The Effects And Mechanism Of CCK-8 On Short-term Desensitization Of ?-opioid Receptor Induced By Morphine

Morphine has been used as clinically analgesic drugs for one hundred years,but it is limited in use because of its tolerance and dependence. Opioid addiction has become a healthy problem which can not be ignored.There are 2.34 million drug addicts in China by 2015,and more than 40% of them are opioid addicts.The mechanism of analgesia in morphine is clear. Morphine combined with ?-opioid receptor, which leads to the decomposition of G protein subunits, inhibits AC(adenylate cyclase) and cAMP( Cyclic Adenosine monophosphate), then calcium ion channel is closed, potassium ion and sodium ion channels is open,which causes analgesic effect. However, the mechanism of tolerance and desensitization in morphine is not entirely clear. Much research suggests that it is related with the phosphorylation of ?-opioid receptor, the degressive function of opioid receptor, G protein coupled receptor kinase, PKC(protein kinase C) and so on. Many researchers consider that the tolerance of morphine will undergo short-term desensitization, long-term desensitization and tolerance, and each process has close relationship with the phosphorylation of ?-opioid receptor. CCK(cholecystokinin) as a kind of typical ghrelin, plays an important role by the form of the neurotransmitter and neural adjustable quality.CCK-8(cholecystokinin octapeptide) as the strongest endogenous "resistant of opioid peptides" as we known, regulates the process of tolerance and dependence in morphine by "negative feedback" mechanisms.Our results show that CCK-8 can inhibit the binding force of opioid receptor by activating the CCK2 receptor,and the antagonist of CCK2 receptor can inhibit the formation of morphine dependence. High doses of CCK 8 can increase the release of endogenous opioid by stimulating CCK1 receptor,the antagonist of CCK1 receptor can partially reverse the inhibition of morphine dependence induced by CCK-8. CCK-8 may play different roles in morphine dependence by different subtypes of CCK receptor and different sensitive state of receptor. However,because CCK1 and CCK2 receptors usually exit in the same tissue and cells,we lack the separate experimental model of two receptors.Based on the above research background,We will use the following experimental models. HEK293 cells are largely transferred ?-opioid receptors(HEK293-?). HEK293 cells are largely transferred ?-opioid and CCK1 receptors(HEK293-CCK1). HEK293 cells are largely transferred ?-opioid and CCK2 receptors(HEK293-CCK2).We will study the relationship between phosphorylation and short-term desensitization in ?-opioid receptor, observe the effects of CCK-8 on ?-opioid receptor phosphorylation by different subtypes,and research the mechanisms by which CCK-8 plays roles in short-term desensitization induced by morphine. Part 1:The effects of CCK-8 on sustained phosphorylation of ?-opioid receptor induced by morphine.Objective: Prove that morphine can cause sustained phosphorylation of ?-opioid receptor, observe the different functions of CCK-8 on sustained phosphorylation by different subtypes, prove that sustained phosphorylation of ?-opioid receptor may lead to short-term desensitization.Methods:1 The HEK293-? cells are treated with morphine(100 ?M) or DAMGO(10 ?M) for 30 min. The stimulation is removed for 0.5h?1h?2h?3h?4h?5h?6h. The protein of p-?u-opioid is detected at each time.2 HEK293-CCK1 or HEK293-CCK2 cells are treated with CCK-8(1?M) for 12 h in advance and morphine(100?M)for 30 min,then the stimulation is removed for 3h.3 HEK293-? cells are treated with DAMGO(10?M)for 30 min,then stimulation is removed for 3h(called D3 group). HEK293-? cells are treated with morphine(100?M)for 30 min,then stimulation is removed for 3h(called M3 group). HEK293-CCK2 cells are treated with CCK-8(1?M)for 12 h in advance and morphine(100?M)for 30 min,then stimulation is removed for 3h(called M+C 3 group).The above cells are treated with morphine(10?M) again. The level of G?i and G? is detected by RT-PCR.Results:1 The effects of morphine on the p-?u-opioid protein.Morphine can cause sustained phosphorylation of ?-opioid receptor(P>0.05).The level of phosphorylation stays the same when stimulation isremoved for 3h. The level of phosphorylation of ?-opioid receptor declinesimmediately when DAMGO is removed.2 The effects of CCK-8 on sustained phosphorylation of ?-opioidreceptor induced by morphine.2.1 The phosphorylation of ?-opioid receptor is not detected whenHEK293-CCK1 cells are treated with morphine(P >0.05).2.2 CCK-8 not only declines the level of phosphorylation of ?-opioidreceptor induced by morphine(P <0.01),but also accelerates the process ofphosphorylation declined of ?-opioid receptor.3 The effects of CCK-8 on short-term desensitization induced bymorphine.D3 group,M3 group and M+C3 group are treated with morphine again.The level of mRNA of G?i and G? is not different between D3 group andcontrol(P >0.05). The level of mRNA of G?i and G? in M3 group isdeclined compared to D3 group(P <0.001). The level of mRNA of G?i andG? in M+C 3 group is raised compared to M3 group(P <0.01). Part 2 :The mechanism of CCK-8 in sustained phosphorylation of ?-opioid receptor induced by morphine.Objective:1 Observe whether PKC participates in the mechanism of CCK-8 inphosphorylation of ?-opioid receptor induced by morphine.2 Observe whether ?-opioid receptor can form functional dimmers withCCK1 or CCK2 receptor.1 HEK293-? cells are treated with morphine(100?M) or DAMGO(10?M) for 30 min.The protein of PKC is detected by western blot.2 HEK293-CCK1 or CCK2 cells are treated with CCK-8(1?M)for 12 h in advance and morphine(100?M)for 30 min. The protein of PKC is detected by western blot.3 HEK293-? cells are treated with morphine(100?M)for 30 min,then morphine is removed for 1h,2h,3h,4h,5h. HEK293- CCK2 cells are treated with CCK-8(1?M)for 12 h in advance and morphine(100?M)for 30 min,then stimulation is removed for 1h,2h,3h,4h,5h.The protein of PKC is detected by western blot at each time.4 ?-opioid receptor is connected with Histag antibody.CCK1 receptor is connected with V5 antibody.CCK2 receptor is connected with Flag antibody.HEK293-CCK1 or HEK293-CCK2 cells are treated with morphine(10?M) for 12 h or morphine(100?M)30min. Histag,V5,Flag is detected by co-immunoprecipitation.Results:1 The effects of PKC on phosphorylation of ?-opioid receptor induced by morphine.1.1 Morphine can cause the raise of PKC(P <0.001), but DAMGO can not cause this change(P >0.05)1.2 It can not induce the raise of PKC that HEK293-CCK1 cells treated with morphine(P >0.05).It can induce the raise of PKC that HEK293-CCK1 cells treated with CCK-8(P <0.001).1.3 CCK-8 can decline the level of PKC raised by morphine in HEK293-CCK2 cells.1.4 The level of PKC delays to decline when morphine is removed(P <0.01);CCK-8 can accelerate the process of the decline of PKC raised by morphine.2 The interaction bwteen ?-opioid receptor with CCK1 receptor or CCK2 receptor.?-opioid receptor can form dimmers with CCK1 receptor. It is not Methods: observed that ?-opioid receptor form dimmers with CCK2 receptor.Conclusion:1 The sustained phosphorylation of ?-opioid receptor can cause the short-term desensitization induced by morphine.CCK-8 can release the short-term desensitization by accelerating the process of phosphorylation declined of ?-opioid receptor.2 CCK-8 can accelerate the process of the decline of PKC raised by morphine. It is consistent with the effects of CCK-8 on the process of phosphorylation declined of ?-opioid receptor.3 ?-opioid receptor can form functional dimmers with CCK1 receptor. It may lead to reverse the effects of morphine on phosphorylation and PKC.