The Influence And Mechanism Research Of Different CCK Receptor Subtypes To The Morphine’s Acute And Chronic Effects

Drug abuse has become a serious public health problem all over the world lately. To explore the mechanism of drug dependence, develop the effective drug is one of important items in forensic toxicological research.Opioids are the common drugs of abuse, and continued use results in the development of tolerance and dependence. Opioid dependence is chronic recurrent encephalopathy, characterized by compulsive drug taking, inability to limit the intake of drugs, and the emergence of withdrawal syndrome after cessation of drug taking. Recent studies suggest that non-opioid systems could be important targets for the treatment of opioid dependence. Cholecystokinin(CCK) is a typical neuropeptide that localized in the central and peripheral nervous system. As a neuro-transmitter and modulator, the CCK system regulates a variety of physiological processes, including pain, cognition,reward and learning or memory. Cholecystokinin octapeptide(CCK-8) is one of the most potent endogenous anti-opioid peptide, regulating process of opioid tolerance and dependence by "negative feedback" mechanisms. Studies have shown administration of CCK receptor antagonists can prevent or reverse tolerance to systemic exogenous opioids or electroacupuncture induced analgesia, alleviate morphine withdrawal syndrome, and inhibit the morphine induced conditioned place preference. We previously found that chronic pretreatment with exogenous CCK-8 significantly inhibited naloxoneprecipitated withdrawal symptoms, which is the same effect as CCK-receptor antagonists. These phenomena suggested that high dose exogenous CCK-8 has a significant regulatory role on morphine physical dependence, psychological dependence and relapse process, which were distinct from the role of endogenous CCK. However the mechanism was not clear.CCK-8, through its action at CCK1 and CCK2 receptors, participates in the secretion of pancreatic enzymes, contraction of gallbladder, gut motility,and other physiological functions, such as anxiety, depression, psychosis,cognition, nociception. In addition to CCK1 receptor and CCK2 receptor,CCK receptors include CCKC receptor, glycine extended gastrin receptor(G-Gly receptor) and CCK2 receptor splice variant(CCK-BRi4SV). CCK1 and CCK2 receptors belong to the G- protein coupled receptor family, which are 50% homology. The CCK1 receptor distributed mainly in the peripheral pancreas, gallbladder, pyloric smooth muscle, and is present in discrete regions of the brain and has a low affinity for central CCK. The CCK2 receptor is predominantly localized in the CNS, mainly distributed in the cortex, striatum, basal ganglia, subiculum, tonsil, nucleus accumbens hippocampus, amygdala, hypothalamus, substantia nigra, ventral tegmental area and orther area, belongs to the highly sensitive receptor. Affinity of CCK-8 for CCK2 receptor was obviously higher than that of CCK1 receptor.According to the present study, CCK2 receptor plays a major role in pain perception, opioid dependence, and has nothing to do with the CCK1 receptor.CCK antagonists suppresseed locomotor activity in mice through an interaction with CCK1 receptor. In the nucleus accumbens CCK1 receptors regulate discrimination learning and conditional avoidance response,suggested CCK in medial posterior nucleus accumbens participated in the regulation of learning and memory through CCK1 receptor, but not CCK2 receptor. Researchers have reported different subtypes of CCK receptors in animal behavior research results are distinct, for example, CCK1 receptor may promote the learning and memory, while CCK2 receptor may be associated with the process of forgetting. CCK receptor subtypes and the different sensitive degree may determine the diversity of the biological function of CCK.In our previous studies, we found both CCK1 and CCK2 receptor antagonists could block the inhibition of CCK-8 on binding of opioid receptors. Only CCK2 R antagonist could block morphine dependence, but just CCK1 R antagonist could partially reverse the inhibitory effect of exogenous CCK-8 on morphine dependence. So that, the different subtypes of CCK receptors may have different regulatory effects on morphine dependence.Different modes of action of CCK receptor may be an important mechanism of exogenous CCK-8 intervention of morphine dependence. We just indirectly proved the different effects of two kinds of receptors by two receptor antagonists. There was no direct evidence to reflect the specific functions in the process of morphine dependence. Because lacking of commercialization of high selectivity for specific ligands, CCK1 and CCK2 receptors usually co-expressing in the same tissue or cell, their respective effect can not be exactly determined. Therefore, the present study aims to explore the role of different CCK receptor subtype in the process of acute and chronic morphine action by building an experimental celluar model expressing CCK1 or CCK2 receptor alone. Furthermore, we propose to reveal possible cellular and molecular mechanisms; and provide systematic and reliable theoretical base for the application of CCK-8 in prevention and treatment of morphine dependence and relapse.Part Ⅰ Generation and Identification of Stable MOR, CCK1 R and CCK2 R expressed HEK-293 Cell LinesObjective: To establish HEK293-OPRM1 cell line that stably expresses human μ-opioid receptor, HEK293-CCK1 R cell line that stably co-exprsses human μ-opioid receptor and CCK1 receptor, HEK293-CCK2 R cell line that stably co-exprsses human μ-opioid receptor and CCK2 receptor, and offer the tools for further studying the interaction of CCKR and MOR.Methods: ① Designed the following primers: OPRM1-His tag primers with enzyme Not I, His tag –IRES-Ds Red monomer primers with enzyme Xba I,CCK1R- V5 tag primers with enzyme Eco RI, V5 tag-IRES-EGFP primers with enzyme Xho I, CCK2R- Flag tag primers with enzyme Eco RI, Flag tag-IRES- EGFP primers with enzyme Xho I. Amplify the gene fragment with different plasmid. Purify by electrophoresis. With DNA fragments OPRM1-His tag and His tag- IRES- Ds Red monomer to the template amplification fragment OPRM1 gene- His tag- IRES- Ds Red monomer. With DNA fragments CCK1R-V5 tag and V5 tag- IRES- EGFP to template amplification fragment CCK1R- V5 tag- IRES – EGFP. With DNA fragments CCK2 R –Flag tag and Flag tag- IRES- EGFP to template amplification fragment CCK2 R –Flag tag- IRES – EGFP. Purified by electrophoresis.Double enzyme digestion and transformed the target gene fragment into plasmid to bacteria, positive clones were selected for sequencing verification.② OPRM1 plasmid was transfected into HEK-293 cells and screened the stable expression of OPRM1 gene by Hygromycin, detected by PCR. The CCK1 R, CCK2 R plasmids were transfectted into OPRM1-HEK293 cell line respectively, screened by Zeocin, detected the expression of target gene by PCR.Results: ① DNA sequencing of constructed plasmids were blasted in NCBI, the result was completely consistent with the human MOR receptor,CCK1 R, CCK2 R m RNA sequence.② HEK293-OPRM1 cell line screened by 100μg/ml hygromycin could be seen the red fluorescence with fluorescence microscope. The expression level of OPRM1 gene was 8287.65 times of control group cells, indicating stable cell line was constructed successfully. ③ HEK293-CCK1 R cell line and HEK293-CCK2 R cell line were screened by 300μg/ml zeocin, could be seen green fluorescence with fluorescence microscope. The expression level of CCK1 R gene was 32993.35 times of control group cells, CCK2 R gene was970 times of control group cells. ④ Stably transfected cells after ten passages,MOR, CCK1 R, CCK2 R gene expression is still high.Summary: This part of study successfully established the vectors of P3.1-hygro-OPRM1-Histag-2, P3.1-Zeo-CCK1R-V5tag-1 and P3.1-ZeoCCK2R-flag-1, the stable HEK293- OPRM1 cell line expressing MOR. We successfully established the HEK293-CCK1 R cell lines co-expressing MOR and CCK1 receptor and the HEK293-CCK2 R cell lines co-expressing MOR and CCK2 receptor for the first time.Part Ⅱ The different effects of CCK receptor subtypers on acute and chronic actions of morphineObjective: We aimed to determine the exact effects of CCK receptor subtypes on acute and chronic actions of morphine by comparing the changes of c AMP content in the stable transfected cell lines and the knocked down cells.Methods: ① The stable transfected cell lines were treated with or without10μM morphine for 15 min or prtreated by 10μM morphine for 6h, 12 h, 16 h,24h, and then precipitated by 100μM naloxon. Then we tested the content of c AMP by using a homogeneous time-resolved fluorescence resonance energy transfer(TR-FRET) c AMP detection kit.② The CCK1 R or CCK2 R of the HEK293-OPRM1 cell line was knocked down by transfecting si RNA. The content of c AMP were tested after the knocked-down cells treated with 10μM morphine for 15 min or 4h.Results: ① In HEK293-OPRM1 cells, after 10μM morphine administration for 15 min, 5μM forskolin-induced c AMP accumulation were inhibited robustly, and 100μM naloxon could reversed this inhibition. After chronic morphine treated for 4h, the 5μM forskolin-induced c AMP accumulation increased definitely. After chronic morphine treated for 12 h, c AMP overshoot was induced by treatment of 100μM naloxone for 15 min.② Over-expressing of CCK1 R and CCK2 R attenuated the inhibition on c AMP by acute morphine treated, especially CCK1 R.③The cAMP overshoot was not induced by 10μM morphine pretreated for 6h, 12 h, 16 h and 24 h in HEK293-CCK1 R cells, which suggested overexpressing CCK1 R inhibit morphine dependence.④The c AMP overshoot appeared at 6h in HEK293-CCK2 R cells, earlier than in HEK293-OPRM1 cells, which suggested over-expressing CCK2 R promote morphine dependence.⑤ The inhibition of CCK1 R expression attenuated the acute morphine effects. The inhibition of CCK1 R and CCK2 R expression enhanced the production of c AMP precipitated by naloxon at 4h, especially si CCK1 R.Summary: ① CCK1 R mediated anti-morphine acute and chronic effects. ② CCK2 R attenuated the acute morphine effects, but time-dependent promoted morphine dependece development,which showthe two receptors have different effects on chronic morphine.Part Ⅲ Mechanism of CCK receptor subtypes on acute and chronic effects of morphineObjective: We aimed to observe the effects of CCK receptor subtypes on MOR phosphorylation, CREB and ERK1/2 activation after acute or chronic morphine treated.Methods: To test the changes of p-MOR, p-CREB, CREB, p-ERK1/2and ERK1/2 by western blot after acute or chronic morphine administrated the stable transfected cell lines.Results: ① Over-expressing CCK1 R attenuated the inhibition of p-CREB, up-regulation of p-MOR and p-ERK1/2 induced by acute morphine treated, but over-expressing CCK2 R attenuated the effects on p-CREB and p-MOR, did not affected the action of ERK1/2. ② Only p-ERK1/2 incresed in HEK293-OPRM1 cells, but both p-CREB and p-ERK1/2 increased by over expressin CCK2 R at 6h. At 12 h both p-CREB and p-ERK1/2 increased in HEK293-OPRM1 cells and HEK293-CCK2 R cells. Over-expressing CCK1 R reversed the regulation of CREB and ERK1/2 activation by chronic morphine.Summary:1 CCK1 R inhibited the regulation of ERK1/2 and CREB by acute and chronic morphine action.2 CCK2 R attenuated the acut morphine inhibition of CREB activation, but time-dependent promoted the up-regulation of activation of CREB and ERK1/2 by chronic morphine treated.3 CCK1 R and CCK2 R attenuated the acut morphine induced phosphorylation of MOR, especially CCK1 R.Conclusions:1 Established the stable HEK293-OPRM1 cell line expressing MOR successfully. Established the stable HEK293-CCK1 R cell line co-expressing MOR and CCK1 R, the stable HEK293-CCK2 R cell line co-expressing MOR and CCK2 R successfully for the first time in the world.2 Both CCK receptors attauated the inhibition of c AMP by acute morphine treated especially CCK1 R. CCK1 R blocked the c AMP over shoot by naloxon precipitated but CCK2 R promoted the c AMP over shoot.3 CCK receptors attauated the inhibition of p-CREB by acute morphine treated especially CCK1 R. CCK1 R blocked the regulation of CREB and ERK1/2 activation, but CCK2 R promote the up-regulation of CREB and ERK1/2 activation induced by chronic morphine action.4 CCK receptors inhibited the morphine-induced phosphorylation of MOR, especially CCK1 R.