Effects Of CCK-8 On Ca²⁺-CaM-CaMKⅡ Signal Transduction In Morphine Withdrawal Rat

Long-term opioid abuse leads to physical and psychological dependence. Uptake of a large amount of exogenous opiate-like compounds (EOC) inhibits the production and release of the endogenous opioid peptides (EOP). Once the drug is discontinued, both EOP and EOC will lack and severe functional disorder will appear,such as withdrawal symptoms and craving for drugs. The physical and psychological dependence are the main reasons for drug abuse, the study to its mechanism is a hot issue. However, the mechanism of opiate addiction is still unclear. For a long time, the change of the endogenous opioid peptides can not explain the phenomenon of opioid addiction. Therefore, researches point to postsynaptic cellular mechanism of morphine dependence. The change of intracellular calcium (Ca2+), calmodulin(CaM) and Ca2+/ calmodulin- dependent protein kinaseⅡ(CaMKⅡ), play important roles in morphine dependence and withdrawal.Cholecyetokinin (CCK), a typical brain-gut peptide, is discovered initially in the gut as a gastrointestinal hormone with the function of contracting gallbladder and mediating pancreatic secretion, and subsequently localized in the central and peripheral nervous system as a neurotransmitter or neuro -modulator. CCK is identified as several different size of the peptide. The sulfated carboxyl-terminal octapeptide (sCCK-8) is the biologically predominant active form localized in the small intestine, blood and CNS system. It is well known that CCK exerts a variety of physiological actions through its cellular surface receptors. CCK1 receptor is mainly distributed in peripheral tissue; CCK2 receptor mainly located in the central nervous system. Researches showed that CCK-8 as an anti-opioid substances can fight against opioid-induced increase in food, morphine analgesia, electroacupuncture. CCK receptor antagonists are not only applied to symptomatic treatment of withdrawal symptoms, but also to relapse prevention, but the mechanism is unclear. Cortex, hippocampus, caudate putamen, are important brain regions thought to account for morphine dependence. Here, we established the models of morphine dependent rats by subcutaneous injection of morphine in gradually increasing doses. The withdrawal syndrome was precipitated with naloxone. The study is to elucidate the molecular mechanism of CCK-8 and its receptor antagonists by detecting the changes of intracellular calcium, calmodulin, CaMKⅡ.Methods: Sixty-six healthy male Wistar rats, 190-210g, were randomly divided into eleven groups: N.S control group morphine dependent group (MOR), naloxone precipitated withdrawal group (MOR+NAL), chronic CCK-8 treatment group (CCK-8+Mor+Nal), chronic CCK1 receptor antagonist L- 364,718 treatment group (L-364,718+Mor+Nal), chronic CCK2 receptor antagonist LY-288,513 treatment group (LY- 288,513+Mor+Nal), chronic vehicle group (vehicle+Mor+Nal), acute CCK-8 treatment group (Mor+ CCK-8+Nal), acute CCK1 L-364,718 treatment group (Mor+L-364,718+Nal), acute CCK2 LY-288,513 treatment group (Mor+LY-288,513+Nal), and acute vehicle group (Mor+Vehicle+Nal). The morphine dependent model in rats was establishshed by subcutaneous injection of morphine. The daily dose gradually increased as following: 10, 20, 30, 40, 50mg·kg-1, twice a day, at 8:00 and 20:00, for 5 days. On day 6, 50 mg·kg-1 morphine was injected at 8:00. With- drawal syndromes was precipitated by intraperitoneal injection of naloxone (5mg·kg-1). Rats were euthanized to isolate cortex, hippocampus and caudate putamen. Cytoplasmic free calcium ([Ca2+]i), CaM activity and CaMKⅡprotein expression were measured by flow cytometry and Western blot, respectively.Results:1 Effects of CCK-8 and its receptor antagonists on morphine withdrawal symptomsThe body weight loss of morphine withdrawal rats increased, compared with Mor group (P<0.01). Withdrawal symptoms apppered, such as wet dog shakes, teeth chattering, tears, salivation and other symptoms, suggesting that the models were successful. CCK-8 and its receptor antagonists could decrease the body weight loss and attenuate other withdrawal symptoms.2 Effects of CCK-8 and its receptor antagonists on the intracellular [Ca2+]i in different brain regions of morphine withdrawal ratsCompared with N.S group, [Ca2+]i of Mor group in cortex , hippocampus, caudate putamen increased (P<0.01), but [Ca2+]i decreased in Nal group, compared with Mor group (P<0.01).Compared with Nal group, chronic CCK-8 and its receptor antagonist treatment increased [Ca2+]i in hippocampus, caudate putamen (P<0.05 or P<0.01). No change were observed in cortex.. Acute CCK-8 receptor antagonists treatment increased [Ca2+]i (P<0.05 or P<0.01) in hippocampus, caudate putamen, while no change in cortex. Vehicle treatment and acute CCK-8 treatment could not induce changes in [Ca2+]i in cortex, hippocampus and caudate putamen (P>0.05).3 Effects of CCK-8 and its receptor antagonists on the intra- cellular CaM activity in different brain regions of morphine withdrawal ratsIn cortex, hippocampus and caudate putamen, CaM activity increased in Mor group, compared with N.S group (P<0.01), but it decreased in Nal group, compared with Mor group (P<0.01).Chronic CCK-8 and its receptor antagonists treatment increased CaM activity, compared with Nal group (P <0.05 or P<0.01). Acute CCK-8 receptor antagonists treatment increased CaM activity (P<0.01) in hippocampus, caudate putamen, while no change in cortex. Vehicle treatment and acute CCK-8 treatment could not induce changes in CaM activity in cortex, hippocampus and caudate putamen (P>0.05). 4 Effects of CCK-8 and its receptor antagonists on CaMKⅡprotein expression in different brain regions of morphine withdrawal ratsCaMKⅡprotein expressions in cortex, hippocampus, caudate putamen were higher in Mor group than in N.S group (P<0.01).Chronic CCK-8 and its receptor antagonists treatment increased CaMKⅡprotein expression, compared with Nal group (P<0.01). Acute CCK-8 receptor antagonists treatment increased CaMKⅡprotein expression in hippocampus (P<0.01), while no change in cortex or caudate putamen (P>0.05). Vehicle treatment and acute CCK-8 treatment could not induce change on CaMKⅡprotein expression in cortex, hippocampus, caudate putamen(P>0.05)Conclusion:CCK-8 and its receptor antagonists could significantly inhibit the naloxone-precipitated symptoms through regulating Ca2+-CaM-CaMKⅡsignaling pathway, with significant specifi- city in different brain regions.