The Role Of G-protein Coupled Receptors In Alleviation Of Morphine Induced Analgesic Tolerance By Oxytocin

As an ancient agent, morphine has being widely used in clinical pain treatment untilrecently because of its powerful analgesic effect. However, chronic morphineadministration always leads to the development of tolerance, dependence and addictioneven worse. All of these side-effects have limited the use of morphine clinically.The dorsal root of spinal cord is the primary nerve centre of nociceptive stimulusafferent. At this level, morphine functionalize as an inhibitory agent by reducing theexcitability of interneurons, which is result in an analgesic effect. It has been confirmedthat morphine educes its effect mainly through μ opioid receptors and opioid receptors.Binding to morphine will change the conformation of the receptors and activate theG-protein downstream. The dissociatied G i inhibits the activation of adenylate cyclase,which makes the down-regulation of cAMP; On the other hand, the βγ-subunit wouldactivate the inward rectification potassium ion channels coupled to G-protein, which leadsto a cell inactivation, as well.It has been reported that chronic morphine induced tolerance is associated with itsfailure to induce the desensitization, internalization and recycle of MOR. Therefore areversed procedure may convert the tolerance of morphine.As one of the most important receptors, G-protein coupled receptor is significant tokeep homeostasis. According to statistics, nearly half percent of the prescribed drugs aretargeted to G-protein coupled receptors. But either positive or negative effect of thesedrugs is decided by the different distribution of receptors, and so is the interaction betweendifferent receptors. More and more studies indicated that GPCRs not only functionalize asmonomers, but also form dimmers or oligomers. It has been reported that opioid receptor(DOR) and μ opioid receptor (MOR) heterodimer could induce internalization of MORunder the co-stimulation of morphine and DAMGO, two selective agonists of opioidreceptor.Regarding internalization and recycle of opioid receptor is related to morphineinduced tolerance, we have tried to find some ways to modulate this process may be reducethe development of tolerance.What we have found in past5years is that chronic morphine could inhibit thesynthesis and release of oxytocin of rat supraoptic nucleus. Our another study indicatedthat chronic lithium salt administration notably reduced the development of morphine mediated tolerance, and this effect would be totally blocked by OVT, a selective antagonistof oxytocin receptor (OTR). All above research imply that OTR is likely to be involved inthe development of tolerance, but the mechanism has not been known. Opioid receptor andoxytocin receptor are both members of G protein coupled receptors, but they are differentin G-protein downstream. Our previous research had reported that oxytocin receptors couldform homo-oligomers, which are sensitive to reducing agent DTT. However, if there is adimer between MOR and OTR has not been reported within opioid pharmacologicalregulation.Based on our previous work, we try to observe the influence of MOR-OTRhetero-dimers in receptor internalization, and explore their roles in morphine tolerance. Wehope our work could provide experimental and theoretical evidence for analgesic drugexploitation.Built rat mouse analgesic tolerance models by intrathecal injected of morphine, andobserved the effect of oxytocin on it. It’s showed that after7days morphine administrationconstantly, the rats had a typical tolerance symptom, but the oxytocin could delay andinhibit the development of tolerance. Immunohistochemistry results showed that MOR andOTR had a co-localization in rat spinal cord dorsal horn; Co-imμnoprecipitation andbimolecular fluorescence complement experimental results also showed that there was aninteraction between MOR and OTR in rat spinal cord. In order to detect the significance ofMOR-OTR interaction, we constructed the MOR-OTR co-expressing cell stains. UtilizedSNAP/CLIP dyeing, associated with the cell imaging technique, we found that morphineand under-stimulant doses of oxytocin co-administration could effectively promote theinternalization of MOR, which is different from morphine and oxytocin treated alone. Inorder to explore the functional fragments mediated the interaction, we constructed GSTfusion protein expression plasmids, expressed purified CT, ICL3, ICL2, ECL3GST fusionproteins, respectively. GST-Pull down research indicated that ICL3might mediate theinteraction between MOR and OTR.Conclusions1、 Intrathecal injection of oxytocin could improve the analgesic efficient of morphine,postponed the appearance of tolerance.2、 It was first reported that there was a co-localization of MOR and OTR in rat spinalcord dorsal horn, so was the interaction between them in spinal cord. 3、 In vitro co-expression system, under-stimulant doses of oxytocin administration couldeffectively induced MOR internalization with morphine.4、 The third intra cellular loop of MOR may be involved in the interaction between MORand OTR.