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The role of estrogen receptors and nociceptive signaling pathway of primary sensory neurons

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The role of estrogen receptors and nociceptive signaling pathway of primary sensory neurons

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THE ROLE OF ESTROGEN RECEPTORS AND NOCICEPTIVE SIGNALING
PATHWAY OF PRIMARY SENSORY NEURONS

by

Tae Hoon Cho

A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(CHEMICAL ENGINEERING)

August 2012

Copyright 2012 Tae Hoon Cho
ii

Dedication

To my Lord,
and his loyal worker, my father, the late Hyosun Cho and my mother, Youngae Kim

iii

Acknowledgements

Don ’t be afraid, for I am with you. Do not be dismayed, for I am your God.
I will strengthen you. I will help you. I will uphold you with my victorious right hand.
(Isaiah 41: 10)
I thank you, Lord, with all my heart. You made me, sent me here, and gave me
this achievement. I will sing to you, Lord, and praise your holy name forever.
I have many individuals who I am grateful to. Thank you, Dr. Pin Wang, for
your kind and consistent guidance and support for me over the my Ph.D. years. I am
highly honored that you are my advisor. I thank all my committee members for your
support: Dr. Katherine Shing, and Dr. Li Zhang. In addition to your time, I am deeply
grateful for your encouragement that this is a valuable project to undertake.
Most importantly, I give thanks to my family, who all stand on the sideline and
cheer me on: Mom (Youngae Kim), mother-in-law (Mija Shin), father-in-law (Myungdal
Kim), brother (Taebong Cho), sister-in-law (Sunyoung Park), and cousin (Mindong
Cho). And, my father, the late Hyosun Cho, who has supported me both materially and
morally. I really miss you.
Finally, I would like to thank Young Kyoung, my wife, from the bottom of my
heart. You have always shared the pleasures and pains with me. I believe you are the
most precious gift God gave me. Moreover, I would like to share this unforgettable
moments with my children Peter (Yedong) and Linda (Shua).

iv

Table of Contents

Dedication ii

Acknowledgements iii

List of Figures vii

Abstract xi

Chapter 1. INTRODUCTION 1
1.1 Characteristics of the estrogens 1
1.1.1 Origin of Estrogens 2
1.1.2 Understanding of cellular mechanisms of estrogens 4
1.1.3 Pharmacological properties of estrogen receptors 9
1.2 The peripheral nervous system 10
1.2.1 The somatic nervous system 10
1.2.2 The visceral nervous system 10
1.3 The nociceptive system 11
1.3.1 Functional characteristics of nociceptors 12
Table 1: Subtypes of Aδ and C Fibers 12
1.3.2 Transduction of nociception 16
1.3.3 The effect of estrogen on ATP-induced [Ca
2+
]
i
signaling in primary
sensory neurons. 19
1.3.4 Opioid receptors 21
1.4 Function of the TRPV1 receptors in DRG neurons 24
1.5 Visceral sensitization of DRG neurons 25
1.6 Calcium Imaging in sensory DRG neurons 26
1.7 Significance 27

Chapter 2. INTERACTION BETWEEN P2X3 AND ERα/ERβ IN ATP-MEDIATED
CALCIUM SIGNALING IN MICE SENSORY NEURONS 29
2.1 Introduction 30
2.2 Results 32
2.2.1 Expression of P2X3 in DRG neurons 32
2.2.2 Effect of E2 and pharmacological profile of E2-mediated modulation
on ATP-induced [Ca
2+
]
i
in DRG neurons. 35
2.2.3 E2 action on voltage-gated calcium channels (VGCC) in DRG neurons 38
2.2.4 Effect of E2-BSA on ATP-induced [Ca
2+
]
i
response in DRG neurons 40
2.2.5 17β-estradiol attenuation of ATP and α,β-me ATP-induced [Ca
2+
]
i

influx in DRGs from ERαKO and ERβKO mice 41
2.3 Discussion 45

v

2.4 Materials and Methods 49
2.4.1 Animals 49
2.4.2 Primary culture of DRG neurons 50
2.4.3 Western Blot Analysis 50
2.4.4 Immunohistochemistry 51
2.4.5 [Ca
2+
]
i
fluorescence imaging 52
2.4.5 Statistical analysis 53

Chapter 3. ESTROGEN RECEPTOR-α MEDIATES RAPID ESTRADIOL
ATTENUATION OF CAPSAICIN-INDUCED CALCIUM
SIGNALING IN MOUSE DORSAL ROOT GANGLION NEURONS 55
3.1 Introduction 56
3.2 Results 57
3.2.1 Expression of the TRPV1 in dorsal root ganglion (DRG) neurons 57
3.2.2 Pharmacological profile of E2 modulation on capsaicin-induced [Ca
2+
]
i

flux in DRG neurons 60
3.2.3 E2-mediated attenuation of capsaicin-induced [Ca
2+
]
i
transients in DRG
neurons from ERαKO, and ERβKO mice 63
3.2.4 PGE2 potentiation of the capsaicin-induced [Ca
2+
]
i
fluxes in DRG from
Wt, ERαKO, and ERβKO 65
3.3 Discussion 69
3.4 Materials and Methods 71
3.4.1 Animals 71
3.4.2 Primary culture of DRG neurons 71
3.4.3 Western Blot Analysis 72
3.4.4 Immunohistochemistry 73
3.4.5 Fluorescence Imaging analysis 74
3.4.6 Statistical analysis 75

Chapter 4. ESTROGEN RECEPTOR-α MEDIATES VISCERAL NOCICEPTION:
INVOLVEMENT OF TPRV1 AND P2X2/3IN MOUSE
DORSAL ROOT GANGLION NEURONS 76
4.1 Introduction 77
4.2 Results 81
4.2.1 E2 modulation of ATP-induced calcium fluxes regulate visceral and
cutaneous nociceptive signaling in mouse DRG neurons through
separate pathways 81
4.2.2 The effect of E2 modulation of P2X2/3 and TRPV1 receptors mediated
[Ca
2+
]
і
response in both retrogradely labeled visceral and cutaneous
DRG neurons 86
4.2.3 The role of ERα/ERβ in E2 modulation on α,β-meATP- and
capsaicin-induced [Ca
2+
]
i
transients in retrogradely labeled visceral and
cutaneous mouse DRG neurons 88
4.2.4 Comparison of the effects of E2 on PGE
2
potentiation of TRPV1-mediated
calcium response in retrogradely labeled visceral and cutaneous
DRG neurons from Wt, ERαKO, and ERβKO mice 92
vi

4.3 Discussion 96
4.4 Materials and Methods 99
4.4.1 Animals 99
4.4.2 Primary culture of DRG neurons 99
4.4.3 Retrograde labeling 100
4.4.4 Fluorescence imaging analysis 102
4.4.5 Statistical analysis 103

Chapter 5. ESTROGEN MODULATION BETWEENµ-OPIOID RECEPTOR
(MOR) AND ERα/ERβ IN MOUSE SENSORY NEURONS 104
5.1 Introduction 105
5.2 Results 108
5.2.1 Expression of μ-, κ-, and δ-opoid receptors in DRG neurons 108
5.2.2 Pharmacological profile of DAMGO-mediated modulation on
KCl-induced [Ca
2+
]
i
in DRG neurons 110
5.2.3 Modulation of evoked calcium signals by mu-, kappa-, and delta-opioid
receptors 112
5.2.4 Effect of E2 on DAMGO-mediated KCl-induced [Ca
2+
]
i
response
in mouse DRG neurons 114
5.2.5 E2 inhibits MOR effects by attenuating PGE2-induced [cAMP]
i

activation and decrease the number of MOR binding sites in mouse
DRG neurons 116
5.2.6 Internalization of μ-opioid receptor (MOR) coupling to G-protein
in wild type, ERαKO, and ERβKO mice 119
5.3 Discussion 121
5.4 Materials and Methods 124
5.4.1 Animals 124
5.4.2 Primary culture of DRG neurons 124
5.4.3 Western Blot Analysis 125
5.4.4 Fluorescence imaging analysis 126
5.4.5 Receptor binding Assay 127
5.4.6 Agonist-stimulated [
35
S]GTPγS binding Assay 128
5.4.7 Statistical analysis 129

REFERENCES 130

vii

List of Figures

Figure 1. 1: Flow chart of sex steroid synthesis from cholesterol ...................................... 2

Figure 1. 2: Schematic representation of various intracellular signaling pathways
of estrogen ....................................................................................................... 8

Figure 1. 3: The major receptor groups and molecular events involved in activation
and sensitization of visceral afferent ............................................................. 15

Figure 1 .4: Simplified schematic of nociceptive pathways and genes involved in
perception and modulation of pain ................................................................ 18

Figure 1. 5: The mechanism of estrogen effect on ATP-induced [Ca2+]i signaling
in primary sensory neurons ............................................................................ 20

Figure 1. 6: The structure of opioid G-coupled protein receptors with seven
transmembrane ............................................................................................... 22

Figure 1. 7: Model of alternative possibilities for viscero-visceral cross-
sensitization in the DRG neurons .................................................................. 26

Figure 2. 1: Western blot analysis of ERs in DRG lysates ............................................... 32

Figure 2. 2: Western blot analysis of DRG lysates shows reduced expression of
P2X3 in both knock-out mice ........................................................................ 33

Figure 2 .3: Expression of P2X3 receptors in DRG neurons from Wt, ERαKO,
and ERβKO in vivo using fluorescent microscopy ....................................... 34

Figure 2. 4: 17β-estradiol inhibits ATP-induced [Ca
2+
]
і
transients
in wild type mice .......................................................................................... 36

Figure 2. 5: Summary of ATP-induced [Ca
2+
]
i
influxes in control, in the presence
of E2-β, E-6-BSA, E2-α, and ICI 182,780 .................................................... 38

Figure 2. 6: Contribution of voltage-gated Ca
2+
channels (VGCC) to E2 inhibition
on ATP-induced [Ca
2+
]
і
................................................................................ 39
viii

Figure 2 .7: The effect of E-6-BSA on ATP-induced [Ca
2+
]
і
fluxes in ERαKO and
ERβKO mice .................................................................................................. 41

Figure 2 .8: The effect of E2 on ATP-induced [Ca
2+
]
і
transients in ERαKO and
ERβKO mice .................................................................................................. 42

Figure 2. 9: The effect of 17β-estradiol (E2) on α,β-meATP-induced [Ca
2+
]
і

transients in Wt, ERαKO, and ERβKO mice................................................. 44

Figure 3. 1: Western blot analysis of DRG lysates shows reduced expression of
TRPV1 in both knock-out mice ..................................................................... 58

Figure 3. 2: Expression of TRPV1 receptors in dorsal root ganglion neurons
from Wt, ERαKO, and ERβKO in vivo ......................................................... 59

Figure 3. 3: 17β-estradiol inhibits capsaicin-induced [Ca
2+
]
і
fluxes
in wild type mice ........................................................................................... 61

Figure 3. 4: Summary data of control, 17β-estradiol (E2), E2 + ICI 182,780,and
E-6- BSA effects on capsaicin-induced [Ca
2+
]
і
fluxes change
in DRG neurons ............................................................................................. 62

Figure 3. 5: The effect of 17β-estradiol (E2) on capsaicin-induced [Ca
2+
]
і
transients
in estrogen receptor α knockout (ERαKO) and estrogen receptor β
knockout (ERβKO) mice ............................................................................... 64

Figure 3. 6: 17β-estradiol blocks prostaglandin E2 (PGE2) enhancement of
capsaicin-induced [Ca
2+
]
і
transients in Wt mice ........................................... 66

Figure 3. 7: The effect of 17β-estradiol (E2) on PGE2 of capsaicin-induced [Ca
2+
]
і

transients in estrogen receptor α knockout (ERαKO) and
estrogen receptor β knockout (ERβKO) mice ............................................... 68

Figure 4. 1: Retrogradely labeled DRG neurons and percentage of distribution in
DRG neurons through the L1 ~S3 levels ....................................................... 83

Figure 4. 2: Retrogradely labeled visceral- and somatic dorsal root ganglion
(DRG) neurons ............................................................................................... 85

Figure 4 .3: E2 effects of α,β-me ATP-/capsaicin-induced [Ca
2+
]
і
transients in
visceral and cutaneous sensory DRG neurons from wild type mice ............. 87
ix

Figure 4. 4: The effect of E2 on α,β-me ATP-induced [Ca
2+
]
і
transients in visceral
and somatic DRG neurons from estrogen receptor-α knockout
(ERαKO) and estrogen receptor-β knockout (ERβKO) mice ..................... 89

Figure 4 .5: The E2 effects with visceral and somatic DRG neurons on
capsaicin-induced [Ca
2+
]
і
fluxes in ERαKO and ERβKO mice .................... 90

Figure 4 .6: Summary of α,β-me ATP-/capsaicin-induced [Ca
2+
]
і
transients of
control and the presence of E2 in visceral and somatic DRG neurons
from wild type, ERαKO, and ERβKO mice .................................................. 91

Figure 4. 7: The effect of 17β-estradiol (E2) on PGE2 potentiation of capsaicin-
induced [Ca
2+
]
і
fluxes in visceral and somatic DRG neurons from
wild type, ERαKO, and ERβKO mice ........................................................... 94

Figure 4 .8: Summary of PGE2 pontentiation of capsaicin-induced [Ca
2+
]
і

transients of control and the presence of E2 in visceral and somatic
DRG neurons from wild type, ERαKO, and ERβKO mice ........................... 95

Figure 4 .9: Retrograde labeling experiments. Schema of marking colon/uterine-
innervating neurons of the DRG via retrograde labeling ............................. 101

Figure 5. 1: Western blot analysis of DRG lysates shows reduced expression of
μ-, κ-, and δ-opioid receptors in both knock-out mice ................................ 109

Figure 5. 2: DAMGO blocks KCl-induced [Ca
2+
]
і
fluxes in wild type mice ................ 111

Figure 5. 3: Inhibition of depolarization-induced [Ca
2+
]
i
fluxes by opioids ................... 112

Figure 5 .4: Summary of KCl-induced [Ca
2+
]
i
influxes in control, in the presence
of DAMGO, U- 69,593, DPDPE, and DAMGO with CTAP. DAMGO
significantly decreased [Ca
2+
]
і
response to KCl whereas μ-opioid
receptor antagonist CTAP inhibited DAMGO effect .................................. 113

Figure 5. 5: E2 inhibits KCl-induced [Ca
2+
]
i
responses in wild type mice ..................... 115

x

Figure 5. 6: E2 decreases μ-opioid receptor (MOR)-mediated responses
in the mouse DRG ....................................................................................... 118

Figure 5. 7: Effects of estrogen-benzoate (EB) on DAMGO, a selective MOR
agonist, [
35
S]GTPγS-binding in membrane and microsomal DRG
fractions from wild type, ERαKO, and ERβKO mice ................................. 120

xi

Abstract

Clinical studies suggest the comorbidity of functional pain syndromes such as
irritable bowel syndrome (IBS), chronic pelvic pain (CPP), fibromyalgia, and
somatoform disorders approaches 40% to 60%. The incidence of episodic or persistent
visceral pain associated with these functional disorders is two to three times higher
women than in men. One of the possible explanations for this phenomenon is the
estrogen modulation of pain transmission. While a central site of this modulation has
been shown previously, here we proposed to study a peripheral site, the dorsal root
ganglion (DRG). In DRG neurons, 17 β-estradiol (E2) rapidly inhibits intracellular
calcium ([Ca
2+
]
i
) flux induced by ATP, a putative nociceptive signal. This proposal,
"Estrogen Receptors mediate Nociceptive Signaling in Primary Sensory Neurons in
Female Mice" will test a general hypothesis that E2 acting on primary afferent
nociceptors has both pro-nociceptive and anti-nociceptive effects depending on which
signals converge upon DRG. First, the role of different estrogen receptors (ERs) in E2
activation of purinergic (P2X3) and vanilloid (TRPV1) receptors will be studied in wild
type, estrogen receptor-α, and estrogen receptor- β knock-out mice. Second, since we
hypothesize that E2 may act differently on visceral then on cutaneous nociceptors, we
will compare the [Ca
2+
]
i
response to activation of P2X3 and TRPV1 receptors in
retrogradely-labeled visceral and cutaneous DRG neurons from knock-out and wild type
mice. Third, E2 may negatively modulate opioid analgesia by interfering with μ-opioid
receptor (MOR). Pharmacological manipulations will be used to determine how ER
activation modulates Ca
2+
channel and MOR functions. Receptor binding will determine
xii

if E2 alters the number and affinity of MOR in the DRG and the site-specific regulation
of MOR coupling to G-proteins. Together these experiments will define a new site(s) and
mechanism of E2 modulation of nociceptive signaling. Furthermore, they will provide
important information about the action of E2 on primary sensory neurons for a better
understanding of sex-differences observed in the clinical presentation of functional pain-
associated syndromes. Nociceptive systems implicated in the etiology of functional
disorders, which often are complicated by comorbid depression will have a major impact
on health-related quality of life in patients with functional pain disorders, significantly
reducing therapeutic interventions.

1

Chapter 1. INTRODUCTION

1.1 Characteristics of the estrogens
Estrogens have remarkably wide range of functions in the human body. Their
functioning as the primary sex hormone in female are a group of steroid in the estrous
cycle. Their name comes from Gr. Oistros (sexual desire) and gennan (to produce). In
addition to their sexual differentiation functions, estrogens are associated with
acceleration of metabolism, increasing of bone formation, maintenance of vessel and
skin, hemostasis, water/salt retention. They are also used for hormonal therapy in
postmenopausal women or transgender females as an oral contraceptive. Allen and Doisy
(1923) found an ovarian estrogenic hormone estrogens as a gonadic hormone in women
have an important role in fundamental cellular mechanisms. Estrogens can easily pass
through the cell membrane and bind to membrane-mediated estrogen receptors
(Whitehead and Nussey, 2001). Prossnitz et al. (2007) reported that G-protein coupled
receptor, GPR30 which is a estrogen binding protein was activated by estrogens. Gonadal
hormones are necessary for reproduction, but it appears that no region in the body, no
neuronal circuit, and virtually no cell is unaffected by them. Thus, increased attention
toward these hormones appears to be obligatory.

2

1.1.1 Origin of Estrogens
Systemic synthesis of estrogens
Estrogens are in the cholesterol-based steroid hormone family. Cholesterol is
transformed into pregnenolone from all derivatives of steroid hormones. Different
hormones such as estradiol, cortisol, aldosterone, and testosterone are produced by
different enzymes and receptors in the steroid cells (Ghayee & Auchus, 2007). Estrogens
have two precursors such as androstenedione and testosterone which are transformed to
estradiol and estrone by the enzyme aromatase.

Figure 1. 1: Flow chart of sex steroid synthesis from cholesterol.

3

Aromatase, one of the cytochrome P450 superfamilies, has several functions such
as aromatization of androgens and production of estrogens. The primary origin of
estradiol which is the most influential and biologically important of the estrogens is the
ovaries while aromatase can be found in gonads, brain, fat, and muscle. Also, estrogens
can be produced by follicle-stimulating hormone (FSH), Luteinizing hormone (LH), and
placenta in the ovaries. Once stimulation of hormones in the cells, they spontaneously
bring on cholesterol mobilization and initiate synthesis of steroids. Small amount of
estrogens produced in brain, live , adrenal glands, and breasts serve as a secondary source
of estrogens which have an important role in postmenopausal women.
Local synthesis of estrogens in the brain
All of the necessary steroidogenic enzymes and activities needed to synthesize
estrogens from cholesterol have been isolated in various parts of the brain that may work
as an autonomic steroidogenic organ (Micevych et al., 2008; Micevych and Mermelstein,
2008). Both estrogens and androgens are locally synthesized from cholesterol in the adult
hippocampal neurons (Hojo et al., 2008). Testosterone and androstenedione are converted
to estrone and estradiol by aromatase enzyme (Cornil et al., 2006). Also, aromatase
enzyme exists in pre-synaptic terminals in regions of the brain (Balthazart et al., 1990;
Naftolin et al., 1996; Evrard et al., 2000; McCarthy, 2008) indicating that estrogens could
play a role in a paracrine manner (Evrard and Balthazart, 2004; Cornil et al., 2006; Boon
et al., 2010). The process of aromatization is slow in the brain while it may be locally
stimulated by estrogens and androgens or other neuronal stimuli. Glutamate transmission
in endogenous variations restrain rapid production of dopamine that in turn, may
inactivate glutamate transmission and the level of dopamine may be lowered (Kaplan,
4

1995; Baillien and Balthazart, 1997; Hojo et al., 2004; Cornil et al., 2006). Therefore,
rapidly changing levels of locally produced estrogens in the brain may instantly modify
neuronal functions. The actual level of estrogens in specific regions of the brain is not
known but local estradiol levels in the hypothalamus are higher than in the circulation
(Bixo et al., 1997) and might well exceed 100 nM (Cho and Chaban, 2011).
1.1.2 Understanding of cellular mechanisms of estrogens
Estrogens can diffuse freely from the blood through the cell membrane or into the
cytoplasm of target tissues. There is the complexity of the many different cellular
signaling pathways and transcriptional mechanisms affected by estrogens. Therefore,
estrogens are now known to influence the expression of a wide range of genes in the
reproductive tract as well as in other areas (Gruber et al., 2002).
Estrogen Receptor α (ERα) and Estrogen Receptor (ERβ)
Although estrogens might act through all mechanisms, both estrogen receptors by
estrogen behavior and placing estrogen receptors in varied tissues have played an
important role in understanding the effects of estrogens. In autoradiography studies,
estrogen receptors were discovered in neuron cells and neuron mediated stem cells as
well as in different brain areas (Jung-Testas et al., 1992; Brännvall et al., 2002; Pfaff and
Keiner, 1973; Keefer et al., 1973; Stumpf and Sar, 1976; Pérez et al., 2003). The estrogen
receptors which have various interactions with co-regulatory proteins and the cross-
talking with transcriptional factors on the ligand such as 17α- and 17β-estradiol and on
the estrogen response elements (ERE) in DNA can produce various stimulation of
estrogens (Shiau et al., 1998; Loven et al., 2001; Gruber et al., 2004). ERα (ESR1) was
first cloned in 1985 (Walter et al., 1985) and ERβ (ESR2) was cloned ten years later
5

(Kuiper et al., 1996). There is a high homology between ERα and ERβ in the DNA-
binding domain (97%), but a moderate homology in the ligand-binding domain (55%),
leading to somewhat lower endogenous ligand binding affinities to ERβ than ERα
(Kuiper et al., 1997). The two subtypes of estrogen receptors are distributed in slightly
different areas in DNA-binding domain of the body because they use different subsets of
co-regulators and slightly different categorization in the cell.
The two estrogen receptors, estrogen receptor α (ERα) and estrogen receptor β
(ERβ), belong to nuclear receptors of the steroid/thyroid superfamily which have
structural and functional similarities such as a ligand-dependent transcription factor that
mediates gene expression. ERα is broadly distributed in the brain, but it prevails in the
hypothalamus regions that control the autonomic nervous system. ERβ is observed in the
cerebellum cortex, the medial preoptic area, septum, amygdala, and thalamus in the brain
(Shughrue et al., 1997). However, ERα and ERβ are co-expressed in the preoptic area
(Shughrue and Merchenthaler, 2001). During the estrous cycle, they vary in the amount
of estrogen receptors. For example, the amount of mRNA estrogen receptor is low during
high levels of estrogen in a region of the hypothalamus, whereas ERs mRNA is high in
the nucleus arcuatus (Shughrue et al., 1992; Axelsson et al., 2007).
The ERα includes three major functionally distinct domains. The first is a DNA
binding domain which is the sequence-specific domain that identify estrogen response
elements (EREs) in the target gene regions by using a zinc-finger mechanism. The second
is a ligand binding domain. The last is the NH
2
-termianl domain that is correlated with
transcriptional activation for specific proteins. Apart from 17β-estradiol (E2), ERα was
up-regulated by some factors in the brain (Pinzone et al., 2004; Wilson et al., 2008), but
6

there is one exception signal transducers and activators of transcription 5 (Stat5) which is
activated by prolactin and that produces the expression of ERα in the hypothalamus
(Frasor et al., 2001; Zanoli et al., 2009). Once ERα and ERβ are co-expressed, ERα-
mediated gene expression was blocked by ERβ and they have shown contrasting behavior
(Barkhem et al., 2004; Fox et al., 2008). In addition, there are various estrogen receptor
isoforms caused by alternative RNA splicing. These ER isoforms display dimerization
with the original estrogen receptor subtype for activation of transcription (Leung et al.,
2006).
Non-genomic mechanism of estrogen action
The classic signaling pathways of steroids take minutes to hours in gene
expression and new protein synthesis but estrogens have modulated different
physiological behaviors in peroptic nerve cells within seconds and can stimulate rapid
Ca
2+
changes (Falkenstein et al.,2000; Perret et al., 2001; Guo et al., 2002). Various cells
respond rapidly to 17β-estradiol (E2) due to a non-genomic mechanism of action.
In general, the effects of non-genomic estrogen are considered to be mediated by
ERα that is separate from the plasma membrane or cytoplasm within the signaling
pathways (Papas et al., 1995; Clarke et al., 2000; Falkenstein et al., 2000; Manavathi and
Kumar, 2006) as well as a G protein-coupled receptor, GPR30 (Filardo et al., 2000;
Thomas et al., 2005; Revankar et al., 2005). Several non-genomic estrogen activities
might be elucidated by the existing classic ERs (Abraham et al., 2004; Chaban and
Micevych, 2005; Song et al., 2005; Pedram et al., 2006), but estrogen receptor X, a
specific estrogen receptor in brain, has been proposed (Toran-Allerand et al., 2002).
However, this new putative receptor has been expressed only in glial cells and can be
7

activated by the inactive stereoisomer, 17α-estradiol. The initial evidence in these studies
shows that transduction of signaling pathways is launched by 17β-estradiol conjugated to
membrane-impermeable molecules such as bovine serum albumin. For example, rapid
activation of extracellular signal-regulated kinases (ERK) occurs in response to estradiol-
BSA (E-6-BSA) in cells that comprise a membrane localization of ERα, but not in ERα-
negative cells (Chen et al., 2004; Micevych and Mermelstein, 2008) as well as various
intracellular signaling pathways including mitogen-activated protein (MAP) kinase, the
activation of transcription factors including cAMP response element-binding protein
(CREB), protein kinase C (PKC) pathways, and direct transition between G-coupled
proteins and Ca
2+
-channels (McEwen, 2001; Marambaud et al., 2009). All this data
indicate that various pathways are affected by estrogens and in turn, influence estrogen
receptors and also they usually need high concentrations of estradiol to be evoked. The
stimulation of membrane-initiated estrogen can be reason for activation of other
intracellular pathways correlated with activation of transcription factors (TF) and turn in,
stimulate downstream gene transcription.
In fact, since the level of plasma hormone seems to be changed to slow to
appropriate these rapid activation pattern, local estrogens synthesis may be a necessity for
the rapid signaling pathways of estrogen receptor. There are many effects of estradiol
with the rapid responses of non-classic mechanisms. For example, these pathways in the
female brain have been involved in the regulation of lordosis behavior (Micevych and
Mermelstein, 2008) and they show several signaling pathways in dorsal root ganglions
(Chaban and Micevych, 2005). Therefore, estrogen actions are determined by their
structure and ligand synthesis, different subtypes of ERs, different subsets of co-
8

regulators, and different transcriptional factors as well as cross talking between all
signaling pathways. This study will provide a wide range of important signaling
pathways including responses of estrogen in cells by tissue-specific mechanisms and in
turn, initiate several responses such as gene expression, signaling pathways of cytoplasm,
activation of membrane-associated receptors, changes of ion channels.

Figure 1. 2: Schematic representation of various intracellular signaling pathways of estrogen. I) Activity of classical
transcription, II) Activity of non-classical transcription, III) Non-genomic estrogen signaling by membrane-initiated
activation, IV) Transcriptional response by membrane-initiated activation.
9

1.1.3 Pharmacological properties of estrogen receptors
ERs were distributed nociceptive regions through peripheral never system (PNS)
and central nerve system (CNS). For example, ERs are expressed in dorsal root ganglia
(Taleghany et al., 1999; Papka and Storey-Workley, 2002) and dorsal horn neurons of the
spinal cord (Amandusson et al., 1999; Williams and Papka, 1996; Papka and Mowa
2003). Chaban and Micevych (2005) showed that DRG neurons exist in both ERα and
ERβ in vitro and Papka and Story-Workley (2010) demonstrated DRG neurons also
express both ERα and ERβ in vivo.
Sex hormones and 17β-estradiol (E2) in particular, may directly influence the
functions of primary afferent neurons since both ERs are present on small-diameter DRG
neurons (Papka and Storey-Workley, 2002). Despite the wide effects of E2

in the nervous
system, the scheme of pain modulation in sex hormones is still poorly understood.
However, a recent study has demonstrated multiplicity of E2 behaviors in membranes,
cytoplasms and nuclei (Nadal et al., 2001). These findings indicate that E2 may modulate
sensory input signals at the primary afferent level. Also, E2 can modify gene
transcription, resulting in pro-nociceptive (reducing β-endorphin expression) or anti-
nociceptive (increasing enkephalin expression) changes of endogenous opioid peptides
(Priest et al., 1995; Marrawi et al., 2007), opioid receptors (Amandusson et al., 1999;
Micevych et al., 1997; Micevych and Sinchak, 2001; Gupta et al., 2008) and, by
increasing levels of CCK, an anti-nociceptive and anti-opioid molecule (Wiesenfeld-
Hallin et al., 1999; Micevych et al., 2002; Bekhit, 2010). In addition, E2 can modulate
cellular activity by opening of ion channel and signaling of second messenger by
stimulating G-coupled proteins (Mermelstein et al., 1996; Eckersell et al., 1998; Kelly
10

and Wagner, 1999; Chaban et al., 2003), the signal transduction pathways traditionally
associated with membrane receptor activation. Many of these effects have been attributed
to membrane-associated receptors (Levin, 1999).
1.2 The peripheral nervous system
The peripheral nervous system is composed of the nerves and ganglia and
separated from the somatic nervous system and the visceral nervous system. The
afferents (called sensory neurons) contain changes of sensory stimuli in the internal and
external environments using sensory receptors including nociceptors, mechanosensors,
chemosensors, thermosensors, and photosensors and transport more interpretation
information to the central nervous system (CNS).
1.2.1 The somatic nervous system
Somatic nervous system is connected with the spontaneous body movements and
perception of external stimuli such as touch, sight, and hearing. The somatic sensory
neurons in the spinal ganglia stimulate the intestine, the lower limb muscles and the skin.
The somatic nervous system comprises many kinds of the neurons associated with sense
organ, skin, and skeletal muscles and is composed of efferent nerves which depend on
sending signals to the brain for muscle contraction.
1.2.2 The visceral nervous system
The visceral nervous system controls the level of consciousness and the
homeostasis of the body, and depends on the unconscious actions throughout the body.
This system influences heart rate, respiration rate, digestion, perspiration, salivation,
urination, diameter of the pupils, and sexual arousal. It can be classified as the
11

sympathetic and parasympathetic nervous system, relating to stimulation and energy
generation or normal activity like digestion.
The sympathetic nervous system communicates with the postsynaptic ganglia in
the sympathetic chain that starts the thoracic and lumbar portions existing next to the
spinal cord. On the other hand, the parasympathetic nervous system transports in the
cranial and sacral nerves to end up in postganglionic neurons from which innervating to
the target organs. These functions of the visceral nervous system can usually be split into
sensory (afferent) and motor (efferent) subsystems. However, there exist inhibitory and
excitatory synapses between sensory neurons in these systems. The peripheral nervous
system is sometimes regarded part of the visceral nervous system, and an independent
system.
1.3 The nociceptive system
Pain is "an unpleasant sensory and emotional experience associated with actual or
potential tissue damage, or depicted in terms of such damage" according to the
International Association for the Study of Pain (IASP). A normal transmitting pain
system is absolutely fundamental to sustain our body. In cellular and molecular science,
the nociceptive system is able to show gene regulation, expression, and intracellular
mechanisms. This system is much more homologous to the different interoceptive
homeostatic systems than the classic exteroceptive somatosensory systems (Craig,
2003a).
Traditionally, there are two main views on the core of pain. The first, pain is
adapted by specific pathways which are made up of different peripheral neurons and
12

central neurons, whereas the other can be sent signals by a particular activation shape in
varied neurons that respond to other sensory stimuli (Craig, 2003b).
1.3.1 Functional characteristics of nociceptors
Sherrington (1906) clarified nociceptors as receptors and they response to stimuli
that cause tissue damage. There are two main groups of nociceptors which are placed on
free nerve endings of either thinly myelinated axons or unmyelinated axons based on
functional characteristics: myelinated Aδ-fibres nociceptors and unmyelinated C-fibres
nociceptors. Differences caused by their morphology have different latencies and
conducting rates. Thinly myelinated fibers have more rapidly conducting Aδ-fibres than
unmyelinated which slowly conduct C-fibres (Julius and Basbaum, 2001). According to
these findings, it has been assumed that Aδ-fiber nociceptors mediate first pain such as
acute and sharp pain, and C-fiber nociceptors mediate delayed and dull pain by noxious
stimuli (Dubner and Bennett, 1983; Djouhri and Lawson, 2004).
Table 1: Subtypes of Aδ and C Fibers

(Ref. from Rocha et. al., Rev. Bras. Anestesiol. 57(1), 2007)
13

There are different types of nociceptive receptor which are activated in various
manners, such a noxious heat, strong mechanical stimuli, and low pH (Weidner et al.,
1999; Craig, 2003b; Cortright et al., 2007; Smith and Lewin 2009). Based on their
different responses to sensory systems of noxious stimuli, nociceptors can be categorized
into thermal, mechanical and chemical nociceptors. Most of nociceptors have responded
by multiple effectors such as thermal, mechanical, and chemical stimuli, but others are
triggered only by a subset of these stimulus modalities (Caterina and Julius, 1999).
Thermal nociception
The activated nociceptors by thermal stimuli have a wide range of temperature
from 38
o
C to 53
o
C (Spray, 1986). There are two nociceptic transducers that react to
sensory nerve endings by heat stimuli. One is TRPV1 which has a heat threshold that
coexist with thermal pain temperature at 42°C and the other is reconciliated by many
transient receptor potential (TRP) channels. Thermal stimuli have been transduced by
varied depths of skin in threshold responses depending on different nociceptors and
increasing temperature rates (Tillman et al., 1995a and 1995b). The thermal threshold of
C-fiber and Aδ-fiber (type II) nociceptors which were activated by thermal stimuli with
long term or very slow change of temperature is between 39° and 41°C (Tillman et al.,
1995a; Tracey et al., 2003), and the thermal threshold of Aδ-fiber (type I) nociceptors is
between 40~50°C (Treede et al., 1998; Meyer et al., 2006; Zhu and Lu, 2010).
Furthermore, the thermal threshold of human pain by heat stimuli is between 41°C and
49°C and is very correlated with C-fiber nociceptors (Campbell et al., 1988; Meyer et al.
2006). According to these findings, they provide an important function of nociceptors in
pain sensation by thermal stimuli.
14

Mechanical nociception
Mechanical nociceptors rely on the types of nociceptors activated or the
characteristics of the nociceptors. Just like thermal or chemical nociception, these
reactions are processed by mechanical stimuli and they have varied properties of sensory
data, so that they have a chance to be a transducer for thermal stimulus. The Aδ-fiber
nociceptors have higher peak response than C-fiber nociceptors excluding high level of
pressure and force (Slugg et al., 2000). For long-term exposure, there is the pain relating
with C-fiber by with mechanical stimulus, but for short time with revealing mechanical
stimulus, there exist the sharp pain caused by Aδ-fiber nociceptors.
Chemical nociception
Generally, there are mainly two chemical agents which are related with activation
of nociceptors and creation of pain such as exogenous agents and endogenous mediators
of inflammation (Reichling and Levine, 1999; Kwan et al., 2006). The TRP channels
response a broad range of chemical nociceptors, but especially, TRPV1 have been
discovered as a mediator by exogenous capsaicin and it can be activated by heat and
protons. TRPV1 which expresses on neuron cells might be a different form of sensory
information nociceptors (Caterina et al., 1997; Kim et al., 2007; Pingle et al. 2007). These
evidences indicate that chemical agents certainly cause pain which is from inflammation
by tissue injury.

15

Figure 1. 3: The major receptor groups and molecular events involved in activation and sensitization of visceral
afferent. (a) Normal activation. TRPV1 sensitive to H+, noxious heat and capsaicin; mDEG, m Degenerin (a receptor
for mechanical transduction); P2X
3
, purinergic receptor; AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic
acid receptor; NMDA, N-methyl-Daspartate receptor. (b) Sensitization. At the dorsal horn synapse increase levels of
afferent neurons activation lead to the activation of the NMDA receptor by the removal of the magnesium ion block.
BK, bradykinin; PGE
2
, prostaglandin E
2
; G, G protein; PKC, protein kinase C; Src, tyrosine kinase Src. (Ref. from
modified Matthews and Azia, 2005).

16

1.3.2 Transduction of nociception
The rate of occurrence for pain disorders such as irritable bowel syndrome (IBS),
interstitial cystitis (IC), chronic pelvic pain (CPP), fibromyalgia, and others are higher in
women than men (Berkley, 1997; Heitkemper and Jarrett, 2001; Lee et al., 2002). The
published literature indicated that 50~60% of publications with IBS have psychiatric
disorders such as panic disorder, post-traumatic stress disorder (PTSD), generalized
anxiety disorder (GAD), social anxiety disorder, and major depression (Lydiard, 2001).
These facts suggest a model which has comparable changes in central stress circuitries by
pathophysiology. A diverse type of variables such as real (physical) and perceived
(psychological) variables play an major role in the development and modulation of IBS
symptoms. A large number of publications and IBS patients have a changed
understanding of visceral sensation like hyperalgesia. IBS is 2~3 times more prevalent
and intense in woman than in men (Toner and Akman, 2000; Mayer et al., 2001a; Mayer
et al., 2001b). Experimental acute pain studies such as pressure pain and electrical
stimulation show that women demonstrate higher sensitivity, pain threshold and tolerance
during the menstrual cycle (Riley et al., 1998; Ring et al., 2009). 17β-estradiol may have
different behaviors on signaling of nociception, perception of pain, and pain symptoms
(Berkley, 1997; Chaban, 2008b). The fact that estrogen receptors are located on DRG
neurons and ATP-induced [Ca
2+
]
i
are attenuated by 17β-estradiol estradiol strongly
suggested that 17β-estradiol peripherally modulates processing of visceral pain (Chaban,
2008; Cho and Chaban, 2011).
External primary afferent nerves are activated by turning on chemosensitive
receptors and ion channels on peripheral terminals, and transition of neuronal irritability.
17

Nociceptors are transmitted by small- and medium- sized DRG sensory neurons that are
known to be detected by potentially damaging physical and chemical stimuli (Szallasi
and Blumberg, 1999; Wood and Perl, 1999; Woolf and Salter, 2000). Many publications
have shown that the terminals of visceral afferent neurons have no organs at terminals or
morphological specialization, but they were activated by different chemical stimuli
(Devor, 1999). The release of ATP by noxious stimuli in target cells activates C-fiber
nociceptors which act as noxious stimulus mediators (Burnstock, 2000; Chaban, 2008b).
The understanding of visceral sensation was enhanced by signal transduction of visceral
afferent neurons. For example, Acute or chronic pelvic pain in women or abdominal pain
from IBS are forms of the visceral sensitization (Giamberardino MA, 1999; Zhou et al.,
2009). Visceral pain occurs during different behaviors based on neurophysiological,
pharmacological, and clinical properties in cutaneous pain (Chang and Heitkemper,
2002). The visceral hyperalgesia is not clear versus cutaneous hyperalgesia in
pathophysiology. We believe that visceral hyperalgesia originated by the sensitization of
peripheral nociceptors because of long-lasting alterations in spinal neurons (Mayer and
Gebhart, 1994). In addition to visceral pain, TRPV1 receptor in sensory DRG neurons
play a major role to transport thermal stimuli and inflammatory pain signals. The
evidence for lacking of TRPV1 gene studies can demonstrates a function of TRPV1 in
chemical-, thermal-, and inflammatory-induced hyperalgesia responses (Davis et al.,
2000; Caterina et al., 2000; Neubert et al., 2008).
Activation of both capsaicin-sensitive TRPV1 and ATP-sensitive P2X3 receptors
caused desensitization correlating to the mobilization of [Ca
2+
]
i
in cultured DRG neurons
(Gschossmann et al., 2000; Chaban, 2008a). [Ca
2+
]
i
changes of capsaicin-sensitive
18

TRPV1 receptor may show a degree of DRG neuron activation by noxious cutaneous
stimuli, but changes of ATP-sensitive P2X
3
receptor in [Ca
2+
]
i
may affect the degree of
DRG neuron sensitization by noxious visceral stimulation since released ATP by noxious
stimulation and tissue damage have been found near the peripheral terminal of primary
sensory afferent neurons (Burnstock, 2001, Chaban, 2008a).

Figure 1. 4: Simplified schematic of nociceptive pathways and genes involved in perception and modulation of pain.

19

1.3.3 The effect of estrogen on ATP-induced [Ca
2+
]
i
signaling in primary sensory
neurons.
Adenosine 5'-triphosphate (ATP) is one of the most common chemical
compounds in living cells and plays a vital role in cellular metabolism. Recently, ATP
has been reported as an extracellular signal involved in peripherally prolonged pain
(hyperalgesia) (Woolf and Salter, 2000; Hamilton et al., 2000; Xu et al., 2008). In
addition, ATP has an important physiological role in peripherally sensory transduction of
noxious stimuli by ATP-sensitive P2X3 receptors on primary afferent neurons (Dunn et
al., 2001; Ma et al., 2005). Pathologically, the released ATP by tissue damages or
inflammation activate P2X3 receptors on innervated primary afferent fibers from afferent
organs (Bodin and Burnstock, 2001; Zhang et al., 2007; Chaban, 2008a). The effects of
ATP are usually the opposite of the neuronal efficiency of adenosine. For instance, ATP
is well known to depolarize DRG neurons by means of activation of P2X3 receptor,
while DRG neurons in high voltage-gated Ca
2+
channels by stimulation of A1-type
adenosine receptors were inhibited by adenosine (Dolphin et al., 1986; Macdonald et al.,
1986; McCool and Farroni, 2001). Once P2X channels were opened, membrane is
depolarized and voltage-gated calcium channels (VGCC) regulate Ca
2+
influx into the
neuron cells (Koshimizu et al., 2000; Schicker et al., 2008; Colombo et al., 2010). In
pharmacological analysis, DRG neurons in P2X3-deficient mice didn't show responses of
any rapid desensitization by ATP or α,β-meATP, these data indicate that these responses
are mediated by the P2X3 receptor, and have reduced pain-related behavior in response to
noxious stimuli (Cockayne et al., 2000; Souslova et al., 2000; Zhong et al., 2001;
Shimizu et al., 2005; Chaban, 2008a).
20

The estrogen at the level of primary sensory neurons is determined by the
properties of the target genes and multiple action of E2 as well as co regulators. A large
body of literatures demonstrated between estrogens and the modulation of nociceptive
signals. One of the possible explanation of this pathway is the estrogen inhibition of
nociceptive signaling which mediated by both P2X3 and TRPV1 via interaction with
mGluR
2/3
. The DRG neurons at peripheral nerve terminals were initiated by painful
stimuli evoking release of excitatory neurotransmitter such as glutamate (Figure 1.5).
ATP-sensitive P2X3 receptors are highly expressed in classified nociceptors. Therefore,
E2 modulation of nociception at the primary sensory neurons may contribute to our
understanding of mechanism of non-genomic gonadal steriods.

Figure 1. 5: The mechanism of estrogen effect on ATP-induced [Ca2+]i signaling in primary sensory neurons. ATP
released by tissue damage acts on P2X3 receptor resulting in activation of the L-type vlotage-gated calcium channel
(VGCC). ERα activates mGluR2/3 which in turn activates Gi/o signaling resulting in inhibition of adenylate-cyclase
(AC). Decreased cAMP connection reduced PKA activation and decreased the coductance of the L-type VGCC (Ref.
from Chaban, 2012).
21

1.3.4 Opioid receptors
Cellular mechanisms of action
Morphine and their derivates whose participated in nociceptive threshold,
nociceptive process control, and modulation of gastrointestinal, endocrine and autonomic
function in an endogenous opioid system are usually used for treatment of acute and
chronic pain. There are multiple opioid receptor subtypes such as morphine (MOR),
ketocyclazocine (KOR), and vas deferens (DOR) and nociceptin orphanin (NOR) FQ
peptide receptor, and they have different anatomical location and pharmacological
profiles. All four opioid receptors are G-protein coupled receptors sharing the similar
seven transmembrane topology. There are classified two receptor groups such as classical
opioid receptors (MOR/KOR/DOR) and the non-classical NOR opioid receptors. Both of
these receptors couple to inhibitory G-proteins. For example, activation of MOR cause (i)
closing of voltage-gated calcium channels (VGCC), (ii) leading to hyperpolarization by
stimulation of potassium outflow, and (iii) reducing cyclic adenosine monophosphate
(cAMP) production by means of inhibition of adenylyl cyclase. According to the above
reasons, these results lead to reduced neuronal cell excitability causing to a reduction in
transmission of nerve impulses along with inhibition of neurotransmitter (i.e. ATP etc.)
release.
22

Figure 1. 6: The structure of opioid G-coupled protein receptors with seven transmembrane. Receptor activation by
opioid receptor ligands leads to initiation of intracellular transduction pathway that include stimulation of potassium
efflux, inhibition of voltage-gated calcium channels (VGCC) and inhibition of adenylyl cylase. In this diagram the G-
protein is denoted α, β, γ but the α-subunit interact K
+
/Ca
2+
channel and adenylate cyclase.
23

17β-estradiol (E2) modulation of opioid inhibition
E2 modulates both anti-nociceptive opioids including enkephalins and β-
endorphin (Priest et al., 1995; Holland et al., 1997; Chaban, 2008a; Lima et al., 2010) and
the pro-nociceptive nociceptin/orphanin FQ systems (Rizzi et al., 2006). E2 increases
nociceptin mRNA expression in the hypothalamus and limbic system (Sinchak and
Micevych, 2003). Moreover, the hyperpolarization of hypothalamic neurons is rapidly
(<20 min) decreased by E2 (Kelly and Wagner, 1999; Qin et al., 2003). These evidences
show that sex steroids present different analgesic responses in women and men,
suggesting that sex-related differences in the analgesic responses were produced by μ-
opioid receptor (MOR) (Miaskowski, 1997; Zubieta et al., 2002). In addition, MOR is
involved in peripheral anti-nociception, analgesia by stress, and behaviors of
systematically administered opiate drugs (e.g. heroin) (Sora et al., 1997; McDonald and
Lambert, 2005).
There are two important action of opioids related with E2. First, E2 affected by
opioids is triggering intracellular signaling cascades. E2 may be performing MOR
through internalization or allosteric interactions (Micevych et al., 1997; Micevych et al.,
2002; Sinchak and Micevych, 2001; Rothman et al., 2007; Schröder et al., 2008). MOR, a
G-coupled protein receptor, demonstrates an internalization which is the expression of
physical desensitization and attenuation of the number of receptor in the plasma
membrane. Maggi (1999) showed that MOR related with MOR ligand that was decreased
by E2 in E2-transfected neuroblastoma cells in vitro. MOR is significantly activated by
E2

but not by KOR, while it may not act on colonic sensory afferents (Su et al. 1998;
Sandner-Kiesling and Eisenach, 2002).
24

1.4 Function of the TRPV1 receptors in DRG neurons
TRPV1 receptor, a member of the TRPV subfamily, is a membrane component of
a subset of primary neurons related with perception of pain known as nociceptors
(Caterina et al., 1997; Benham et al., 2002; Gunthorpe et al., 2002). TRPV1 is has a
specific non-endogenous agonist such as capsaicin which is the active ingredient in
pungent chili peppers (Higashiquchi et al., 2006; Barbero et al., 2006; Kozukue et al.,
2005; Iorizzi et al., 2001). TRPV1 receptors are expressed in widespread areas of the
nervous system, but they are particularly expressed on both dorsal root ganglion (DRG)
and trigeminal ganglion in which they are regarded to be between the prototypes of
detectors by noxious stimuli (Julius and Basbaum, 2001). TRPV1 receptors are highly
expressed in nociceptive neurons of peripheral nervous system, as well as they present in
peripheral nervous endings of central nerve system (Nakagawa and Hiura, 2006; Lauria
et al., 2006; Aoki et al., 2005; Tanimoto et al., 2005; Cui et al., 2006; Funakoshi et al.,
2006).
The cloned TRPV1 receptor is a non-selective cation channel with a high
permeability for Ca
2+
(Caterina et al., 1997; Tominaga and Caterina, 2004). TRPV1
functions as molecular integrator of painful chemical and physical stimuli (noxious heat
(>43º C) and low pH). Various inflammatory mediators such as prostaglandin E2 (PGE2)
and bradykinin potentiate TRPV1. The potentiation of TRPV1 activity can be quantified
by measuring the differences of capsaicin-induced [Ca
2+
]
i
changes before and after
receptor activation (Petruska et al., 2000). Significantly, a subset of DRG neurons
respond to both capsaicin and ATP (Canti et al., 1999) indicating that there may be cross-
activation of these receptors that may underlie the sensitization of visceral nociceptors.
25

1.5 Visceral sensitization of DRG neurons
The receptors mediated transduction of nociceptive signals are expressed on DRG
sensory neurons which mainly contain their intracellular signaling cascades (Gold et al.,
1996; Chaban, 2005). DRG neurons are a valuable preparation in vitro because adult
primary sensory neurons can be studied without the intervention of modulation by
peripheral or central messengers. ATP is known to be sensitive on visceral afferents
(Burnstock, 2001; Christianson et al., 2009). Ji et al. (2008) reported a model of visceral
nociception suggesting that visceral nociceptive processing was controlled by modulation
of estrogen. For example, the visceromotor responses to colonic distension were
measured in female rat with variation of estrous cycle (Sapsed-Byrne et al., 1996; Ji et
al., 2008). There exist three main evidences about estrogen sensitive-visceral afferents.
First, sex steroid hormone levels on pain sensation in cycling females are affected by
visceral pain (Riley et al., 1998; Micevych et al., 2008). Second, there is some evidence
in sex-related differences of prevalence of functional disorders including the viscera (Lee
et al., 2001; Chaban, 2008b); Finally, the population of estrogen-sensitive DRG neurons
appear to be putative visceral afferents (McRoberts et al., 2001; Chaban, 2008b).
Theoretically, a primary afferent neuron responds to a single sensory channel, but some
studies showed that a population of DRG neurons can stimulate both somatic and visceral
tissues. Chaban (2008b) reported that the same primary afferent can innervate both
reproductive and gastrointestinal organs using retrograde labeling from uterus and colon
(DRG neurons have shown both organs specific dyes for retrograde tract tracer). This
evidence indicates that this new subset of dichotomizing fibers provides a novel
sensitization pathway of one viscera by another.
26

Figure 1. 7: Model of alternative possibilities for viscero-visceral cross-sensitization in the DRG neurons. (a): ATP
released by a neuron innervating the inflamed uterus acts on a neighboring neuron sensitizing its responses to colonic
distention. (b): The same neuron innervates the uterus and colon. Uterus inflammation directly sensitizes the neuron to
colonic distention (Ref. modified from Chaban, 2008a).

1.6 Calcium Imaging in sensory DRG neurons
Calcium plays a role in the transduction of signaling pathways. Calcium imaging
is designed to measure the calcium (Ca
2+
) concentration in the medium or a tissue. To
measure Ca
2+
concentration, a different wavelength of fluorescent dyes which is based on
Ca
2+
-binding molecules were used, since it may buffer the changes of Ca
2+

concentration. In addition, calcium imaging use some calcium indicator such as Fura-2,
Indo-1, Aequorin, and Quin-2 molecules that respond to the Ca
2+
ion by changing their
spectral characteristics. For example, fura-2 are now broadly used to measure the Ca
2+

ion concentration in living cells as well as they can be used for measuring of intracellular
calcium levels that play a important role in neuronal signal transduction in energy
production of mitochondria. Raising of intracellular Ca
2+
modulates releasing of
presynaptic neurotransmitter, membrane excitability, activity of various second
27

messenger system, and gene expression (Hartmann et al., 1996). There are three sets of
complex and integrated systems in regulation of intracellular calcium: i) plasma
membrane which is calcium channels and transporters, ii) intracellular calcium buffering
that bind to protein related to calcium, and iii) intracellular storage sites such as
mitochondria and endoplasmic reticulum. The relationship between calcium influx
through membrane calcium channels and neuronal function has been well studied in the
central nervous system. These evidences suggest that modifications of calcium channels
result in changes in intracellular Ca
2+
concentration.
1.7 Significance
P2X and TRPV1 receptors are expressed on adult DRG neurons in short-term
culture (Gold et al., 1996; Simonetti et al., 2006). These receptors respond to putative
nociceptive signals as well as they proceed to react to activation of estrogen receptors and
μ-opioid receptor agonists mimicking in vivo. There is an significant benefit that can be
studied short of endogenous signals with these neurons. Many publications give the idea
that nociceptive responses have been regulated by E2 in functional pain syndromes,
however, it is not clear whether E2 has pro- or anti-nociceptive effects. According to our
hypothesis, different nociceptive responses by modulation of E2 depend on the different
pains, durations, and the other anti-nociceptive mechanisms. E2 modulates DRG neuron
response to ATP, suggesting that visceral afferent nociceptors are modulated by E2,
which may explain the observed clinical and animal sex differences in visceral
hypersensitivity and suggest a potential target for mediating nociception. We propose to
systematically test how E2 responses as an anti- and pro-nociceptive modulator in a
model of visceral functional pain. Future directions of this proposal will include
28

experiments to study the role of peripheral ERs in visceral nociception using in vivo
models. Thus, from a public health perspective, the outcome of this study will have a
substantial impact, because it will increase our knowledge of nociceptive functional
diseases such as IBS, interstitial cystitis, and chronic pelvic pain, and help achieve a
deeper understanding of sex differences presented in clinical aspects of these symptoms
associated with various psychiatric disorders.
Only a thorough understanding of the mechanism implicated in these phenomena
can truly contribute to the design of new and more efficient therapies. Many illnesses
affect women and men differently. Some disorders are more common in women, and
some express themselves with different symptoms. Institute of Medicine (IOM) report,
“Exploring the Biological Contribution to Human Health”, confirmed that differences
between the sexes exist in the prevalence and severity of a broad range of diseases,
disorders and conditions. In calling for greater focus on sex-based biomedical research,
the IOM report identified barriers to the achievement of knowledge about sex differences
including ethical, financial, sociological and scientific factors. In the United States, pain
accounts for nearly 20% of all primary health care visits. Studies have shown that at least
one-third of patients with pain also suffer from depression, and it affects more women
than men. This thesis will address a crucial question in women’s health and in visceral
nociception in particular. Reaching further, this thesis is a liaison between the basic
science work and the clinical aspects that are addressed through other disciplines such as
anesthesiology (pain management), gastroenterology and obstetrics and gynecology
(OB/GYN).
29

Chapter 2. INTERACTION BETWEEN P2X3 AND ERα/ERβ
IN ATP-MEDIATED CALCIUM SIGNALING
IN MICE SENSORY NEURONS

Portions of this Chapter are adapted from:
Taehoon Cho and Victor Chaban, Journal of Neuroendocrinology (2012) 24, 789-797
and Taehoon Cho and Victor Chaban, NeuroReport (in press, 2012)

Emerging evidence support a role of purinergic P2X3 receptors in modulating
nociceptive signaling in sensory neurons. Previously we showed that DRG neurons (L1-
S1) express both ERα and ERβ receptors. In this study we investigated the expression of
P2X3 receptors and the effect of 17β-estradiol (E2) on ATP-induced [Ca
2+
]
i
increase in
DRG neurons collected from C57Bl/6J, ERαKO and ERβKO mice. Our data showed a
significant decrease for P2X3 in ERαKO (all levels) and ERβKO (mostly observed in L1,
L2, L4, and L6). Furthermore, 17β-estradiol (100 nM) significantly attenuated the ATP
(10 µM)-induced [Ca
2+
]
і
in C57Bl/6J mice. ERs antagonist ICI 182,780 (1µM) blocked
this attenuation. Homomeric P2X3 receptors are plentifully expressed in DRG neurons
and contribute to nociceptive signals. α,β-me ATP which is a specific agonist of P2X2/3
receptors showed similar responses to the ATP-induced calcium increase in knock-out
mice. A membrane-impermeable E-6-BSA (1µM) had the same effect as E2 suggesting
30

action on the membrane. In DRG neurons from ERβKO and WT mice E2 attenuated the
ATP/α,β-me ATP-induced [Ca
2+
]
і
fluxes but in DRG neurons from ERαKO mice, this
hormone had no effect suggesting that this attenuation depends on membrane-associated
ERα receptors. Together our data indicate an interaction between P2X3 and membrane-
associated ERα in primary sensory neurons that may represent a novel mechanism to
explain sex differences observed in clinical presentation of visceral nociceptive
syndromes.
2.1 Introduction
Sex-related differences in pain processing and responsiveness have been observed
in clinical studies (Chang and Heitkemper, 2002) as well as in animal models of
nociception (Berkley et al., 2006). Experts generally agree that in both population-based
studies and patient-based studies, women are more likely affected by most chronic
functional pain conditions, including painful bladder syndrome (PBS), irritable bowel
syndrome (IBS), chronic pelvic pain (CPP) and fibromyalgia. The mechanisms
underlying the greater vulnerability of women remains incompletely understood.
17β-estradiol (E2), the most common form of estrogen, interferes with pain
transmission by regulating rapid changes of ion-channel opening and membrane-
associated neurotransmitter receptors (Nadal et al., 2001; Levin, 2002). Estrogen-binding
proteins have been associated with the plasma membrane and transfected chinese hamster
ovary cells with the cDNAs for ERα and ERβ showed that the putative membrane-
associated estrogen receptors are indistinguishable to the nuclear receptors (Razandi et
al., 2003). Localization of estrogen receptor-α (ERα) and estrogen receptor-β (ERβ) in
31

DRG neurons (Papka and Mowa, 2003) suggests that estrogen may regulate nociceptive
signaling at the level of primary afferents.
DRG neurons can be activated or modulated by the activation of chemosensitive
receptors on peripheral terminals and ATP has been implicated in sensory transduction of
noxious stimuli by activating purinergic P2X receptors (Dunn et al., 2001). Once ATP is
released into the intercellular areas, P2X3

receptors are activated on primary afferent
fibers and cell bodies within DRG. Activation of P2X3 receptors results in the
depolarization and opening of voltage-gated Ca
2+
channels (VGCC) (Koshimizu et al.,
2000). A sensation of pain is produced by depolarization of the peripheral nerve
terminals. ATP-sensitive P2X3 and α,β-methylene ATP-sensitive P2X2/3 receptors play
an important role within the nociceptive systems triggering a nociceptive signaling.
Primary DRG neurons culture has been a useful model system for investigating sensory
physiology and putative nociceptive signaling (Gold and Gebhart, 2010). ATP-induced
intracellular calcium concentration ([Ca
2+
]
i
) transients in cultured DRG neurons have
been used to model the response of nociceptors to painful stimuli (Xu and Huang, 2002).
Previously we showed that E2, acting at the level of the plasma membrane,
attenuates ATP-induced [Ca
2+
]
і
fluxes (Chaban et al., 2003). Within the context of our
present hypothesis E2 modulation of visceral nociception and nociceptor sensitization
appear to be regulated by both P2X3 and P2X2/3. Estrogen attenuation of DRG neurons
response to ATP suggests that visceral afferent nociceptors can be modulated by sex
steroids at a new site at the level of primary afferent neurons. In this manuscript we
report that the expression of P2X3 depends on the expression of both ERs and that E2
mediates its effect through membrane-associated ERs.
32

2.2 Results
2.2.1 Expression of P2X3 in DRG neurons
Previously we detected both ERα and ERβ in Wt mouse DRG neurons using
mRNA RT-PCR (Chaban and Micevych, 2005). To conform the expression of ERs in
DRG neurons, both ERα and ER β proteins were expressed in wild type mice but ERα
didn't detect in ERαKO mice and ER β also didn't detect ER βKO mice (Fig. 2.1, n=5,
p<0.05).

Figure 2. 1: Western blot analysis of ERs in DRG lysates. Representative results with ERα and ERβ are shown in (a).
Immunoreactivity Western is shown as a fold change of the mean value from wild type animals. Quantification of
signals from western blots shows statistically significant difference between the intensity of the bands from both knock-
out DRG neurons when compared with wild type animals. Values are expressed as mean ± SEM P<0.05, n=5. *
indicate significant difference from control.

In this study P2X3 receptors expression was examined by Western blot analysis
of lysates from Wt, ERαKO, and ERβKO DRG tissues using a P2X3 specific primary
antiserum. An intense band representing a ~64 kDa protein (P2X3) was seen in DRG
33

lysates from Wt animals. In our experiments we found that there was a dramatic decrease
in intensity of this band using lysates from the both knock out DRG tissues (>4 fold
decrease of control). A representative result of P2X3 receptors is shown and the
standardization ratio statistics of three tests is shown in Fig. 2.2. The average intensities
of the bands in both knock-out mice decreased significantly. When the density in the
control group was standardized to 1.0, the average densities were 0.172 ± 0.08 of ERαKO
and 0.262 ± 0.10 of ERβKO in P2X3 receptors suggesting that P2X3 protein decreased
in DRG from knock-out mice p<0.05 (n=4).

Figure 2. 2: Western blot analysis of DRG lysates shows reduced expression of P2X3 in both knock-out mice.
Quantification of signals from Western blots shows statistically significant difference between the intensity of the
bands from both knock-out DRG neurons when compared with wild type animals.

Our data also show that DRG neurons from Wt, ERαKO, ERβKO express
nociceptive ATP-sensitive P2X3

receptors by using immunohistochemistry (IHC).
Representative neuronal profiles from each group (n=4 in each group) presented in Fig
2.3 (a). The distributions of labeled dorsal root ganglion neurons represent the
34

statistically significant difference between L1, L2, L4, and L6 levels for P2X3 receptors
in both ERαKO and ERβKO DRG neurons when compared with Wt mice. In ERαKO
mice P2X3 receptors expression was reduced in L1-L6 levels but not in S1 (Fig. 2.3 (b),
P<0.05, n=4 in each group).

Figure 2 .3: (a) Expression of P2X3 receptors in DRG neurons from Wt, ERαKO, and ERβKO in vivo using
fluorescent microscopy. DRG sections were incubated in P2X3 primary antibodies. (b) Percentage distribution of
labeled dorsal root ganglion neurons in ERαKO and ERβKO as well as wild type mice with P2X3 through L1-S1
levels. * indicate statistically significant difference from control, P<0.05.
35

2.2.2 Effect of E2 and pharmacological profile of E2-mediated modulation on ATP-
induced [Ca
2+
]
i
in DRG neurons.
Our data suggest that ATP-induced [Ca
2+
]
і
transients in DRG neurons in mice, a
result similar to that observed in rat DRG neurons [11]. Brief 10 second application of
ATP (10μM) by fast superfusion produced equal [Ca
2+
]
і
spikes in 65% of tested neurons..
After a 5-min washout with HBSS, additional stimulation with ATP (10μM) induced a
subsequent [Ca
2+
]
і
transients (Fig 2.4 (a)). Pretreatment with purinergic receptor
antagonist PPADS (5 μM) blocked the ATP-induced [Ca
2+
]
i
transients (data not shown).
Similarly, ATP stimulation in a Ca
2+
-free media with the Ca
2+
chelator, BAPTA (10
mM), eliminated [Ca
2+
]
i
spikes indicating the necessity for P2X3 receptors and
extracellular Ca
2+
(data not shown).
36

Figure 2. 4: 17β-estradiol (E2) inhibits ATP-induced [Ca
2+
]
і
transients in wild type mice. (a) Typical indication of
equal [Ca
2+
]
і
responses to repeated ATP (10 µM) stimulation (indicated by arrow) with 10 min interval under control
condition. (b) Second ATP-induced [Ca
2+
]
і
response rapidly attenuated by E2 (100 nM) in dorsal root ganglion cells.
(c) E-6-BSA (1 µM) inhibited the ATP-induced [Ca
2+
]
i
transient. After wash-out with experimental medium, ATP
response on [Ca
2+
]
i
returned to initial (control) amplitude of stimulation. (d) The effect of E2 does not desensitize upon
repeated application of ATP. (e) Effect of estrogen receptor antagonist ICI 182,780 alone and application of ATP. (f)
ICI 182,780 (1 µM) blocked the E2 attenuation of ATP-induced [Ca
2+
]
і
transients.

37

E2 (100 nM) by itself had no effect on basal [Ca
2+
]
і
, but this hormone attenuated
the ATP-induced [Ca
2+
]
і
transients (Figs 2.4 (b) & 2.5). The effect of E2 was reversible.
After the initial ATP response, five minute incubation with E2 inhibited ATP-induced
[Ca
2+
]
і
transient (440.3±58.3 vs. 280.4±48.8 nM, n=15, p < 0.05). To confirm the
desensitization of E2, we administered application of repeated ATP, indicating that E2
does not desensitize upon of application of repeated ATP (Fig. 2.4 (d). The estrogen
receptor antagonist ICI 182,780 (1 µM) blocked the 17β-estradiol effect on attenuated
ATP-induced [Ca
2+
]
і
transients (Fig 2.4 (f) & Fig. 2.5) and ICI 182,780 demonstrates no
effect by itself (Fig 2.4 (e)).

38

Figure 2. 5: Summary of ATP-induced [Ca
2+
]
i
influxes in control, in the presence of E2-β, E-6-BSA, E2-α, and ICI
182,780. E2 significantly decreased [Ca
2+
]
і
response to ATP whereas estrogen receptor antagonist ICI 182,780 blocked
E2 effect. Values are expressed as mean ± SEM. * indicate statistically significant difference from control, P<0.05.

2.2.3 E2 action on voltage-gated calcium channels (VGCC) in DRG neurons
To clarify contribution of voltage-gated calcium channels (VGCC) to estrogen,
we demonstrated treatment with ω-conotoxin GVIA and ω-agatoxin IVA or nifidipine on
ATP-induced calcium responses in DRG neurons. E2 has been shown selective effects
which inhibited L-type calcium channels. E2 has no effects on the basal [Ca
2+
]
і
, but
attenuated on ATP-induced [Ca
2+
]
і
. In addition, nifedipine, a dihydropyridine calcium
channel blocker, mimicked E2 effects on ATP-induced [Ca
2+
]
і
. Moreover, co-application
39

of E2 and nifedipine didn't enhance, indicating that E2 as well as nifedipine associate
with the same L-type voltage-gated calcium channel (VGCC). Treatment of DRG
neurons either ω-conotoxin GVIA, a selective blocker of N-type calcium channel, or ω-
agatoxin IVA, a selective blocker of P-type calcium channel attenuated the ATP-induced
[Ca
2+
]
і
(Fig. 2.6), suggesting that inhibition of L-type calcium channel and either the N-
type or P-type VGCC modulates more profound attenuation than only inhibition with a
single VGCC.

Figure 2. 6: Contribution of voltage-gated Ca
2+
channels (VGCC) to E2 inhibition on ATP-induced [Ca
2+
]
і
. Separate
experiments were done with blocker of L-, N-, and P-type VGCC: nifedipine (Nif, 10 μM), ω-conotoxin GVIA (ω-Con,
1 μM), and ω-agatoxin IVA (ω-Aga, 100 nM) respectively. The VGCC antagonists were used by themselves and in the
combination with E2 treatment. E2 showed a significant attenuation on ATP-induced [Ca
2+
]
і
fluxes. Nifedipine either
alone or with E2 showed the same inhibition. In addition, the ω-Con or ω-Aga blocked ATP-induced [Ca
2+
]
і
fluxes. E2
and ω-Con or ω-Aga produced more blocking effects than E2 alone. Values are expressed as mean ± SEM (Repeated
from Chaban et al., 2003).

40

2.2.4 Effect of E2-BSA on ATP-induced [Ca
2+
]
i
response in DRG neurons
In the present study, the rapid time course and the higher doses required for ER
activation suggest that E2 inhibition of ATP-induced [Ca
2+
]
i
flux was mediated through a
membrane associated ERs. A membrane impermeable construct, E-6-BSA (17β-estradiol
6-(O-carboxymethyl) oxime-BSA, Sigma) was used to determine whether the E2 actions
on ATP-induced [Ca
2+
]
i
were mediated at the membrane. E-6-BSA (1μM), filtered to
remove any potentially unconjugated estradiol, mimicked the effect of E2 (Fig 2.4 (c) &
Fig. 2.5). Our data indicate that diffusion of E2 into DRG neurons to act on nuclear ERs
was not essential for the E2 inhibition of ATP-induced [Ca
2+
]
i
transients in DRG neurons
in primary culture. To clarify the E-6-BSA diminution of ATP-induced Ca
2+
response
with ER subtypes, we compared E-6-BSA action mediating [Ca
2+
] flux in DRG neurons
from Wt, and knockout mice. The effect of E-6-BSA mimicked in ERβKO mouse DRG
neurons to that performed in Wt mice (n = 38 cells/3 mouse) (Fig 2.7 (b)). However, in
ERαKO mice, E-6-BSA did not block ATP-induced [Ca
2+
]
і
fluxes (n=32 cells/3 mouse)
(Fig. 2.7 (a)), indicating that its diminution relies on ERα.
41

Figure 2 .7: The effect of E-6-BSA on ATP-induced [Ca
2+
]
і
fluxes in ERαKO and ERβKO mice. (a) In ERαKO mouse,
E-6-BSA added for 5 min didn't inhibit ATP-induced [Ca
2+
]
і
flux; (b) In ERβKO mouse, Effect of E-6-BSA mimicked
that observed in Wt mouse. Summary data represented on the right bar graphs. * Statistically significant difference
from control, P<0.05.

2.2.5 17β-estradiol attenuation of ATP and α,β-me ATP-induced [Ca
2+
]
i
influx in
DRGs from ERαKO and ERβKO mice
To confirm which ER subtype mediates the E2 attenuation of ATP-induced
response, we compared estradiol action mediating Ca
2+
signaling in DRG neurons from
Wt, and knockout mice. The effect of E2 was similar in ERβKO mouse DRG neurons to
that observed in Wt mice (n = 125 cells/5 mouse) (Fig. 2.8 (b)). However, in DRG
neurons from ERαKO mice, E2 did not block ATP-induced [Ca
2+
]
і
suggesting that its
42

attenuation depends on ERα (n = 112 cells/5 mouse) (Fig. 2.8 (a)). 17α-Estradiol had no
effect on Wt, ERαKO or ERβKO mice.

Figure 2. 8: The effect of E2 on ATP-induced [Ca
2+
]
і
transients in estrogen receptor-α knockout (ERαKO) and
estrogen receptor-β knockout (ERβKO) mice. (a) In ERαKO mouse, E2 added for 5 min didn't inhibit ATP-induced
[Ca
2+
]
і
transient; (b) In ERβKO mouse E2 stimulation significantly attenuated the ATP-stimulated [Ca
2+
]
і
transient
similar to that observed in Wt mouse. Summary data represented on the right bar graphs. *Statistically significant
difference from control, P<0.05.

We also used α,β-me ATP, a specific agonist of P2X2/3, to confirm the
observations presented with ATP (n=105 cells/each group 6 mouse). Even the properties
of P2X2/3 receptors are not similar to those of the P2X3 receptors both P2X3 and
P2X2/3 receptors may be a target of action for estradiol (Fig. 2.9). Our experiments used
a combination of techniques to determine that DRG neurons in culture can be used to
study the cellular response to a putative nociceptive signal, ATP. Our data suggest that in
primary DRG neuronal cultures, E2 attenuates the ATP/α,β-meATP -induced [Ca
2+
]
i

43

responses and interferes with the membrane-associated ERα. In our experiments we
noticed significant decrease in number of responsive neurons in both knock-out mice.
Fewer than 20% of tested cells responded to ATP/α,β-meATP stimulation which
correspond to the fact that that both ERαKO and ER βKO exhibit decrease in their
expression of P2X3 receptors (Fig. 2.2).
44

Figure 2. 9: The effect of 17β-estradiol (E2) on α,β-meATP-induced [Ca
2+
]
і
transients in Wt, ERαKO, and ERβKO
mice. (a) Wt mouse as a control, (b) ERαKO mouse, E2 added for 5min didn't inhibit α,β-meATP-induced [Ca
2+
]
і

transient in control vs. after E2 treatment. (c) In ERβKO mouse, E2 stimulation significantly attenuated the α,β-
meATP-stimulated [Ca
2+
]
і
transient similar to that observed in Wt mouse. Summary data represented on the right bar
graphs. Values are expressed as mean ± SEM. Δ[Ca
2+
]
і
were determined by subtracting the [Ca
2+
]
і
peak levels from
the basal [Ca
2+
]
і
levels. *Statistically significant difference from control, p < 0.05.

45

2.3 Discussion
Visceral pain sensations associated with Irritable Bowel Syndrome, Pain Bladder
Syndrome and Chronic Pelvic Pain are different from cutaneous pain based on clinical,
neurophysiologic and pharmacological characteristics. The pathophysiology of visceral
hyperalgesia is less well-known than its cutaneous counterpart, and our understanding of
visceral hyperalgesia is colored by comparison to cutaneous hyperalgesia, which is
believed to arise as a consequence of the sensitization of peripheral nociceptors due to
long-lasting changes in the excitability of spinal neurons (Mayer et al., 2008). Many
chronic functional syndromes characterized by recurring symptoms of abdominal
discomfort or pain and pathophysiological alterations in the absence of detectable organic
disease are more prevalent in women. In general, pain thresholds are lower in women
than in men and pain symptoms vary with reproductive cycle. In women, estrogen may
be a causative factor, inducing inflammatory diseases that may contribute to nociception
associated with functional pain syndromes. Functional syndromes lack a specific
pathology in the affected organ but may respond to a viscero-visceral cross-sensitization
in which increased nociceptive input from an inflamed organ (i.e., uterus) sensitizes
neurons that receive convergent input from an unaffected organ (i.e., colon) (Berkley,
2005). The site of visceral cross-sensitivity is unknown. One explanation is a central
nervous system E2 modulation and convergence. Data from our laboratory and others
suggest that E2-induced modulation of viscero-visceral cross-sensitization occurs in the
dorsal root ganglion (Chaban et al., 2003; Chaban and Micevych, 2005; Xu et al., 2008;
Sarajari and Oblinger, 2010). The localization of ERs in DRG neurons (Papka and Mowa,
2003) and the attenuation of ATP-induced [Ca
2+
]
i
strongly suggest that E2 modulates
46

visceral pain processing peripherally. Visceral nociception and nociceptor sensitization
appear to be regulated by ATP. More than half the small diameter DRG neurons
(presumably nociceptors) in culture respond to ATP and were estrogen-sensitive
(Chaban, 2010; Chaban and Micevych, 2005). ATP effect was blocked by PPADS,
indicating an involvement of purinoreceptors. The involvement of the P2X3 receptor in
the ATP response was proven by using selective P2X3 agonist α,β-meATP therefore ERα
interacts with P2X3 in DRGs.
Sex hormones and E2, in particular, may directly influence the functions of
primary afferent neurons. Both subtypes of estrogen receptors (ERα and ERβ) are present
in small-diameter DRG neurons (Papka and Storey-Workley, 2002). While several
actions of E2 have been demonstrated in the nervous system, the mechanisms of E2 pain
modulation remain unclear. ERs were traditionally envisioned as E2-activated
transcription factors. However, E2 has a multiplicity of actions: membrane, cytoplasmic
and nuclear (reviewed in (Nadal et al., 2001)). As we expected, treatment with E2 was
blocked by ICI 182,780 indicating the mechanism through ER. Most of the published
reports in the area of sex and hormone-related differences in pain have addressed the
modulatory effect of E2 on CNS mechanisms of nociception (Aloisi et al., 2000).
Recent studies demonstrate that E2 has a significant role in modulating visceral
sensitivity, indicating that E2 alterations in sensory processing may underlie sex-based
differences in functional pain symptoms (Al-Chaer and Traub, 2002). However, reports
of E2 modulation of visceral and somatic nociceptive sensitivity are inconsistent. For
example, elevated E2 levels have been reported to increase the threshold to cutaneous
stimuli but decrease the percentage of escape responses to ureteral calculosis (Bradshaw
47

and Berkley, 2002). However, nociceptive sensitivity appears to increase when E2 levels
are elevated (Holdcroft, 2000; Bereiter, 2001). Indeed in most clinical studies, women
report more severe pain levels, more frequent pain and longer its duration of pain than
men (Berkley, 1997; Fillingim and Edwards, 2001). To help resolve these inconsistencies
we propose to study E2 actions on the primary afferents. Little is known about E2-
mediated mechanisms in peripheral nervous system, but the fact that DRG neurons
express ER and respond to E2 treatment suggest that they are a potential target for
mediating nociception.
Visceral nociception and nociceptor sensitization appear to be regulated by ATP
(Burnstock, 2009). Large body of literature supports the idea that E2 modulates
nociceptive responses in pelvic pain syndromes, however, whether E2 is pro- or anti-
nociceptive remains unresolved. Recent study by R. Traub laboratory showed that spinal
ERα mediates the pro-nociceptive effect of E2 on visceral signal processing through
activation of the MAPK pathway (Ji et al., 2011). Recently, our data showed that that
membrane-associated ERα-initiated signaling involves interaction with mGluRs (Chaban
et al., 2011). Within the context of our hypothesis E2 modulation of nociceptive response
depends on the type of pain, its durations and the involvement of other anti-nociceptive
mechanisms. The P2X3 receptor subtype has been found to be involved in peripheral pain
signal transduction but, to date, changes in the expression and function of P2X3 receptors
from DRG neurons in the varied gonadal hormone levels have not been well documented.
E2 modulates DRG neurons response to ATP suggesting that visceral afferent
nociceptors are modulated by E2 in the DRG. Although estradiol doses of 10–100 nM are
commonly used in neuronal preparations are higher than those achieved during the
48

preovulatory peak of estradiol (physiological concentrations of 100pM). Lower levels of
estradiol typically provide homeostatic (negative) feedback, whereas during the female
reproductive cycle, exposure to the sustained high level of estradiol in the circulation at
the end of the follicular phase elicits a neurobiological switch to positive feedback action.
In contrast to the marked inhibition of ATP-induced calcium signaling by 100 nM
estradiol in nociceptors, low physiological doses of estradiol (100 pM) had no effects that
may reveal differences between rapid effects (5-10 min) independent of gene
transcription, and longer-term actions (24 - 48 hrs) that may suggest differences in
transcription. Indeed, our data support the idea that estradiol can signal through both
genomic mechanisms and rapid changes in signaling cascades. The short latency (<5
min) of responses and 100 nM dose strongly argues for a mechanism that does not rely on
changes in gene expression that may involve different receptor subtype activation.
In this study we obtained the same effect with E-6-BSA as with E2 proving that
rapid action of E2 occurs at the membrane site. The DRG is an important site of visceral
afferent convergence and cross-sensitization. Mouse DRG neurons in culture retain the
same responses to a rapid application of E2 as rat cultured DRG, providing investigators
with an additional tool of ER 'knock-outs' for studying the effects of estradiol. In this
study we showed that P2X3 receptors expression significantly decreased in ER knock-out
mice and that E2 acts through an ERα in modulating the ATP-mediated [Ca
2+
]
і
response
in DRG, since its effect was eliminated in ERαKO mouse and retained in ERβKO. This
result shows an important non-reproductive role of ERα in modulating ATP-induced Ca
2+

signaling at the level of the primary afferent neuron, thereby modulating the sensitivity to
painful stimuli in the periphery. To test if E2 directly modulates [Ca
2+
]
i
responses in
49

visceral nociceptors in a future experiments DRG from retrogradely labeled colonic
afferents will be compared with retrogradely labeled cutaneous DRG neurons.
Nociceptive systems implicated in the etiology of functional disorders may be
affected by E2 and often are complicated by co-morbid disorders, all of which may pose
health risks. Treatment strategies for patients should consider this modulation in response
to therapy. Sex steroids have been suggested to play an important role in pain regulation.
E2 effects on visceral nociceptive signaling have been observed in clinical studies, but
most of the research has been focused on the CNS. Our data reveal a new mechanism
how E2 modulates primary sensory neurons response to ATP suggesting that visceral
afferent nociceptors are modulated by E2 in the DRG. Therefore, our study supports the
potential of the P2X3 receptor as a target for pain therapy in females.
2.4 Materials and Methods
2.4.1 Animals
We have used 6~8 week old female wild type (Wt, C57Bl/6J), ERαKO
(B6.129P2-Esr1
tm1Ksk
/J), and ERβKO (B6.129P2-Esr2
tm1Unc
/J) mice (Jackson Laboratory,
Bar Harbor, ME). Upon arrival mice were housed in microisolator caging and maintained
on a 12-h light/dark cycle in a temperature-controlled environment with access to food
and water ad libitum for two weeks. All studies were carried out in accordance with the
guidelines of the Institutional Animal Care and Use Committee (IACUC) of Charles R.
Drew University and the NIH Guide for the Care and Use of Laboratory Animals. In
some experiments we used animals from our breeding colony.

50

2.4.2 Primary culture of DRG neurons
The isolation procedure and primary culture of mouse lumbosacral DRG has been
published in detail (Chaban et al., 2003). DRG tissues were obtained from C57Bl/6J (30
g), ERαKO and ERβKO (Jackson Laboratory; 20 g) transgenic types. Briefly,
lumbosacral adult DRGs (level L1-S1) were collected under sterile technique and placed
in ice-cold medium Dulbecco’s Modified Eagle’s Medium (DMEM; Sigma-Aldrich St.
Louis, MO). Adhering fat and connective tissue were removed and each DRG was
minced with scissors and placed immediately in a medium consisting of 5 ml of DMEM
containing 0.5 mg/ml of trypsin (Sigma, type III), 1 mg/ml of collagenase (Sigma, type
IA) and 0.1 mg/ml of DNAase (Sigma, type III) and kept at 37°C for 30 minutes with
agitation. After dissociation of the cell ganglia, soybean trypsin inhibitor (Sigma, type
III) was used to terminate cell dissociation. Cell suspensions were centrifuged for one
minute at 1000 rpm and the cell pellet were resuspended in DMEM supplemented with
5% fetal bovine serum, 2 mM glutamine-penicillin-streptomycin mixture, 1 μg/ml
DNAase and 5 ng/ml NGF (Sigma). Cells were placed on Matrigel
®
(Invitrogen,
Carlsbad, CA)- coated 15-mm coverslips (Collaborative Research Co., Bedford, PA) and
kept at 37° C in 5% CO
2
incubator for 24h, given fresh media and maintained in primary
culture until used for experimental procedures.
2.4.3 Western Blot Analysis
The expressions of P2X3 receptors in L1-S1 DRGs were studied by using Western blot
analyses. Tissues from Wt (C57Bl/6J), ERαKO, and ERβKO mice were quick frozen in
tubes on dry ice during collection. L1-S1 DRG were combined, homogenized by
mechanical disruption on ice-cold RIPA buffer plus protease inhibitors and incubated on
51

ice for 30 minutes. Homogenates were then spun at 5000g for 15 minutes and
supernatants collected. Total protein was determined on the supernatants using the BCA
microtiter method (Pierce, Rockford, IL, USA). Samples containing equal amounts of
protein (40µg) were electrophoresed under denaturing conditions using Novex Mini-cell
system (San Diego, CA, USA) and reagents (NuPage 4–12% Bis-Tris gel and MOPS
running buffer). After electrophoretic transfer onto nitrocellulose membrane using the
same system, the membrane was blocked with 5% non-fat dry milk (NFDM) in 25 mM
TRIS buffered saline, pH 7.2, plus 0.1% Tween 20 (TBST) for 1 hour at room
temperature, followed by incubation with polyclonal rabbit antibody against P2X3
receptor (1:1000, Neuromics, Edina, MN) for overnight at 4
o
C. The membrane was then
washed in TBST plus NFDM, and proteins were visualized using a horseradish
peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, CA). Following a
final wash in TBST without NFDM, the membrane was incubated with ECL+
(Amersham, Arlington Heights, Ill., USA) substrate for HRP. Membranes were probed
with primary antibody and corresponding secondary antibodies, signals were scanned and
quantified by Image J version 1.28U and NIH Image 1.60 scan software. Following
enhanced chemiluminescence detection of proteins, the membranes were stripped with
stripping buffer (Pierce, Rockford, IL, USA) and re-hybridized with β-actin antibody as a
loading control. At least three independent cell preparations were used.
2.4.4 Immunohistochemistry
For tissue collection DRG from bilateral spinal levels L1-S2 were removed and
fixed in 4% paraformaldehyde for overnight at 4
o
C. DRGs were rinsed in Delbecco’s
Phosphate Buffered Saline (DPBS) and kept in sucrose (20%, 4
o
C) for cryoprotection (48
52

h), after which excess liquid was removed. Each DRG was mounted in Tissue-Tek
®
OCT
embedding medium (Sakura Finetek, Torrance, CA), and sectioned at -20
o
C in a
MICROM H505E (Viocompare, San Francisco, CA) cryostat. Sections were cut at 20µm
and collected in PBS. Endogenous tissue peroxidase activity was quenched by soaking
the sections for 10 min in 3% hydrogen peroxide solution in 0.01 M PBS. The specimens
were washed and then treated for 60 min in blocking solution, 0.01 M PBS containing
0.5% Triton X-100 and 1% normal donkey serum (NDS) at room temperature. They were
processed with P2X3 receptor antibody (1:5000, Neuromics, Edina, MN) for overnight
at 4
o
C, washed in 0.01 M phosphate-buffered saline (PBS) and 0.01M Tris Buffered
Saline (TBS), followed by incubation in solutions of donkey anti-rabbit fluorophore-
conjugated secondary antibodies (1:200, Invitrogen) in 0.01M Tris Buffered Saline (TBS)
for 3 hours at room temperature. Cells showing no apparent or only faint membrane ⁄
intracellular labeling were considered to be negative for P2X3. P2X3-positive neurons
showed diffuse membrane⁄ intracellular labeling were mounted and coverslipped with
Aqua Poly Mount (Polisciences, Warrington, PA). Images from at least three sections in
each level were taken using Leica DMLB M130X microscope. The total numbers of
DRG neurons expressing P2X3 were counted. Immunohistochemical signal percent was
measured by computerized image analysis (Image Pro-Plus, Media Cybernetics, Silver
Spring, MD, USA).
2.4.5 [Ca
2+
]
i
fluorescence imaging
Ca
2+
fluorescence imaging was carried out as previously described (Chaban et al,
2001; Chaban, 2010). DRG neurons were loaded with fluorescent dye 5 mM Fura-2 AM
(Invitrogen, Carlsbad, CA) for 45 min at 37°C in HBSS supplemented with 20 mM
53

HEPES, pH 7.4. The coverslips were mounted in a RC-26 recording chamber P-4
(Warner Instruments, Hamden, CT) and placed on a stage of Olympus IX51 inverted
microscope (Olympus America, Center Valley, PA). Observations were made at room
temperature (20-23°C) with 20X UApo/340 objective. Neurons were bathed and perfused
with HBSS buffer using with using gravity at a rate of 1-2 ml/min. Fluorescence intensity
at 505 nm with excitation at 334 nm and 380 nm were captured as digital images
(sampling rates of 0.1-2 s). Regions of interest were identified within the soma from
which quantitative measurements were made by re-analysis of stored image sequences
using Slidebook
®
Digital Microscopy software. [Ca
2+
]
i
was determined by ratiometric
method of Fura-2 fluorescence from calibration of series of buffered Ca
2+
standards.

E2
was applied acutely for five minutes onto the experimental chamber. Application of drugs
was achieved by superfusion in a rapid mixing chamber and Perfusion Fast-Step system
SF-77B (Warner Instruments) to add drugs in 100-500 ms interval.
We calculated actual [Ca
2+
]
i
in areas of interest in each neurons with the formula:
[Ca
2+
]
i
=K
d
X (R-R
min
)/(R
max
-R)Xβ
Where K
d
is the indicator's dissociation constant of the fluoroprobe; R is ratio of
fluorescence intensity at two different wavelengths (340/380 nm for fura-2); R
max
and
R
min
are the ratio at fura-2 with an saturated Ca
2+
and free Ca
2+
. β is the ratio of the
denominators of the minimum and maximum conditions.
2.4.5 Statistical analysis
The amplitude of [Ca
2+
]
i
response represents the difference between baseline
concentration and the transient peak response to drug stimulation. Significant differences
in response to chemical stimulation were obtained by comparing [Ca
2+
]
i
increases during
54

the first stimulation with the second. All of the data are expressed as the mean ± SEM.
Statistical analysis was performed using Statistical Package for the Social Sciences 18.0
(SPSS, Chicago, IL, USA). To assess the significance among different groups, data were
analyzed with one-way ANOVA followed by Schéffe post hoc test. A p <0.05 was
considered statistically significant.

55

Chapter 3. ESTROGEN RECEPTOR-α MEDIATES
RAPID ESTRADIOL ATTENUATION OF
CAPSAICIN-INDUCED CALCIUM SIGNALING
IN MOUSE DORSAL ROOT GANGLION NEURONS

Portions of this Chapter are adapted from:
Taehoon Cho and Victor Chaban, NeuroReport (in press, 2012) & Journal of
Neuroscience (Submitted 2012)

The incidence of episodic or persistent pain associated with many "functional"
disorders such as irritable bowel syndrome, painful bladder syndrome, fibromyalgia is
two to three times higher in women than in men. One of the possible explanations for this
phenomenon is the estrogen modulation capsaicin-sensitive TRPV1 receptors at the level
of primary sensory neurons. In this study we demonstrated a significant decrease for
TRPV1 expression in ERαKO (all levels) and ERβKO (mostly observed in L1, L2, and
S1) compared with Wt. 17β-estradiol (E2, 100 nM) significantly attenuated the capsaicin
(100 nM)-induced [Ca
2+
]
і
fluxes in Wt (355.83 ± 57.23 nM to 180.67 ± 38.20 nM, n=105
from 10 mice) and ERβKO (n=98 from 6 mice) but not in ERαKO (n=110 from 7 mice).
Estrogen receptors antagonist ICI 182,780 (1 µM) inhibited E2-mediated attenuation and
membrane-impermeable E2-BSA construct had a similar effect to E2. Prostaglandin E2
56

potentiated capsaicin-induced [Ca
2+
]
i
fluxes by almost 2~3 fold from 202.76 ± 4.35 nM
to 525.02 ± 1.92 nM (n=101 from 7 mice) with prolonged duration of the response. The
effect of this inflammatory mediator was ERα-dependent. Together our data suggest a
novel interaction between capsaicin-sensitive TRPV1 receptors and membrane-associated
ERα in primary sensory DRG neurons that may help to understand a mechanism of sex
difference observed in clinical presentations of many functional nociceptive disorders.
3.1 Introduction
Primary DRG neurons in vitro have been a useful model system for investigating
sensory physiology and putative nociceptive signaling (Chaban et al., 2003; Gold and
Gebhart, 2010).The expression of the transient receptor potential subfamily 1 (TRPV1)
receptors is widespread in several areas of the nervous system but it is particularly strong
in dorsal root ganglion (DRG) (McCleskey, 2007). The small (diameter <25 μm) and
medium (diameter <40 μm) capsaicin-sensitive DRG neurons mediate nociceptive
signaling suggesting that TRPV1 expressing neurons are nociceptors. TRPV1 can be
activated by exgenous (capsaicin and ethanol), endogenous (noxious heat (>45
o
C),
proton of pH (<5.3), and anandamide) factors (Caterina et al., 1997; Zygmunt et al., 1999;
Trevisani et al., 2002), and is regulated by inflammatory mediators that activate G-
protein coupled receptors including prostaglandin E2 (PGE2), breadykinin, ATP
(Tominaga et al., 2001; Vellani et al., 2001; Hu et al., 2002; Rathee et al., 2002;
Mohapatra et al., 2003) and receptor-tyrosine kinases such as NGF (Shu & Mendell,
1999; Chuang et al., 2001) whose effects are mediated through an increase of [Ca
2+
]
і
. and
indirectly sensitized to cause hyperalgesia. Evidence for TRPV1’s role in the
transmission of nociceptive modalities comes from studies showing that mice lacking
57

TRP1 gene have deficits in thermal- or inflammatory-induced hyperalgesia (Davis et al.,
2000). Activation of TRPV1 receptors results in the sensation of pain produced by
depolarization of the peripheral nerve terminals and the prostaglandin E2 (PGE2) is the
main pro-inflammatory autacoid contributing to one of the key characteristics of
inflammation, pain hypersensitivity. PGE2 sensitization may elicit a large influx of Ca
2+

via certain ligand-gated cation channels such as TRPV1. PGE2

could also potentiate the
response of the capsaicin-induced [Ca
2+
]
i
responses which might be one of the
mechanisms for the sensitization of neurons to pain signals. In the view of this fact, the
expression and activity of TRPV1 receptors could be a useful tool to examining the
possible effects of pain modulators.
17β-estradiol (E2) may play a key role in pain modulation by inhibiting transient
receptor TRPV1 activation by capsaicin in nociceptor neurons (Xu et al., 2008). In this
report we showed that expression of TRPV1 relies on the expression of both estrogen
receptor-α (ERα) and estrogen receptor-β and that E2 attenuates capsaicin-induced [Ca
2+
]
і

fluxes through membrane-associated ERα.
3.2 Results
3.2.1 Expression of the TRPV1 in dorsal root ganglion (DRG) neurons
In our experiments we test TRPV1 receptors expression by western blot analysis
of lysates from wild type (Wt), ERαKO, and ERβKO DRG tissues using a TRPV1
specific primary antiserum (Fig. 3.1 (a)). An intense band representing a ~130 kDa
(TRPV1) was seen in DRG lysates from wild type animals. There was a dramatic
decrease in intensity of this band using lysates made from the both knock out DRG
tissues when compared with wild type control animals (>2~3 fold decrease of control Fig.
58

3.1 (b)). The average intensities of the bands in both knock-out mice decreased
significantly. When the density in the control group was standardized to 1.0, the average
densities were 0.59 ± 0.06 of ERαKO and 0.391 ± 0.04 of ERβKO in TRPV1 receptors,
suggesting that TRPV1 protein decreased in DRG, P<0.05, n=4. Our study show that
nociceptive capsaicin-sensitive TRPV1 receptors express in DRG neurons.

Figure 3. 1: Western blot analysis of DRG lysates shows reduced expression of TRPV1 in both knock-out mice. Equal
amounts of lysates (40 µg) generated from ERαKO and ERβKO DRG neurons, as well as from wild type mice, were
electrophoresed under denaturing conditions and probed with a anti-TRPV1 antibodies (n=4 per group). Representative
results with TRPV1 are shown in (a). (b) Both DRG neurons of knock-out mice show a significant reduction in band
intensity, 2~3 fold changes of knock-out compared with neurons from wild type (control) DRG. Immunoreactivity is
shown as a fold change of the mean value from wild type animals. Quantification of signals from western blots shows
statistically significant difference between the intensity of the bands from both knock-out DRG neurons when
compared with wild type animals. Values are expressed as mean ± SEM P<0.05, n=4. * indicate significant difference
from control.

59

The sections of DRGs were immunostained with primary antibodies against
TRPV1. Neuronal profiles from each four mouse with ERαKO, ERβKO as well as wild
type mice (n=4 in each group) were quantified for each fluorescent probe. The TRPV1
receptors present in DRG neurons (Fig. 3.2 (a)). The distributions of labeled dorsal root
ganglion neurons represent the statistically significant difference between L1, L2, S1
levels of TRPV1 receptors in both ERαKO and ERβKO DRG neurons, comparing with
wild type mice. And also there was statistically significant difference L3 and L4 of
TRPV1 in wild type compared to ERαKO (Fig. 3.2 (b); P<0.05, n=4 per group).

Figure 3. 2: Expression of TRPV1 receptors in dorsal root ganglion neurons from Wt, ERαKO, and ERβKO in vivo. (a)
DRG sections were incubated in TRPV1 primary antibodies (1:5,000, Neuromics). (b) Percentage distribution of
labeled DRG neurons in ERαKO and ERβKO as well as wild type mice with TRPV1 through lumbar spine level 1
through sacral spine level 1. There was some statistically significant difference between the intensity from knock-out
DRG neurons when compared with wild type animals. Values are expressed as mean ± SEM P<0.05, n=4. * indicate
significant difference from control.
60

3.2.2 Pharmacological profile of E2 modulation on capsaicin-induced [Ca
2+
]
i
flux in
DRG neurons
Brief 3 second application of TRPV1 agonist capsaicin (100 nM) by fast
superfusion produced [Ca
2+
]
і
spikes which were almost completely blocked by 100 nM
capsazepine (CPZ.), a TRPV1-selective antagonist (data not shown). After a 10 min
washout with HBSS, addtitonal stimulation with capsaicin (100 nM) induced a
subsequent [Ca
2+
]
і
fluxes that were similar to the previous stimuli (Fig. 3 (a)). The E2 by
itself had no effect on basal [Ca
2+
]
і
, but E2 (100 nM) attenuated the peak of capsaicin-
induced [Ca
2+
]
і
transients in dose-dependent manner (Fig. 3 (b)). In the presence of 100
nM of E2, capsaicin-induced [Ca
2+
]
і
transients was 180.67 ± 38.20 nM, but under control
condition, capsaicin-induced [Ca
2+
]
і
transients was 355.83 ± 57.23 nM . (Fig. 3 (c) & Fig.
4, p<0.05, n=105 cells from 10 mice). The effect of E2 was reversible. The estrogen
receptor antagonist ICI 182,780 (1 µM) blocked the E2 effect on attenuated capsaicin-
induced [Ca
2+
]
і
transients (Fig 3 (f) & Fig. 4) and ICI 182,780 demonstrates no effect by
itself (Fig 3 (e)).
61

Figure 3. 3: 17β-estradiol (E2) inhibits capsaicin-induced [Ca
2+
]
і
fluxes in wild type mice. (a) Typical indication of
equal [Ca
2+
]
і
responses to repeated capsaicin (100 nM) stimulation (indicated by arrow) with 10 min interval under
control condition. (b) Demonstration of dose dependence of E2 effect. (c) The first capsaicin-induced [Ca
2+
]
і
response
rapidly attenuated by E2 on DRG neurons. (d) E-6-BSA (1 µM) inhibited the capsaicin-induced [Ca
2+
]
i
transient. (e)
Effect of estrogen receptor antagonist ICI 182,780 alone and application of capsaicin. (f) Effect of estrogen receptor
antagonist ICI 182,780 (1 µM) on E2 attenuation of capsaicin-induced [Ca
2+
]
і
transients.

62

To verify the dependence on Ca
2+
influx and release from intracellular calcium
stores the our next experiments were conducted in the presence of BAPTA (10 mM), the
Ca
2+
chelator, prior to the administration of capsaicin. Capsaicin stimulation in a Ca
2+
-
free media with 10 mM BAPTA eliminated [Ca
2+
]
i
fluxes (data not shown).

Figure 3. 4: Summary data of control, 17β-estradiol (E2), E2 + ICI 182,780,and E-6-BSA effects on capsaicin-induced
[Ca
2+
]
і
fluxes change in DRG neurons. The amplitudes of Δ [Ca
2+
]
і
changes were determined by subtracting the
[Ca
2+
]
і
fluxes from the basal [Ca
2+
]
і
levels during capsaicin stimulation. * indicate significant difference from control,
p<0.05.

A membrane impermeable construct, E2-BSA was used to determine whether the
E2 actions on capsaicin-induced [Ca
2+
]
i
were mediated at the membrane. E2-BSA (1μM),
filtered to remove any potentially unconjugated estradiol, mimicked the effect of E2 (Fig
3 (d) & Fig. 4). Our data indicate that diffusion of E2 into DRG neurons to act on nuclear
ERs was not essential for the E2 suppression of capsaicin-induced [Ca
2+
]
i
fluxes in
primary cultured DRG neurons.
63

3.2.3 E2-mediated attenuation of capsaicin-induced [Ca
2+
]
i
transients in DRG
neurons from ERαKO, and ERβKO mice
To identify the role of both ERα and ERβ in E2 modulation of capsaicin-induced
[Ca
2+
]
i
transients, we tested DRG neurons from Wt, ERαKO and ERβKO mice. We
found the similarity of E2 effect in ERβKO mouse on DRG neurons to that observed in
Wt mouse (n=98 cells/6 mice) (Fig. 3.5 (b)). In contrast, ERαKO mouse show that E2 did
not inhibit capsaicin-induced [Ca
2+
]
i
transients, indicating that it depends on ERα (n=110
cells/7 mice) (Fig. 3.5 (a)). 17α-estradiol had no effect on Wt, ERαKO, or ERβKO mouse
(data not shown). Our data indicated that DRG neurons in culture can be used to study
the cellular response to a putative nociceptive signal, capsaicin and E2 attenuates the
capsaicin-induced [Ca
2+
]
i
responses and interferes with the membrane-associated ERα.
64

Figure 3. 5: The effect of 17β-estradiol (E2) on capsaicin-induced [Ca
2+
]
і
transients in estrogen receptor α knockout
(ERαKO) and estrogen receptor β knockout (ERβKO) mice. (a) In ERαKO mouse, E2 added for 5 min didn't inhibit
capsaicin-induced [Ca
2+
]
і
fluxes in control vs. after E2 treatment (p > 0.05; n=110 cells/7 mice). (b) In ERβKO mouse
E2 stimulation significantly attenuated the capsaicin-stimulated [Ca
2+
]
і
fluxes similar to that observed in Wt mouse (p
< 0.05; n = 98 cells/6 mice). Summary data represented on the right bar graphs. Values are expressed as mean ± SEM.
Δ [Ca
2+
]
і
were determined by subtracting the [Ca
2+
]
і
peak levels from the basal [Ca
2+
]
і
levels. *Significant difference
from control, p < 0.05.

65

3.2.4 PGE2 potentiation of the capsaicin-induced [Ca
2+
]
i
fluxes in DRG from Wt,
ERαKO, and ERβKO
To investigate the effect of TRPV1 sensitization observed during inflammatory
response we investigate the effect of inflammatory mediator PGE2 (100 nM) on
capsaicin-induced [Ca
2+
]
i
fluxes. After 5 minutes pretreatment with PGE2 (100 nM), the
[Ca
2+
]
i
fluxes evoked by capsaicin were elevated from 202.76 ± 4.35 nM in control to
525.02 ± 1.92 nM after the treatment (n=101 cells from 7 mice, Fig. 3.6 (a) & (d)).
However E2 (100 nM) attenuated PGE2-enhanced capsaicin-induced [Ca
2+
]
і
transients to
338.72 ± 2.3 nM (Fig. 3.6 (b) & (d)). The ERα/ERβ antagonist ICI 182,780 (1 μM),
blocked the E2 effect to 489.12 ± 33.58 nM (Fig. 3.6 (c) & (d)).
66

Figure 3. 6: 17β-estradiol blocks prostaglandin E2 (PGE2) enhancement of capsaicin-induced [Ca
2+
]
і
transients in Wt
mice. (a) The effect of PGE2 (100 nM, 5 min) pretreatment on the [Ca
2+
]
і
transients evoked by application of capsaicin
(100 nM, 3 s) in the DRG neurons. (b) The potentiating effect of PGE2

on capsaicin-induced [Ca
2+
]
і
response rapidly
attenuated by E2 in dorsal root ganglion cells. E2 blocked the PGE2 effect and (c) ICI 182,780 (1 µM), estrogen
receptor antagonist, blocked the E2 effect of capsaicin-induced [Ca
2+
]
і
transients. (d) Summary of data of control, E2
only on pretreatment with PGE2, and E2 on PGE2 with ICI 182,780 effect on capsaicin-induced [Ca
2+
]
і
changes in
DRG neurons. The amplitudes of [Ca
2+
]
і
changes were determined by subtracting the [Ca
2+
]
і
fluxes from the basal
[Ca
2+
]
і
levels during capsaicin stimulation. Addition 17β-estradiol (E2, 100 nM) on pretreatment with PGE2
significantly decreased [Ca
2+
]
і
response to capsaicin while estrogen receptor antagonist ICI 182,780 (1 µM) blocked
E2 effect. Values are expressed as mean ± SEM P<0.05, n=101 cells/7 mice. * indicate significant difference from
control, P<0.05.
67

Based on our previous data (Fig. 3.5), we expect that ERα may be involved in this
mechanism. To identify that we compared E2 action mediating [Ca
2+
]
і
signaling in DRG
neurons from Wt, ERαKO, and ERβKO mice. The effect of PGE2 after E2 pretreatment
was similar in ERβKO mice DRG neurons to that observed in Wt mice (n=95 cells /6
mice, Fig. 3.7 (b)). However, as expected, in DRG neurons from ERαKO mice, E2 did
not block capsaicin-induced [Ca
2+
]
і
suggesting that its attenuation depends on ERα (n=99
cells/6 mice, Fig. 3.7 (a)).
68

Figure 3. 7: The effect of 17β-estradiol (E2) on PGE2 of capsaicin-induced [Ca
2+
]
і
transients in estrogen receptor α
knockout (ERαKO) and estrogen receptor β knockout (ERβKO) mice. (a) In ERαKO mouse, E2 added for 5min didn't
inhibit capsaicin-induced [Ca
2+
]
і
transient in control vs. after E2 pretreatment with PGE2; p > 0.05; n=99 cells/6 mice).
(b) In ERβKO mouse E2 pretreatment with PGE2 significantly attenuated the capsaicin-stimulated [Ca
2+
]
і
fluxes
similar to that observed in Wt mouse p < 0.05; n = 95 cells/6 mice). Summary data represented on the right bar graphs.
Values are expressed as mean±SEM. Δ[Ca
2+
]
і
were determined by subtracting the [Ca
2+
]
і
peak levels from the basal
[Ca
2+
]
і
levels. *Significant difference from control, p < 0.05.
69

3.3 Discussion
Defining the site(s) and mechanisms through which sex steroid hormones such as
estradiol modulate visceral nociception is an important step in understanding sex-related
differences in pain perception and for designing appropriate therapies. Sex steroids have
been suggested as a plausible mechanism of pain regulation. E2 effects on visceral
nociceptive signaling have been observed in clinical studies, but most of the research has
been focused on the CNS (Micevych et al., 2002; Ji et al., 2003; Berkley et al., 2006;
Giamberadino et al., 2010). On the other hand, we focused on the nociceptive signaling
mechanisms in the PNS, and how E2 might modulate encoding of nociceptive stimuli that
contribute to the development of many functional pain syndromes. Our previous results
showed that DRG express TRPV1 receptors (Chaban, 2008) and their activation by
capsaicin induce mobilization of [Ca
2+
]
і
(Gschossmann et al., 2000). Present data suggest
that E2 modulates TRPV1 receptor-mediated changes in [Ca
2+
]
і
may represent a novel
mechanism that explain sex difference in response to putative nociceptive signals
observed in clinical presentations of pain-related syndromes.
In the contest of visceral pain associated with functional disorders, the TRPV1
receptor plays an important role in transducing thermal and inflammatory pain signals.
TRPV1 functions as molecular integrator of painful chemical and physical stimuli
(noxious heat (>43
o
C) and low pH). Various inflammatory mediators such as
prostaglandin E2 (PGE2) potentiate TRPV1. The potentiating of TRPV1 activity can be
quantified by measuring the differences of capsaicin-induced [Ca
2+
]
і
changes before and
after this receptor activation (Petruska et al., 2000). Evidence for TRPV1's role in there
pathogenesis come from studies showing that mice lacking TRP1R gene have deficits in
70

thermal- or inflammatory-induced hyperalgesia (Davis et al., 2000). Although large
numbers of clinical and animal studies have indicated that there are sex and estrous cycle
differences in pain perception of various pain models (Bradshaw and Berkley, 2002) it
remains unclear whether that occurs in capsaicin-induced pain responses. Present study
shows that application of capsaicin produced a rapid rise of [Ca
2+
]
і
in small (diameter
d<25 μm) and medium (diameter d<40 μm) in size of DRG neurons and after
pretreatment with PGE2 this response was noticeable higher. PGE2 is synthesized and
released in response to tissue damage, brings to hyperalgesia, and is associated with
inflammatory symptoms. The response was inhibited by capsazepine (300 nM), an
antagonist of the TRPV1, and relied on the extracellular [Ca
2+
]
і
transients. The
potentiating of TRPV1 is thought to be important in inflammatory pain processes such as
allodynia and hyperalgesia. Furthermore, pretreatment with PGE2 can also enhance the
[Ca
2+
]
і
transients induce by ATP, phenylbiguanide, and KCl (Gu et al., 2003).
Significantly, subset of DRG neurons responds to both capsaicin and ATP (unpublished
data) indicating that there may be cross-activation of these receptors that may underlie
the sensitization of visceral nociceptors. Capsaicin-induced TRPV1 receptor-mediated
changes [Ca
2+
]
і
may represent a level of DRG activation to noxious cutaneous stimulation
while ATP-induced changes in [Ca
2+
]
і
may reflect the level of DRG neuron sensitization
to noxious visceral stimuli since ATP is released by noxious stimuli and tissue damage
near the primary afferent nerve terminals (Burnstock, 2001, 2010).
Previously, we showed that E2 rapidly acts through an ERα in modulating P2X3
receptors (Cho & Chaban, 2011). Similarly, E2 also modulates TRPV1 receptor-mediated
[Ca
2+
]
і
responses through ERα since its effect was eliminated in ERαKO and retained in
71

ERβKO and Wt mice. The present results demonstrate an important non-reproductive
role of ERα in modulating capsaicin-induced Ca
2+
signaling at the level of the primary
afferent neurons, thereby modulating the sensitivity to painful stimuli in the periphery.
The E2 decreases capsaicin-induced [Ca
2+
]
і
responses and prostaglandin E2 (PGE2)
potentiation of this response. The mechanism underlying the effect of E2 on the TRPV1-
mediated nociceptive response needs further investigation using in vivo models for a
better understanding of how E2 potentiates capsaicin-induced nociception which may
offer a new clinical therapeutic strategy for pain management.
3.4 Materials and Methods
3.4.1 Animals
We have used 6~8 week old female wild type (Wt, C57Bl/6J), ERαKO
(B6.129P2-Esr1
tm1Ksk
/J), and ERβKO (B6.129P2-Esr2
tm1Unc
/J) mice (Jackson Laboratory,
Bar Harbor, ME). Upon arrival mice were housed in microisolator caging and maintained
on a 12-h light/dark cycle in a temperature-controlled environment with access to food
and water ad libitum for two weeks. All studies were carried out in accordance with the
guidelines of the Institutional Animal Care and Use Committee (IACUC) of Charles R.
Drew University and the NIH Guide for the Care and Use of Laboratory Animals. In
some experiments we used animals from our breeding colony.
3.4.2 Primary culture of DRG neurons
The isolation procedure and primary culture of mouse lumbosacral DRG has been
published in detail (Chaban et al., 2003). DRG tissues were obtained from C57Bl/6J (30
g), ERαKO and ERβKO (Jackson Laboratory; 20 g) transgenic types. Briefly,
lumbosacral adult DRGs (level L1-S1) were collected under sterile technique and placed
72

in ice-cold medium Dulbecco’s Modified Eagle’s Medium (DMEM; Sigma-Aldrich St.
Louis, MO). Adhering fat and connective tissue were removed and each DRG was
minced with scissors and placed immediately in a medium consisting of 5 ml of DMEM
containing 0.5 mg/ml of trypsin (Sigma, type III), 1 mg/ml of collagenase (Sigma, type
IA) and 0.1 mg/ml of DNAase (Sigma, type III) and kept at 37°C for 30 minutes with
agitation. After dissociation of the cell ganglia, soybean trypsin inhibitor (Sigma, type
III) was used to terminate cell dissociation. Cell suspensions were centrifuged for one
minute at 1000 rpm and the cell pellet were resuspended in DMEM supplemented with
5% fetal bovine serum, 2 mM glutamine-penicillin-streptomycin mixture, 1 μg/ml
DNAase and 5 ng/ml NGF (Sigma). Cells were placed on Matrigel
®
(Invitrogen,
Carlsbad, CA)- coated 15-mm coverslips (Collaborative Research Co., Bedford, PA) and
kept at 37° C in 5% CO
2
incubator for 24h, given fresh media and maintained in primary
culture until used for experimental procedures.
3.4.3 Western Blot Analysis
The expressions of TRPV1 receptors in L1-S1 DRGs were studied by using
Western blot analyses. Tissues from Wt (C57Bl/6J), ERαKO, and ERβKO mice were
quick frozen in tubes on dry ice during collection. L1-S1 DRG were combined,
homogenized by mechanical disruption on ice-cold RIPA buffer plus protease inhibitors
and incubated on ice for 30 minutes. Homogenates were then spun at 5000g for 15
minutes and supernatants collected. Total protein was determined on the supernatants
using the BCA microtiter method (Pierce, Rockford, IL, USA). Samples containing equal
amounts of protein (40 µg) were electrophoresed under denaturing conditions using
Novex Mini-cell system (San Diego, CA, USA) and reagents (NuPage 4–12% Bis-Tris
73

gel and MOPS running buffer). After electrophoretic transfer onto nitrocellulose
membrane using the same system, the membrane was blocked with 5% non-fat dry milk
(NFDM) in 25 mM TRIS buffered saline, pH 7.2, plus 0.1% Tween 20 (TBST) for 1 hour
at room temperature, followed by incubation with polyclonal rabbit antibody against
TRPV1 receptor (1:1000, Neuromics, Edina, MN) for overnight at 4
o
C. The membrane
was then washed in TBST plus NFDM, and protein were visualized using a horseradish
peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, CA). Following a
final wash in TBST without NFDM, the membrane was incubated with ECL+
(Amersham, Arlington Heights, Ill., USA) substrate for HRP. Membranes were probed
with primary antibody and corresponding secondary antibodies, signals were scanned and
quantified by Image J version 1.28U and NIH Image 1.60 scan software. Following
enhanced chemiluminescence detection of proteins, the membranes were stripped with
stripping buffer (Pierce, Rockford, IL, USA) and re-hybridized with β-actin antibody as a
loading control. At least three independent cell preparations were used.
3.4.4 Immunohistochemistry
For tissue collection DRG from bilateral spinal levels L1-S2 were removed and
fixed in 4% paraformaldehyde for overnight at 4
o
C. DRGs were rinsed in Delbecco’s
Phosphate Buffered Saline (DPBS) and kept in sucrose (20%, 4
o
C) for cryoprotection (48
h), after which excess liquid was removed. Each DRG was mounted in Tissue-Tek
®
OCT
embedding medium (Sakura Finetek, Torrance, CA), and sectioned at -20
o
C in a
MICROM H505E (Viocompare, San Francisco, CA) cryostat. Sections were cut at 20µm
and collected in PBS. Endogenous tissue peroxidase activity was quenched by soaking
the sections for 10 min in 3% hydrogen peroxide solution in 0.01 M PBS. The specimens
74

were washed and then treated for 60 min in blocking solution, 0.01 M PBS containing
0.5% Triton X-100 and 1% normal donkey serum (NDS) at room temperature. They were
processed with TRPV1 receptor antibody (1:5000, Neuromics, Edina, MN) for overnight
at 4
o
C, washed in 0.01 M phosphate-buffered saline (PBS) and 0.01M Tris Buffered
Saline (TBS), followed by incubation in solutions of donkey anti-rabbit fluorophore-
conjugated secondary antibodies (1:200, Invitrogen) in 0.01M Tris Buffered Saline (TBS)
for 3 hours at room temperature. Cells showing no apparent or only faint membrane ⁄
intracellular labeling were considered to be negative for TRPV1. TRPV1-positive
neurons showed diffuse membrane⁄ intracellular labeling were mounted and coverslipped
with Aqua Poly Mount (Polisciences, Warrington, PA). Images from at least three
sections in each level were taken using Leica DMLB M130X microscope. The total
numbers of DRG neurons expressing TRPV1 were counted. Immunohistochemical signal
percent was measured by computerized image analysis (Image Pro-Plus, Media
Cybernetics, Silver Spring, MD, USA).
3.4.5 Fluorescence Imaging analysis
Ca
2+
fluorescence imaging was carried out as previously described (Chaban et al.,
2011). DRG neurons were loaded with fluorescent dye 5 mM Fura-2 AM (Invitrogen,
Carlsbad, CA) for 45 min at 37°C in HBSS supplemented with 20 mM HEPES, pH 7.4.
The coverslips were mounted in a RC-26 recording chamber P-4 (Warner Instruments,
Hamden, CT) and placed on a stage of Olympus IX51 inverted microscope (Olympus
America, Center Valley, PA). Observations were made at room temperature (20-23°C)
with 20X UApo/340 objective. Neurons were bathed and perfused with HBSS buffer
using with using gravity at a rate of 1-2 ml/min. Fluorescence intensity at 505 nm with
75

excitation at 334 nm and 380 nm were captured as digital images (sampling rates of 0.1-2
s). Regions of interest were identified within the soma from which quantitative
measurements were made by re-analysis of stored image sequences using Slidebook
®

Digital Microscopy software. [Ca
2+
]
i
was determined by ratiometric method of Fura-2
fluorescence from calibration of series of buffered Ca
2+
standards.

E2 was applied acutely
for five minutes onto the experimental chamber. Repeated application of drugs was
achieved by superfusion in a rapid mixing chamber into individual neurons for specific
intervals (100-500 ms). We calculated actual [Ca
2+
]
i
in areas of interest in each neurons
with the formula: [Ca
2+
]
i
=K
d
x (R-R
min
)/(R
max
-R) x β, Where K
d
is the indicator's
dissociation constant of the fluoroprobe; R is ratio of fluorescence intensity at two
different wavelengths (340/380 nm for fura-2); R
max
and R
min
are the ratio at fura-2 with
an saturated Ca
2+
and free Ca
2+
. β is the ratio of the denominators of the minimum and
maximum conditions.
3.4.6 Statistical analysis
The amplitude of [Ca
2+
]
i
response represents the difference between baseline
concentration and the transient peak response to drug stimulation. Significant differences
in response to chemical stimulation were obtained by comparing [Ca
2+
]
i
increases during
the first stimulation with the second. All of the data are expressed as the mean ± SEM.
Statistical analysis was performed using Statistical Package for the Social Sciences 18.0
(SPSS, Chicago, IL, USA). To assess the significance among different groups, data were
analyzed with one-way ANOVA followed by Tukey's multiple comparison test. A p
<0.05 was considered statistically significant.

76

Chapter 4. ESTROGEN RECEPTOR-α MEDIATES
VISCERAL NOCICEPTION:
INVOLVEMENT OF TPRV1 AND P2X2/3
IN MOUSE DORSAL ROOT GANGLION NEURONS

Portions of this Chapter are adapted from:
Taehoon Cho and Victor Chaban, Neuron (Submitted 2012)

Defining the visceral nociception sites and mechanisms through modulation of
sex steroid hormones is an important steps in understanding the sex differences in pain
perception and in planning suitable therapies for patient populations. Estrogen has been
shown to participate in the modulation of female sensitivity to clinical and experimentally
induced pain. Clinical studies further suggest that persistent visceral pain associated with
functional disorders such as irritable bowel syndrome (IBS), interstitial cystitis (IC), and
chronic pelvic pain (CPP) are in general two to three times higher among women than
men. One of possible mechanisms may be the convergence of nociceptive stimuli and
estrogen input on primary sensory neurons which innervate viscera. E2 shows
preferentially, on visceral afferents, to modulate the transmission of visceral pain
signaling. In this study we identified visceral and cutaneous afferent neurons and
compared E2 modulation of P2X2/3 and TRPV1 mediated calcium changes in cultured
77

retrograde labeled visceral and cutaneous DRG neurons. Data from this study
demonstrated that E2 significantly decreased the α,β-me ATP-/capsaicin-induced [Ca
2+
]
i

transients in C57BI/6J and ERβKO mice. However, this hormone had no detectable effect
in ERαKO mice indicating that this situation depends on membrane associated ERα. In
addition, Prostaglandin E2 potentiated capsaicin-induced calcium fluxes showed a 2~3
fold amplitudes with a prolonged duration of the response. The effect of this
inflammatory mediator also was found to be estrogen receptor alpha-dependent. It was
also noted that there are no effect and smaller E2 effect with/without PGE2 on α,β-
meATP/capsaicin-induced [Ca
2+
]
і
responses in retrograde labeled somatic DRG neurons
when compared with visceral DRG neurons. In summation, our data suggests that a novel
communication exists between P2X2/3 or TRPV1 and membrane-associated ER-α in
primary visceral sensory neurons and is a novel mechanism to understand sex differences
observed in clinical studies of many functional diseases related with visceral nociception.
4.1 Introduction
Responses of sex-related differences in pain are associated with synergy among
biological, psychological, social-cultural factors including internal hormonal
circumstances, integration of nociceptive inputs, cultural effects, and modulation of the
sensory neurons. Pain sensation originated by internal organs is a basic human
physiological concern and visceral pains are considered as the most common incidence in
the functional pain syndromes including irritable bowel syndrome (IBS), interstitial
cystitis (IC), chronic pelvic pain (CPP), painful bladder syndrome (PBS), and
fibromyalgia. Diseases of the visceral organ pain account for a significant portion of
major complaints by patients that suffer from these conditions with 40~60% of co-
78

morbidity in women (Unruh, 1996). To date, the most common pain studies have
concentrated on somatic tissues which have contributed the development of pain
management. Interestingly, visceral pain diseases which are affected from different organ
often co-exist with other visceral pain related diseases. Many clinical researchers have
found that many patients who have functional pain syndromes consistently complain
about pelvic pain or interstitial cystitis (Whitehead et al., 2002), this is contrary to many
patients with interstitial cystitis that present with functional pain syndromes (Alagiri et
al., 1997; Francis et al., 1997). Furthermore, clinical studies have shown that women
seem to feel more frequently experience pain, have longer episodes of pain, develop less
tolerance, and self-report more severe pain than men do (Fillingim and Maixner, 1995;
Berkley, 1997; Riley et al., 1998; Arendt-Nielsen et al., 2004). The incidence of
persistent or episodic visceral pain involving differences in action of sex hormones and
reproductive functions are reported two to three higher in women than men.
Regulation of nociceptive signaling keeps a balance inhibition between (anti-
nociception) and facilitation (pro-nociception) in the normal state of the organism. Most
primary visceral afferent neurons have cell bodies in dorsal root ganglia (DRG). Visceral
afferents encode different types of information such as direct stimulation of chemo-
sensitive receptors, modulation of neuronal excitability, and activation of ion channels on
the peripheral terminals. The nociceptors are utilize small (diameter d<25 μm) and
medium (diameter d<40 μm) DRG neurons which detected potentially damaged tissues
from noxious stimuli.
Visceral pain is different from somatic pain clinically in presentation as well as
possess distinctly different neurophysiological and pharmacological characteristics.
79

Defining the neural mechanism of transmission of visceral pain is a critical step in
understanding pain as a perception and will lend toward developing a scheme to identify
appropriate clinical therapeutic interventions. On the basis of previous research findings,
there are two important mechanisms that which participate in visceral sensory
innervations of noxious stimuli: (1) viscero-visceral convergence was demonstrated by
sensitizing visceral primary afferent neurons innervating different organs and converging
on the same sensory neurons (Giamberardino et al., 2002; Frokjaer et al., 2005a; Frokjaer
et al., 2005b; Bielefeldt et al., 2006), (2) viscero-somatic convergence has been shown by
stimulation of somatic afferents, in sites of pain referral, which is a consequence of
viscero-somatic convergence (Bielefeldt et al., 2006). The pattern of viscero-visceral
hyperalgesia, between different organs, may involve the sensory neurons of viscero-
somatic convergence (Giamberardino et al., 2010). For examples, ATP can be released by
noxious stimuli, from intraganglionical target organs during inflammation, which can
cross-sensitize ATP responses of colonic DRG neurons within the reproductive system.
Although there is one hypothesis that each primary sensory neuron has a unique sensory
channel, some of researches have reported that DRG neuron, that innervate different
visceral organs (i.e. colon and uterus), can perceive the same sensory input and the
inflammatory process occurring in the uterus enhanced sensation of visceral nociceptive
perception, which is a typically reported in patients with different functional disorders
such as IBS (Malykhina et al., 2006; Chaban, 2008; Li et al., 2008).
In the context of visceral pain, the capsaicin-sensitive DRG neurons has been
shown to mediate visceral nociceptive syndromes, suggesting that TRPV1 expressing
neurons are nociceptors. Evidence for TRPV1’s role in the transmission of nociceptive
80

modalities have been elucidated in studies which demonstrated that mice lacking TRP1R
gene have deficits in thermal- or inflammatory-induced hyperalgesia (Davis et al., 2000).
It is strongly attenuated and the response of primary sensory neurons to the same stimuli
is clearly severely impaired (Caterina et al., 2000). Furthermore, DRG neurons can also
be modulated by the activation of chemo-sensitive receptors on peripheral terminals and
ATP has been implicated in sensory transduction of noxious stimuli by activating
purinergic P2X receptors (Dunn et al., 2001). Once ATP is released into the intercellular
areas, P2X3

receptors are activated on primary afferent fibers and cell bodies within
DRG. Activation of TRPV1 and P2X3 receptors results in the production of a cation
current with a subsequent depolarization and opening of voltage-gated Ca
2+
channels
(VGCC) (Koshimizu et al., 2000). Also these events contribute to [Ca
2+
]
i
changes in
cultured DRG neurons. The [Ca
2+
]
i
changes in stimulation of TRPV1 receptors may
indicate the sensitization level of DRG neuron to noxious somatic stimuli, while [Ca
2+
]
i

changes in activation of P2X3 receptors may demonstrate the level of DRG neuron
sensation to noxious visceral stimulation because ATP is released by damaged tissues and
noxious stimuli near sensory nerve endings (Burnstock, 2001).
In this study, we investigated subsets of dichotomizing visceral DRG neurons
which innervate, colon and uterus, and express nociceptive TRPV1, and P2X3/P2X(2/3)
receptors in the mouse. Our hypothesis is that E2 acts preferentially on visceral sensory
neurons to modulate the transmission of visceral pain stimuli. Retrograde tract tracing
was used to identify visceral and somatic sensory DRG neurons in the mouse. E2
modulation of ATP/α,β-me ATP- and capsaicin-induced [Ca
2+
]
i
fluxes were compared in
cultured retrograde-labeled visceral and somatic DRG neurons.
81

4.2 Results
4.2.1 E2 modulation of ATP-induced calcium fluxes regulate visceral and cutaneous
nociceptive signaling in mouse DRG neurons through separate pathways
Our previous study demonstrated that DRG neurons innervate viscera and are
about 5-10%. A corollary of that hypothesis is that cutaneous pain may show different
action of pain modulation compared with visceral pain. Even though there are no changes
in cutaneous modulation of pain, pathologically visceral modulation of pain such as
inflammation and IBS is found to be more prevalent in women (Lee et al., 2001). This
experiment is designed to compare the pharmacology of retrogradely labeled visceral
DRG neurons with retrogradely labeled cutaneous DRG neurons. Within the context of
our hypothesis, nociceptive responses by E2 modulation depend on the type of pain, and
its duration.
To identify the mechanism of viscero-visceral hyperalgesia, between organs,
retrograde labeling was used to demonstrate tissue and cultured DRG neurons receiving
sensory signaling input from different visceral organs such as the colon and uterus. We
injected fluorogold (FG) into distal colon tissue and tetramethylrhodamine (TMR) into
uterus, specially into the muscle wall by abdominal incision and somatic afferent neurons
(Alexa Fluor 647) via subcutaneous injection into mouse hind paw.
In vivo, both colonic and uterine afferents were shown in L1~L6 and S1~S3 DRG
tissues (Fig. 4.1). To verify the population of DRG neurons that innervate both colon and
uterus in DRG tissues, DRG cells that projected to the colon and uterine were co-labeled
in the same DRG (Fig. 4.1 (a)). The neuronal population of the retrogradely labeled
profiles for colon and uterine DRG is clearly demonstrated in bar graphs (Fig. 4.1 (b)).
82

These study suggests that the colonic and uterine specific DRG were located in L1
through S3 levels. The distribution of DRG innervating both the colon and the uterus
represent a novel subset of dichotomizing afferents (5~15%) innervating both the colon
and the uterus.

83

Figure 4. 1: Retrogradely labeled DRG neurons and percentage of distribution in DRG neurons through the L1 ~S3
levels. (a) Colon-specific DRG neurons labeled with Fluorogold (FG, yellow) injected into the distal colon. (b) Uterus-
specific DRG neurons labeled with tetramethylrhodamine (TMR, red) injected into the uterus. (c) DRG neurons that
project both to the colon and to the uterus dually labeled with both FG and TMR (orange). DRG neurons innervating
both colon and uterus indicated by arrows (Ref. from Chaban et al., 2007).

84

In addition, in vitro retrogradely labeled visceral cultured DRG neurons that
project to the colon or uterine were also co-localized in the same DRG neuron,
suggesting a subpopulation of DRG neurons that innervate both the colon and the uterus
(Fig. 4.2 (a)). These DRG neurons innervating both the colon and the uterus represent a
new group of dichotomizing sensory afferents. In addition, E2 (100 nM) by itself had no
effect on basal [Ca
2+
]
і
, but the ATP-induced [Ca
2+
]
і
fluxes were decreased by this
hormone significantly. After the initial ATP response, incubation with E2 for five minute
inhibited ATP-induced [Ca
2+
]
і
transient (Fig.4.2 (b), 408.72 ± 47.50 vs. 219.16 ± 38.64
nM, n=69 cells/each group 5 mice, p < 0.05). On the other hand, retrogradely labeled
cutaneous DRG neurons that were injected into the mouse hind paw were shown in the
DRG cells (Fig 4.2 (c)). These neurons did not effectively block ATP-induced [Ca
2+
]
і
and
showed low amplitudes of E2 effects on ATP-induced [Ca
2+
]
і
response (Fig. 4.2 (d),
196.73 ± 21.32 vs. 189.05 ± 15.69 nM, n=55 cells/each group 5 mice, p < 0.05).
85

Figure 4. 2: Retrogradely labeled visceral- and somatic dorsal root ganglion (DRG) neurons. (a) Colonic DRGs were
injected with Fluorogold (blue) into the distal colon, uterus-specific DRGs were injected with tetramethylrhodamine
(red) into the uterus, and DRGs labeled with both dyes co-localized. (b) Cholera toxin conjugated Alexa Fluor 647 was
injected (red) subcutaneously into five sites at the mouse hind paw. (c) 17β-estradiol (E2, 10 µM) inhibits ATP-induced
[Ca
2+
]
і
transients in viscerally labeled DRG neuron from wild type mice. (d) E2 did not block ATP-induced [Ca
2+
]
і

and showed low amplitudes of E2 effects on ATP-induced [Ca
2+
]
і
fluxes in somatic labeled DRG neurons. Visceral
DRG sensory neurons innervating both uterus and colon, and cutaneous DRG neurons indicated by arrows.

86

4.2.2 The effect of E2 modulation of P2X2/3 and TRPV1 receptors mediated [Ca
2+
]
і

response in both retrogradely labeled visceral and cutaneous DRG neurons
DRG neurons innervating viscera demonstrated greater amplitudes of E2 effects
on ATP-induced [Ca
2+
]
і
response (Fig. 4.2) suggesting that neurons innervating visceral
organs may express and regulate ER differently. ER is more tightly coupled to P2X2/3
and TRPV1. To identify E2 modulation of calcium responses, from retrogradely labeled
visceral and somatic neurons, we used α,β-me ATP, a specific agonist of P2X2/3, to
confirm the observations presented with ATP (n=69 cells/each group 7 mouse). Both
P2X2/3 and P2X3 receptors share similar properties and seem to be a target for estrogen
(Fig. 4.3 (a) & Fig. 4.6 (a)), 382.60 ± 40.09 vs. 216.84 ± 38.64 nM, n=63 cells/each
group 7 mouse, p<0.05).
87

Figure 4 .3: E2 effects of α,β-me ATP-/capsaicin-induced [Ca
2+
]
і
transients in visceral and cutaneous sensory DRG
neurons from wild type mice. (a) α,β-me ATP-induced [Ca
2+
]
і
response rapidly attenuated by E2 (100 nM) in visceral
DRG neurons. (b) The first capsaicin-induced [Ca
2+
]
і
response also rapidly decreased by E2 on visceral DRG neurons.
E2 didn't inhibit (c) α,β-me ATP/ (d)capsaicin-induced [Ca
2+
]
і
response in cutaneous DRG neurons and this showed
low intensities on α,β-me ATP/capsaicin-induced [Ca
2+
]
і
response.
88

Moreover, TRPV1 receptors can be activated by capsaicin and regulated by
inflammatory mediators including PGE2 and bradykinin (Dray, 2008), through an
increase of [Ca
2+
]
і
in mouse DRG neurons. The E2 demonstrated no effect on calcium
changes by itself, however, E2 (100 nM) showed low intensity of capsaicin-induced
[Ca
2+
]
і
transients (Fig. 4.3 (b)). The superfusion with capsaicin (100 nM) induced [Ca
2+
]
і

increases to 354.33 ± 41.11 nM, but this effect was diminished to 199.81 ± 44.18 nM by
E2. There was statistically significant difference in the effect of TRPV1 in wild type mice
(Fig. 4.6 (b), n=76 cells/each group 8 mouse, p<0.05). However, there was no effect
noted and smaller E2 effect on α,β-meATP/capsaicin-induced [Ca
2+
]
і
responses in
retrogradely labeled somatic DRG neurons compared with visceral DRG neurons (Fig.
4.3 (c) and (d) & Fig. 4.6 (c) and (d), n=58 cells/each group 6 mice, p<0.05). Our results
suggest that the E2 effects on TRPV1 seemed to be one of the primary signaling
pathways for E2-mediated changes of TRPV1 activity and the pain perception of visceral
afferents.
4.2.3 The role of ERα/ERβ in E2 modulation on α,β-meATP- and capsaicin-induced
[Ca
2+
]
i
transients in retrogradely labeled visceral and cutaneous mouse DRG
neurons
To verify the role of ERα and ERβ, in the potentially different response from
visceral and somatic DRG neurons, visceral DRG neurons were retrograde labeled with
FG for colonic afferents and TMR for uterine afferents. ERα and ERβ were stimulated
with either α,β-me ATP or capsaicin and compared to differences from retrograde labeled
somatic DRG neurons in knock-out mice. The E2 effects in DRG neurons from ERβKO
mouse were found to be similar to wild type mice (Fig. 4.4 (b) & Fig. 4.6 (a), n=65
89

cells/7 mice). However, in ERαKO mice it was noted that there were no E2 effects on
α,β-meATP-induced [Ca
2+
]
і
fluxes (Fig. 4.4 (a) & Fig. 4.6 (a), n=58 cells/7 mice). 17α-
Estradiol, a different isomer of 17β-estradiol, had no effect on Wt, ERαKO or ERβKO
mice (data not shown).

Figure 4. 4: The effect of E2 on α,β-me ATP-induced [Ca
2+
]
і
transients in visceral and somatic DRG neurons from
estrogen receptor-α knockout (ERαKO) and estrogen receptor-β knockout (ERβKO) mice. (a) In ERαKO mouse, E2
added for 5 min didn't inhibit α,β-me ATP-induced [Ca
2+
]
і
transient in visceral DRG sensory neurons; (b) In ERβKO
mouse E2 stimulation significantly attenuated the α,β-me ATP-stimulated [Ca
2+
]
і
transient similar to that observed in
visceral DRG neurons from Wt mice. E2 didn't decrease and somatic DRG neurons showed low amplitudes on α,β-me
ATP-induced [Ca
2+
]
і
in (d) ERαKO and (e) ERβKO mice.
90

We also demonstrated capsaicin-induced [Ca
2+
]
і
changes in knock-out mice to
examine the effect of capsaicin in wild type mice. We also found the similarity of E2
effects in ERβKO to that presented in wild type mice (Fig. 4.5 (b) & Fig. 4.6 (b), n=61
cells/7 mice), but E2 did not show any differences in ERαKO mice on capsaicin-induced
[Ca
2+
]
і
fluxes (Fig. 4.5 (a) & Fig. 4.6 (b), n=69 cells/7 mice).

Figure 4 .5: The E2 effects with visceral and somatic DRG neurons on capsaicin-induced [Ca
2+
]
і
fluxes in ERαKO and
ERβKO mice. (a) E2 didn't inhibit capsaicin-induced [Ca
2+
]
і
fluxes in visceral DRG sensory neurons from ERαKO
mice. (b) E2 stimulation significantly attenuated the capsaicin-stimulated [Ca
2+
]
і
fluxes similar to that observed in
visceral DRG neurons from wild type mice. In somatic DRG neurons, there is no significant attenuation and low
amplitudes on capsaicin-induced [Ca
2+
]
і
in (d) ERαKO and (e) ERβKO mice compared with visceral DRG neurons.
91

Our study used a combined approach to determine whether the primary cultured
DRG neurons can be used to research the cellular reaction of a nociceptive α,β-meATP-
sensitive P2X2/3 and capsaicin-sensitive TRPV1 receptors. However, in retrograde
labeled somatic DRG neurons, E2 did not inhibit and showed low intensity on α,β-
meATP (Fig. 4.4 (c), (d), & Fig. 4.6 (c), n=49 cells/6 mice)/capsaicin-induced [Ca
2+
]
і

fluxes (Fig. 4.5 (c), (d), & Fig. 4.6 (d), n=55 cells/6 mice). Our data suggests that sensory
DRG neurons innervating viscera demonstrate greater amplitudes of E2 effects on the
α,β-meATP/capsaicin-induced [Ca
2+
]
i
fluxes and E2 interferes with the membrane-
associated ERα in viscerally-specific neurons.

Figure 4 .6: Summary of α,β-me ATP-/capsaicin-induced [Ca
2+
]
і
transients of control and the presence of E2 in
visceral and somatic DRG neurons from wild type, ERαKO, and ERβKO mice. Summary data represented on the right
bar graphs. E2 significantly decreased [Ca
2+
]
і
response to (a) α,β-me ATP/(b) capsaicin in wild type and ERβKO mice
whereas somatic DRG neurons didn't show statistically significant E2 effect on (c) α,β-me ATP/(d) capsaicin-induced
[Ca
2+
]
і
transients and there are low intensities calcium changes compared visceral DRG neurons. Values are expressed
as mean ± SEM. * indicate statistically significant difference from control, P<0.05.
92

4.2.4 Comparison of the effects of E2 on PGE
2
potentiation of TRPV1-mediated
calcium response in retrogradely labeled visceral and cutaneous DRG neurons from
Wt, ERαKO, and ERβKO mice
The action of E2 is important to determine whether physiological properties of
ERs expressed in visceral neurons and and to identify which ER mediates E2 effects in
visceral compared with somatic DRG neurons. Methodologically it is very difficult to
study receptor activation on the neuritis of DRG neurons. This method requires an
underlying assumption that receptors located on peripheral terminals of colonic primary
afferent neurons have the same functional properties as those expressed on the soma of
DRG neurons (Gold et al., 1996). Hence in vitro DRG neurons are the best model for the
proposed experiments. It is hypothesized that ERs on colonic primary afferent neurons
may have different functional properties compared with those expressed on somatic
afferent neurons. The potentiation of TRPV1 is considered to be important in the pain
related process of inflammation. Moreover, E2 is more likely to magnify higher
intensities of PGE2-mediated TRPV1 activation in retrogradely labeled visceral DRG
neurons than in somatic DRG neurons.
To confirm whether ERα or ERβ play an important role in E2 pretreatment before
the addition of PGE2 (100 nM) on capsaicin-induced calcium responses, retrogradely
labeled DRG neurons from Wt, ERαKO, and ERβKO mice were used. In retrogradely
labeled visceral DRG neurons, after pretreatment with PGE2, the E2 mediated [Ca
2+
]
i

transients by capsaicin were fould to be elevated from a control condition (data not
shown). This study suggested that capsaicin-induced [Ca
2+
]
i
transients were enhanced
after the PGE2 pretreatment by almost 2~3 fold. However, the E2 by itself had no effect
93

on basal [Ca
2+
]
і
, but E2 (100 nM) in the presence of an inflammatory mediator, PGE2,
attenuated the intensities of capsaicin-induced [Ca
2+
]
і
transients in wild type (Fig. 4.7 (a)
& Fig. 4.8 (a), 356.83 ± 42.38 nM, n=67 cells/7 mice, p<0.05) . Specifically, we tested
the role of ERβ in the context of the influence of PGE2-mediated capsaicin-induced
[Ca
2+
]
і
fluxes. The effect of PGE2 after E2 pretreatment was found to be similar in
ERβKO mouse DRG neurons to that observed in Wt mice (Fig. 4.7 (c) & Fig. 4.8 (a),
339.07 ± 54.83 nM, n=58 cells/7 mice, p<0.05) but, E2 did not block capsaicin-induced
[Ca
2+
]
і
in DRG neurons from ERαKO mice suggesting that its diminution depends on
ERα (Fig. 4.7 (b) & Fig. 4.8 (a), 260.12 ± 39.42 nM in control to 583.52 ± 47.52 nM,
n=72 cells/7 mice, p<0.05). However E2 didn't work on capsaicin-induced [Ca
2+
]
і

responses in the retrogradely labeled somatic DRG neurons (Fig. 4.7 (b) & Fig. 4.8 (b)).
94

Figure 4. 7: The effect of 17β-estradiol (E2) on PGE2 potentiation of capsaicin-induced [Ca
2+
]
і
fluxes in visceral and
somatic DRG neurons from wild type, ERαKO, and ERβKO mice. E2 pretreatment with PGE2 significantly decreased
capsaicin-induced [Ca
2+
]
і
fluxes in visceral DRG neurons from (a) wild type and (c) ERβKO mice. (b) In visceral DRG
neurons from ERαKO mice, E2 didn't inhibit PGE2-potentiated capsaicin-induced [Ca
2+
]
і
fluxes. In somatic DRG
neurons, there are no significant attenuation and low amplitudes on capsaicin-induced [Ca
2+
]
і
in (d) wild type, (e)
ERαKO and (f) ERβKO mice compared with visceral DRG neurons.
95

Our data suggest that visceral DRG neurons demonstrate higher intensities of E2
effects on PGE2-mediated capsaicin-induced [Ca
2+
]
i
fluxes and E2 interfere with the
membrane-associated ERα in viscerally-specific neurons. PGE2 could potentiate the
response of the TRPV1 and E2 effects on TRPV1 under pathological conditions such as
inflammation. This data confirmed that the role of ERα and not ERβ in the E2
modulation of PGE2-mediated TRPV1 activation in visceral DRG neurons. This
relationship can be seen in patients suffering from pathological abdominal pain, in
clinical studies, for an array of functional disorders.

Figure 4 .8: Summary of PGE2 pontentiation of capsaicin-induced [Ca
2+
]
і
transients of control and the presence of E2
in visceral and somatic DRG neurons from wild type, ERαKO, and ERβKO mice. Summary data represented on the
right bar graphs. E2 significantly decreased [Ca
2+
]
і
response to (a) PGE2 potentiated capsaicin in ERαKO mice but
somatic DRG neurons had no statistically significant E2 effect on (b) PGE2 potentiated capsaicin and there are low
intensities calcium changes compared visceral DRG neurons. Values are expressed as mean ± SEM. * indicate
statistically significant difference from control, P<0.05.
96

4.3 Discussion
Visceral hyperalgesia is considered to be an important signaling pathways of
functional pain syndromes. The mechanisms of viscero-visceral cross-sensitization plays
an important role in the demonstration of visceral pain syndromes affecting different
organs and how often they coexist in different organs. Although it is usually understood
that each primary sensory neuron is a single channel, several studies have demonstrated
that DRG neurons can innervate both visceral and somatic organs. This study, using
retrograde labeling from uterus and colon, gave important insight into the way same
sensory neuron can innervate both reproductive and gastrointestinal organs. These
dichotomized fibres provide a novel mechanism for sensitization of one internal organ by
another. Our study clearly showed that DRG neurons innervate both colon and uterus in
short-term culture express capsaicin-sensitive TRPV1 receptors and ATP-sensitive
P2X3/α,β-meATP-sensitive P2X2/3 receptors which mediate the response to putative
nociceptive signals. Our findings suggest that sensory information to the sensory neurons
may initiate in different viscera. Current scientific literatures suggests that the importance
of E2 modulation of nociceptive pain processing in functional pain disorders. Within our
hypothesis, E2 modulation depends on pain types, durations, and other anti-nociceptive
signaling pathways. Thus, E2 modulates short-cultured sensory DRG neurons to
capsaicin and ATP/α,β-meATP, suggesting that visceral nociceptive neurons are
modulated by E2. This modulation can manifest in clinical findings, animal sex-
differences associated with visceral hyperalgesia, and a potential target for mediating
nociception. In addition, E2 modulation of viscero-visceral cross sensitization appear in
DRG neurons (Chaban et al., 2003; Chaban and Micevich, 2005; Sarajari and Oblinger,
97

2010) and P2X3/P2X2/3 and TRPV1 receptors in retrogradely labeled visceral DRG
neurons attenuated ATP/α,β-meATP/capsaicin-induced [Ca
2+
]
і
fluxes, but retrogradely
labeled somatic DRG neurons did not inhibit [Ca
2+
]
і
responses, suggesting that
nociceptive signals modulate peripherally visceral pain sensitization in vitro.
Although large numbers of clinical and animal studies have indicated that there
are sex and estrous cycle differences in pain perception of various pain models. The
relationship remains unclear whether sex and estrous cycle differences in ATP/capsaicin-
induced acute pain also occur in mice. Furthermore, the PGE2 is synthesized and released
in response to damaged tissues, contributing to hyperalgesia, and is also involved in the
acute and chronic inflammatory response related reactions. The E2 is anti-nociceptive
and has been shown to decrease capsaicin-induced [Ca
2+
]
і
responses without PGE2
present. In our previous studies it was shown that PGE2 enhanced the Ca
2+
responses
induced by ATP and capsaicin. This data indicated that the sensitizing actions of PGE2
on retrogradely labeled visceral DRG neurons are mediated through the pain signaling
pathway such as cAMP/PKA. Also, recent studies show that E2 acts through an ERα in
modulating the TRPV1 receptor-mediated [Ca
2+
]
i
response in retrogradely labeled
visceral DRG neurons, since its effect was eliminated in ERαKO mouse and retained in
ERβKO. These results demonstrate an important non-reproductive role of ERα in
modulating capsaicin-induced Ca
2+
signaling at the level of the visceral afferent neuron,
thereby modulating the sensitivity to painful stimuli in the periphery.
The abdominal pain related with irritable bowel syndromes and acute and
chronic/repeated pelvic pain in women are examples of visceral pain tha is associated
with sensitization (Giamberardino, 1999). Patients have reported pain associated with
98

irritable bowel syndrome acuity and frequently seek consult with their physician to
manage the pain related with this condition. Hence, from a public health view, the results
of study will have an important impact and will help to improve our knowledge of
nociceptive functional disorders including irritable bowel syndrome, chronic pelvic pain,
and interstitial cystitis. Moreover, the analysis of data from this project will help
accomplish a deeper understanding of sex-related differences in clinical studies in
various related psychiatric disorders. Only through the understanding of implicated
mechanisms can designs of new and more efficient clinical therapies be rendered.
Women and men clearly respond to many illnesses differently. It has been documented
that differences between sexes and diseases, disorders, and clinical conditions exist. In
the sex-related biomedical sciences, clinical and scientific communities will set clearly
defined boundaries in terms of accomplishments of knowledge related to sex-related
differences in ethical, sociological, financial, and scientific models. Pain is approximately
20% of the reason primary complaint reported in health care visits at present. In addition,
about 33% of patients that report pain symptoms suffer from co-morbid condition such as
depression two to three times higher among women than men. Further approaches in pain
research and therapy should address this important difference realated to visceral
nociception among women. Furthermore, this study has the potential impact both clinical
study and basic science research efforts especially in anesthesiology (pain management),
gastroenterology, obstetrics, and gynecology.

99

4.4 Materials and Methods
4.4.1 Animals
We have used 6~8 week old female wild type (Wt, C57Bl/6J), ERαKO
(B6.129P2-Esr1
tm1Ksk
/J), and ERβKO (B6.129P2-Esr2
tm1Unc
/J) mice (Jackson Laboratory,
Bar Harbor, ME). Upon arrival mice were housed in microisolator caging and maintained
on a 12-h light/dark cycle in a temperature-controlled environment with access to food
and water ad libitum for two weeks. All studies were carried out in accordance with the
guidelines of the Institutional Animal Care and Use Committee (IACUC) of Charles R.
Drew University and the NIH Guide for the Care and Use of Laboratory Animals. In
some experiments we used animals from our breeding colony.
4.4.2 Primary culture of DRG neurons
The isolation procedure and primary culture of mouse lumbosacral DRG has been
published in detail. DRG tissues were obtained from C57Bl/6J (30 g), ERαKO and
ERβKO (Jackson Laboratory; 20 g) transgenic types. Briefly, lumbosacral adult DRGs
(level L1-S1) were collected under sterile technique and placed in ice-cold medium
Dulbecco’s Modified Eagle’s Medium (DMEM; Sigma-Aldrich St. Louis, MO).
Adhering fat and connective tissue were removed and each DRG was minced with
scissors and placed immediately in a medium consisting of 5 ml of DMEM containing
0.5 mg/ml of trypsin (Sigma, type III), 1 mg/ml of collagenase (Sigma, type IA) and 0.1
mg/ml of DNase (Sigma, type III) and kept at 37°C for 30 minutes with agitation. After
dissociation of the cell ganglia, soybean trypsin inhibitor (Sigma, type III) was used to
terminate cell dissociation. Cell suspensions were centrifuged for one minute at 1000 rpm
and the cell pellet were resuspended in DMEM supplemented with 5% fetal bovine
100

serum, 2 mM glutamine-penicillin-streptomycin mixture, 1 μg/ml DNAase and 5 ng/ml
NGF (Sigma). Cells were placed on Matrigel
®
(Invitrogen, Carlsbad, CA)- coated 15-mm
coverslips (Collaborative Research Co., Bedford, PA) and kept at 37°C in 5% CO
2

incubator for 24h, given fresh media and maintained in primary culture until used for
experimental procedures.
4.4.3 Retrograde labeling
Retrograde labeling was used to confirm DRG neurons that had innervated both
visceral organs: uterus and colon. All surgical protocols were performed under sterile
environments within the designated animal surgery room. For colon afferents, the
descending colon of female mice (Wild type, ERαKO, and ERβKO) was exposed under
anesthesia with isoflurane. Fluorogold (FG, 1.5% in .9% saline) was injected into the
intestinal muscle wall into five to six different sites (1 - 2 µl / site, total of 10 µl / mice)
by using a Hamilton syringe (Hamilton Co., Reno NV) with a 31-gauge needle. In
another experiments, we used uterus-specific DRG neurons in which
tetramethylrhodamine (TMR, 1.0% in .9% saline) tracer was injected into the uterus
(total of 20 µl / mice). Injection sites were carefully swabbed and the colon and uterus
was extensively rinsed with 0.9% sodium chloride solution. The abdomen was sutured
and the animals monitored for signs of pain or discomfort during survival period. Both
dyes are injected into respective place into colon and uterus of the same animals.
Immediately after surgery, mouse were injected with sterile saline to prevent dehydration
and were given analgesics for pain relief and antibiotics. For somatic labeling, 50 µl
cholera toxin conjugated to Alexa Fluor 647 (2.5% solution in PBS; Molecular Probes,
Eugene, OR) was injected subcutaneously into five sites at the mouse hind paw. All
101

mouse were allowed to survive 7 - 10 days to allow for maximal transport of retrograde
neuron tracer and housed in groups of two under 12/12 hours light cycle with food and
water available ad libitum.

Figure 4.9: Retrograde labeling experiments. Schema of marking colon/uterine-innervating neurons of the DRG via
retrograde labeling.
102

4.4.4 Fluorescence imaging analysis
Ca
2+
fluorescence imaging was carried out as previously described (Chaban,
2011). DRG neurons were loaded with fluorescent dye 5 mM Fura-2 AM (Invitrogen,
Carlsbad, CA) for 45 min at 37°C in HBSS supplemented with 20 mM HEPES, pH 7.4.
The coverslips were mounted in a RC-26 recording chamber P-4 (Warner Instruments,
Hamden, CT) and placed on a stage of Olympus IX51 inverted microscope (Olympus
America, Center Valley, PA). Observations were made at room temperature (20-23°C)
with 20X UApo/340 objective. Neurons were bathed and perfused with HBSS buffer
using with using gravity at a rate of 1-2 ml/min. Fluorescence intensity at 505 nm with
excitation at 334 nm and 380 nm were captured as digital images (sampling rates of 0.1-2
s). Regions of interest were identified within the soma from which quantitative
measurements were made by re-analysis of stored image sequences using Slidebook
®

Digital Microscopy software. [Ca
2+
]
i
was determined by ratiometric method of Fura-2
fluorescence from calibration of series of buffered Ca
2+
standards.

E2 was applied acutely
for five minutes onto the experimental chamber. Repeated application of drugs was
achieved by superfusion in a rapid mixing chamber into individual neurons for specific
intervals (100-500 ms). We calculated actual [Ca
2+
]
i
in areas of interest in each neurons
with the formula:
[Ca
2+
]
i
=K
d
x (R-R
min
)/(R
max
-R) x β
Where K
d
is the indicator's dissociation constant of the fluoroprobe; R is ratio of
fluorescence intensity at two different wavelengths (340/380 nm for fura-2); R
max
and
R
min
are the ratio at fura-2 with an saturated Ca
2+
and free Ca
2+
. β is the ratio of the
denominators of the minimum and maximum conditions.
103

4.4.5 Statistical analysis
The amplitude of [Ca
2+
]
i
response represents the difference between baseline
concentration and the transient peak response to drug stimulation. Significant differences
in response to chemical stimulation were obtained by comparing [Ca
2+
]
i
increases during
the first stimulation with the second. All of the data are expressed as the mean ± SEM.
Statistical analysis was performed using Statistical Package for the Social Sciences 18.0
(SPSS, Chicago, IL, USA). To assess the significance among different groups, data were
analyzed with one-way ANOVA followed by Tukey's multiple comparison test. A p
<0.05 was considered statistically significant.

104

Chapter 5. ESTROGEN MODULATION BETWEEN
µ-OPIOID RECEPTOR (MOR) AND ERα/ERβ
IN MOUSE SENSORY NEURONS

Portions of this Chapter are adapted from:
Taehoon Cho and Victor Chaban, Journal of Neuroscience (Submitted 2012)

The endogenous opioid systems play an important role for the regulation of sexual
receptivity which is tightly stimulated by the sequential release of estrogen in female
mice. E2 induces internalization of rapid mu-opioid receptor (MOR) which is mediated
by E2 release of endogenous opioid peptides in DRG neurons and internalization has
been used as a neurochemical indicator of E2 activity in the central nervous system. We
have investigated the coupling between opioid receptors and calcium channel in mouse
DRG neurons using calcium imaging. The MOR selective agonist DAMGO (1 µM)
attenuated the 25 mM KCl-induced [Ca
2+
]
і
transients (382.3 ± 67.4, n=36 cells/5 mice).
The kappa-selective opioid receptor agonist U-69,593 (1 µM) also regulated inhibition
but it was much less effective (367.6 ± 38.4, n=29 cells/5 mice). However, the delta
selective opioid receptor DPDPE (1 µM) showed no significant effect in DRG neurons
(379.1 ± 6, n=31 cells/5 mice). To confirm E2 attenuation of opioid effects, we used the
changes of affinity number of opioid receptors (B
max
) and ligand affinities (K
d
) from
105

cultured DRG neurons. E2 have down-regulated of the number of MOR binding sites,
diminished the MOR affinities, or declined the coupling of the MOR to its G-protein. E2
activated downstream signaling mechanisms that interact with opioid signaling. The level
of [
35
S]GTPγS binding was higher in oil and EB-treated mice compared with binding to
the membranes. E2 significantly increased DAMGO-induced [
35
S]GTPγS binding in the
membrane fraction but not in the microsomal fraction. Moreover, E2 shows to be
working through estrogen receptor alpha (ERα) to stimulated MOR internalization, since
E2 did not induce MOR internalization in estrogen receptor alpha knock-out (ERαKO)
mice. However, estrogen modulated MOP internalization in wild type and estrogen
receptor beta knock-out (ERβKO) mice. E2-induced MOR internalization indicate that
this situation requires the activation of a non-genomic estrogen receptor mechanism
involving ERα and may offer a new clinical therapeutic strategy for pain management.
5.1 Introduction
A potent opioid analgesia mediated peripheral opioid receptors exists after
inflammation and tissue injury (Lewanowitsch et al., 2006; Shinoda et al., 2007). Opioid
receptors are expressed on C-fibers of primary sensory neurons associated in pain
modulation and transmission in the periphery, the brain, and the spinal cord. In addition,
opioid receptors inhibit the activation and sensitization of these C-fibers and prevent the
release of pain transmitters. Opioid receptors in dorsal root ganglion (DRG) neurons are
expressed on body of neuron cells are carried to the peripheral sensory nerve endings at
the inflammatory site (Coggeshall et al., 1997; Jason and Stein, 2003; Truong et al.,
2003).
106

The properties of opioids in physiology are related with mu (μ)-, kappa (κ)-, and
delta (δ)-opioid receptor subtypes by separated gene and protein expression, tissue
patterns, functional characteristics, and side effects (Riviѐre and Junien, 2000). Opioid
produce anti-nociceptive potency during tissue injury or inflammation and that this
produced potency is demonstrated for μ-opioid receptor (MOR) agonists (Stanfa et al.,
1992; Mecs et al., 2009). The μ-opioid receptor used to the primary site of responses for
most common opioids such as morphine, heroin, methadone, and fentanyl. The
endogenous opioid peptide β-endorphin during inflammation in central and peripheral
nervous system are commonly used to enhance sensitivity to opiate compounds through
activation of mu opioid receptor which is seven membranes of the G protein-coupled
receptor superfamily (Zubieta et al., 2001; Stein et al., 2009). The opioid receptors which
expressed on peripheral afferent axons in inflammatory pain may produce the opioid
mediated anti-nociception in neuropathic pain. Activation of peripheral μ-opioid
receptors bring to attenuation of neuropathic pain with minimal side effects of central
nervous systems (Kabli and Cahill, 2007; Guan et al., 2008; Obara et al., 2009),
indicating that peripheral acting mu opioid receptors may address an alternative therapy
plan for the neuropathy pain. Nevertheless, the changes of μ-opioid receptor expression
with peripheral nerve injury in the peripheral nervous systems are too difficult to
understand these phenomena.
In the present study, depending on the absence or presence of opioid receptor
agonists, E2 can modulate anti-nociceptive or pro-nociceptive opioid (Chaban, 2008;
Lima et al., 2010). A mechanism for E2 pro-nociception decrease nociceptin mRNA
expression (Sinchak and Micevych, 2003) and attenuate the opioid actions in intracellular
107

signaling, suggesting that sex steroids present to mediate different analgesic responses in
women and men and actually sex-related differences by activation of μ-opioid receptor
emerge greater analgesia in men than women (Miaskowski 1997; Zubieta et al., 2002). In
culture DRG neurons, E2 reverse μ-opioid receptor attenuation of KCl-induced calcium
fluxes, suggesting that E2 has direct connection on opioid-mediated signal transduction.
The effect of μ-opioid receptor agonist is inhibited by E2 which modulates suppression of
DAMGO in KCl-induced [Ca
2+
]
i
transients. There are two main properties of opioid
associated with E2. E2 can trigger intracellular signaling cascades and may be acting on
μ-opioid receptor through internalization or allosteric interaction (Micevych et al., 2002;
Rothman et al, 2007; Schröder et al., 2008). The μ-opioid receptor is internalized
following activation of opioid agonist. This internalization is the attenuation of number of
opioid receptors in plasma membrane by the physical manifestation of desensitization.
Therefore, E2 consequently attenuate the number of opioid receptors to the
responsiveness to μ-opioid receptor ligand. E2 rapidly significantly decrease the effects
of μ-opioid receptor agonists to hyperpolarize guinea pig hypothalamic neurons (Kelly
and Wagner, 1999). The number of literature showed mu opioid receptor has significantly
been responded by E2, but not by κ-opioid receptor (Su et al., 1998; Sandner-Kiesling
and Eisenach, 2002). While colonic afferents may more sensitive to κ-opioid receptor, E2
may preferentially act on μ-opioid receptor. This study compared E2 actions on kappa
opioid receptors and mu opioid receptor and regulation of intracellular signaling
pathways. The effects of E2 on functional activity of μ-opioid receptor and κ-opioid
receptor are characterized by visualization of calcium fluxes. DRG neurons allowed to
108

test in vitro if E2 respond directly to μ-opioid receptor expressing neurons cells, or if
effects of E2 are mediated through activation of endogenous opioid systems.
5.2 Results
5.2.1 Expression of μ-, κ-, and δ-opoid receptors in DRG neurons
In our previous studies, we have shown both ERα and ERβ in Wt mouse DRG
neurons using mRNA RT-PCR (Chaban & Micevych, 2005). In this study expression of
μ-, κ-, and δ-opoid receptors were examined by western blot analysis of whole cell
lysates from Wt, ERαKO, and ERβKO DRG tissues using μ-, κ-, and δ-opoid receptors
specific primary antiserum. An intense band representing a 45 kDa (μ-opioid receptor),
46 kDa (κ-opioid receptor), and 48 kDa (δ-opioid receptor) protein were seen in DRG
lysates from Wt animals. In our experiments we found that there was a dramatic decrease
in intensity of this band using lysates from the both knock out DRG tissues (> 2 fold
decrease of control) in μ-opioid receptor. A representative result of μ-, κ-, and δ-opioid
receptors are shown and the standardization ratio statistics of three tests is shown in
Figure 5.1. The only average intensities of the bands of μ-opioid receptor in both knock-
out mice decreased significantly, but both κ- and δ-opioid receptors didn't show
statistically significant differences. When the density in the control group was
standardized to 1.0, the average densities were 0.539 ± 0.10 of ERαKO and 0.465 ± 0.05
of ERβKO in μ-opioid receptors suggesting that μ-opioid protein decreased in DRG from
knock-out mice p<0.05 (n=5).
109

Figure 5. 1: Western blot analysis of DRG lysates shows reduced expression of μ-, κ-, and δ-opioid receptors in both
knock-out mice. Equal amounts of lysates (40 μg) generated from ERαKO and ERβKO DRG neurons, as well as from
wild type mice, were electrophoresed under denaturing condition (n=5 per group). Representative results with μ-, κ-,
and δ-opioid receptors are shown in (a). Demonstrate quantification of signals from (b) μ-, (c) κ-, and (d) δ-opioid
receptors by statistically significant difference between the intensity of the bands from both knock-out DRG neurons in
opioid receptors when compared with wild type animals.
110

5.2.2 Pharmacological profile of DAMGO-mediated modulation on KCl-induced
[Ca
2+
]
i
in DRG neurons
Figure 5.2 demonstrates typical [Ca
2+
]
i
transients from cultured DRG neurons
evoked by exposure to 25 mM KCl. KCl induced a spike increase in [Ca
2+
]
i
and this
spike could be evoke several times in the same DRG neuron if it obtain separated 10
minutes washing step. To determine whether DAMGO attenuated [Ca
2+
]
i
fluxes by
blocking calcium channels, our data demonstrated K
+
-induced [Ca
2+
]
i
fluxes in DRG
neurons in wild type mice. Brief 3 second application of KCl (25 mM) by fast
superfusion produced equal [Ca
2+
]
i
fluxes in 69% of tested neurons. After 10 minutes
washout with HBSS, additional applications with KCl (25 mM) induced a subsequent
[Ca
2+
]
i
fluxes (Fig. 5.2 (a)). The MOR selective agonist DAMGO (1 µM) by itself had no
effect on basal [Ca
2+
]
i
fluxes, but DAMGO suppressed the K
+
-induced [Ca
2+
]
i
fluxes in a
dose-dependent manner (Fig. 5.2 (b) and (c)). The DAMGO effects were reversible. The
DAMGO mediated inhibition in DRG cells was completely reversed K
+
-induced [Ca
2+
]
i
fluxes by addition of the highly selective MOR antagonist CTAP (1 µM) (Fig. 5.2 (d) &
Fig. 5.4, 382.3 ± 67.4 vs. 214.6 ± 35.9 nM, n=36 cells/5 mice , p < 0.05).
111

Figure 5. 2: DAMGO blocks KCl-induced [Ca
2+
]
і
fluxes in wild type mice. (a) Typical indication of equal [Ca
2+
]
і

responses to repeated KCl (25 mM) stimulation (indicated by arrow) with 10 min interval under control condition. (b)
Effect of DAMGO was reversible. Second KCl-induced [Ca
2+
]
і
fluxes rapidly attenuated by DAMGO (1 μM) in dorsal
root ganglion cells. (c) Dose dependence of DAMGO effect in a single DRG neuron. DAMGO (1 nM, 10 nM, 100 nM,
and 1 μM) added 5 min prior to KCl gradually decreased KCl-induced [Ca
2+
]
і
fluxes. (d) Effect of μ-opioid receptor
antagonist CTAP (1 µM) blocked the DAMGO attenuation of KCl-induced [Ca
2+
]
і
fluxes.

112

5.2.3 Modulation of evoked calcium signals by mu-, kappa-, and delta-opioid
receptors
Our data suggest that KCl-induced [Ca
2+
]
і
transients in DRG neurons in mice, a
result similar to that observed in ATP-mediated calcium changes in mouse DRG neurons
(Cho & Chaban, 2011). Increments of calcium transients evoked by depolarization which
was used 3 second exposure to 25 mM KCl-induced [Ca
2+
]
і
were measured using fura-2
based calcium imaging system. Figure 5.2 show that the μ-selective opioid receptor
agonist DAMGO (1 µM) attenuated the 25 mM KCl-induced [Ca
2+
]
і
transients (382.3 ±
67.4, n=36 cells/5 mice).

Figure 5. 3: Inhibition of depolarization-induced [Ca
2+
]
i
fluxes by opioids. (a) The U-69,593 (1 µM), a κ-opioid
receptor agonist, attenuated much less K+-induced [Ca
2+
]
i
fluxes compared with DAMGO. (b) DPDPE (1 µM), a δ-
opioid receptor agonist, didn't change K+-induced [Ca
2+
]
i
fluxes.

113

The kappa-selective opioid receptor agonist U-69,593 (1 µM) also regulated
inhibition but it was much less effective (Fig. 5.3 (a) & Fig. 5.4, 367.6 ± 38.4, n=29
cells/5 mice). However, the delta selective opioid receptor DPDPE (1 µM) showed no
significant effect in DRG neurons (Fig. 5.3 (b) & Fig. 5.4, 379.1 ± 6, n=31 cells/5 mice).
These data suggest that only mu-opioid receptor agonists could inhibit calcium increasing
in DRG neurons and a greater proportion of DRG neurons were found to be mu-opioid
receptor sensitive.

Figure 5 .4: Summary of KCl-induced [Ca
2+
]
i
influxes in control, in the presence of DAMGO, U- 69,593, DPDPE, and
DAMGO with CTAP. DAMGO significantly decreased [Ca
2+
]
і
response to KCl whereas μ-opioid receptor antagonist
CTAP inhibited DAMGO effect. Values are expressed as mean ± SEM. * indicate statistically significant difference
from control, P<0.05.

114

5.2.4 Effect of E2 on DAMGO-mediated KCl-induced [Ca
2+
]
i
response in mouse
DRG neurons
E2 (100 nM) by itself had no significant different effects on basal [Ca
2+
]
і
, but
estrogen attenuated the KCl-induced [Ca
2+
]
і
fluxes. E2 showed reversible effect on
calcium changes. After the first application with KCl, 5-min incubation with E2 blocked
KCl-induced [Ca
2+
]
і
fluxes (387.5 ± 39.1 vs. 184.8 ± 28.6 nM, n=33 cells/5 mice, p <
0.05). To confirm the desensitization of E2, we administered application of repeated KCl
indicating that E2 does not desensitize upon of application of repeated KCl (Fig. 5.5
(a)&(d)). The ER antagonist ICI 182,780 (1 µM) inhibited the E2 effect on attenuated
KCl-induced [Ca
2+
]
і
fluxes (Fig. 5.5 (b)&(d)) and ICI 182,780 demonstrates no effect by
itself (data not shown). In addition, to verify the E2 effect on DAMGO-induced [Ca
2+
]
і
fluxes, we observed MOR on the membrane in cultured DRG neurons. In vitro DRG
neurons retain functional MOR based on the observation that E2 attenuated MOR
inhibition of DAMGO-induced [Ca
2+
]
і
fluxes (Fig. 5.5 (c)&(d)). In cultured DRG
neurons, E2 reverses µ-opioid receptor (MOR) attenuation of KCl-induced [Ca
2+
]
і
fluxes
suggesting that E2 has direct action on opioid-mediated signal transduction.
115

Figure 5. 5: E2 inhibits KCl-induced [Ca
2+
]
i
responses in wild type mice. (a) The second KCl-induced [Ca
2+
]
i
response
rapidly attenuated by E2 on DRG neurons. (b) Effect of ICI 182,780, a estrogen receptor antagonist, on E2 attenuation
of KCl-induced [Ca
2+
]
i
fluxes. ICI 182,780 (1 µM) inhibited the E2 attenuation of KCl-induced [Ca
2+
]
і
fluxes. (c)
Effect of μ-opioid receptor DAMGO (1 µM) inhibited the E2 attenuation of KCl-induced [Ca
2+
]
і
fluxes. (d) Summary
data represented bar graph on KCl-induced [Ca
2+
]
і
fluxes in presence of E2, E2 with ICI 182,780, and E2 with
DAMGO. * Statistically significant difference from control, P<0.05.

116

5.2.5 E2 inhibits MOR effects by attenuating PGE2-induced [cAMP]
i
activation and
decrease the number of MOR binding sites in mouse DRG neurons
One of the best model for several responses of primary afferent neurons to pro-
inflammatory stimuli is prostaglandin E2 (PGE2). The FICRhR probe which is the
recombinant fluorescein and rhodamine-labeled cAMP-dependent protein kinase allowed
to image of [cAMP]
i
on PGE2-stimulated DRG neurons. In this study DRG neurons were
grown on and coverslips and then micro-injection with FICRhR (2 μM). PGE2 rapidly
increased [cAMP]
i
levels, which did not return to baseline for the duration of the
experiment (data not shown). The time course of the PGE2-stimulated [cAMP]
i
was
reduced by the μ-opioid receptor agonist
3
H-D-Ala
2
, N-Me-Phe
4
, glycinol
5
-enkephalin
(DAMGO, 10 nM). E2 (100 nM) alone did not increase [cAMP]
i
levels but prevented the
ability of MOR agonist (DAMGO) on the PGE2-induced [cAMP]
i
level (Fig.5.6 (b)).
These data are suggest that E2 block potentiality of MOR agonist suppression of
nociceptive responses to hypothalamic neurons. To confirm E2 attenuation of opioid
effects, we used the changes of affinity number of opioid receptors (B
max
) and ligand
affinities (K
d
) from cultured DRG neurons. E2 could down-regulate the number of MOR
binding sites, diminish the MOR affinities, or decline the coupling of the MOR to its G-
protein. First, we demonstrated that
3
H-DAMGO binding to DRG membranes from
control mice and pretreated with estradiol benzoate (EB) mice. DRG membranes were
homogenized in Tris-HCl buffer (50 mM, pH 7.5) from mice who are treated with either
estradiol benzoate (EB) or not. This experiment is processing for radio-receptor binding
by incubating membrane homogenates with 0.1 nM ~ 25.0 nM of presence of
3
H-
DAMGO in the presence or absence of DAMGO (1 μM). The number of
3
H-DAMGO
117

binding sites (B
max
) was significant reduced in DRG membranes EB-treated mice
compared with control mice, but MOR binding affinity (K
d
) was not affected (Fig. 5.6
(a)). Together our data suggested that MOR internalization is rely on the emission of
endogenous opioid and opioid peptides. In addition, The attenuation of MOR binding
sites in DRG membrane suggested that E2 attenuates reactivity sites to MOR-induced
responses in the DRG.
118

Figure 5. 6: E2 decreases μ-opioid receptor (MOR)-mediated responses in the mouse DRG. (a) Effect of E2 on number
of MOR binding sites (B
max
) and MOR binding affinity (K
d
) of mouse DRG membranes. (b) E2 decreases MOR
attenuation of PGE2-induced [cAMP]
i
in mouse DRG neurons. DAMGO blocked PGE2-induced [cAMP]
i
. However,
E2 pretreatment for 5 minutes attenuated the MOR-induced attenuation of PGE2-induced [cAMP]
i
accumulation
(Repeated from Chaban, 2008b).

119

5.2.6 Internalization of μ-opioid receptor (MOR) coupling to G-protein in wild type,
ERαKO, and ERβKO mice
E2 modulates signal transduction mechanisms which are mediated by rapid non-
genomic pathways through G proteins either directly or indirectly, where estrogen
receptors are associated with plasma membrane or distributed in cytoplasm. E2 has been
reported to act on μ-opioid receptors (MOR) specifically but not on κ-opioid receptors
(KOR) (Sandner-Kiesling & Eisenach, 2002). To identify receptor which is activated G
protein by μ-opioid receptor, we used the [
35
S]GTPγS-binding assay. This assay is based
on the fact that calculations of receptor coupling are relative and depend on a number of
technical factors including affinity of the G protein from GDP to GTP and the kinetics of
[
35
S]GTPγS-binding. Changes in [
35
S]GTPγS-binding were analyzed by calculating a
catalytic amplication factor (B
max
of [
35
S]GTPγS-binding/B
max
of receptors). The mice
were treated with estradiol benzoate (50 µg) or oil systematically for 4hrs and then
processed for [
35
S]GTPγS-binding using 1 µM DAMGO as a MOR agonist. E2 activated
downstream signaling mechanisms that interact with opioid signaling. The level of
[
35
S]GTPγS binding was higher in oil and EB-treated mice compared with binding to the
membranes. E2 significantly increased DAMGO-induced [
35
S]GTPγS binding in the
membrane fraction but not in the microsomal fraction (Fig. 5.7 (a)). In addition, E2
changes the functional coupling of MOR in the membrane fraction, but not in the
microsomal fraction, suggesting internalization. These data suggest that E2 may increase
the potency of MOR such that when E2 is raised, active endogenous peptides of MOR are
released and bind more efficiently to the blockade of second messenger cascade within
120

MOR neurons. In the absence of E2, activation of MOR ligand and its binding to second
messenger is reduced.

Figure 5. 7: Effects of estrogen-benzoate (EB) on DAMGO, a selective MOR agonist, [
35
S]GTPγS-binding in
membrane and microsomal DRG fractions from wild type, ERαKO, and ERβKO mice. EB increased G-protein
coupling in membrane fractions from (a) wild type and (c) ERβKO, but not microsomal fractions. However, (b)
ERαKO didn't change between membrane and microsomal fractions. Values are expressed as mean ± SEM. * indicate
statistically significant difference from control, P<0.05.
121

In this study E2 did not induce MOR internalization in estrogen receptor alpha
knock-out (ERαKO) mice (Fig. 5.7 (b)) but estrogen modulated MOR internalization in
wild type and estrogen receptor beta knock-out (ERβKO) mice (Fig. 5.7 (c)). These data
indicate that ERα is required for E2-mediated MOR internalization and can induce rapid
activation of E2.
5.3 Discussion
Depending on the presence or absence of opioid receptor agonists, E2 can be
either anti-nociceptive or pro-nociceptive. A mechanism for E2 pro-nociception is
attenuation of opioid actions on intracellular signaling. The recent research have been
published the receptor and intracellular signaling that mediates a novel activity of E2 to
fast change synaptic transmission. E2 pretend to show through a specific receptor
because E2 depends on physiologically relevant concentration and is not same as the
biologically interactive isomer 17α-estradiol which is not against μ-opioid receptor
(MOR). E2 has been reported to react on MOR significantly, but not on KOR (Sandner-
Kiesling & Eisenach, 2002), since E2 may change opioid receptor activity by regulating
Ca
2+
, or K
+
channels, a lack of effect in the present assay can support a separate E2 action
that did not involve the functional coupling of the opioid receptors to their G-proteins.
However, colonic afferents may not respond to MOR (Su et. al., 1998). The transmission
of nociceptive signaling pathways is modulated by activation of broad of G protein-
coupled receptors such as opioid receptors and opioid receptor-like receptors. Opioid
receptors agonists block pain neurotransmission by inhibition of presynaptic calcium
channels. E2 can stimulate cellular action by opening ion channels and changing second
messenger signaling by triggering G-proteins, the transduction signaling pathways
122

associated with activation of membrane receptors. Many of E2 effects have been
described to membrane-related receptors. These data suggest that E2 is responding to
modulate L-type voltage gated calcium channel (VGCC). Many clinical studies have
reported the a variety of μ-opioid receptor (MOR) activities associated with nociception
for acute and chronic pain conditions. Estrogen receptors expressed through central nerve
system and peripheral nerve system including specific sites such as dorsal horn neurons
of the spinal cord and DRG neurons that mediate nociception. DRG neurons express both
ERα and ERβ in vitro and in vivo. These data suggest E2 may modulate sensory signals at
the primary sensory neuron level. One outstanding way of E2 modulation is related with
interaction of opioid systems. E2 can modulate anti-nociceptive (enkephalins, β-
endorphin) alteration of endogenous opioid receptors by changing gene transcriptions.
In our study the technique of fura-2 based calcium imaging provide to measure a
sensitive MOR function. Depolarization of cultured DRG neurons with 25 mM K
+
results
in calcium influx. In cultured DRG cells, E2 reverses MOR attenuation of K
+
-induced
[Ca
2+
]
і
, fluxes suggesting that E2 has direct interaction with opioid-mediated signal
transduction. Application of a MOR agonist DAMGO onto opioid sensitive cultured
DRG neurons has been presented to result in inhibition of KCl-induced [Ca
2+
]
і
, fluxes by
opioid receptor-mediated suppression of calcium channels. Our previous studies found
MOR staining on the membrane in cultured DRG neurons. DRG neurons in vitro retain
functional MOR based on the previous observation that E2 attenuated MOR suppression
of DAMGO-induced Ca
2+
increases. However both a kappa-opioid receptor agonist U-
69,593 and a delta-opioid receptor agonist DPDPE did not inhibit KCl-induced [Ca
2+
]
і
,
fluxes. Therefore, we confirmed that E2 has been shown to act on MOR significantly.
123

MOR is a G-protein-coupled receptor, which is internalized following agonist activation.
This internalization is the physical manifestation of desensitization whereby the number
of receptors in the plasma membrane is reduced. To determine whether opioid binding is
affected by E2 or not, we tested the effects of E2 on MOR physiology, receptor numbers
(B
max
) and ligand affinities (K
d
), which are measured in membranes and microsomal
fractions (endosomes) from cultured DRG neurons. E2 treatments reduced the number of
MOR binding sites in vivo (Eckersell et al., 1998; Sinchak & Micevych, 2001) and it is
likely that the observed attenuation of MOR binding sites is due to release of endogenous
opioids in spinal cord. Therefore, E2 treatment of DRG neurons did not reduce number of
opioid receptors in cultured DRG membranes because there are no endogenous peptides
to internalize opioid receptors. This study suggest that E2 may not act on MOR directly
since E2 treatment of plasma membrane preparations did not alter the B
max
or the K
d

(Micevych et al., 2002). Therefore, it is likely that E2 may activate downstream signaling
mechanisms that interact with opioid signaling in DRG neurons. E2 decreased the
number of MOR in membrane fraction but increased in the microsomal fraction since this
data suggest MOR internalization.
E2 shows to be working through estrogen receptor alpha (ERα) to stimulated
MOR internalization, since E2 did not induce MOR internalization in estrogen receptor
alpha knock-out (ERαKO) mice. However, estrogen modulated MOP internalization in
wild type and estrogen receptor beta knock-out (ERβKO) mice. In terms of E2-mediated
MOR internalization, an ERα associated with the membrane that initiates downstream of
intracellular mechanisms. Together these studies indicate that ERα is required for E2-
mediated MOR internalization suggesting that this form of ER is play an important role
124

to mediate activation of MOR circuits. E2-induced MOR internalization suggests that this
situation requires the activation of a non-genomic estrogen receptor mechanism involving
ERα and may offer a new clinical therapeutic strategy for pain management.
5.4 Materials and Methods
5.4.1 Animals
We have used 6~8 week old female wild type (Wt, C57Bl/6J), ERαKO
(B6.129P2-Esr1
tm1Ksk
/J), and ERβKO (B6.129P2-Esr2
tm1Unc
/J) mice (Jackson Laboratory,
Bar Harbor, ME). Upon arrival mice were housed in microisolator caging and maintained
on a 12-h light/dark cycle in a temperature-controlled environment with access to food
and water ad libitum for two weeks. All studies were carried out in accordance with the
guidelines of the Institutional Animal Care and Use Committee (IACUC) of Charles R.
Drew University and the NIH Guide for the Care and Use of Laboratory Animals. In
some experiments we used animals from our breeding colony.
5.4.2 Primary culture of DRG neurons
The isolation procedure and primary culture of mouse lumbosacral DRG has been
published in detail (Chaban et al., 2003). DRG tissues were obtained from C57Bl/6J (30
g), ERαKO and ERβKO (Jackson Laboratory; 20 g) transgenic types. Briefly,
lumbosacral adult DRGs (level L1-S1) were collected under sterile technique and placed
in ice-cold medium Dulbecco’s Modified Eagle’s Medium (DMEM; Sigma-Aldrich St.
Louis, MO). Adhering fat and connective tissue were removed and each DRG was
minced with scissors and placed immediately in a medium consisting of 5 ml of DMEM
containing 0.5 mg/ml of trypsin (Sigma, type III), 1 mg/ml of collagenase (Sigma, type
IA) and 0.1 mg/ml of DNAase (Sigma, type III) and kept at 37°C for 30 minutes with
125

agitation. After dissociation of the cell ganglia, soybean trypsin inhibitor (Sigma, type
III) was used to terminate cell dissociation. Cell suspensions were centrifuged for one
minute at 1000 rpm and the cell pellet were resuspended in DMEM supplemented with
5% fetal bovine serum, 2 mM glutamine-penicillin-streptomycin mixture, 1 μg/ml
DNAase and 5 ng/ml NGF (Sigma). Cells were placed on Matrigel
®
(Invitrogen,
Carlsbad, CA)- coated 15-mm coverslips (Collaborative Research Co., Bedford, PA) and
kept at 37° C in 5% CO
2
incubator for 24h, given fresh media and maintained in primary
culture until used for experimental procedures.
5.4.3 Western Blot Analysis
The expressions of P2X3 receptors in L1-S1 DRGs were studied by using
Western blot analyses. Tissues from Wt (C57Bl/6J), ERαKO, and ERβKO mice were
quick frozen in tubes on dry ice during collection. L1-S1 DRG were combined,
homogenized by mechanical disruption on ice-cold RIPA buffer plus protease inhibitors
and incubated on ice for 30 minutes. Homogenates were then spun at 5000g for 15
minutes and supernatants collected. Total protein was determined on the supernatants
using the BCA microtiter method (Pierce, Rockford, IL, USA). Samples containing equal
amounts of protein (40µg) were electrophoresed under denaturing conditions using
Novex Mini-cell system (San Diego, CA, USA) and reagents (NuPage 4–12% Bis-Tris
gel and MOPS running buffer). After electrophoretic transfer onto nitrocellulose
membrane using the same system, the membrane was blocked with 5% non-fat dry milk
(NFDM) in 25 mM TRIS buffered saline, pH 7.2, plus 0.1% Tween 20 (TBST) for 1 hour
at room temperature, followed by incubation with polyclonal rabbit antibody against mu-
opioid receptor (1:1000, Neuromics, Edina, MN) for overnight at 4
o
C. The membrane
126

was then washed in TBST plus NFDM, and proteins were visualized using a horseradish
peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, CA). Following a
final wash in TBST without NFDM, the membrane was incubated with ECL+
(Amersham, Arlington Heights, Ill., USA) substrate for HRP. Membranes were probed
with primary antibody and corresponding secondary antibodies, signals were scanned and
quantified by Image J version 1.28U and NIH Image 1.60 scan software. Following
enhanced chemiluminescence detection of proteins, the membranes were stripped with
stripping buffer (Pierce, Rockford, IL, USA) and re-hybridized with β-actin antibody as a
loading control. At least three independent cell preparations were used.
5.4.4 Fluorescence imaging analysis
Ca
2+
fluorescence imaging was carried out as previously described (Chaban et al.,
2011). DRG neurons were loaded with fluorescent dye 5 mM Fura-2 AM (Invitrogen,
Carlsbad, CA) for 45 min at 37°C in HBSS supplemented with 20 mM HEPES, pH 7.4.
The coverslips were mounted in a RC-26 recording chamber P-4 (Warner Instruments,
Hamden, CT) and placed on a stage of Olympus IX51 inverted microscope (Olympus
America, Center Valley, PA). Observations were made at room temperature (20-23°C)
with 20X UApo/340 objective. Neurons were bathed and perfused with HBSS buffer
using with using gravity at a rate of 1-2 ml/min. Fluorescence intensity at 505 nm with
excitation at 334 nm and 380 nm were captured as digital images (sampling rates of 0.1-2
s). Regions of interest were identified within the soma from which quantitative
measurements were made by re-analysis of stored image sequences using Slidebook
®

Digital Microscopy software. [Ca
2+
]
i
was determined by ratiometric method of Fura-2
fluorescence from calibration of series of buffered Ca
2+
standards.

E2 was applied acutely
127

for five minutes onto the experimental chamber. Application of drugs was achieved by
superfusion in a rapid mixing chamber and Perfusion Fast-Step system SF-77B (Warner
Instruments) to add drugs in 100-500 ms interval.
We calculated actual [Ca
2+
]
i
in areas of interest in each neurons with the formula:
[Ca
2+
]
i
=K
d
x (R-R
min
)/(R
max
-R) x β
Where K
d
is the indicator's dissociation constant of the fluoroprobe; R is ratio of
fluorescence intensity at two different wavelengths (340/380 nm for fura-2); R
max
and
R
min
are the ratio at fura-2 with an saturated Ca
2+
and free Ca
2+
. β is the ratio of the
denominators of the minimum and maximum conditions.
5.4.5 Receptor binding Assay
Cultured DRG neurons from wild type, ERαKO, and ERβKO mice were grown in
standard (Falcon
®
, Franklin Lakes, NJ) flasks for receptor binding experiments. DRG
cultures were treated with a water soluble E2 (Sigma-Aldrich, St Louis MO) or steroid-
free DMEM for six hours (Eckersell et al., 1998). Approximately 10
6
cells were used for
each experiment (n=6/group). Cells are placed into assay buffer (50 mM Tris-HCl, pH
7.4 at 4°C) and disrupted using a Polytron homogenizer at 4°C and centrifuged with
46,000 g for 30 minutes at 4°C. Pellets were resuspended in an equal volume of assay
buffer, incubated for 60 minutes on ice to dissociate endogenous ligands, centrifuged
with 50,000 g, resuspended in buffer, and stored in aliquots of 1mL at -80°C. Protein
concentration was determined from small aliquots by the bicinchoninic acid (BCA)
protein assay. For receptor binding assays, ligands were obtained from Multiple Peptide
Systems (San Diego, CA). Opioid receptor binding assay were done within 30 days after
membrane preparation. Microsomal and membrane fractions were homogenized and
128

diluted to a concentration of 2~3 mg/mL. Binding assay was conducted in total volume of
0.5 mL at 22°C. The binding solution contain 400 µL membranes or microsomes, 50 µL
[
3
H]-U69,593 (PerkinElmer Inc, USA) as a KOR agonist and [
3
H]-DAMGO
(PerkinElmer Inc, USA) as a MOR agonist. Following incubation, samples were filtered
through Whatman GF/C filters presoaked in 0.1% BSA, washed with 3 x 3 mL ice-cold
buffer and the filters counted with a scintillation counter.
5.4.6 Agonist-stimulated [
35
S]GTPγS binding Assay
DRG neurons from wild type, ERαKO, and ERβKO mice was homogenized in 20
volume buffers (50 mM Tris-HCl, 3 mM MgCl
2
, 1 mM EGTA, pH 7.4). The
homogenates was centrifuged twice with 48,000 g for 15 minutes at °C and resuspended
with assay buffer (50 mM Tris-HCl, 3 mM MgCl
2
, 0.2 mM EGTA, 100 mM NaCl, pH
7.4). The [
35
S]GTPγS-binding assay is used to identify receptor activated G-proteins
based on the fact that in the inactive states, the α subunit of the G-protein has a relatively
high affinity for GDP over GTP. Activation of the receptors by an agonist shifts the α
subunit to a higher affinity state for GTP. Addition of [
35
S]GTPγS, MOR, and KOR
agonists-activated binding of G-protein to the [
35
S]GTPγS, or non-hydrolysable form of
GTP to the α subunit. Basal binding was assessed in the absence of agonist, and
nonspecific binding was measured with unlabeled GTPγS. For the Scatchard analysis of
the effects of E2, comparison of coupling was done by calculating a catalytic
amplification factor obtained by dividing the B
max
of DAMGO and U-69,593 stimulated
[
35
S]GTPγS binding by the B
max
of receptor binding. Membranes (10 µg protein) were
incubated for 1 hour with 20 M GDP, 0.05 nM [
35
S]GTPγS, and 0 to 20 nM GTPγS, with
and without DAMGO and U69,593 in 1 mL total volume. The reaction was terminated by
129

vacuum filtration through Whatman GF/B glass filters followed by three washes with
cold Tri buffer. Bound radioactivity was determined by liquid scintillation
spectrophotometry at 95% efficiency after overnight extraction in Ecolite Scintillation
fluid. Data were presented as mean ± SEM of at least four determinations which are
performed in triplicate.
5.4.7 Statistical analysis
The amplitude of [Ca
2+
]
i
response represents the difference between baseline
concentration and the transient peak response to drug stimulation. Significant differences
in response to chemical stimulation were obtained by comparing [Ca
2+
]
i
increases during
the first stimulation with the second. All of the data are expressed as the mean ± SEM.
Statistical analysis was performed using Statistical Package for the Social Sciences 18.0
(SPSS, Chicago, IL, USA). To assess the significance among different groups, data were
analyzed with one- or two-way ANOVA followed by Schéffe post hoc test. A p <0.05
was considered statistically significant.

130

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Abstract (if available)

Abstract Clinical studies suggest the comorbidity of functional pain syndromes such as irritable bowel syndrome (IBS), chronic pelvic pain (CPP), fibromyalgia, and somatoform disorders approaches 40% to 60%. The incidence of episodic or persistent visceral pain associated with these functional disorders is two to three times higher women than in men. One of the possible explanations for this phenomenon is the estrogen modulation of pain transmission. While a central site of this modulation has been shown previously, here we proposed to study a peripheral site, the dorsal root ganglion (DRG). In DRG neurons, 17β-estradiol (E2) rapidly inhibits intracellular calcium ([Ca²⁺]i) flux induced by ATP, a putative nociceptive signal. This proposal, ""Estrogen Receptors mediate Nociceptive Signaling in Primary Sensory Neurons in Female Mice"" will test a general hypothesis that E2 acting on primary afferent nociceptors has both pro-nociceptive and anti-nociceptive effects depending on which signals converge upon DRG. First, the role of different estrogen receptors (ERs) in E2 activation of purinergic (P2X3) and vanilloid (TRPV1) receptors will be studied in wild type, estrogen receptor-α, and estrogen receptor-β knock-out mice. Second, since we hypothesize that E2 may act differently on visceral then on cutaneous nociceptors, we will compare the [Ca²⁺]i response to activation of P2X3 and TRPV1 receptors in retrogradely-labeled visceral and cutaneous DRG neurons from knock-out and wild type mice. Third, E2 may negatively modulate opioid analgesia by interfering with μ-opioid receptor (MOR). Pharmacological manipulations will be used to determine how ER activation modulates Ca2+ channel and MOR functions. Receptor binding will determine if E2 alters the number and affinity of MOR in the DRG and the site-specific regulation of MOR coupling to G-proteins. Together these experiments will define a new site(s) and mechanism of E2 modulation of nociceptive signaling. Furthermore, they will provide important information about the action of E2 on primary sensory neurons for a better understanding of sex-differences observed in the clinical presentation of functional pain-associated syndromes. Nociceptive systems implicated in the etiology of functional disorders, which often are complicated by comorbid depression will have a major impact on health-related quality of life in patients with functional pain disorders, significantly reducing therapeutic interventions.

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Creator Cho, Tae Hoon (author)

Core Title The role of estrogen receptors and nociceptive signaling pathway of primary sensory neurons

School Andrew and Erna Viterbi School of Engineering

Degree Doctor of Philosophy

Degree Program Chemical Engineering

Publication Date 05/24/2012

Defense Date 04/30/2012

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

Tag 17beta-estradiol,ATP,calcium,Capsaicin,DRG,estrogen receptor alpha,estrogen receptor beta,OAI-PMH Harvest

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Wang, Pin (committee chair), Shing, Katherine (committee member), Zhang, Li I. (committee member)

Creator Email taecho@usc.edu,uscbioman@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-42498

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