 1986; Kennedy 1989). Calcium channel activity is often regulated
by phosphorylation/dephosphorylation o f its subunits via various kinases.
Protein kinase C and calcium/calmodulin-dependent kinases have been
implicated as potential mediators of calcium-regulated neuronal adaptive
responses, such as long-term potentiation (Bliss et al., 1993). In sensory
neurons, protein kinase C inhibits calcium channel activation whereas in
sympathetic neurons protein kinase C enhances calcium channel activation (Zhu
et al., 1994).
Three reasons governed the selection of investigating the mechanism of
V laR activation o f L-type calcium channels in cortical neurons. First, our
previous work has demonstrated that V laR activation in cultured cortical
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neurons leads to activation of the phosphatidylinositol signal pathway, uptake of
calcium from the extracellular medium, and induction of complex intracellular
calcium signaling (Son et al., 1998). Furthermore, our findings of Vi agonist-
induced calcium influx (Son et al., 1998) indicated V laR regulation of calcium
channels in cultured cortical neurons. Second, presence of multiple calcium
channel subtypes has been reported in cortical neurons including L-type calcium
channels (Zhang et al., 1995). In immunocytochemical studies, L-type calcium
channels were localized primarily to neuronal cell bodies and to the base of
proximal dendrites (Ahlijanian et al., 1990; Westenbroek et al., 1990). Lastly,
calcium entry through calcium channels induces gene expression which can lead
to structural and functional changes that underlie long-term adaptive responses
(Ginty et al., 1 992; Goelet et al., 1 986). Moreover, L-type calcium channels
have been demonstrated to be involved in mechanism(s) of learning and
memory (Murphy et al., 1991). Therefore, in the present study, we pursued to
investigate the mechanism of V laR regulation of L-type calcium channel
activation in cultured cortical neurons.
Methods and materials
Cell culture preparation
Cultures of cortical neurons were prepared following the method
described by Son and Brinton. (1998). Cortices were dissected from the brains
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of embryonic day 18 (El 8 : with EO as breeding day) Sprague-Dawley rat
fetuses. The tissue was treated with 0.05% trypsin in Hank’s Balanced salt
solution (50 mM KC1, 3 mM KH2PO4, 80 mM NaCl, 0.9 mM NaH2P0 4 - 7 H2 0 ,
10 mM Dextrose, 0.3 M HEPES) for 5 minutes at 37 °C. Following incubation,
trypsin was inactivated with cold phenol red free Dulbeccos Modifies Eagle
Medium (DMEM; Gibco) supplemented with 10 mM NaHC03, 10% fetal
bovine serum, 5 U/ml penicillin and 5 pg/ml streptomycin, and 10% F I2
nutrient medium for 3 minutes. Tissue was then washed with Hank’s Balanced
salt solution (2 x) and dissociated by repeated passage through a series of fire
polished constricted Pasteur pipettes. Cells were plated at a concentration of 1 x
106 cells/ml onto polyethylenimine (PEI) coated (10 pg/ml; Sigma. St. Louis) 35
mm Petri dishes. Cells were grown in 2 ml of phenol red containing Neurobasal
Medium (which does not promote glial cell proliferation; Gibco #320-1103),
B27 medium supplement (Gibco #680-7504), 25 pM glutamate, 0.5 mM
glutamine, 5 U/ml penicillin, and 5 pg/ml streptomycin and maintained in a 37
°C 5% CO2 incubator.
Assessment o f f 3 H I IP 1 accumulation
After 2 days of incubation, 1 ml of the media was aspirated off and
replaced with 0.5 ml of media containing 4 pCi/ml of [3H]myo-inositol (specific
activity = 23.45 Ci/mmol). Preliminary studies indicated that 24 hours of
incubation with [ HJmyo-inositol was optimal for incorporation into the cell
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lipids. Cells were rinsed twice with 1 ml o f Krebs Ringer bicarbonate (KRB)
buffer (124 mM NaCl, 5 mM KC1, 1.3 mM MgCl2-6H20 , 1.2 mM KH2P 0 4, 26
mM NaHCC>3, 10 mM dextrose, 1 mM CaCl2 ) then pre-incubated in 1 ml of
KRB for 20 m in at 37 °C. F ollowing thepreincubationperiod, solution was
exchanged for KRB + 10 mM LiCl (inositol phosphatase inhibitor) + test
peptides at 20 min at 37 °C for the dose response analysis and at varying time
points for the time course analysis. Peptides were dissolved in KRB solution
(without LiCl) immediately prior to use.
The reaction was terminated by the addition of 750 pi of ice cold
methanol, cells were scraped from the petri dishes with a cell scraper, and
transferred to test tubes containing 1 ml of chloroform and 0.5 ml of deionized
distilled water. An additional 750 pi of ice cold methanol was added to the petri
dishes then transferred to the same test tubes. Chloroform samples were
vortexed then centrifugated (5 min, 2000 rpm). The aqueous phase was
transferred to test tubes containing 4 ml o f deionized distilled water, vortexed,
and centrifuged (5 min, 2000 rpm). Five ml o f the sample was filtered through 1
ml Dowex columns (Bio-rad, MO), which had been generated using 1 ml of 1 M
ammonium formate/0.1 M formic acid. Columns were washed with 5 ml of
distilled deionized water (2x), which was discarded, followed by 2.5 ml of 1 M
ammonium formate/0.1 M formic acid. This eluate containing the inositol
phosphates was collected and 1 ml o f the 2.5 ml eluate was counted by
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scintillation in 5 ml of scintillation fluid. In order to present comparable data
cross experiments, [3H]IPi accumulation data were analyzed by determining the
ratio of aqueous CPM/organic CPM and expressed as a percent o f basal
accumulation.
Calcium imaging
Cortical neurons to be used in calcium imaging studies were cultured at a
density of lxlO 6 cell/ml onto (+)poly-L-lysine coated coverslips then placed on
to coverslip clamp chamber MS-502S (ALA Scientific Instruments; NY) for the
calcium imaging analysis. Unless stated otherwise, neurons were used 7 days
following seeding. Neurons were briefly washed with Krebs buffer (137 mM
NaCl, 5.3 mM KC1, 1.0 mM MgCl2-6H20 , 1.2 mM KH2P 0 4, 10 mM HEPES, 25
mM Dextrose, and 1.5 mM CaCl2 ) then loaded with fura-2 acetoxymethyl ester
(5 pmol / L; Molecular Probes, Inc., OR) by incubating for 45 minutes at 37 °C.
Excess fura-2 dye was removed by washing with Krebs buffer and incubated for
30 minutes at 37 °C for neurons to equilibrate. Five micromolar glutamate was
used at the end of each experiment as positive control. Fluorescence
measurements o f intracellular calcium concentration were performed using
InCyt2™, Fluorescence Imaging System (Intracellular Imaging, Inc., OH).
Neurons w ere p laced on a stage o f an inverted microscope (MT-2, Olympus)
equipped with epifluorescence optics (20X, Nikon). Fluorescence was excited
at wavelengths of 340 and 380 nm alternatively using rotating 4-wheel filter
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changer. To minimize the background noise of the fura-2 signal, successive
values (16 sample images, 8 background images), were averaged (~13 images /
min).
45Ca2 + uptake
Cultures were similar in age and cellular density to those used for
[3 H]EPi accumulation. Cultures were washed in Krebs buffer for 20 min at 37
°C. Following this wash, V) agonist + 1.0 (iCi 45Ca2+ per 2 ml were
simultaneously added to the cultures (specific activity of 45Ca2 + = 30.7 mC/mg).
Following the incubation period with V) agonist, the treatment solution was
decanted and cultures were washed two times with 2 ml o f Krebs buffer. After
decanting the last Krebs wash, 45Ca2 + uptake was terminated by the addition of 1
ml 7 % ice cold trichloroacetic acid to each culture dish and incubated for 45
min at 4 °C. Trichloroacetic acid extracts were removed and placed into
scintillation vials for counting and 1 ml o f NaOH added to the cultures to
solubilize protein for analysis of protein content by the Lowry method (Lowry et
al., 1951).
Protein isolation
Cells were plated at a concentration of 1 x 106 cells/ml onto
polyethylenimine coated (10 pg/ml; Sigma. St. Louis) 100 mm Petri dishes in
Neurobasal media and maintained in a 37 °C 5% CO2 incubator. After 7 days,
cells were treated with and without 250 nM Vi agonist for 1 minute, media was
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discarded, washed w ith cold PBS solution, then 2 m l o f 1 ysis buffer (20 m M
Tris-HCl; pH 7.4, 2 mM EDTA, 0.5 mM EGTA, 0.25 M sucrose, 50 mg / ml
PMSF) was added. Cells were scraped and transferred to ultracentrifuge tube
(Beckman, CA). Ultracentrifuge tubes were balanced, put into Ti-70 rotor
(Beckman, C A), then c entrifuged at 1 00,000 g fo r 1 hour in Beckman L8-70
Ultracentrifuge. Then supernatant that contains the cytosolic proteins were
collected. The pellet which contains the membrane proteins were dissolved with
1 ml lysis buffer and 0.2% Triton X-100, centrifuged at 100,000 g for 1 hour,
then supernatant was collected to collect the membrane proteins.
Protein kinase C assay
PepTag Assay for Non-Radioactive Detection of Protein Kinase C from
Promega was used. Protein kinase C was diluted to 2.5 pg / ml in protein kinase
C buffer and protein kinase C activator 5X solution was sonicated using a probe
sonicator for 20-30 seconds until it was warm. For protein kinase C assay,
reaction mixtures were prepared containing 5 pi PepTag Protein Kinase C
Reaction 5X Buffer, 2 pg PepTag C l Peptide, 5 pi Protein Kinase C Activator
5X Solution, 1 pi Peptide Protection Solution, 5 pi protein sample, and 4 pi
deionized water. Four microliter o f 2.5 pg / ml protein kinase C was substituted
for the sample proteins for protein kinase C positive control. For protein kinase
C negative control, sample protein was replaced with deionized water. At time
zero, reaction mixtures were removed from the ice and incubated in 30 °C water
66
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bath for 2 minutes. Then sample or protein kinase C was added and incubated at
30 °C for 30 minutes. Reaction was stopped by placing the tubes in a boiling
water bath or 95 °C heating block for 10 minutes. Cytosolic and membrane
protein samples were loaded onto 0.8% agarose gel in 50 mM Tris-HCl, pH 8.0
then ran at 100 V for 15 to 20 minutes to separated the phosphorylated and
nonphosphorylated PepTag Peptides. To visualize the separation, ultraviolet
light was used to view the bands then photograph was taken for the qualitative
assay.
To quantitate kinase activity, negatively charged phosphorylated bands
from the gel were excised, keeping the total volume at approximately 250 pi.
Excised bands were placed into a 1.5 ml graduated microcentrifuge tube and
heated at 95 °C until the gel slice was melted. 125 pi of the hot agarose was
transferred to a tube containing 75 upl of Gel Solubilization Solution (that has
been warmed to room temperature and mixed w ell), 1 0 0 upl of glacial acetic
acid and 200 u p l o f d istilled w ater. Once in the a cidified G el S olubilization
Solution, the agarose should remain liquid for several hours. The samples were
vortexed then 500 pi of the solution was transferred to a 0.5 ml cuvette. The
absorbance was read at 570 nm. The spectrophotometer was zeroed with
liquefied agarose without PepTag Peptide.
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Data analysis
[3H]IPi accumulation data are presented as mean percent of basal ±
S.E.M., as determined by the ratio of aqueous cpm/organic cpm. 45Ca2 + uptake
data are presented as mean percent of basal ± S.E.M. Statistical analysis was
performed by a Student’s t-test or by a one-way analysis o f variance (ANOVA)
followed by Newman-Keuls post hoc analysis.
Results
The L-type calcium channel blocker, nifedipine, was used to investigate
whether Vi agonist-induced calcium entry in cortical neurons was via L-type
calcium channel. Results of these e xperiments d emonstrated that 250 nM Vi
agonist induced calcium influx (141.9 % ± 6.2, p < 0.05) whereas pretreatment
with 20 pM nifedipine blocked V) agonist-induced calcium influx (99.0 % ±
7.2) in cultured cortical neurons (Figure 10).
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Figure 10. The L-type calcium channel antagonist, nifedipine, blocked V] agonist-
induced uptake of extracellular Ca2 + in cortical neurons. Cultures of cortical neurons
were pretreated with 20 pM nifedipine for 20 minutes, followed by 5-second exposure
to 250 nM Vi agonist or 10 pM BayK 8644. Values represent mean ± S.E.M. from one
experiment and are representative of three separate analyses with 7-8 cultures per
condition per experiment, **/><0 .0 1 .
To confirm the selectivity of nifedipine, (-)S-BayK 8644, a L-type
channel activator, was used. Ten micromolar (-)S-BayK 8644 induced calcium
influx (142.3 % ± 7.0,/? < 0.05 whereas 20 pM nifedipine effectively blocked 10
pM (-) S-BayK 8644-induced calcium influx (96.2 % ± 4.6) in cultured cortical
neurons (Figure 10). Calcium imaging studies demonstrated that 20 pM
nifedipine blocked the Vi agonist-induced rise in intracellular calcium
concentration (Figure 11).
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Figure 11. Blockade of Vi agonist-induced rise in intracellular calcium concentration by
L-type calcium channel antagonist, nifedipine, in cultured cortical neurons. Cultures of
seven-day old neurons were loaded with fura-2 for 30 minutes (A), then exposed to 250
nM Vi agonist (B). Another set of neurons was pretreated with 20 pM nifedipine for 30
minutes (C), then exposed to 250 nM Vi agonist and 20 pM nifedipine (D).
Calcium channels can either be activated directly by a ligand or
indirectly by intermediate components of the signaling cascade such as
retrograde signal. U-73122 is a phospholipase C inhibitor that blocks the
production of inositol phosphates whereas BIS I is a protein kinase C inhibitor
that allows continuous production of inositol phosphates by inhibiting the
negative feedback m echanism o f t he p hosphoinositide s ignaling p athway. U -
73122 and BIS I were used to determine the mechanism o f Vf agonist-induced
calcium channel activation in cortical neurons by inhibiting various steps along
the phosphatidylinositol signal pathway. Results from 45Ca2 + uptake
experiments demonstrated that 1 pM BIS I and 5 pM U-73122 both blocked V;
agonist-induced calcium influx, 90.2% ±3.5 and 97.8% ± 3.9, respectively
70
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(Figure 12). In addition, results o f the experiments to confirm the efficiency of
BIS I and U-73122 showed that 1 pM BIS I and 240 nM V) agonist allowed
greater accumulation of inositol phosphates (206.94% ± 13.91, _p<0.001; Figure
13) compared to 250 nM Vi agonist alone (176.30% ± 15.07, /K0.001; Figure
13) whereas 5 pM U-73122 effectively blocked the accumulation of inositol
phosphates (83.7% ± 4.5; Figure 13).
prdnaibdim with preincubction with
1 uMBisitubtyleimcle I 5uMU-73122
Figure 1 2. Inhibitory effects o f bisindolylmaleimide I ( PKC inhibitor) and U-73122
(PLC inhibitor) on V] agonist-induced calcium channel activation. Neuronal cultures
were pretreated with 1 pM BIS I for 20 min, then exposed to 250 nM V] agonist and 1
pM BIS I simultaneously for 5 sec. Another set of cultures were pretreated the with 5
pM U -73122 for 20 min followedbyexposure to 250 nM V) agonist and 5 pM U -
73122 for 5 sec. Values are mean ± S.E.M. from one experiment and are representative
of data obtained in two separate analyses with 7-8 cultures per condition per
experiment, **/?<0.01, *p<0.05.
71
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2 4 0
Figure 13. Effects of bisindolylmaleimide I (PKC inhibitor) and U-73122
(phospholipase C inhibitor) on Vi agonist-induced [3H]IPi accumulation. Cultures were
pre-treated with 1 pM BIS I for 20 min, then exposed to 250 nM V) agonist and 1 pM
BIS I for 60 min. In separate set of cultures, neurons were pre-treated with 5 pM U-
73122 for 20 min followed by exposure to 250 nM V) agonist and 5 pM U-73122 for
60 min. Values are mean ± S.E.M. from one experiment and are representative of data
obtained in two separate analyses with 7-8 cultures per condition per experiment, ***
/X 0 .0 0 1 .
To further confirm the role of protein kinase C in L-type channel
regulation, experiments to determine whether V ia vasopressin receptor
activation led to protein kinase C activation in cortical neurons were performed.
Results of these studies demonstrated that as early as 1-minute exposure to 250
nM Vj agonist induction of protein kinase C activity occurred (Figure 14A).
Quantitative analysis indicated a 54.5% increase in phosphorylation activity of
72
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protein kinase C in cortical neurons treated with 250 nM Vi agonist (3.4 U/ml;
Figure 14B) compared to control neurons (2.2 U/ml; Figure 14B).
1 min
Non-phosphorylated — >
Phosphorylated ->
B
3.5 i
Ctrl V I Po.Ctrl
Condition
Figure 14. Vi agonist induction of protein kinase C activation. Proteins were collected
from seven-day old cortical neurons then protein kinase C activity was assayed. Peak
activation of protein kinase C occurred following 1 min exposure to 250 nM Vi agonist.
Exogenous protein kinase C was used in positive control experiment. A: Lower band
below the loading well shows protein kinase C activity, phosphorylation of PepTag
peptide. The equal fluorescent levels of the upper band of non-phosphoiylated PepTag
peptide demonstrate the quantitative control for the amount of Peptag peptide used for
each conditions. B: Quantitative determination of protein kinase C activity by
cytofluorometry. Ctrl: control, Vi: 250 nM V] agonist, Po. Ctrl: positive control.
73
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Lastly, the effect of protein kinase C inhibition on Vf agonist-induced
rise in intracellular calcium concentration was conducted using intracellular
calcium imaging. Results o f these determinations showed that blockade of
protein kinase C activity inhibited Vi agonist-induced rise in intracellular
calcium concentration in cultured cortical neurons (Figure 15).
C o n tro l * 5 « H B IS I
; ty iff:?.
Figure 15. Inhibition of Vi agonist-induced intracellular calcium increase by protein
kinase C inhibition in cultured cortical neurons. (A) Seven-day old neurons were
loaded with fura-2AM (A), then exposed to 250 nM V) agonist (B). Another set of
neurons was pretreated with 5 pM bisindolylmaleimide I (protein kinase C inhibitor)
for 30 minutes (C), then exposed to 250 nM V) agonist with 5 pM bisindolylmaleimide
1(D).
Discussion
The purpose o f this study was to identify the calcium channel involved in
Vi agonist-induced calcium influx and to determine the mechanism underlying
74
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activation. Results of the present study demonstrate that Vi agonist-induced
calcium influx occurs through an L-type calcium channel mechanism.
Furthermore, we have found that the protein kinase C arm o f the VI receptor
activated phosphatidylinositol signaling pathway is responsible for the L-type
calcium channel activation in cultured cortical neurons. Results of the present
study add additional insights into understanding the mechanisms underlying VI
receptor-induced rises in intracellular calcium. Moreover, these data provide the
first demonstration of VI vasopressin receptor activation of L-type calcium
channels in cortical neurons and the first to demonstrate that regulation of these
calcium channels by VI vasopressin receptor activation is a protein kinase C
dependent process.
Thus far, there are two common modes for calcium signaling in neurons.
First is the “frVinduced calcium release” model where calcium contained within
intracellular stores is released to the cytosol when IP3 binds to its receptor
(Berridge, 1993). Second is the “calcium-induced calcium release” model
where small influx of calcium through voltage-operated calcium channels
triggers an explosive release of stored calcium from the intracellular stores
(Berridge, 1993). Data from the current study and that of previous studies (Son
et al., 1998) continue to indicate that Vi agonist-induced calcium signaling in
cultured cortical neurons occurs via the “calcium-induced calcium release”
model where blockade of calcium influx from the extracellular compartment
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prevents a rise in intracellular calcium despite Vi agonist-induced generation of
IP3.
In addition to changes in the level of intracellular calcium, the route of
calcium entry and its intracellular localization give rise to the activation of
specific biochemical signaling pathways that mediate particular biological
responses. By controlling the route of calcium entry, neuronal calcium channels
regulate a wide variety of cellular functions including spike patterning,
neurotransmitter release, and gene transcription (Holliday et al., 1991; Wheeler
et al., 1994; Mintz et al., 1995; Bito et al., 1997; Hemandez-Lopez et al., 1997).
Influx o f calcium during cortical plasticity development where calcium rise has
been demonstrated to underlie the signal transduction events leading to long
term alterations of synaptic efficacy (Geiger et al., 1986; Murphy et al., 1991;
Singer, 1985).
L-type calcium channels have been demonstrated to be abundant in
neurons (Ahlijanian et al., 1990; Giffm et al., 1991; Nakazawa et al., 1999;
Westenbroek et al., 1990), have long-opening characteristic (Ishibashi et al.,
1997; Zhang et al., 1993), and have been implicated to play a role in learning
and memory models such as long-term potentiation and neuronal plasticity
(Deyo et al., 1989; Murphy et al., 1991; Yamada et al., 1996). In pituitary
adenoma cells, vasopressin has been shown to increase calcium currents through
L-type calcium channels (Mollard et al., 1988). Moreover, the abundance of L-
76
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type calcium channels in neuronal cells including cortical neurons has been
reported further suggesting an important role of L-type calcium channels in
vasopressin-induced calcium influx (Zhang et al., 1995). Our results from
calcium imaging analysis and calcium uptake experiments using nifedipine have
demonstrated Vi agonist induction of calcium regulation via L-type calcium
channels in cultured cortical neurons.
It is now becoming increasingly apparent that the influx of calcium
through voltage-sensitive calcium channels can be modulated by receptor-
mediated events. A number of cell surface receptors are coupled either directly
to calcium channels (Fasolato et al., 1994) or through the intermediate G-
proteins (Hescheler et al., 1993; Strubing et al., 1997). Modulation of calcium
channels by both, phosphorylation/dephosphorylation and G-protein-dependent
regulation has been widely described (Netzer et al., 1994). It has been
established that G proteins play a role and diffusible second messengers may be
involved in some cases of calcium channel regulation (Heschler et al., 1993;
Walker et al., 1998).
Another important mechanism regulating calcium channel function is
phosphorylation by Ca2+ /phospholipid-dependent protein kinase C. In addition
to the presence in high concentrations of protein kinase C in neuronal tissue
(Tanaka et al., 1992), pivotal role in controlling cellular functions, and
involvement in the modulation of signal transduction (Nishizuka, 1992), protein
77
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kinase C has been implicated in the modulation of calcium channel function
where protein kinase C phosphorylate G-protein and/or calcium channel
subunits (Shistik et al., 1999; Swartz et al., 1993). Several studies have reported
L-type calcium channels modulation by protein kinase C activation (Bourinet et
al., 1992 and 1994; Singer-Lahat et al., 1992; Y ang et al., 1993). Moreover,
protein kinase C has been demonstrated to mediate the enhancement of L-type
calcium channels by vasopressin (Zhang et al., 1995). In cortical neurons, our
results indicate that Vi agonist-induced calcium influx was mediated by
downstream components of the phosphatidylinositol signaling cascade. More
specifically, the application of protein kinase C inhibitor effectively blocked Yi
agonist-induced calcium influx and increase in intracellular calcium
concentration which may suggest a possible phosphorylation of G-proteins
and/or calcium channel subunits b y proteinkinase C in m o d u latio n o f L-type
calcium channels in cultured cortical neurons. Recent evidence of G-protein a-
and p-subunit modification by protein kinase C also suggested that the G-protein
could be a “programmable messenger” that after interaction with protein kinase
C would interact weakly with calcium channels (Chen et al., 1996; Puri et al.,
1997). In rat sympathetic neurons, the activation of protein kinase C
antagonizes G-protein mediated inhibition of calcium channels by shifting
calcium channels from the “reluctant” state to the “willing” state (Zhu et al.,
1994).
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Though protein kinase C has been implicated in the regulation of
calcium channels in many types of central and peripheral neurons (Swartz, 1993;
Zhu et al., 1994), the molecular mechanisms of calcium channel modulation by
protein kinase C are poorly understood, in part due to the fact that most neurons
coexpress multiple types of calcium currents with overlapping
electrophysiological and pharmacological characteristics. However, recent
findings suggest that the N-terminus of L-type calcium channel ocic subunit acts
as an inhibitory gate, and its removal enhances channel activation where protein
kinase C may increase the current by attenuating the inhibitory action of the N-
terminus (Shistik et al., 1998 and 1999). Thus, protein kinase C may
phosphorylate a site at a ic which interacts directly or allosterically resulting in
weakening the inhibitory gating effect of N-terminus or protein kinase C may
phosphorylate an unknown auxiliary protein that may obstructs the inhibitory
gating effect of N-terminus. We are currently in pursuit of identifying which
protein kinase C isoform(s) regulates vasopressin-induced calcium channel
activity. For future studies, we will be determining the direct or indirect effect
of protein kinase C on calcium channels by identifying intermediate proteins
that are phosphorylated by protein kinase C in cultured cortical neurons. In
conclusion, our studies suggest that V \ agonist induces calcium influx via L-type
calcium channel activation and that V ia vasopressin receptor activation ofL-
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type calcium channel is mediated by protein kinase C in cultured cortical
neurons.
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CHAPTER FOUR
Mechanism of Vasopressin-Induced Phosphatidylinositol Signal Desensitization
and V ia Vasopressin mRNA Regulation by Protein Kinase C Activation
in Cultured Cortical Neurons
Abstract
Recent studies of cortical neurons have demonstrated that V ia
vasopressin receptor (VlaR) activation leads to the induction of
phosphatidylinositol signal pathway and a rise in intracellular calcium
concentration (Son and Brinton, 1998a). We have sought to understand the
dynamics of V laR adaptation and have therefore investigated both the
characteristics of and the mechanisms that underlie desensitization of the V laR
in cortical neurons. To characterize vasopressin-induced signal desensitization,
both of the effectors, phosphatidylinositol signaling c ascade and regulation of
V laR mRNA expression were analyzed. Time course analysis of VI
-3
vasopressin receptor agonist (Vi agonist)-induced accumulation o f [ H]IPi
demonstrated that phosphatidylinositol signaling desensitized within 2 hours of
pre-exposure to 250 nM Vi agonist (94.0% ± 2.4; p=0.77) compared to non-pre-
exposed (136.5% ± 5.7; p<0.001) and remained desensitized for 24 hours in the
presence of 250 nM Vi agonist. In addition, intracellular calcium imaging
analysis demonstrated that 250 nM Vi agonist-induced rise in intracellular
calcium was diminished after 30-minute pre-exposure to 250 nM Vi agonist.
86
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Analysis o f V laR mRNA regulation by Vi agonist revealed a gradual down
regulation o f V laR mRNA expression following 2 to 6 hours of exposure.
Mechanistically, inhibition of protein kinase C blocked Vi agonist-induced
phosphatidylinositol signal desensitization and significantly reduced Vi agonist-
induced down-regulation of V laR mRNA expression. These results
demonstrate that in cultured cortical neurons the V ia vasopressin receptor
effector, phosphatidylinositol signaling, undergoes sustained desensitization that
is accompanied by down-regulation of V laR mRNA expression in response to
prolonged exposure to Vj agonist. Protein kinase C controls both the
desensitization of the phosphatidylinositol signaling and the down-regulation of
V ia vasopressin receptor mRNA expression.
Introduction
The neurohypophysial hormone vasopressin whose actions are mediated
by stimulation of specific G protein-coupled receptors is classified into V r
vascular (V ia), V2-renal (V2), and V 3-pituitary (V lb) vasopressin receptors
(Thibonnier et al., 1994 and 1998). Within the nervous system, vasopressin
impacts a broad spectrum of functions including those regulating homeostasis
(DeWied et al., 1988 and 1993; Kasting et al., 1992), the stress response
(DeWied et al., 1988 and 1993; Jezova et al., 1995; McEwen et al., 1987), and
learning and memory (Brinton, 1990; DeWied et al., 1988 and 1993), The V ia
87
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vasopressin receptor is widely distributed throughout the central nervous system
and belongs to Gq -protein-coupled receptors with seven transmembrane
domains. B inding o f vasopressin to the V laR activates phospholipase C and
produces inositol 1,4,5-trisphosphate and diacylglycerol, which increase
intracellular calcium concentration and activate protein kinase C, respectively
(Briley et al., 1994; Son and Brinton, 1998a and 1998b).
Desensitization is a key adaptive response of second-messenger
pathways and is manifested by diminished responsiveness to persistent agonist
stimulation. Many peptide hormones including vasopressin have been shown to
elicit desensitization in target cells (Catt et al., 1979; Sibley et al., 1987; Sibley
and Lefkowitz, 1985). Like most G-protein-coupled receptors already studied,
V ia vasopressin receptor undergoes desensitization when stimulated by its
agonist which results in phosphatidylinositol and calcium signal desensitization
in native tissues and in cell lines expressing the receptor (Cantau et al., 1988;
Collins and Rozengurt, 1984; Grier et al., 1989; Nathanson et al., 1994; Plevin
and Wakelam, 1992). In addition, work from several investigators suggests that
receptor internalization may also contribute to V laR desensitization (Cantau et
al., 1988; Fishman et al., 1985; Lutz et al., 1991). However, the mechanism of
down-regulation of V laR which may last up to hours has not been extensively
studied.
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The most frequently invoked mechanism for receptor desensitization is
phosphorylation of threonine and serine residues by protein kinase A or C and/or
by G receptor kinase (Ancellin et al., 1997; Lohse, 1993). The VlaR, like other
transmembrane G protein receptors, has a number of threonine and serine
residues which are putative sites fo r phosphorylation a n d m ay b e in v o lv e d in
desensitization. Results from Morel’s group using Xenopus oocytes transfected
with V ia vasopressin receptors suggest that desensitization of the V laR is
mediated by protein kinase C activation (Ancellin et al., 1997). In addition, our
previous findings have demonstrated a time dependent decrease in V i agonist-
induced inositol phosphate accumulation which was inhibited by protein kinase
C inhibitor in cortical neurons (Son and Brinton, 1998b). Based on these
findings, we have investigated the mechanism of Vi agonist-induced signal
desensitization and the possible role of protein kinase C in down-regulation of
V ia vasopressin receptors in cultured cortical neurons.
Methods and materials
Cell culture preparation
Cultures of cortical neurons were prepared following the method
described by Brinton et al. (Son and Brinton, 1998a). Cortices were dissected
from the brains of embryonic day 18 (El 8 : with EO as breeding day) Sprague-
Dawley rat fetuses. The tissue was treated with 0.05% trypsin in Hank’s
89
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Balanced salt solution (50 mM KC1, 3 mM KH2PO4, 80 mM NaCl, 0.9 mM
NaH2P 0 4 -7H2 0 , 10 mM Dextrose, 0.3 M HEPES) for 5 minutes at 37 °C.
Following incubation, trypsin was inactivated with cold phenol red free
Dulbeccos Modifies Eagle Medium (DMEM; Gibco) supplemented with 10 mM
NaHC03, 10% fetal bovine serum, 5 U/ml penicillin and 5 pg/ml streptomycin,
and 10% F12 nutrient medium for 3 minutes. Tissue was then washed with
Hank’s Balanced salt solution (2x) and dissociated by repeated passage through
a series of fire polished constricted Pasteur pipettes. Cells were plated at a
concentration of 1 x 106 cells/ml onto polyethylenimine (PEI) coated (10 jig/ml;
Sigma, St. Louis) 35 mm Petri dishes. Cells were grown in 2 ml of phenol red
containing Neurobasal Medium (which does not promote glial cell proliferation;
Gibco #320-1103), B27 medium supplement (Gibco #680-7504), 25 pM
glutamate, 0.5 mM glutamine, 5 U/ml penicillin, and 5 pg/ml streptomycin and
maintained in a 37 °C 5% CO2 incubator.
Assessment o f [ H lIPi accumulation
After 2 days of incubation, 1 ml of the media was aspirated off and
replaced with 0.5 ml of media containing 4 pCi/ml of [3H]myo-inositol (specific
activity = 23.45 Ci/mmol). Preliminary studies indicated that 24 hours of
incubation with [3H]myo-inositol was optimal for incorporation into the cell
lipids. Cells were rinsed twice with 1 ml o f Krebs Ringer bicarbonate (KRB)
buffer (124 mM NaCl, 5 mM KC1, 1.3 mM MgCl2-6H20 , 1.2 mM KH2P 0 4, 26
90
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mM NaHC0 3 , 10 mM dextrose, 1 mM CaCB) then pre-incubated in 1 ml of
KRB for 2 0 m in at 3 7 °C. F ollowing the preincubationperiod, solution was
exchanged for KRB + 10 mM LiCl (inositol phosphatase inhibitor) + test
peptides at 20 min at 37 °C for the dose response analysis and at varying time
points for the time course analysis. Peptides were dissolved in KRB solution
(without LiCl) immediately prior to use.
The reaction was terminated by the addition of 750 pi of ice cold
methanol, cells were scraped from the petri dishes with a cell scraper, and
transferred to test tubes containing 1 ml of chloroform and 0.5 ml of deionized
distilled water. An additional 750 pi o f ice-cold methanol was added to the petri
dishes then transferred to the same test tubes. Chloroform samples were
vortexed then centrifuged (5 min, 2000 rpm). The aqueous phase was transferred
to test tubes containing 4 ml of deionized distilled water, vortexed, and
centrifuged (5 min, 2000 rpm). Five ml o f the sample was filtered through 1 ml
Dowex columns (Bio-rad, MO), which had been generated using 1 ml of 1 M
ammonium formate/0.1 M formic acid. Columns were washed with 5 ml of
distilled deionized water (2x), which was discarded, followed by 2.5 ml of 1 M
ammonium formate/0.1 M formic acid. This eluate containing the inositol
phosphates was collected and 1 ml of the 2.5 ml eluate was counted by
scintillation in 5 ml o f scintillation fluid. In order to present comparable data
cross experiments, [ H]IPi accumulation data were analyzed by determining the
91
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ratio of aqueous CPM/organic CPM and expressed as a percent of basal
accumulation.
Calcium imaging
Cortical neurons to be used in calcium imaging studies were cultured at a
density of lxlO 6 cell/ml onto (+)poly-L-lysine coated coverslips then placed on
to coverslip clamp chamber MS-502S (ALA Scientific Instruments; NY).
Unless stated otherwise, neurons were used 7 days following seeding. Neurons
were briefly washed with Krebs buffer (137 mM NaCl, 5.3 mM KC1, 1.0 mM
MgCl2-6 H2 0 , 1.2 mM KH2PO4, 10 mM HEPES, 25 mM Dextrose, and 1.5 mM
CaC^) then loaded with fura-2 acetoxymethyl ester (5 pmol / L; Molecular
Probes, Inc., OR) by incubating for 45 minutes at 37 °C. Excess fura-2 dye was
removed by washing with Krebs buffer followed by incubation for 30 minutes at
37 °C for dye distribution and equilibration. Fluorescence measurements of
intracellular calcium concentration were performed using InCyt2™,
Fluorescence Imaging System (Intracellular Imaging, Inc., OH). Neurons were
placed on a stage of an inverted microscope (MT-2, Olympus) equipped with
epifluorescence optics (2OX, Nikon). Fluorescence was excited at wavelengths
of 340 and 380 nm alternatively using rotating 4-wheel filter changer. To
minimize the background noise of the fura-2 signal, successive values (16
sample images, 8 background images), were averaged (~13 images / min).
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RNA isolation and RT-PCR
The procedures for RNA isolation and RT-PCR for V ia vasopressin
receptor mRNA w ere followed as described in Yamazaki et a l (1997). T otal
RNA was extracted and purified by the guanidinium thiocyanate-phenol-
chloroform method of Chomczynski and Sacchi (Chomczynski, 1987). RNA
pellets were washed briefly in ice-cold 70% ethanol, dried at room temperature,
and dissolved in 10 to 20 pi RNAse-free water. From 10 pg o f purified total
RNA sample, poly (A)+ RNA was selectively converted to cDNA by reverse
transcription, using the avian murine virus reverse transcriptase (AMV RT,
Boehringer-Mannheim, Indianapolis, IN, USA), in the presence of oligo dT (0.1
pg/pl), dNTP mixture (10 mM each of dATP, dGTP, and dCTP), dithiothreitol
(100 mM), and RNAse inhibitor (40 U/pl; Boehringer-Mannheim). As positive
control, 3 ng of plasmid DNA containing a 390 base-pair insert corresponding to
the fifth through seventh transmembrane domains of the rat V ia vasopressin
receptor gene (rV 1 aR, kindly provided by Dr. S.J. Lolait) was used. A set of 20-
mer oligonucleotides based on the published sequence of V ia vasopressin
receptor was used as primers to amplify a 350 base-pair cDNA fragment (Morel,
1992). The primers were as follows:
Primer 1: 5’- TAC GTG ACC TGG ATG ACC AG - 3 ’ (bp 901 to 920)
Primer 2: 5 ’- AGC AAC GCC GTG A TT GTG A T - 3 ’ (bp 1255 to
1275)
93
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PCR o f the sample cDNA (10 pi) was carried out in the presence of
dNTP (2.5 mM each of dATP, dGTP, dTTP, and dCTP), and Taq polymerase (5
U; Boehringer-Mannheim), in addition to the primers (lpg), buffer containing
1.5 mM MgCl2 (Boehringer-Mannheim), and deionized water to make up a total
volume of 25 pi. Amplification was performed at 94°C for 40 s, 56°C for 1 min,
and 72°C for 1 min, for 35 cycles.
Southern blot analyses
Ten microliter cDNA samples of RT-PCR products were size-
fractionated on a 1% agarose gel, denatured, and transferred onto nylon
membranes (BioRad). Membranes were incubated for 4 hours at 42°C, with a
hybridization buffer consisting of 5X SSPE, 5X Denhardt’s, 0.1% SDS, 100
pg/ml ssDNA, and 50% formamide (Sambrook, Molecular Cloning).
Subsequently, hybridization to a purified radiolabeled probe was performed.
Hybridization autoradioerayhv
A radiolabeled probe was made by using an oligolabeling kit in which
the V ia vasopressin receptor plasmid was incubated with [a- PjdCTP
according to manufacturer’s protocol (oligolabeling kit; Pharmacia, NJ, USA).
It was purified and added, at a mean specific activity of 1.5 X 109 cpm/pg, to
the membrane incubating in buffer solution and hybridization was performed
overnight at 42°C. Membranes were washed under high stringency conditions
94
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(0.1X SSPE at 55°C for 15 min) and exposed to X-ray film (Kodak X-Omat) at
-70°C for 24 hours.
Data analysis
[3 H]EPi accumulation data are presented as mean percent of basal ±
S.E.M., as determined by the ratio of aqueous cpm/organic cpm. 45Ca2+ uptake
data are presented as mean percent o f basal ± S.E.M. Statistical analysis was
performed by a Student’s f-test or by a one-way analysis of variance (ANOVA)
followed by Newman-Keuls post hoc analysis.
Results
To characterize the vasopressin induction of desensitization process,
cultures of cortical neurons were pre-exposed to 250 nM Vi agonist for various
time periods followed by determination of V) agonist-induced [3H]IPi
accumulation. Results of these analyses demonstrated a desensitization of V i
agonist-induced phosphatidylinositol signaling as early as 2 hours of pre
exposure to 250 nM Vi agonist (94.0% ± 2.4; jo=0.77) compared to control
cortical neurons (136.5% ± 5.7; /K0.001; Figure 16A). Furthermore,
desensitization of V) agonist-induced phosphatidylinositol signaling was
sustained for 24 hours during exposure to 250 nM V) agonist in 7 day-old
cultured cortical neurons (Figure 16B).
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A
250 nMV
liine(hrs)
B
13 5
1 2 0
3 1 0 5
Time (his)
Figure 16. Time course for 250 nM V] agonist-induced [3 H]IPi accumulation
desensitization in cultured cortical neurons. Cultures were pre-exposed to 250 nM V)
agonist then re-exposed at different time periods, (A) 6-hr time course; (B) 24-hr time
course. Values are from one experiment and are representative of two separate
experiments, with six to eight cultures per condition per experiment. Data points
represent mean ± S.E.M., * p< 0.05, ** p < 0.01.
Since the phosphatidylinositol signal pathway leads to an increase in
intracellular calcium concentration, the effect of V i agonist exposure on calcium
signaling was investigated to determine the impact of desensitization on Vi
agonist-induced calcium signaling in cultured cortical neurons. Analyses of
intracellular calcium using fura-2AM demonstrated that 250 nM Vi agonist
induced a rise in intracellular calcium concentration (Figure 17A and 17B)
whereas pre-exposure to 250 nM Vi agonist for 30 minutes resulted in a marked
reduction of Vi agonist-induced rise in intracellular calcium concentration
(Figure 17C and 17D).
96
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Figure 17. Desensitization of Vi agonist-induced increase in intracellular calcium
concentration. Seven-day old cortical neurons were loaded with fura-2AM. One set of
neurons was incubated in Krebs buffer for 30 minutes (A), then exposed to 250 nM V]
agonist (B). Another set of neurons was pretreated with 250 nM Vi agonist for 30
minutes (C), then exposed to 250 nM Vi agonist (D).
To determine the role of protein kinase C in Vj agonist-induced
phosphatidylinositol signal desensitization, the protein kinase C inhibitor,
bisindolylmaleimide I (BIS I), was used to block protein kinase C and the
impact on [3H]IPi accumulation was assessed. Results of these analyses
demonstrated that pre-exposure to 5 pM BIS I prior to 250 nM Vi agonist
exposure significantly blocked the desensitization of Vi agonist-induced [3H]IPi
accumulation in cortical neurons (Figure 18).
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500
■ M G u t d
l 1 250nM V , agonist
i W 250nM V , agonist
+ 5 u M B IS I
450-
400-
s? 350-
¥ 300'
1 250-
S 200-
r ioo-
Cbntrd 4 2 6
lime (his)
Figure 18. Time course for 250 nM Vi agonist-induced [3 H]IPi accumulation
desensitization/recovery in cultured cortical neurons. Cultures were pre-exposed to 250
nM Vi agonist, washed at different time periods for 30 mins, and re-exposed to 250 nM
V, agonist or 250 nM V) agonist + 5 pM BIS I. Values are from one experiment and
are representative o f three s eparate e xperiments, with five c ultures per condition p er
experiment. Data points represent mean ± S.E.M., ** p< 0.01, *** p< 0.001.
To determine if desensitization of the phosphatidylinositol and calcium
signaling were accompanied by a decrease in the V laR mRNA, regulation of
V laR mRNA expression was investigated using RT-PCR. Results of Southern
blot analysis by RT-PCR revealed a gradual decrease in V ia vasopressin
receptor mRNA within 2, 4, and 6 hours of exposure to 250 nM Vi agonist
(Figure 19A). Quantitative measure o f Vi agonist-induced V laR mRNA down-
regulation relative to invariant /?-actin mRNA expression demonstrated a 48.5 %
98
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decrease in V laR mRNA following 6-hour exposure to 250 nM Vj agonist in
cultured cortical neurons (Figure 19B).
A
V laR mRNA -»
P-actin mRNA — >
Cont. 2 hrs 4 hrs
vnapM i
0 hr 2 hrs 4 hrs 6 hrs
Time
Figure 19. Southern blot analysis of regulation of Vi agonist-induced V ia vasopressin
receptor mRNA expression levels in cortical neurons by RT-PCR. Cultures of cortical
neurons were exposed to 250 nM Vi agonist and total mRNA was collected at 0, 2, 4,
and 6 hour time points. Reverse transcriptase-polymerase chain reaction was used to
amplify the V ia vasopressin receptor mRNA (A). Expression levels of p-actin mRNA
were used as invariant internal standard to quantify the V ia vasopressin receptor
mRNA using Scion Image software.
To determine protein kinase C regulation o f V laR mRNA, cultures of
cortical neurons were pre-incubated with 5 pM BIS I prior to 250 nM Vi agonist
then RT-PCR was performed at 0, 2, 4 and 6 hour time points to determine the
99
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V laR mRNA expression. Results of quantitative RT-PCR analysis showed a
69% decrease compared to control in V laR mRNA levels after the 6-hour
exposure to 250 nM Vi agonist alone. However, inhibition of protein kinase C
effectively reduced the magnitude of V laR mRNA down-regulation by 65%
m a x im u m at 6 hours compared to V i agonist-treated cortical neurons at 6 hours
(Figure 20).
120% i
100% -
1 80% -
O
u
C m
| 60% -
1 40%
c
> 20% ~
0% -
0 hr 2 hrs 4 hrs 6 hrs
Time
Figure 20. Quantitative RT-PCR analysis of r egulation o f V 1 a vasopressin r eceptor
mRNA expression by protein kinase C. Cultures of cortical neurons were exposed to
250 nM Vi agonist or 250 nM Vi agonist + 5 pM BIS I, then total mRNA was collected
at 0, 2, 4, and 6 hour time points. Reverse transcriptase-polymerase chain reaction was
used to amplify the V ia vasopressin receptor mRNA. Expression levels of P-actin
mRNA was used as invariant internal standard.
— 250 nM VI agonist + 5 uM BIS 1
- 2 5 0 nM VI agonist
100
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Discussion
The purpose of this study was to investigate the adaptive process of the
V laR in cortical neurons and to determine the mechanism underlying the
desensitization process. Results o f this study demonstrated that Vi agonist
induced phosphatidylinositol and calcium signal desensitization correlated with
a gradual decrease in Vi agonist-induced V laR mRNA expression in cultured
cortical neurons. Using the protein kinase C inhibitor, bisindolylmaleimide I,
the data demonstrated that desensitization of both phosphatidylinositol and
calcium signaling and down-regulation of V laR mRNA expression levels were
mediated by protein kinase C activity.
Consistent with Son and Brinton’s previous observation (Son et al.,
1998b), prolonged exposure to Vi agonist has induced desensitization of
calcium and phosphatidylinositol signaling in cultured cortical neurons. Cantau
et al. have found that desensitization consists o f a rapid uncoupling step
followed by receptor internalization where loss o f vasopressin binding sites
begins to occur over 15-30 min in WRKi cells, although impaired inositol
phosphate production occurs within 2-5 min in this cell type (Cantau et al.,
1988). Several studies have also demonstrated that desensitization is the
outcome of a rapid (in minutes) and reversible uncoupling of the receptor-G
protein complex which results in attenuation of signaling cascade, followed by
sequestration and/or internalization of receptors from the cell surface (Ferguson
101
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et al., 1998; Fishman et al., 1985; Hack et al., 1998; Lutz et al., 1990 and 1991;
Preisser et al., 1999; Zhang et al., 1997). Receptor internalization occurs with a
half time of 3-6 min in hepatocytes (Fishman et al., 1985) and can be visualized
within 4 min in A10 cells by using fluorescently tagged ligand (Lutz et al., 1990
and 1991). However, when V laR internalization is inhibited, vasopressin-
induced phosphatidylinositol turnover is greatly potentiated (Lutz et al., 1993).
Thus, these findings suggest that internalization is an early and critical event in
desensitization, although this does not eliminate the possibility that a receptor
kinase-mediated uncoupling step occurs simultaneously. However, later phase
of vasopressin induction of V laR desensitization may be the result of decreased
V laR density upon prolonged exposure to vasopressin since receptor down-
regulation displays a much longer time-course (hours to days) and is
characterized by a decrease in receptor density. Our RT-PCR and Southern blot
analyses demonstrate that prolonged exposure to Vi agonist reduces V laR
mRNA expression in cultured cortical neurons.
In Xenopus oocytes, V ia vasopressin receptor undergoes rapid
desensitization which can be mimicked by diacylglycerol analog and increased
protein kinase C activity which suggest that desensitization of the V ia
vasopressin receptor is mediated by protein kinase C (Ancellin et al., 1997).
V ia vasopressin receptor has a number of threonine and serine residues that are
putative sites for phosphorylation and may be involved in desensitization. This
102
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is further supported in HEK293 cells where activation of the V ia vasopressin
receptor by vasopressin promotes phosphorylation of the receptor by protein
kinase C (Innamorati et al., 1998). Several sites between V ia vasopressin
receptor interaction and increased intracellular calcium may be modulated by
protein kinase C. Aiyar et al. reported that treatment of A10 VSMC with
phorbol dibutyrate suppressed vasopressin-induced phosphatidylinositol
turnover and calcium efflux without altering cell binding of vasopressin (Aiyar
et al., 1987). They suggested that direct stimulation of protein kinase C might
uncouple V ia vasopressin receptor activation and G-protein activation. In
addition, altered V ia vasopressin receptor mRNA transcription by protein
kinase C activation demonstrated to be responsible for the reduced hepatic V ia
vasopressin receptor density in diabetes mellitus (Phillips et al., 1995) may
explain the role of protein kinase C in V ia vasopressin receptor mRNA
regulation by vasopressin in cortical neurons. Therefore, the mechanism of the
reduced V laR mRNA expression could be due to reduced transcription or
instability and increased degradation o f the V laR mRNA. Results of RT-PCR
analyses to determine the role of protein kinase C in the regulation of V laR
mRNA levels indicated that inhibition of protein kinase C reduced the down-
regulation of V laR mRNA induced by vasopressin in cultured cortical neurons.
Our previous studies have demonstrated Vi agonist-induced signaling
was mediated by protein kinase C in cultured cortical neurons (Son and Brinton,
103
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1998a and b). There are several possible phosphoproteins that can regulate Vi
agonist-induced signal desensitization. (1) V ia vasopressin receptor
phosphorylation has been reported to regulate receptor internalization (Ancellin
et al., 1997; Innamorati et al., 1998 and 1997; Ozcelebi et al., 1995). Thus,
receptor internalization may result in rapid desensitization o f VI agonist-
induced calcium and phosphatidylinositol signaling (Son and Brinton, 1998b).
(2) Phospholipase C is phosphorylated by protein kinase C as a negative
feedback mechanism of the phosphatidylinositol signaling pathway (Bird et al.,
1993). Our previous studies have demonstrated that protein kinase C inhibition
resulted in diminishing the negative feedback mechanism of the
phosphatidylinositol signaling (Son and Brinton, 1998a). (3) G-protein subunits
are often phosphorylated which result in uncoupling from the receptor
(Clapham, 1995 and 1996). Thus, uncoupling o f the receptor-G-protein
complex may result in rapid desensitization of the signaling pathways.
Together, our results indicate that desensitization of vasopressin-induced
signaling is regulated by protein kinase C at both the early and later phase of the
desensitization process. Moreover, results of this study documents that the
mechanism regulating desensitization of V ia vasopressin receptor in brain is
common to that found in other cell types. We are currently investigating the
protein kinase C isoform(s) responsible for regulating the early and late phases
of Vi agonist-induced signal desensitization and V ia vasopressin receptor
104
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mRNA desensitization in cultured cortical neurons. Present findings provide
insights in understanding the mechanism of signal desensitization at an early and
later phase which could be essential in providing strategic approaches in
treatment of drug tolerance development at different stages.
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CHAPTER FIVE
Overview and Summary
Research into human and animal memory has expanded at an enormous
rate in recent decades, spanning fields from epidemiology and psychology to
single-cell neurophysiology and molecular biology. The pioneering work by
DeWied and his colleagues from the 1960s (DeWied, 1964, 1966 and 1969)
have demonstrated memory-enhancing effects of vasopressin in the brain.
During the past several decades, the mnemonic effects of arginine vasopressin
on memory consolidation and retrieval have been demonstrated in a variety of
behavioral paradigms not only in rodents, but also in primates, including
humans (DeWied, 1988; DeWied et al., 1993; Jolles, 1987; Weingartner et al,
1981). However, its mnemonic effects at the cellular and molecular levels
remain to be elucidated.
More than a decade ago, detection of vasopressin recognition sites has
been demonstrated in the cerebral cortex (Brinton et al., 1984; Chen et al.,
1993). One of the hallmark characteristics of the cerebral cortex is its
integrative capacity. The cerebral cortex mediates complex integrative functions
such as abstract reasoning, planning, language, sensory perception and is
generally held to be the site of long-term memory storage (Brinton, 1998;
Goldman-Rakic, 1988; Posner et al., 1988; Ojeman, 1991; Chapman, 1959).
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Later, cloning of the V ia vasopressin receptor subtype by Lolait and his
colleagues made possible studies of vasopressin-induced signals in brain at the
cellular and molecular levels (Morel et al., 1992). In recent years, our
laboratory has demonstrated that four cortical regions (ventral, caudal, dorsal,
and rostral) expressed mRNA for V ia vasopressin receptor in three different cell
types (neurons, astrocytes, and oligodendrocytes) of the cerebral cortex
(Yamazaki et al., 1997). In addition, our laboratory has demonstrated
neurotrophic effects of vasopressin in cortical neurons (Chen et al., 1996).
However, the effector mechanism of vasopressin in the cerebral cortex has yet
been determined.
During thep ast 6 years, m y efforts with the help from m y colleagues
were focused in determining the effector mechanism of vasopressin action and
to characterize the vasopressin-induced signaling pathway in the cerebral cortex.
As a result, we have demonstrated that vasopressin action is mediated by V ia
vasopressin receptor subtype in neurons derived from the cerebral cortex of the
rat brain. In cortical neurons, vasopressin induced rapid calcium influx and
phosphatidylinositol hydrolysis in a time- and dose-dependent m anner which
resulted in a rise in intracellular calcium concentration. However, the initial
calcium influx through L-type calcium channels was found to be a critical step
in the induction of vasopressin-induced calcium signaling. Furthermore, the
regulatory role of protein kinase C was also demonstrated where the activity of
i l l
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protein kinase C prevented over-stimulation of cortical neurons through a
negative feedback mechanism and desensitization o f vasopressin-induced
signaling.
In addition to the neurotrophic effects of vasopressin in cortical neurons
(Chen et al., 1996), the expression o f V ia vasopressin receptor mRNA
throughout cerebral cortex has been demonstrated (Yamazaki et al., 1997). If
vasopressin influences memory formation and retrieval by affecting structural
plasticity in brain, then it would be of importance to determine the effector
mechanisms of vasopressin within the region of plasticity. V ia vasopressin
receptor has been characterized as Gq-protein coupled receptor that act through
phosphatidylinositol hydrolysis to mobilize intracellular calcium. Our earlier
studies have demonstrated that vasopressin induces calcium signaling via V ia
vasopressin receptor in hippocampal neurons (Brinton et al., 1994); however,
the mechanism of vasopressin action in cerebral cortex has not been established.
Results of our study, along with the results of Vi agonist-induced neurotrophism
have demonstrated the functionality of V ia vasopressin receptors in the cerebral
cortex. Results of our investigation indicated that Vi agonist activates
phosphatidylinositol hydrolysis leading to an accumulation of [3 H]IPi that is
independent of extracellular calcium. Moreover, V ia vasopressin receptor
activation induced a rapid and significant influx of extracellular calcium which
resulted in an immediate and sustained increase in intracellular calcium
112
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concentration. Thus, the influx of extracellular calcium induced by Vi agonist
plays a key role in triggering the release of calcium from the intracellular stores
in cultured cortical neurons (Son et al., 1998). Therefore, our data
demonstrating that V) agonist-induced rise in intracellular calcium concentration
is dependent upon the presence of extracellular calcium are consistent with the
“calcium-induced calcium release” model (Berridge, 1993).
Calcium entry through calcium channels induces gene expression which
can 1 ead to structural and functional changes that underlie 1 ong-term adaptive
responses (Ginty et al., 1992; Goelet et al., 1986). Presence of multiple calcium
channel subtypes has been reported in cortical neurons (Zhang et al., 1993).
Specifically, L-type calcium channels have been demonstrated to be abundant in
neurons (Ahlijanian et al., 1990; Giffm et al., 1991; Nakazawa et al., 1999;
Westenbroek et al., 1990), have long-opening characteristic (Ishibashi et al.,
1997; Zhang et al., 1995), and have been implicated to play a role in learning
and memory models such as long-term potentiation and neuronal plasticity
(Deyo et al., 1989; Murphy et al., 1991; Yamada et al., 1996). In pituitary
adenoma cells, vasopressin has been shown to increase calcium currents through
L-type calcium channels (Mollard et al., 1988). To investigate the mechanism
underlying the dependency on vasopressin-induced calcium signaling, we have
pursued to investigate the mechanism of L -type c alcium channel activation in
cortical neurons. Our results from calcium imaging analysis and calcium uptake
113
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experiments using nifedipine, L-type calcium channel blocker, have
demonstrated Vi agonist induction of calcium regulation via L-type calcium
channels in cultured cortical neurons.
One important mechanism of regulating calcium channel function is
phosphorylation by Ca2 + /phospholipid-dependent protein kinase C where the
modulation of calcium channels by both, phosphorylation/dephosphorylation
and G-protein-dependent regulation has been widely described (Netzer et al.,
1994). In addition to the presence in high concentrations o f protein kinase C in
neuronal tissue (Tanaka et al., 1992), pivotal role in controlling cellular
functions, and involvement in the modulation of signal transduction (Nishizuka,
1992), protein kinase C has been implicated in the modulation o f calcium
channel function where protein kinase C phosphorylate G-protein and/or
calcium channel subunits (Shistik et al., 1999; Swartz et al., 1993). Several
studies have reported L-type calcium channels modulation by protein kinase C
activation (Bourinet et al., 1992 and 1994; Singer-Lahat et al., 1992; Yang et al.,
1993). Moreover, protein kinase C has been demonstrated to mediate the
enhancement of L-type calcium channels by vasopressin (Zhang et al., 1995).
Our results indicated that in cultured cortical neurons, V ia vasopressin receptor
activation regulates influx of extracellular calcium via L-type calcium channel
activation through a protein kinase C dependent mechanism. More specifically,
the application o f protein kinase C inhibitor effectively blocked Vi agonist-
114
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induced calcium influx and increase in intracellular calcium concentration which
may suggest a possible p hosphorylation of G -proteins and/or calcium channel
subunits by protein kinase C in modulation of L-type calcium channels in
cultured cortical neurons.
Consistent with Son and Brinton’s previous observation (Son et al.,
1998b), prolonged exposure to Vi agonist has induced desensitization of
calcium and phosphatidylinositol signaling in cultured cortical neurons. In
addition, our RT-PCR and Southern blot analyses demonstrated that prolonged
exposure to V) agonist reduces V ia vasopressin receptor mRNA expression in
cultured cortical neurons. Cantau et al. have found that desensitization consists
o f a rapid uncoupling step followed by receptor internalization where loss of
vasopressin binding sites begins to occur over 15-30 min in WRKi cells,
although impaired inositol phosphate production occurs within 2-5 min in this
cell type (Cantau et al., 1988). However, when V ia vasopressin receptor
internalization is inhibited, vasopressin-induced phosphatidylinositol turnover is
greatly potentiated (Lutz et al., 1993). Thus, these findings suggest that
internalization is an early and critical event in desensitization whereas later
phase of vasopressin induction of V ia vasopressin receptor desensitization may
be the result of decreased receptor density upon prolonged exposure to
vasopressin since receptor down-regulation displays a much longer time-course
(hours to days).
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V ia vasopressin receptor has a number of threonine and serine residues
that are putative sites for phosphorylation and may be involved in
desensitization. In Xenopus oocytes, V ia vasopressin receptor undergoes rapid
desensitization which can be mimicked by diacylglycerol analog and increased
protein kinase C activity which suggested that desensitization of the V ia
vasopressin receptor is mediated by protein kinase C (Ancellin et al., 1997).
Aiyar et al. reported that treatment of A10 VSMC with phorbol dibutyrate
suppressed vasopressin-induced phosphatidylinositol turnover and calcium
efflux without altering cell binding of vasopressin (Aiyar et al., 1987). They
suggested that direct stimulation of protein kinase C might uncouple V ia
vasopressin receptor activation and G-protein activation. In addition, altered
V ia vasopressin receptor mRNA transcription by protein kinase C activation
demonstrated to be responsible for the reduced hepatic V ia vasopressin receptor
density in diabetes mellitus (Phillips et al., 1995) may explain the role of protein
kinase C in V ia vasopressin receptor mRNA regulation by vasopressin in
cortical neurons. Therefore, the mechanism of the reduced V ia vasopressin
receptor mRNA expression could be due to reduced transcription or instability
and increased degradation of the V ia vasopressin receptor mRNA. Results of
RT-PCR analyses to determine the role of protein kinase C in the regulation of
V ia vasopressin receptor mRNA levels indicated that inhibition of protein
kinase C reduced the magnitude of V ia vasopressin receptor mRNA down-
116
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regulation induced by vasopressin in cultured cortical neurons. Together, our
results indicate that desensitization of vasopressin-induced signaling is regulated
by protein kinase C at both the early and later phase of the desensitization
process.
Thus far, our results have demonstrated Vi agonist-induced signaling is
mediated by protein kinase C in cultured cortical neurons (Son and Brinton,
1998a and b). There are several possible phosphoproteins that can regulate Vi
agonist-induced signal desensitization. (1) V ia vasopressin receptor
phosphorylation has been reported to regulate receptor internalization (Ancellin
et al., 1997; Innamorati et al., 1998 and 1997; Ozcelebi et al., 1995). Thus,
receptor internalization may result in rapid desensitization of VI agonist-
induced calcium and phosphatidylinositol signaling (Son and Brinton, 1998b).
(2) Phospholipase C is phosphorylated by protein kinase C as a negative
feedback mechanism of the phosphatidylinositol signaling pathway (Bird et al.,
1993). Our previous studies have demonstrated that protein kinase C inhibition
resulted in diminishing the negative feedback mechanism of the
phosphatidylinositol signaling (Son and Brinton, 1998a). (3) G-protein subunits
are often phosphorylated which result in uncoupling from the receptor
(Clapham, 1995 and 1996). Thus, uncoupling o f the receptor-G-protein
complex may result in rapid desensitization of the signaling pathways.
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In conclusion, our investigation of the mechanism of vasopressin
enhancement o f cognitive function in the cerebral cortex via V la vasopressin
receptors has led to the following novel findings: (1) In cerebral cortical
neurons, vasopressin induces calcium influx and phosphatidylinositol hydrolysis
that lead to a rise in intracellular calcium concentration via V ia vasopressin
receptor activation. (2) Vasopressin-induced calcium signaling follows
“calcium-induced calcium release” model where initial calcium influx through
L-type calcium channel is required for the increase in intracellular calcium
concentration. (3) Vasopressin-induced L-type calcium channel activation is
mediated by protein kinase C. And (4) desensitization of vasopressin-induced
signaling and V ia vasopressin receptors are mediated via protein kinase C
regulation in cortical neurons. Future research in determining the vasopressin-
specific gene expression will be helpful in delineating the mechanism of
vasopressin function in the cerebral cortex. In addition, investigation of the
identity o f protein kinase C isoform(s) regulated by V ia vasopressin receptors
can further elucidate the regulation of the early and late phases of Vi agonist-
induced signal desensitization and V ia vasopressin receptor mRNA
desensitization in cultured cortical neurons. Understanding the mechanism of
vasopressin function at the biochemical and molecular levels may provide
insights in developing therapeutic strategies for various cognitive disorders. In
addition, present findings provide insights in understanding the mechanism of
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signal desensitization at an early and later phase which could be essential in
providing strategic approaches in treatment of drug tolerance development at
different stages. Results of our study provide the first documentation of the
effector mechanism o f V ia vasopressin receptor and its regulation in the
cerebral cortex. Furthermore, these data demonstrate that V ia vasopressin
receptors in the cerebral cortex are functional entities and open the intriguing
possibility that vasopressin can influence the complex functions of the cerebral
cortex.
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Creator Son, Michael Chang (author)

Core Title Dynamic regulation of vasopressin, a mnemonic neuropeptide, induction of calcium signaling and V1a vasopressin receptors in the rat cerebral cortex

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Degree Program Molecular Pharmacology and Toxicology

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