G Protein-Coupled Estrogen Receptor-mediated Calcium Signaling And Its Regulatory Role In Morphine Analgesia And Tolerance

Estrogens exert a broad spectrum and important regulatory actions in the human body.Except the regulation of reproductive endocrine system function,they also have a complex and extensive influence in the central nervous system,including regulating hyperalgesia,learning and memory,coordination of movement,mood and emotion.In general,women show greater pain sensitivity,less tolerance to pain,and lower sensivitity to opioid analgesia,indicating that estrogens play an important role in pain regulation,but the exact mechanism is not so clear.Estrogen receptors are divided into two categories:1)Classical nuclear estrogen receptor ERαand ERβ,which mediate genomic effects;2)Non-classical estrogen receptors such as the G protein coupled receptor 30（GPR30）,also known as GPER,which mediate the rapid nongenomic effects.The subcellular localization and the signaling mechanisms of the non-classical estrogen receptors remain ambiguous.Our previous study had found that microinjection of estrogen into the rostral ventromedial medulla（RVM）,an important part of the endogenous pain modulation pathway,facilitated colorectal nociception and attenuated the analgesic effect of morphine.The effect appeared to be mediated by GPR30 and the down-stream signaling might involve calcium-dependent phosphorylation of theμ-type opioid receptor（MOR）.In the current study,we aimed to delineate the mechanisms underlying GPR30 mediated inhibition of opioid signaling using a neuronal cell model that endogenously express GPR30 and MOR.In addition,we employed Gpr30gene knockout mice to explore the role of GPR30 signaling in morphine analgesia and tolerance.The following are the main findings of this study:1.Estrogen stimulates intracellular calcium signaling in SH-SY5Y cells1.1 SH-SY5Y cells co-express GPR30 and MOR.Activation of GPR30 results in calcium rise.Using qPCR we detected higher GPR30 mRNA expression in SH-SY5Y cells than in Neuro-2a cells.Western blot and immunofluorescence assay confirmed SH-SY5Y cells co-express GPR30 and MOR,with GPR30 localizing at a perinuclear orgnaelle.17β-estrogen（E2,1μM）and the GPR30 selective agonist G-1（1μM）induced rapid intracellular calcium rise,which was blocked by the GPR30 antagonist G15（3μM）in SH-SY5Y cells,Neuro-2a cells failed to show calcium response to E2 or G-1.1.2 GPR30 mediated calcium rise was due to PLC/IP3 dependent store releaseIn the calcium free extracellular solution,activation of GPR30 also induced an increase in intracellular calcium.Broad-spectrum voltage-dependent calcium channel blocker CdCl2（200μM）had no effect on E2 or G-1-induced calcium rise.Following store depletion using thapsigargin（1μM）,neither E2 nor G-1 evoked a calcium rise,suggesting that GPR30-mediated calcium rise was due to store release rather than calcium entry.IP3 receptor inhibitor 2-APB（3μM）and the PLC inhibitor U73122（3μM）virtually abolished E2/G-1-induced calcium signal.1.3 Estrogen and G-1 stimulates c-fos expression in SH-SY5Y cells.Treatment of SH-SY5Y cells with 0.01μM E2 or 1μM G-1 lead to activation of c-Fos expression in a time-dependent manner.The effect was blocked by G15,U73122 and 2-APB,indicating that GPR30-mediated calcium signal also stimulates indirect non-genomic effects.1.4 Activation of GPR30 results in PKC activation and MOR phosphorylationFollowing incubation with E2 or G-1,membrane expression of PKCαand PKCεwere increased.Concommitantly,phosphorylated MOR expression was also elevated.The results indicate that GPR30-mediated calcium rise led to activation of PKC and subsequent phosphorylation of MOR.The above results revealed novel signaling mechanisms mediated by GPR30:PLC/IP3-dependent calcium rise followed by PKC activation and MOR phosphorylation.Furthermore,the calcium signal was also transmitted to the nucleus to impact on gene expression.GPR30-mediated calcium signaling likely plays an important role in estrogenic effects in the nervous system.2.The role of GPR30-mediated MOR phosphorylation in morphine analgesia and morphine tolerance2.1 Pharmacological activation of GPR30 attenuates morphine analgesia;inhibition of GPR30 enhances morphine analgesia.In the heat-induced tail flick model,systematic application of G-1 1 mg/kg caused a right-ward shift of the dose-response curve for morphine analgesia,whereas G15 0.5mg/kg led to a left-ward shift of the curve,indicating that activation of GPR30decreased morphine analgesic effect,whereas inhibition of GPR30 enhanced the analgesic effect.Similar results were obtained in the formalin-induced acute inflammatory pain model.2.2 Morphine analgesia was enhanced in Gpr30 gene knockout miceAgain using the heat-induced tail flick model,we compared the dose-response curve for morphine analgesia in WT and Gpr30 gene knockout(Gpr30^-/-)mice.Regardless of sex（male or female）,the Gpr30^-/-mice showed greater sensitivity to morphine analgesia than the WT mice.Similar results were obtained in the formalin pain model.2.3 GPR30 gene knockout mice were resistant to morphine toleranceWe studied the effect of pharmacological inhibition or genetic ablation of GPR30 on the development of morphine tolerance in mice.Mice were subcutaneously injected with morphine（10mg/kg）twice a day for 7 days and their heat-induced tail flick latency was measured after the first injection of morphine on day 1,3,5 and 7.WT mice developed morphine tolerance rapidly,G15 delayed the occurrance of tolerance and Gpr30^-/-mice were virtually resistant to morphine tolerance.Furthermore,pMOR expression in RVM following 7 days of morphine treatment was significantly lower in Gpr30^-/-mice than their WT counterparts.Thus,GPR30-mediated MOR phosphorylation may play an important role in morphine tolerance.In conclusion,the present study revealed a novel GPR30-mediated estrogenic signaling in which activation of GPR30 was followed by calcium mobilization,PKC activation and MOR phosphorylation.Such signaling mechanisms likely play a major role in the development of morphine tolerance.We propose that GPR30 and its up-stream or down-stream signals may potentially be targeted to conteract morphine tolerance and other adverse effects of opioid analgesics.