Effects Of Exogenous CCK-8on Morphine Dependence And Relapse And Its Mechanism

Opioids are not only potent analgesics but also drugs of abuse. Chronic use of opioids results in the development of tolerance and dependence. Opioid abuse will decrease the health level of individuals, resulting in a rising social crime rate and threatening the public stability seriously. The study of drug abuse is an important item in forensic toxicological research. Currently, the drug abuse in China is quite severe. It is of deep practical significance and social value to develop effective drugs for abuse treatment.Opioid dependence is a chronic relapsing disorder in the brain characterized by compulsive drug taking, inability to limit the intake of drugs, and the emergence of withdrawal syndrome after cessation of drug taking. Nevertheless, the exact mechanisms underlying opioid dependence are still unknown. 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. Cholecystokinin octapeptide (CCK-8) is the most potent endogenous anti-opioid peptide. Studies have shown that CCK-8blocked the binding of opioid to its receptor, and morphine or endogenous enkephalins enhanced the K+-evoked overflow of CCK from rat substantia nigra as feedback. CCK receptor antagonist administrations have been shown to prevent tolerance to systemic exogenous opioids and electroacupuncture induced analgesia. Besides the anti-opioid functions described above, as a neuro-transmitter and modulator, the CCK system regulates a variety of physiological processes, including pain, cognition, reward and learning or memory. CCK and CCK receptor mRNA are expressed in the cerebral cortex, amygdala, nucleus accumbens (NAc) and ventral tegmental area (VTA), important areas thought to modulate the rewarding effects of the addictive properties of drugs. Based on this profile, we conclude that the CCK system may be involved in the rewarding effects of drugs. Mitchell et al. examined the effects of CCK antagonists on the rewarding effect of morphine by means of the conditioned place preference (CPP) paradigm. They reported that endogenous CCK was necessary for the expression of morphine CPP. Additionally, a CCK receptor antagonist suppresses morphine dependence and withdrawal syndrome, as well as blocks reinstatement of cocaine or morphine induced CPP. These results suggest an important role of endogenous CCK system in the process of opioid denpendence.Interestingly, we previously found that pretreatment with exogenous CCK-8significantly attenuated withdrawal symptoms, appearing to exert the same effect as CCK receptor antagonists. Other studies also have reported that CCK receptor activation is able to reverse tolerance to morphine antinociception. These results suggest that the regulating effect of exogenous CCK-8is distinct from the role of endogenous CCK, and the mechanism was not clear. We suppose that the conflicting phenomena may be related to the specific dose-response curve of CCK-8and sensitivity of CCK receptor. The mechanism of opioid dependence involves many aspects including the opioid receptor, neurotransmitter, neuropeptide, intracellular signal transduction, ion channel, nerve nucleus and neural circuit, learning and memory as well as rewarding effect, etc. As one of the most essential neurobiological mechanisms during the process of drug dependence and withdrawal, the neurotransmitter plays an important role in the process of drug addiction. However, the relation of CCK-8with the neurotransmitter is not clear. The mechanism of CCK-8regulation in the long term adaption of drug addiction needs to be studied.Based on these profiles, this study aimed to evaluate the influence of CCK-8on cellular morphine dependence and morphine induced CPP to clarify the exact effects of CCK-8in vivo and vitro. Furthermore, the interaction of CCK and opioid system, as well as the neurochemistry mechanism was explored to provide systematic and reliable theoretical base for the application of CCK-8in prevention and treatment of morphine dependence.1Effects of exogenous CCK-8on psychological morphine dependence and relapse in ratsObjective:We aimed to determine the exact effects of CCK-8at various doses on the acquisition, expression and extinction of morphine induced conditioned place preference (CPP), as well as reinstatement of CPP and locomotor sensitization for investigating the influence of exogenous CCK-8on psychological morphine dependence and relapse, respectively.Methods:Morphine (10mg/kg, i.s.) and CCK-8(0.01,0.1,1μg, i.c.v.) were injected before each programed training for seven days to build the rat model of CPP (unbiased design), and to investigate the the ability of morphine and CCK-8itself in CPP establishment. Rats were treated with CCK-8(0.01,0.1,1μg, i.c.v.) before morphine administration to observe the effect of CCK-8on the acquisition of morphine induced CPP. Subsequently, CCK-8(0.01,0.1,1μg, i.c.v.) was given15min before CPP test to observe the effect of CCK-8on the expression of morphine induced CPP. After CPP test, the10days’extinction procedure without any treatment was performed for evaluating the effect of CCK-8on the extinction of morphine induced CPP. Thirdly,3mg/kg morphine was administered after the acquisition and extinction of CPP to induce the reinstatement of CPP, and CCK-8was given15min before CPP reinstatement to investigate the effect of CCK-8on the process of relapse. The fourth, rats were treated with10mg/kg morphine for7days and with no treatment for another10days. The total distance in30min was recorded in the locomotor test after the above treatment. CCK-8was given15min before morphine administration in the stage of locomotor sensitization expression to observe the effect of CCK-8on morphine induced locomotor sensitization. At last, the rats were treated with CCK-8(0.01,0.1,1μg, i.c.v.), and the spontaneous locomotor test was performed. The total distance in30min was recorded to observe the effect of CCK-8on the spontaneous locomotion.Results:①CCK-8(0.01,0.1,1μg, i.c.v.), administered alone, induced neither CPP nor place aversion.②CCK-8(0.01,0.1,1μg, i.c.v.) significantly blocked the acquisition of CPP and reduced the CPP scores when administered with10mg/kg morphine.③The highest dose of CCK-8(1μg) administered before CPP testing increased CPP and, along with lower doses (0.1μg), reduced its extinction.④3mg/kg morphine successfully provoked the reinstatement of CPP, and CCK-8(0.1、1μg, i.c.v.) effectively inhibited the reinstatement of morphine induce CPP when administered with lowe dose morphine while0.01μg CCK-8did not.⑤CCK-8(0.1、1μg, i.c.v.) significantly attenuated the expression of morphine induced locomotor sensitization. Similarly,0.01μg CCK-8has no effect on it.⑥The highest dose (1μg) of CCK-8suppressed locomotor activity and significantly decreased the total distance in30min.Summary:This part of study successfully established the CPP model in rats, and provides the first behavioral evidence for the inhibitory effects of exogenous CCK-8(0.01,0.1,1μg) on the acquisition of morphine induced CPP. Meanwhile, we found that CCK-8(0.1,1μg) significantly attenuated the reinstatement of CPP elicited by low dose morphine, as well as morphine induced locomotor sensitization. Above results reveal significant regulation of exogenous CCK-8on the process of psychological morphine dependence and relapse.2Effects of exogenous CCK-8on cellular morphine dependence in SH-SY5Y cells and its receptor mechanismObjective:We aimed to observe the exact effect of exogenous CCK-8on cellular morphine dependence, and the dose-response relationship of CCK-8Furthermore, the role of CCK receptor sensitivity in CCK-8regulation on morphine dependence was investigated.Methods:To determine if the SH-SY5Y cell was suitable for establishing the cellular model of the present study, the co-expression of μ opioid receptor, CCK1and CCK2receptors in SH-SY5Y cell was detected by utilizing RT-PCR. The retinoic acid (RA) differentiated SH-SY5Y cell was treated with10μM morphine for48h to build the cellular model of morphine dependence, and then cAMP overshoots and acute morphine withdrawal was induced by treatment of10μM naloxone for15min. Moreover, cAMP overshoot was used as an evaluation index for observing the effect of CCK-8and CCK1/2receptor antagonists on cellular morphine dependence.Results:①μ-opioid receptor, CCK1and CCK2receptors, and endogenous CCK were co-expressed in SH-SY5Y cell, which showed that SH-SY5Y cell was suitable for building the cellular model.②RA differentiated SH-SY5Y cells were treated with morphine (10μM) for48h, and the content of cAMP obviously increased. After that, naloxone (10μM) made the cAMP content up-regulated3.09±0.28fold and induced a cAMP overshoot, indicating that cellular morphine dependence had been built. Moreover, CCK receptor and endogenous CCK were up-regulated after chronic morphine exposure.③CCK2receptor antagonist (LY-288,513) at1-10μM inhibited the naloxone-precipitated cAMP overshoot, but CCK1receptor antagonist (L-364,718) did not.④CCK-8(0.1-1μM), a strong CCK receptor agonist, dose-dependently inhibited naloxone-precipitated cAMP overshoot in SH-SY5Y cells when co-pretreated with morphine. L-364,718completely blocked the inhibitory effect of exogenous CCK-8on the cAMP overshoot at1-10μM, while LY-288,513did not. Therefore, the CCK2receptor in SH-SY5Y cells appears to be necessary for low concentrations of endogenous CCK to potentiate morphine dependence. An additional inhibitory effect of CCK-8at higher concentrations seems to relate to the CCK1receptor.Summary:This study reveals the difference between exogenous CCK-8and endogenous CCK in the effect on morphine dependence development, and provides the first evidence for participation of the CCK1receptor in the inhibitory effects of exogenous CCK-8on morphine dependence.3Effects of CCK-8on the endogenous opioid systemObjective:We proceed to the observation of different concentrations of CCK-8regulation on endogenous opioid system of celluar morphine dependence model in SH-SY5Y cells. Furthermore, we preliminary discussed the interaction between CCK-8and endogenous opioid system in the process of morphine dependence and its mechanism.Methods:The radio-ligand binding assay was utilized to observe the effect of CCK-8at various concentrations on the binding characteristic ofμ opioid receptor (MOR) in rat brain and SH-SY5Y cell membrane lysate. The effect of CCK-8at various concentrations on the expression of PENK and POMC gene in SH-SY5Y cell were studied by Real-Time PCR. The SH-SY5Y cell model of chronic morphine dependence was established. The change of the expression of PENK and POMC and the effects of CCK-8on the expression of PENK and POMC gene in morphine treated SH-SY5Y cell were also investigated.Results:①The Bmax of μ opioid receptor in rat brain was82.9±7.2pmol·mg-1Pro, and Kd was1.38±0.05. CCK-8suppressed dose-dependently the binding of [3H]DAMGO to μ opioid receptor, as well as reduced the Bmax of μ opioid receptor binding action with no effect on Kd.②The Bmax of μ opioid receptor in RA differentiated SH-SY5Y cells was378.3+48.9pmol·mg-1Pro, and Kd was0.79±0.09. CCK-8suppressed dose-dependently the binding of [3H]DAMGO to μ opioid receptor, and this effect could be reversed by CCK1and CCK2receptor antagonist (L-364,718, LY-288,513).③The expression of CCK1, CCK2receptor and endogenous CCK mRNA in RA differentiated SH-SY5Y cells were up-regulated to a level of2.51±0.56,6.10±0.91and4.87±1.08times as the level before10μM morphine incubation for48h.④RA differentiated SH-SY5Y cells were treated with10-10M-10-6M CCK-8for48h. The results revealed that10-7and10-6M CCK-8increased the expression of PENK and POMC in RA differentiated SH-SY5Y cells, and this effect could be reversed by CCK1receptor antagonist (L-364,718).⑤The expression of PENK and POMC in RA differentiated SH-SY5Y cells were down-regulated by10μmol·L-1morphine incubation for48h as compared with control group. Furthermore,10-8,10-7and10-6M CCK-8co-treated with morphine significantly up-regulated the deceased PENK and POMC expression as compared with morphine group. Summary:①Low concentration (10-10,10-9M) of CCK-8could suppress μ opioid receptor in SH-SY5Y cells by activating CCK receptor, but no effect on PENK, POMC gene expression.②High concentration (10-8-10-6M) of CCK-8could up-regulate the expression PENK, POMC gene, and improve the imbalance of endogenous opioid system through increasing endogenous opioid peptide release.4Effects of CCK-8on monoamine and amino acid neurotransmitters in SH-SY5Y cell cultural supernatantObjective:We detected the content of DA,5-HT, NE, Glu, GABA and its metabolin in the SH-SY5Y cell-cultural supernatant with the high performance liquid chromatography mass-spectrometric (LC-MS) technique, for inspecting the influence of acute morphine and CCK-8treatment on the content of neurotransmitters in cultural supernatant. Furthermore, the neurobiological mechanism during the process of the CCK-8adjustment of morphine addiction was clarified.Methods:We have cultured the SH-SY5Y cells, changed into HBSS buffer solution for cell culture6days after RA differentiation, added the corresponding medicines for stimulation after24h and taken out100μl of supernate after10,20,40,60min respectively. Detect the content of the nine neurotransmitters--DA, DOPAC, HVA,5-HT,5-HIAA, NE, MHPG, Glu and GABA and their metabolin and inspect the influence of acute morphine and CCK-8treatment on the content of neurotransmitters in cultural supernatant.Results:①SH-SY5Y cells after RA differentiation are cultured with100μM of morphine and part of supernate is taken out after10,20,40,60min respectively for LC-MS detection, and the result show that the content of monoamine neurotransmitters—DA, NE and5-HT and their metabolin in the cell-cultural supernatant declines substantially in10to20min and gradually declines to and maintains at a relatively low level. In the meantime, the release of excitatory amino acid (Glu) also declines substantially and the release of inhibitory amino acid (GABA) rises. This shows that the direct effect of activated opiate receptors on the neuron is inhibitory.②Cells are cultured with100μM of morphine and10-6M CCK-8and it is found out that the contents of DA, NE,5-HT and Glu and their metabolin in the cultural supernatant are decreasing but obviously higher that the transmitter level in the cell supernate treated with morphine only; the content of GABA also rises to a certain extent but obviously lower than that in the cell supernate treated with morphine only. This shows that CCK-8can effectively antagonize the inhibitory effect of acute morphine treatment on cells.③SH-SY5Y cells are treated acutely with CCK-8only, and results show that CCK-8can decrease the contents of DA and its metabolin in the cell-cultural supernatant which reaches the lowest level after about20min, and then gradually rises; besides, with the acute treatment of CCK-8, the content of GABA rises and that of Glu declines while there is no obvious change to the contents of NE and5-HT and their metabolin.Summary:In this part of the experiment, we have inspected, based on the SH-SY5Y model, the influences of the acute joint and individual treatment by CCK-8and morphine on the monoamine and amino acid neurotransmitters. Results show that the direct effect of the acute individual morphine or CCK-8treatment on the SH-SY5Y cell is inhibitory but CCK-8can antagonize the inhibitory effect of the acute morphine treatment, indicating that CCK-8can apply the "anti-opioid" effect on the neurotransmitter level through the interaction between receptors.Conclusions:The present study observed the effect of exogenous CCK-8on the process of morphine dependence in vivo and in vitro. We also explored the receptor and neurochemical mechanism, and investigated the regulation of CCK-8on the endogenous opioid system. We can reach the conclusion as follows.1Exogenous CCK-8given intracerebroventricularly inhibited the acquisition of morphine induced CPP. Meanwhile, we found that CCK-8significantly attenuated the reinstatement of CPP elicited by low dose morphine, as well as blocked morphine induced locomotor sensitization. Above results reveal significant regulation of exogenous CCK-8on the process of psychological morphine dependence and relapse.2The effect of exogenous CCK-8on morphine dependence development is distinct from that of endogenous CCK. The endogenous CCK potentiated the morphine dependence via CCK2receptor, while exogenous CCK-8attenuated it by activating CCK1receptor.3Low concentration CCK-8could suppress the binding feature of μ opioid receptor by activating CCK receptor, but no effect on PENK, POMC gene expression. High concentration of CCK-8could up-regulate the expression PENK, POMC gene, and improve the imbalance of endogenous opioid system through increasing endogenous opioid peptide release.4The acute individual morphine or CCK-8treatment exerts a direct inhibitory effect on the SH-SY5Y cell. Furthermore, CCK-8can antagonize the inhibitory effect of the acute morphine treatment, indicating that CCK-8can apply the "anti-opioid" effect on the neurotransmitter level through the interaction between receptors.