GPR30 estrogen receptor agonists induce mechanical hyperalgesia in the rat.

GPR30 estrogen receptor agonists induce mechanical
hyperalgesia in the rat
Julia Kuhn,1,2 Olayinka A. Dina,3 Chandan Goswami,1 Vanessa Suckow,1 Jon D. Levine3 and Tim Hucho1
1Department for Molecular Human Genetics, Max Planck Institute for Molecular Genetics, Ihnestrasse 73, 14195 Berlin, Germany
2Freie Universita¨t Berlin, Fachbereich fu¨r Biologie, Chemie und Pharmazie, 14195 Berlin, Germany
3University California in San Francisco, San Francisco, CA 94143, USA
Keywords: estrogen, intracellular signalling, nociception, pain
Abstract
We evaluated the signalling pathway by which estrogen acts in peripheral tissue to produce protein kinase Ce (PKCe)-dependent
mechanical hyperalgesia. Specific agonists for the classical estrogen receptors (ER), ERa and ERb, did not result in activation of
PKCe in neurons of dissociated rat dorsal root ganglia. In contrast, G-1, a specific agonist of the recently identified G-protein-coupled
estrogen receptor, GPR30, induced PKCe translocation. Involvement of GPR30 and independence of ERa and ERb was confirmed
using the GPR30 agonist and simultaneous ERa and ERb antagonist ICI 182,780 (fulvestrant). The GPR30 transcript could be
amplified from dorsal root ganglia tissue. We found estrogen-induced as well as GPR30-agonist-induced PKCe translocation to be
restricted to the subgroup of nociceptive neurons positive for isolectin IB4 from Bandeiraea simplicifolia. Corroborating the cellular
results, both GPR30 agonists, G-1 as well as ICI 182,780, resulted in the onset of PKCe-dependent mechanical hyperalgesia if
injected into paws of adult rats. We therefore suggest that estrogen acts acutely at GPR30 in nociceptors to produce mechanical
hyperalgesia.
Introduction
Awide variety of endogenous substances have been shown to result in
sensitization of nociceptive neurons (Hucho & Levine, 2007; Woolf &
Ma, 2007). Also, gonadal hormones influence pain. This is apparent,
e.g. in well described sex-dependent pain behaviour and sex-
dependent properties of analgesics, both in humans and in animals
(Coyle et al., 1996; Gear et al., 1996; Berkley, 1997; Mogil et al.,
1997; Fillingim & Ness, 2000; Cairns et al., 2001; Tall et al., 2001;
Joseph & Levine, 2003; Hucho et al., 2006; Werhagen et al., 2007).
Estrogen, which in addition to being the prototypic ‘female’ hormone
also plays a crucial role in the male organism, has attracted
considerable attention. The basal mechanical thresholds of male and
female rats differ in an estrogen-dependent manner (Khasar et al.,
2005), and the extent of mechanical hyperalgesia induced by the
inflammatory mediator epinephrine is strongly modulated by systemic
estrogen (Khasar et al., 2005). Also, beyond the quantitative
modulation, estrogen determines the coupling of inflammatory
mediators to intracellular signalling cascades (Dina et al., 2001;
Hucho et al., 2006). While systemic hormone levels potentially induce
indirect effects, which in turn might underlie the behaviourally
observed difference, we recently described estrogen acting directly on
the primary afferent nociceptor (Hucho et al., 2006).
Beside systemic long-term effects as indicated above, immediate
action of hormones on pain sensitivity has barely been documented.
This is in contrast to large numbers of reports documenting estrogen-
induced rapid stimulation of second messengers in various cellular
systems (Gu & Moss, 1996; Migliaccio et al., 1996; Zhou et al., 1996;
Le Mellay et al., 1997; Ansonoff & Etgen, 1998; Beyer & Raab, 1998;
Ahmad et al., 1999; Simoncini et al., 2000; Kousteni et al., 2002; Lu
et al., 2004). Recently, we found estrogen to have fast and therefore
potentially nontranscriptional effects on nociceptive neurons. Appli-
cation of estrogen translocates protein kinase Ce (PKCe), within
seconds, toward the plasma membrane (Hucho et al., 2006). This in
turn abolishes the induction of PKCe translocation if attempted by
successive activation of an as-coupled G-protein-coupled receptor
(GPCR; b2-adrenergic receptor) or its downstream signalling molecule
Epac (Hucho et al., 2006). Consistent with these findings, in
behavioural experiments intradermal injection of estrogen alone
results in PKCe-dependent mechanical hyperalgesia (Hucho et al.,
2006).
The actions of estrogen are classically considered to be mediated by
estrogen receptor (ER) ERa and ⁄ or ERb; these are mostly localized in
the nucleus, where they have been associated with transcriptional
regulation. Nevertheless, many of the rapid effects of estrogen are
independent of the classical ER-mediated initiation of transcription
(Kousteni et al., 2001, 2002; Filardo et al., 2002; Lu et al., 2004;
Manavathi & Kumar, 2006). Both classical estrogen receptors, ERa
and ERb, are known to be expressed in dorsal root ganglia (DRG)
neurons in male and female rats (Taleghany et al., 1999; Papka &
Storey-Workley, 2002), but their functional role in nociception is not
understood. Recently, a novel estrogen receptor was identified, the
integral membrane protein GPR30 (Revankar et al., 2005; Thomas
et al., 2005; Bologa et al., 2006). Being a GPCR, GPR30 also can
rapidly initiate fast second-messenger cascades. Having found estro-
gen to produce mechanical hyperalgesia and to activate PKCe we set
out to identify which of the three receptors mediates the observed
rapid action of estrogen on nociceptive neurons.
Correspondence: Dr Tim Hucho, as above.
E-mail: hucho@molgen.mpg.de
Received 2 October 2007, revised 2 January 2008, accepted 4 February 2008
European Journal of Neuroscience, Vol. 27, pp. 1700–1709, 2008 doi:10.1111/j.1460-9568.2008.06131.x
ª The Authors (2008). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd

Materials and methods
Animals
Behavioural experiments were performed on male Sprague–Dawley
rats (200–300 g; Charles River Laboratories, Hollister, CA, USA).
Animals were housed in a controlled environment under a 12-h
light : dark cycle. Food and water were available ad libitum. Care and
use of animals conformed to National Institutes of Health guidelines.
The UCSF Committee on Animal Research approved the experimental
protocols.
Cell biological experiments were performed on male Sprague–
Dawley rats (200–300 g; Harlan Winkelmann, Borchen, Germany).
Care and use of animals were in accordance with the European
Communities Council Directive of 24 November 1986 (86 ⁄ 609 ⁄ EEC)
and were approved by the LAGeSo, Berlin.
All efforts were made to minimize the number of animals used.
DRG cultures
Cultures of dissociated DRG were prepared from male Sprague–
Dawley rats as described previously (Hucho et al., 2005); rats were
killed by CO2 intoxication and L1–L6 DRGs were removed,
desheathed, pooled and incubated with collagenase (final concentra-
tion 0.125%, 1 h, 37 �C), followed by trypsin digestion (final
concentration 0.25%, 8 min, 37 �C). Triturating with a fire-polished
Pasteur pipette separated cells. Axon stumps and dead cells were
removed by centrifugation (5 min, 100 g). Cells were resuspended in
12 mL of Neurobasal A medium with B27, plated at 0.5 mL per
culture (0.5 DRG-equivalents) onto polyornithine- and laminin-
precoated glass coverslips (12 mm diameter) and incubated overnight
in 24-well plates at 37 �C in 5% CO2. Variability between batches of
Neurobasal A medium and its B27 supplement, resulting in varying
translocation of PKCe after stimulation, was eliminated by titration of
the media to a pH of 7.45.
Cell stimulation
After being in culture for 15–20 h cells were stimulated. To ensure
homogeneous dispersion of the stimulants, 250 out of 500 lL medium
was removed, mixed thoroughly with the stimulant as indicated in the
Results section, and replaced into the same culture. Negative controls
were treated alike but without the addition of any reagent. After
treatment, the cells were washed once with phosphate-buffered saline
(PBS) and fixed with paraformaldehyde (4%, 10 min) at room
temperature (RT). All stock solutions of the respective reagents were
dissolved in 100% dimethylsulfoxide (DMSO; final concentration on
cells, 0.2%).
Immunocytochemistry
Paraformaldehyde-fixed cells were permeabilized with 0.1% Triton
X-100 (10 min, RT), followed by three washes with PBS (5 min,
RT). After blockage of nonspecific binding sites (5% bovine serum
albumin (BSA) and 10% normal goat serum in PBS; 1 h, RT), the
cultures were probed with primary antibodies against target proteins
(antibody concentrations against target proteins as indicated in the
Materials and Methods, Antibodies section) in 1% BSA in PBS
(overnight, 4 �C), washed three times (1% BSA in PBS; 5 min,
RT), and incubated with secondary FITC-coupled antiserum (1 h,
RT). After three final washes (PBS; 5 min, RT), the cultures were
mounted with Fluoromount-G (Southern Biotech ⁄ Biozol) containing
DAPI (5 lg ⁄mL). For confocal imaging Alexa-488-labelled chicken
antirabbit IgG was used instead of FITC-coupled antiserum.
Staining with isolectin from Bandeiraea simplicifolia (IB4) was
performed in a solution containing BSA, 1%; Ca2+, 0.1 mm; Mg2+,
0.1 mm; and Mn2+, 0.1 mm in PBS (1 h, RT) and followed by
three washing steps in the same solution (5 min, RT) before
mounting with DAPI ⁄ Fluoromount-G.
Evaluation of PKCe translocation
Cells were evaluated with a Zeiss Axioplan 2 microscope, using a
63· oil-immersion objective. Fifty randomly selected cells per
culture were evaluated, by the observer, for translocation of PKCe
toward the plasma membrane as evident from a clear rim-like
immunofluorescent signal around the neurons. Data are plotted as
mean ± SEM percentage of translocating cells per evaluated culture
based on the number of evaluated cultures. All counting was done
in a blind manner. Not more than two cultures treated alike were
derived from the same animal. All treatments were repeated with
DRG neurons from different rats, on at least two separate days.
Confocal images were taken on an inverted Zeiss LSM 510 Meta
with a 63· objective, and plasma membrane staining was analysed
with the Zeiss LSM image examiner software.
Testing of mechanical nociceptive threshold
The nociceptive flexion reflex was quantified using the Randall–
Selitto paw pressure device (Analgesymeter; Stoelting, Wood Dale,
IL, USA), which applies a linearly increasing mechanical force to the
dorsum of a rat’s hind paw. The nociceptive mechanical threshold was
defined as the force in grams at which the rat withdrew its paw. The
protocols for this procedure have been described previously (Taiwo
et al., 1989; Dina et al., 2003). In the week preceding the experiments,
rats were familiarized with the testing procedure at 5 min intervals for
a period of 1 h per day for 3 days. Baseline paw-withdrawal threshold
was defined as the mean of six readings before test agents were
injected. Each paw was treated as an independent measure, and each
experiment was performed on a separate group of rats. Each group of
rats was treated with the agonists and ⁄ or inhibitors injected intrader-
mally on the dorsum of the hind paw. Measurement of nociceptive
threshold was taken 30 min after administration. The reagents (see
description in the Results section) were injected as described
previously (Khasar et al., 1995, 1999). Because it is less membrane-
permeant, injections of the PKCe inhibitor (eV1-2; Johnson et al.,
1996) were always preceded by administration of 2.5 lL of distilled
water in the same syringe, separated by a small air bubble, to produce
a hypo-osmotic shock, thereby enhancing cell membrane permeability
to the drug (Khasar et al., 1995; Khasar et al., 1999). All experiments
were performed at the same time of day. In the week preceding the
experiments, rats were familiarized with the testing procedure at 5-min
intervals for a period of 1 h per day for 3 days.
RT-PCR
For RNA extraction rats were killed with CO2 and DRGs and brain
from male Sprague–Dawley rats were harvested. Total RNA was
extracted using the Nucleospin RNA ⁄ Protein Kit (Macherey-Nagel,
Du¨ren, Germany). DNAse treatment was done for 15 min at 37 �C
with RQ1 DNAse (Promega, Mannheim, Germany). RNA was
precipitated (0.1 volume 3 m Na acetate, 2.5 volumes ethanol;
20 min, 20 �C and 10 min, 20 800 g), washed with 70% ethanol
GPR30 agonists induce mechanical hyperalgesia 1701
ª The Authors (2008). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd
European Journal of Neuroscience, 27, 1700–1709

(5 min, 20 800 g), dried and resuspended in RNAse-free water. cDNA
was created using SuperScript III First Strand Synthesis SuperMix for
qRT PCR (Invitrogen, Karlsruhe, Germany). A GPR30-specific
fragment was amplified with the following primers: 5¢-TCTTCA-
TCAGCGTCCACCTAC-3¢ (forward) and 5¢-TTGTCCCTGAAGG-
TCTCTCC-3¢ (reverse). As loading control GAPDH was amplified
with the primers 5¢-CGTTGTGGATCTGACATGC-3¢ (forward) and
5¢-CCCTGTTGCTGTAGCCATATT-3¢ (reverse). PCR conditions
were as follows: 2 min at 94 �C, 35 cycles of 30 s at 94 �C, 30 s at
58 �C, 60 s at 72 �C and a final 10 min at 72 �C. The PCR product
was analysed on a 2% agarose gel and imaged with a Herolab EASY
440K gel documentation system.
Statistical analysis
All statistical comparisons were made with one-way anovas followed
by Dunnett’s test for comparisons with one control value, or the
Tukey–Kramer post hoc test for multiple comparisons, respectively.
P < 0.05 was considered statistically significant.
Chemicals
BSA, l-glutamine, poly l-ornithine hydrochloride, Xgal, DMSO,
paraformaldehyde, Triton X-100 and glutamate were purchased from
Sigma (Taufkirchen, Germany), collagenase P from Roche
(Mannheim, Germany), trypsin from Worthington Biochemical
Corporation (Freehold, NJ, USA), Neurobasal A (without phenol
red), B27 supplement, laminin, minimum essential medium and
glutamax were purchased from Invitrogen (Germany, UK), DMEM
was purchased from Bio Whittaker (Lonza, Belgium), trypsin and
EDTA from Clonetics (Cambrex, US) and normal goat serum from
Dianova (Hamburg, Germany).
Drugs
17-b-estradiol, water-soluble, was purchased from Sigma (Taufkir-
chen, Germany) and 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN),
4,4¢,4¢¢-(4-Propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT) and
ICI 182,780 from Tocris (UK). Dr Eric R. Prossnitz, University of
New Mexico Health Sciences Center, Albuquerque, New Mexico,
USA, kindly provided G-1. PKCe inhibitory peptide eV1-2 was
purchased from Calbiochem (La Jolla, CA, USA).
Antibodies
Dr Robert Messing, University of California San Francisco, kindly
provided PKCe-specific rabbit serum (final concentration 1 : 1000).
Alexa-488-labelled chicken antirabbit IgG was purchased from
Molecular Probes ⁄ Invitrogen (Karlsruhe, Germany; final concentra-
tion 1 : 5000). FITC-coupled antirabbit IgG was purchased from
Dianova (Hamburg, Germany; final concentration 1 : 500). TRITC-
labelled IB4 was purchased from Sigma; final concentration
1 : 10 000.
Results
ERa and ERb agonists did not translocate PKCe
Recently, we found that estrogen induces mechanical hyperalgesia if
injected into the paw of male adult rats (Hucho et al., 2006). This
effect was dependent on PKCe. On a cellular level, activation of PKC
can be monitored by its translocation to a target membrane in response
to stimulation (Cesare et al., 1999; Schaefer et al., 2001; Hucho et al.,
2005, 2006) and, indeed, estrogen-induced hyperalgesia could be
correlated with PKCe translocation in sensory neurons 90 s after
estrogen treatment of cultures of dissociated DRG (Hucho et al.,
2006). We set out to identify which estrogen receptor mediates the
cellular and the behavioural effects. As rapid actions can be mediated
Fig. 1. The ERa- and ERb-specific agonists PPT and DPN did not translocate
PKCe. (a) Treatment of dissociated DRG neurons with the ERa-specific
agonist PPT at concentrations of 1 and 10 nm, for periods from 30 s up to
5 min, did not induce significant PKCe translocation to the plasma membrane.
(b) Application of 10 and 100 nm of the ERb agonist DPN for 30 s up to
5 min also did not affect PKCe localization in cultured DRG neurons (n ¼ 6
cultures; **P < 0.001 compared with negative controls).
1702 J. Kuhn et al.
ª The Authors (2008). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd
European Journal of Neuroscience, 27, 1700–1709

by both classical estrogen receptors ERa and ERb (Manavathi &
Kumar, 2006), we investigated whether their activation results in
PKCe translocation in primary sensory neurons. We treated dissoci-
ated DRG neurons with the ERa agonist PPT and the ERb agonist
DPN. Using 1 nm PPT (Boulware et al., 2005; Jelks et al., 2007), no
PKCe translocation was observed following periods of between 30 s
and 5 min of stimulation. Also, a 10-fold increase in PPT to a
concentration of 10 nm did not result in observable activation of PKCe
(Fig. 1a; n ¼ 6 cultures).
To test whether ERb played a role in estrogen-induced PKCe
activation, we treated cells with the ERb-specific agonist DPN (10 and
100 nm; Boulware et al., 2005; Jelks et al., 2007) for periods from
30 s to 5 min but, again, a significant PKCe translocation was not
observed (Fig. 1b; n ¼ 6 cultures). These results indicate that neither
ERa nor ERb are involved in the process of estrogen-induced
activation of PKCe in primary sensory neurons.
G-1 induced PKCe activation
As agonists of ERa and ERb did not result in translocation of PKCe,
we tested whether instead the recently identified estrogen receptor
GPR30 mediates the observed estrogen-induced translocation. We
used the estrogen derivative G-1, which has been reported to activate
GPR30 while not mediating signal transduction through ERa or -b
(Bologa et al., 2006). We added G-1 to the culture medium at a
concentration (100 nm) that has been shown to result in GPR30-
specific cellular responses (Bologa et al., 2006; Albanito et al., 2007;
Brailoiu et al., 2007). Paralleling the response to estrogen, indeed, we
observed rapid translocation of PKCe to the plasma membrane after
G-1 stimulation (Fig. 2a and c). PKCe signal intensity profiles from
confocal images show a clear increase in signal intensity at the plasma
membrane of stimulated cells in contrast to untreated cells (Fig. 2b).
The largest number of cells showed plasma membrane localization
after 30 s of stimulation (17.3 ± 3.0%), leveling off after 60 s
(9.0 ± 2.3%) and returning to baseline after 90 s (Fig. 3a; n ¼ 6
cultures per treatment).
GPR30 was expressed in DRG
As our pharmacological experiments suggest the involvement of
GPR30 in estrogen-induced mechanical hyperalgesia, we tested for
expression of GPR30 mRNA in DRG. The novel estrogen receptor
GPR30 is expressed in, among others (Revankar et al., 2005), the
normal adult central nervous system (Brailoiu et al., 2007; Sakamoto
et al., 2007). Therefore, brain served as positive control for
endogenous GPR30 expression. RT-PCR data of total RNA prepara-
tions from both DRG and brain extract from adult male rats show a
clear band at the expected size (Fig. 4).
(a)
(b)
(c)
Fig. 2. The GPR30 agonist G-1, like estrogen, translocates PKCe in DRG neurons to the plasma membrane. (a) Confocal images of DRG neurons show
translocation of PKCe to the plasma membrane after treatment with estrogen (10 nm, 90 s) or G-1 (100 nm, 30 s). White lanes indicate positions of intensity
measurement shown in (b). (b) Intensity profiles of cells shown in (a) display the increased plasma membrane staining after estrogen or G-1 stimulation in contrast
to untreated cells. (c) Orthogonal image analysis of Z-sections confirms that PKCe translocation to the plasma membrane after treatment with estrogen or G-1
occurrs in all focal planes. Scale bars, 10 lm.
GPR30 agonists induce mechanical hyperalgesia 1703
ª The Authors (2008). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd
European Journal of Neuroscience, 27, 1700–1709

PKCe translocation was IB4(+)-neuron specific
We detected translocation of PKCe in only � 20% of neurons in the
cultures. This suggests a mechanism specific for a subpopulation of
sensory neurons. Previously we have shown that epinephrine trans-
locates PKCe only in the IB4(+) subgroup of nociceptors (Hucho
et al., 2005). Whether estrogen also induces translocation specifically
in this subset of nociceptive neurons is not known. Therefore we
performed double staining experiments with PKCe and the marker for
nonpeptidergic nociceptors, IB4, on estrogen-stimulated cultures. The
majority of PKCe-translocating cells showed strong IB4 staining
(Fig. 5a). After estrogen treatment, 86.8 ± 4.9% of PKCe-translocat-
ing neurons were positive for IB4. Application of the estrogen
derivative G-1 also resulted in PKCe translocation in � 20% of the
neurons. We therefore tested whether the translocation occurred in the
same subpopulation of neurons; indeed, 85.3 ± 2.1% of the cultured
sensory neurons responsive to G-1 were positive for IB4 (Fig. 5b;
n ¼ 6 cultures per treatment).
ICI 182,780 induced PKCe translocation
Targeting the same mechanism with more than one reagent corrob-
orates pharmacological approaches. While G-1 has, so far, been shown
to specifically activate GPR30 but not ERa or -b, this might reflect
first of all the very brief period since the initial publication of this
compound (Bologa et al., 2006). However, ICI 182,780 has also
recently been shown to bind and activate GPR30 at concentrations
between 1 and 10 lm (Filardo et al., 2002; Boulware et al., 2005;
Thomas et al., 2005). Importantly, ICI 182,780 is first of all known to
inhibit estrogen functions mediated by the classical estrogen receptors
ERa and ERb (DeFriend et al., 1994; Molinari et al., 2000; Chan
et al., 2007). Therefore, we complemented our pharmacological
studies by using this GPR30 agonist and simultaneous inhibitor of
ERa and ERb. After only 30 s treatment with 1 lm ICI 182,780 there
was PKCe activation in a significant number of cells (13.1 ± 1.0%); as
with G-1, translocation was completely reversed after 90 s of
incubation (Fig. 3b; n ¼ 6 cultures).
G-1 induced PKCe-dependent mechanical hyperalgesia
Injection of estrogen into the hind paw of male rats rapidly induces
PKCe-dependent mechanical hyperalgesia (Hucho et al., 2006).
Which receptor mediates this effect in vivo is unknown. Our cellular
data indicate that estrogen acts through GPR30 but not through ERa
or -b. To test whether G-1 is also able to substitute for estrogen in
behavioural experiments, we injected it (dissolved to 10 mg ⁄mL in
100% DMSO, diluted to final concentrations of 1–1000 ng in 2.5 lL
in PBS; final concentration of DMSO 10%) into the hind paw. While
neither spontaneous pain nor redness or swelling was observed, G-1
induced concentration-dependent mechanical hyperalgesia. While
Fig. 3. GPR30 agonists induced PKCe-translocation in DRG neurons within
30 s of stimulation. (a) Quantification of PKCe in dissociated DRG neurons,
showing PKCe staining at the plasma membrane after treatment with 100 nm
G-1; clear time-dependence is observed. While in a highly significant number
of neurons plasma membrane staining for PKCe was detected after 30 s of
stimulation (17.3 ± 3.0%), plasma membrane staining returned to baseline by
90 s of stimulation (n ¼ 6 cultures; **P < 0.001 compared with negative
controls). (b) Treatment of cultured DRG neurons with the ERa and ERb
antagonist and GPR30 agonist, ICI 182,780 (1 lm), for 30 or 60 s led to PKCe
translocation to the plasma membrane while, following longer treatment (90 s
and 5 min), no translocation was observed (n ¼ 6 cultures; **P < 0.001
compared with negative controls).
bp DR
G
GPR30
GAPDH
br
ai
n
w
/o
R
T
H
2O
300
200
100
300
200
Fig. 4. GPR30 mRNAwas expressed in DRG and brain of male rats. Analysis
of GPR30 mRNA expression in DRG and brain derived from male rats by RT-
PCR. Lane 1, total RNA from DRG; lane 2, total RNA from brain; lane 3,
reaction without reverse transcriptase; lane 4, water control.
1704 J. Kuhn et al.
ª The Authors (2008). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd
European Journal of Neuroscience, 27, 1700–1709