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Gpr30 estrogen receptor agonists induce mechanical hyperalgesia in the rat

European Journal of Neuroscience, Vol. 27, pp. 1700–1709, 2008 GPR30 estrogen receptor agonists induce mechanicalhyperalgesia in the rat Julia Kuhn,1,2 Olayinka A. Dina,3 Chandan Goswami,1 Vanessa Suckow,1 Jon D. Levine3 and Tim Hucho11Department for Molecular Human Genetics, Max Planck Institute for Molecular Genetics, Ihnestrasse 73, 14195 Berlin, Germany2Freie Universita¨t Berlin, Fachbereich fu¨r Biologie, Chemie und Pharmazie, 14195 Berlin, Germany3University California in San Francisco, San Francisco, CA 94143, USA Keywords: estrogen, intracellular signalling, nociception, pain We evaluated the signalling pathway by which estrogen acts in peripheral tissue to produce protein kinase Ce (PKCe)-dependentmechanical hyperalgesia. Specific agonists for the classical estrogen receptors (ER), ERa and ERb, did not result in activation ofPKCe in neurons of dissociated rat dorsal root ganglia. In contrast, G-1, a specific agonist of the recently identified G-protein-coupledestrogen receptor, GPR30, induced PKCe translocation. Involvement of GPR30 and independence of ERa and ERb was confirmedusing the GPR30 agonist and simultaneous ERa and ERb antagonist ICI 182,780 (fulvestrant). The GPR30 transcript could beamplified from dorsal root ganglia tissue. We found estrogen-induced as well as GPR30-agonist-induced PKCe translocation to berestricted to the subgroup of nociceptive neurons positive for isolectin IB4 from Bandeiraea simplicifolia. Corroborating the cellularresults, both GPR30 agonists, G-1 as well as ICI 182,780, resulted in the onset of PKCe-dependent mechanical hyperalgesia ifinjected into paws of adult rats. We therefore suggest that estrogen acts acutely at GPR30 in nociceptors to produce mechanicalhyperalgesia.
A wide variety of endogenous substances have been shown to result in Le Mellay et al., 1997; Ansonoff & Etgen, 1998; Beyer & Raab, 1998; sensitization of nociceptive neurons (Hucho & Levine, 2007; Woolf & Ahmad et al., 1999; Simoncini et al., 2000; Kousteni et al., 2002; Lu Ma, 2007). Also, gonadal hormones influence pain. This is apparent, et al., 2004). Recently, we found estrogen to have fast and therefore e.g. in well described sex-dependent pain behaviour and sex- potentially nontranscriptional effects on nociceptive neurons. Appli- dependent properties of analgesics, both in humans and in animals cation of estrogen translocates protein kinase Ce (PKCe), within (Coyle et al., 1996; Gear et al., 1996; Berkley, 1997; Mogil et al., seconds, toward the plasma membrane (Hucho et al., 2006). This in 1997; Fillingim & Ness, 2000; Cairns et al., 2001; Tall et al., 2001; turn abolishes the induction of PKCe translocation if attempted by Joseph & Levine, 2003; Hucho et al., 2006; Werhagen et al., 2007).
successive activation of an as-coupled G-protein-coupled receptor Estrogen, which in addition to being the prototypic ‘female' hormone (GPCR; b2-adrenergic receptor) or its downstream signalling molecule also plays a crucial role in the male organism, has attracted Epac (Hucho et al., 2006). Consistent with these findings, in considerable attention. The basal mechanical thresholds of male and behavioural experiments intradermal injection of estrogen alone female rats differ in an estrogen-dependent manner (Khasar et al., results in PKCe-dependent mechanical hyperalgesia (Hucho et al., 2005), and the extent of mechanical hyperalgesia induced by the inflammatory mediator epinephrine is strongly modulated by systemic The actions of estrogen are classically considered to be mediated by estrogen (Khasar et al., 2005). Also, beyond the quantitative estrogen receptor (ER) ERa and ⁄ or ERb; these are mostly localized in modulation, estrogen determines the coupling of inflammatory the nucleus, where they have been associated with transcriptional mediators to intracellular signalling cascades (Dina et al., 2001; regulation. Nevertheless, many of the rapid effects of estrogen are Hucho et al., 2006). While systemic hormone levels potentially induce independent of the classical ER-mediated initiation of transcription indirect effects, which in turn might underlie the behaviourally (Kousteni et al., 2001, 2002; Filardo et al., 2002; Lu et al., 2004; observed difference, we recently described estrogen acting directly on Manavathi & Kumar, 2006). Both classical estrogen receptors, ERa the primary afferent nociceptor (Hucho et al., 2006).
and ERb, are known to be expressed in dorsal root ganglia (DRG) Beside systemic long-term effects as indicated above, immediate neurons in male and female rats (Taleghany et al., 1999; Papka & action of hormones on pain sensitivity has barely been documented.
Storey-Workley, 2002), but their functional role in nociception is not This is in contrast to large numbers of reports documenting estrogen- understood. Recently, a novel estrogen receptor was identified, the induced rapid stimulation of second messengers in various cellular integral membrane protein GPR30 (Revankar et al., 2005; Thomas systems (Gu & Moss, 1996; Migliaccio et al., 1996; Zhou et al., 1996; et al., 2005; Bologa et al., 2006). Being a GPCR, GPR30 also canrapidly initiate fast second-messenger cascades. Having found estro- Correspondence: Dr Tim Hucho, as above.
gen to produce mechanical hyperalgesia and to activate PKCe we set out to identify which of the three receptors mediates the observed Received 2 October 2007, revised 2 January 2008, accepted 4 February 2008 rapid action of estrogen on nociceptive neurons.
ª The Authors (2008). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd GPR30 agonists induce mechanical hyperalgesia Materials and methods 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 Behavioural experiments were performed on male Sprague–Dawley performed in a solution containing BSA, 1%; Ca2+, 0.1 mm; Mg2+, rats (200–300 g; Charles River Laboratories, Hollister, CA, USA).
0.1 mm; and Mn2+, 0.1 mm in PBS (1 h, RT) and followed by Animals were housed in a controlled environment under a 12-h three washing steps in the same solution (5 min, RT) before light : dark cycle. Food and water were available ad libitum. Care and mounting with DAPI ⁄ Fluoromount-G.
use of animals conformed to National Institutes of Health guidelines.
The UCSF Committee on Animal Research approved the experimentalprotocols.
Evaluation of PKCe translocation Cell biological experiments were performed on male Sprague– Cells were evaluated with a Zeiss Axioplan 2 microscope, using a Dawley rats (200–300 g; Harlan Winkelmann, Borchen, Germany).
63· oil-immersion objective. Fifty randomly selected cells per Care and use of animals were in accordance with the European culture were evaluated, by the observer, for translocation of PKCe Communities Council Directive of 24 November 1986 (86 ⁄ 609 ⁄ EEC) toward the plasma membrane as evident from a clear rim-like and were approved by the LAGeSo, Berlin.
immunofluorescent signal around the neurons. Data are plotted as All efforts were made to minimize the number of animals used.
mean ± SEM percentage of translocating cells per evaluated culturebased on the number of evaluated cultures. All counting was done in a blind manner. Not more than two cultures treated alike werederived from the same animal. All treatments were repeated with Cultures of dissociated DRG were prepared from male Sprague– DRG neurons from different rats, on at least two separate days.
Dawley rats as described previously (Hucho et al., 2005); rats were Confocal images were taken on an inverted Zeiss LSM 510 Meta killed by CO2 intoxication and L1–L6 DRGs were removed, with a 63· objective, and plasma membrane staining was analysed desheathed, pooled and incubated with collagenase (final concentra- with the Zeiss LSM image examiner software.
tion 0.125%, 1 h, 37 C), followed by trypsin digestion (finalconcentration 0.25%, 8 min, 37 C). Triturating with a fire-polishedPasteur pipette separated cells. Axon stumps and dead cells were Testing of mechanical nociceptive threshold removed by centrifugation (5 min, 100 g). Cells were resuspended in The nociceptive flexion reflex was quantified using the Randall– 12 mL of Neurobasal A medium with B27, plated at 0.5 mL per Selitto paw pressure device (Analgesymeter; Stoelting, Wood Dale, culture (0.5 DRG-equivalents) onto polyornithine- and laminin- IL, USA), which applies a linearly increasing mechanical force to the precoated glass coverslips (12 mm diameter) and incubated overnight dorsum of a rat's hind paw. The nociceptive mechanical threshold was in 24-well plates at 37 C in 5% CO2. Variability between batches of defined as the force in grams at which the rat withdrew its paw. The Neurobasal A medium and its B27 supplement, resulting in varying protocols for this procedure have been described previously (Taiwo translocation of PKCe after stimulation, was eliminated by titration of et al., 1989; Dina et al., 2003). In the week preceding the experiments, the media to a pH of 7.45.
rats were familiarized with the testing procedure at 5 min intervals fora period of 1 h per day for 3 days. Baseline paw-withdrawal threshold was defined as the mean of six readings before test agents wereinjected. Each paw was treated as an independent measure, and each After being in culture for 15–20 h cells were stimulated. To ensure experiment was performed on a separate group of rats. Each group of homogeneous dispersion of the stimulants, 250 out of 500 lL medium rats was treated with the agonists and ⁄ or inhibitors injected intrader- was removed, mixed thoroughly with the stimulant as indicated in the mally on the dorsum of the hind paw. Measurement of nociceptive Results section, and replaced into the same culture. Negative controls threshold was taken 30 min after administration. The reagents (see were treated alike but without the addition of any reagent. After description in the Results section) were injected as described treatment, the cells were washed once with phosphate-buffered saline previously (Khasar et al., 1995, 1999). Because it is less membrane- (PBS) and fixed with paraformaldehyde (4%, 10 min) at room permeant, injections of the PKCe inhibitor (eV1-2; Johnson et al., temperature (RT). All stock solutions of the respective reagents were 1996) were always preceded by administration of 2.5 lL of distilled dissolved in 100% dimethylsulfoxide (DMSO; final concentration on water in the same syringe, separated by a small air bubble, to produce cells, 0.2%).
a hypo-osmotic shock, thereby enhancing cell membrane permeabilityto the drug (Khasar et al., 1995; Khasar et al., 1999). All experimentswere performed at the same time of day. In the week preceding the experiments, rats were familiarized with the testing procedure at 5-min Paraformaldehyde-fixed cells were permeabilized with 0.1% Triton intervals for a period of 1 h per day for 3 days.
X-100 (10 min, RT), followed by three washes with PBS (5 min,RT). After blockage of nonspecific binding sites (5% bovine serumalbumin (BSA) and 10% normal goat serum in PBS; 1 h, RT), the cultures were probed with primary antibodies against target proteins For RNA extraction rats were killed with CO2 and DRGs and brain (antibody concentrations against target proteins as indicated in the from male Sprague–Dawley rats were harvested. Total RNA was Materials and Methods, Antibodies section) in 1% BSA in PBS extracted using the Nucleospin RNA ⁄ Protein Kit (Macherey-Nagel, (overnight, 4 C), washed three times (1% BSA in PBS; 5 min, Du¨ren, Germany). DNAse treatment was done for 15 min at 37 C RT), and incubated with secondary FITC-coupled antiserum (1 h, with RQ1 DNAse (Promega, Mannheim, Germany). RNA was RT). After three final washes (PBS; 5 min, RT), the cultures were precipitated (0.1 volume 3 m Na acetate, 2.5 volumes ethanol; mounted with Fluoromount-G (Southern Biotech ⁄ Biozol) containing 20 min, 20 C and 10 min, 20 800 g), washed with 70% ethanol ª The Authors (2008). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing LtdEuropean Journal of Neuroscience, 27, 1700–1709 J. Kuhn et al.
(5 min, 20 800 g), dried and resuspended in RNAse-free water. cDNA can be monitored by its translocation to a target membrane in response was created using SuperScript III First Strand Synthesis SuperMix for to stimulation (Cesare et al., 1999; Schaefer et al., 2001; Hucho et al., qRT PCR (Invitrogen, Karlsruhe, Germany). A GPR30-specific 2005, 2006) and, indeed, estrogen-induced hyperalgesia could be fragment was amplified with the following primers: 5¢-TCTTCA- correlated with PKCe translocation in sensory neurons 90 s after TCAGCGTCCACCTAC-3¢ (forward) and 5¢-TTGTCCCTGAAGG- estrogen treatment of cultures of dissociated DRG (Hucho et al., TCTCTCC-3¢ (reverse). As loading control GAPDH was amplified 2006). We set out to identify which estrogen receptor mediates the with the primers 5¢-CGTTGTGGATCTGACATGC-3¢ (forward) and cellular and the behavioural effects. As rapid actions can be mediated 5¢-CCCTGTTGCTGTAGCCATATT-3¢ (reverse). PCR conditionswere as follows: 2 min at 94 C, 35 cycles of 30 s at 94 C, 30 s at58 C, 60 s at 72 C and a final 10 min at 72 C. The PCR productwas analysed on a 2% agarose gel and imaged with a Herolab EASY440K gel documentation system.
Statistical analysis All statistical comparisons were made with one-way anovas followedby Dunnett's test for comparisons with one control value, or theTukey–Kramer post hoc test for multiple comparisons, respectively.
P < 0.05 was considered statistically significant.
BSA, l-glutamine, poly l-ornithine hydrochloride, Xgal, DMSO,paraformaldehyde, Triton X-100 and glutamate were purchased fromSigma (Mannheim, Germany), trypsin from Worthington BiochemicalCorporation (Freehold, NJ, USA), Neurobasal A (without phenolred), B27 supplement, laminin, minimum essential medium andglutamax were purchased from Invitrogen (Germany, UK), DMEMwas purchased from Bio Whittaker (Lonza, Belgium), trypsin andEDTA from Clonetics (Cambrex, US) and normal goat serum fromDianova (Hamburg, Germany).
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 ICI 182,780 from Tocris (UK). Dr Eric R. Prossnitz, University ofNew Mexico Health Sciences Center, Albuquerque, New Mexico,USA, kindly provided G-1. PKCe inhibitory peptide eV1-2 waspurchased from Calbiochem (La Jolla, CA, USA).
Dr Robert Messing, University of California San Francisco, kindlyprovided PKCe-specific rabbit serum (final concentration 1 : 1000).
Alexa-488-labelled chicken antirabbit IgG was purchased fromMolecular Probes ⁄ Invitrogen (Karlsruhe, Germany; final concentra-tion 1 : 5000). FITC-coupled antirabbit IgG was purchased fromDianova (Hamburg, Germany; final concentration 1 : 500). TRITC-labelled IB4 was purchased from Sigma; final concentration1 : 10 000.
Fig. 1. The ERa- and ERb-specific agonists PPT and DPN did not translocatePKCe. (a) Treatment of dissociated DRG neurons with the ERa-specific ERa and ERb agonists did not translocate PKCe agonist PPT at concentrations of 1 and 10 nm, for periods from 30 s up to5 min, did not induce significant PKCe translocation to the plasma membrane.
Recently, we found that estrogen induces mechanical hyperalgesia if (b) Application of 10 and 100 nm of the ERb agonist DPN for 30 s up to injected into the paw of male adult rats (Hucho et al., 2006). This 5 min also did not affect PKCe localization in cultured DRG neurons (n ¼ 6 effect was dependent on PKCe. On a cellular level, activation of PKC cultures; **P < 0.001 compared with negative controls).
ª The Authors (2008). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd European Journal of Neuroscience, 27, 1700–1709 GPR30 agonists induce mechanical hyperalgesia by both classical estrogen receptors ERa and ERb (Manavathi & (Bologa et al., 2006). We added G-1 to the culture medium at a Kumar, 2006), we investigated whether their activation results in concentration (100 nm) that has been shown to result in GPR30- PKCe translocation in primary sensory neurons. We treated dissoci- specific cellular responses (Bologa et al., 2006; Albanito et al., 2007; ated DRG neurons with the ERa agonist PPT and the ERb agonist Brailoiu et al., 2007). Paralleling the response to estrogen, indeed, we DPN. Using 1 nm PPT (Boulware et al., 2005; Jelks et al., 2007), no observed rapid translocation of PKCe to the plasma membrane after PKCe translocation was observed following periods of between 30 s G-1 stimulation (Fig. 2a and c). PKCe signal intensity profiles from and 5 min of stimulation. Also, a 10-fold increase in PPT to a confocal images show a clear increase in signal intensity at the plasma concentration of 10 nm did not result in observable activation of PKCe membrane of stimulated cells in contrast to untreated cells (Fig. 2b).
(Fig. 1a; n ¼ 6 cultures).
The largest number of cells showed plasma membrane localization To test whether ERb played a role in estrogen-induced PKCe after 30 s of stimulation (17.3 ± 3.0%), leveling off after 60 s activation, we treated cells with the ERb-specific agonist DPN (10 and (9.0 ± 2.3%) and returning to baseline after 90 s (Fig. 3a; n ¼ 6 100 nm; Boulware et al., 2005; Jelks et al., 2007) for periods from cultures per treatment).
30 s to 5 min but, again, a significant PKCe translocation was notobserved (Fig. 1b; n ¼ 6 cultures). These results indicate that neitherERa nor ERb are involved in the process of estrogen-induced GPR30 was expressed in DRG activation of PKCe in primary sensory neurons.
As our pharmacological experiments suggest the involvement ofGPR30 in estrogen-induced mechanical hyperalgesia, we tested forexpression of GPR30 mRNA in DRG. The novel estrogen receptor G-1 induced PKCe activation GPR30 is expressed in, among others (Revankar et al., 2005), the As agonists of ERa and ERb did not result in translocation of PKCe, normal adult central nervous system (Brailoiu et al., 2007; Sakamoto we tested whether instead the recently identified estrogen receptor et al., 2007). Therefore, brain served as positive control for GPR30 mediates the observed estrogen-induced translocation. We endogenous GPR30 expression. RT-PCR data of total RNA prepara- used the estrogen derivative G-1, which has been reported to activate tions from both DRG and brain extract from adult male rats show a GPR30 while not mediating signal transduction through ERa or -b clear band at the expected size (Fig. 4).
Fig. 2. The GPR30 agonist G-1, like estrogen, translocates PKCe in DRG neurons to the plasma membrane. (a) Confocal images of DRG neurons showtranslocation 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 intensitymeasurement shown in (b). (b) Intensity profiles of cells shown in (a) display the increased plasma membrane staining after estrogen or G-1 stimulation in contrastto untreated cells. (c) Orthogonal image analysis of Z-sections confirms that PKCe translocation to the plasma membrane after treatment with estrogen or G-1occurrs in all focal planes. Scale bars, 10 lm.
ª The Authors (2008). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing LtdEuropean Journal of Neuroscience, 27, 1700–1709

J. Kuhn et al.
Fig. 4. GPR30 mRNA was expressed in DRG and brain of male rats. Analysisof 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.
locates PKCe only in the IB4(+) subgroup of nociceptors (Huchoet al., 2005). Whether estrogen also induces translocation specificallyin this subset of nociceptive neurons is not known. Therefore weperformed double staining experiments with PKCe and the marker fornonpeptidergic nociceptors, IB4, on estrogen-stimulated cultures. Themajority 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 estrogenderivative G-1 also resulted in PKCe translocation in  20% of theneurons. We therefore tested whether the translocation occurred in thesame subpopulation of neurons; indeed, 85.3 ± 2.1% of the culturedsensory 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 shownto specifically activate GPR30 but not ERa or -b, this might reflectfirst of all the very brief period since the initial publication of thiscompound (Bologa et al., 2006). However, ICI 182,780 has alsorecently been shown to bind and activate GPR30 at concentrationsbetween 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 toinhibit estrogen functions mediated by the classical estrogen receptorsERa and ERb (DeFriend et al., 1994; Molinari et al., 2000; Chanet al., 2007). Therefore, we complemented our pharmacologicalstudies by using this GPR30 agonist and simultaneous inhibitor ofERa and ERb. After only 30 s treatment with 1 lm ICI 182,780 therewas PKCe activation in a significant number of cells (13.1 ± 1.0%); as with G-1, translocation was completely reversed after 90 s of . 3. GPR30 agonists induced PKCe-translocation in DRG neurons within 30 s of stimulation. (a) Quantification of PKCe in dissociated DRG neurons, incubation (Fig. 3b; n ¼ 6 cultures).
showing PKCe staining at the plasma membrane after treatment with 100 nmG-1; clear time-dependence is observed. While in a highly significant numberof neurons plasma membrane staining for PKCe was detected after 30 s of G-1 induced PKCe-dependent mechanical hyperalgesia stimulation (17.3 ± 3.0%), plasma membrane staining returned to baseline by90 s of stimulation (n ¼ 6 cultures; **P < 0.001 compared with negative Injection of estrogen into the hind paw of male rats rapidly induces controls). (b) Treatment of cultured DRG neurons with the ERa and ERb PKCe-dependent mechanical hyperalgesia (Hucho et al., 2006).
antagonist and GPR30 agonist, ICI 182,780 (1 lm), for 30 or 60 s led to PKCe Which receptor mediates this effect in vivo is unknown. Our cellular translocation to the plasma membrane while, following longer treatment (90 sand 5 min), no translocation was observed (n ¼ 6 cultures; **P < 0.001 data indicate that estrogen acts through GPR30 but not through ERa compared with negative controls).
or -b. To test whether G-1 is also able to substitute for estrogen inbehavioural experiments, we injected it (dissolved to 10 mg ⁄ mL in PKCe translocation was IB4(+)-neuron specific 100% DMSO, diluted to final concentrations of 1–1000 ng in 2.5 lL We detected translocation of PKCe in only  20% of neurons in the in PBS; final concentration of DMSO 10%) into the hind paw. While cultures. This suggests a mechanism specific for a subpopulation of neither spontaneous pain nor redness or swelling was observed, G-1 sensory neurons. Previously we have shown that epinephrine trans- induced concentration-dependent mechanical hyperalgesia. While ª The Authors (2008). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd European Journal of Neuroscience, 27, 1700–1709 GPR30 agonists induce mechanical hyperalgesia Fig. 5. G-1 and estrogen translocated PKCe inIB4-positive neurons. (a) Confocal images ofdissociated DRG neurons stimulated with estrogen(10 nm, 90 s) or G-1 (100 nm, 30 s). Plasmamembrane localization of PKCe is seen almostexclusively in IB4-positive neurons. Green, PKCe;red, IB4. (b) Quantification of PKCe translocationafter treatment with estrogen or G-1. PKCetranslocation was induced in 15.7 ± 0.8%(estrogen, 10 nm, 90 s) or 20.7 ± 1.6% (G-1,100 nm, 30 s) of stimulated neurons. Estrogen-as well as G-1-induced PKCe translocation wasspecific for IB4-positive neurons (n ¼ 6 cultures;**P < 0.001 compared with negative controls).
Scale bars, 10 lm.
ª The Authors (2008). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing LtdEuropean Journal of Neuroscience, 27, 1700–1709 J. Kuhn et al.
(1 lg G-1) decrease in mechanical nociceptive threshold measured 30 min after administration (Fig. 6a; n ¼ 6 paws per treatment;absolute value of baseline withdrawal threshold of negative controls116 ± 1.4 g).
In previous studies we showed that estrogen-induced nociceptor sensitization is PKCe-dependent (Hucho et al., 2006). To test whetherPKCe is also involved in G-1-induced hyperalgesia, we blocked PKCeby intradermal injection of the PKCe-inhibitory peptide eV1-2 (1 lgin 2.5 lL H2O; Johnson et al., 1996) before stimulation with G-1.
eV1-2 by itself did not affect the nociceptive threshold. However, aswith estrogen, G-1-induced hyperalgesia was abolished completely byinhibition of PKCe through the inhibitory peptide eV1-2 (Fig. 6b; G-1concentration 100 ng, n ¼ 6 paws per treatment).
ICI 182,780 induced PKCe-dependent mechanicalhyperalgesia To substantiate the finding that the novel estrogen receptor GPR30apparently mediates the recently described effect of estrogen onnociceptive neurons, we used a second agonist of GPR30, whichsimultaneously blocks signalling through ERa and -b, ICI 182,780(DeFriend et al., 1994; Molinari et al., 2000; Chan et al., 2007). Thebehavioural experiments established a clear dose-dependence forintradermal (ICI 182,780 dissolved to 10 mg ⁄ mL in 100% DMSO, diluted tofinal concentration in 2.5 lL in PBS; final concentration of DMSO10%) into hind paws of male rats. Neither spontaneous pain norredness or swelling was observed. The maximum decrease innociceptive threshold, by 35.3 ± 2.2%, was observed after injectionof 100 ng ICI 182,780 (Fig. 6a; n ¼ 6 paws; absolute value ofbaseline withdrawal threshold of negative controls 114 ± 1.6 g).
Because estrogen and G-1 led to PKCe-dependent hyperalgesia and ICI 182,780 caused PKCe translocation in vitro, we examined whetherICI 182,780-induced hyperalgesia was also PKCe-dependent. Admin-istration of 1 lg ICI 182,780, 30 min after PKCe-inhibitor treatment,failed to induce mechanical hyperalgesia, demonstrating that, as forestrogen and G-1, PKCe is essential for this process (Fig. 6b; n ¼ 12,10 and 6 paws, respectively).
We previously observed rapid stimulatory effects of estrogen onnociceptive neurons as well as induction of mechanical hyperalgesia(Hucho et al., 2006). Our current study suggests both effects to be Fig. 6. The GPR30 agonists G-1 and ICI 182,780 induced PKCe-dependent mediated by the novel estrogen receptor GPR30. This result is based mechanical hyperalgesia in male rats. (a) Intradermal injection of the GPR30 on a pharmacological approach. We found that two GPR30 agonists, agonists G-1 (dissolved to 10 mg ⁄ mL in 100% DMSO, diluted to final G-1 and ICI 182,780, result in nociceptor sensitization in cell culture concentration of 1–1000 ng in 2.5 lL in PBS; final concentration of DMSO as well as in the animal. G-1 was developed to differentiate between 10%) or ICI 182,780 (dissolved to 10 mg ⁄ mL in 100% DMSO, diluted to final classical ER-mediated and GPR30-mediated mechanisms (Bologa concentration of 0.1–1000 ng in 2.5 lL in PBS; final concentration of DMSO10%) in the hind paw of male rats resulted in concentration-dependent et al., 2006); ICI 182,780, widely used as an antiestrogen, although mechanical hyperalgesia, with a maximal decrease in nociceptive threshold of blocking the classical estrogen receptors ERa and ERb activates (G-1) 41.0 ± 1.2% or (ICI 182,780) 35.3 ± 2.2%. Measurements were taken GPR30 (Filardo et al., 2002; Boulware et al., 2005; Thomas et al., before and 30 min after agonist administration (n ¼ 6 paws per treatment).
2005). Both substances can substitute for estrogen in cellular as well (b) Blocking PKCe by intradermal injection of 1 lg of the PKCe-inhibitorypeptide eV1-2 (in 2.5 lL distilled water) completely abolished hyperalgesia as in behavioural assays, but GPR30 stimulation also resembles induced by the subsequent injection of G-1 (100 ng in 2.5 lL PBS) or estrogen in another important respect. We found that both estrogen and ICI 182,780 (1 lg in 2.5 lL PBS). eV1-2 by itself did not affect nociceptive GPR30-specific agonists act on the same subset of sensory neurons, threshold [n ¼ 6] (G-1, PKCe inhibitor + G-1, PKCe inhibitor + ICI 182,780), the IB4-positive nociceptive neurons. In contrast, neither ERa nor n ¼ 10 paws (ICI 182,780), n ¼ 12 paws (PKCe inhibitor). P < 0.001 for ERb agonists induced PKCe translocation even if used in high agonists compared to agonists + PKCe inhibitor.
1 ng did not induce significant hyperalgesia, 10 ng resulted in The most described signalling aspect of classical estrogen receptors significant, and 100 ng as well as 1 lg in near-maximal mechanical is their ability to initiate gene transcription but, in addition, ERa and hyperalgesia, with a 35.6 ± 0.7% (100 ng G-1) and 41.0 ± 1.2% ERb can induce rapid nongenomic actions. This fast estrogen ª The Authors (2008). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd European Journal of Neuroscience, 27, 1700–1709 GPR30 agonists induce mechanical hyperalgesia signalling can be independent of the transcription initiation site of ERa ger, 1994), bone marrow (Nawata et al., 1995), adipose tissue and skin and -b (Kousteni et al., 2001). Therefore, in principle the observed (Sebastian & Bulun, 2001; Shozu et al., 2003), and not only in females rapid effects of estrogen on nociception could also be mediated by but also in males. Interestingly, the receptive nerve terminals of classical ERs. Nevertheless, ERa and -b agonists did not induce nociceptors end in epithelial tissues, e.g. skin, while the connection to translocation of PKCe. On the contrary, PKCe activation was induced the secondary neuron is established in the central nervous system, i.e.
even in the presence of the ERa and -b inhibitor ICI 182,780.
the spinal cord. Both of these tissues have been reported to express Previously, PKCe has been shown to be activated by aq-coupled aromatase (Harada, 1992; Evrard & Balthazart, 2002; Evrard, 2006).
GPCRs (Cesare et al., 1999) as well as as-coupled GPCRs (Hucho Estrogen can, therefore, be produced in close proximity to nociceptive et al., 2005). In both cases the signalling involves the activation of neurons and, in the context of the data presented here, is therefore phospholipase C and D, resulting necessarily in the production of indeed likely to modulate nociception.
diacylglycerol and phosphatidic acid, two components strongly Locally derived estrogen in peripheral tissue or central nervous activating PKCe (Jose Lopez-Andreo et al., 2003; Hucho et al., system can elicit slow potentially transcriptional as well as fast 2005). GPR30 has also been reported to be coupled to the small signalling cascade-mediated effects. There is, indeed, ample evidence G-protein as (Thomas et al., 2005). Thus, it is exciting to find that of fast induction of various signalling cascades in response to estrogen GPR30 agonists induced PKCe-dependent effects in culture as well as in non-nociceptive cells (Edwards, 2005). To what extent estrogen is in the behavioural experiments. It will therefore be interesting to test produced quickly remains to be measured. One prerequisite for a fast whether GPR30 agonists as well as estrogen also mediate PKCe reaction kinetic is a large pool of the educt, testosterone, which can be activation through the production of cAMP and the cAMP-binding aromatized to estrogen. Indeed, testosterone concentrations are one to protein Epac, as suggested for as-coupled b2-adrenergic receptor three orders of magnitude higher than plasma estrogen levels, assuring activation (Hucho & Levine, 2007).
fast estrogen production (Cornil et al., 2006). Finally, the estrogen A prerequisite for a function of GPR30 in nociception is its producing enzyme, aromatase, also has to be regulated rapidly through expression in the nociceptive neuron. Expression of ERa as well as e.g. phosphorylation, which indeed is the case (Balthazart et al., 2001, ERb in sensory neurons is widely described, though there are still disputes as to whether the two receptors are expressed by the same The above establishes that estrogen induces hyperalgesia in animals neurons or by neighbouring ones (Taleghany et al., 1999; Papka & and activates PKCe, one of the cellular correlates of sensitization in Storey-Workley, 2002). Expression data for GPR30 are so far rare.
sensory neurons. Finally, we would like to point out three aspects of This is partially due to only limited suitability of available antibodies estrogen-dependent pain in humans. First, three mutations in the for immunohistochemical studies. We therefore tested for the presence aromatase gene have been described in male patients. These males of GPR30 mRNA in DRG and found, indeed, GPR30 to be suffer severe symptoms including estrogen-dependent pain syndromes transcribed in this tissue.
(Carani et al., 1997; Bulun, 2000; Maffei et al., 2004). Second, Our data suggest that GPR30 but not the classical ERs mediate the estrogen supplementation in the course of gender transformations observed estrogen effects. This can be substantiated by two additional results in strong pain phenotypes (Aloisi et al., 2007). Third, and most arguments. First, the classical function of the estrogen receptors ERa importantly as it points to the importance of GPR30, widely known and ERb, i.e. initiation of transcription, can be excluded due to the adverse effects of ICI 182,780 (i.e. fulvestrant) in the course of breast rapid onset of action; second, the differential binding constants (Kd) of cancer therapy are pain at the injection site, long-term general pain, the three ERs indicate dependence of PKCe activation on GPR30.
joint pain and headache (Vergote & Abram, 2006).
ERa and ERb are reported to have Kds of  0.1–0.6 nm (Cornil et al., In this study we have elucidated new components underlying the 2006). GPR30 in contrast requires  10-fold higher estrogen concen- sensitizing effects of estrogen on nociception. In addition to bringing a trations as its Kd is 2–6 nm (Cornil et al., 2006). Indeed, 10 nm new receptor into the field of pain research, i.e. GPR30, it might also estrogen was the minimum concentration able to induce PKCe help to advance the procedure accompanying breast cancer therapy.
translocation in sensory neuron cultures (Hucho et al., 2006). That thisconcentration is in the range of the Kd of GPR30 renders it highlylikely that the observed effect of estrogen is indeed mediated by We thank Dr Messing for providing anti-PKCe antiserum, Dr Prossnitz for Under which physiological conditions might GPR30-mediated providing the GPR30 agonist, G-1, and Dr H. H. Ropers for financial support.
pronociceptive estrogen effects occur? Basal plasma levels of estrogenhave been measured as high as 1.1 nm in male rats (Cornil et al.,2006). With Kd values of 0.1–0.6 nm for ERa and -b, this suggests tonic activation of the classical ERs but not of GPR30. We found 10 nm estrogen to be the minimal concentration required to observe DRG, dorsal root ganglia; ER, estrogen receptor; GPCR, G-protein-coupled activation of PKCe in our culture system. This requires that, on top of receptor; IB4, isolectin from Bandeiraea simplicifolia BS-1; PBS, phosphate- the measured systemic concentration of estrogen under physiological buffered saline; PKCe, protein kinase Ce; PPT, 4,4¢,4¢¢-(4-propyl-[1H]-pyraz- conditions in the animal, there should be higher locally restricted ole-1,3,5-triyl)trisphenol; RT, room temperature.
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ª The Authors (2008). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing LtdEuropean Journal of Neuroscience, 27, 1700–1709

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