Mechanism Of AQP4 Participating In Morphine Dependence

Objective: The drug abusen has seriously affected the social stability and economic development. On the degree of harm, spread and influence scope, morphine is a serious type of drugs in our country. Morphine is the main component of opium, which is a potent analgesic. People found that repeated administration of therapeutic doses of morphine, its effect will be weakened, and the tolerance is gradually formed. The dose must be increased to maintain its effect. Drug abusers intend to experience and pursuit morphine euphoric effects and to avoid the discomfort of withdrawal. They gradually present the behavior of dependence and addiction. At present, most scholars believe that the essence of the psychoactive drug dependence, including opioid dependence, is the long-term exposure to substances, the compensatory adaptation occurred in the central nervous system on the molecules, cells and neural network level. The mechanism of morphine addiction is still not fully elucidated at present. Almost all of the neurotransmitter in the central nervous system is participating in different degrees. And this process is not only occurred in neurons, besides glial cells are also involved. Glutamate is the major excitatory neurotransmitter in the central nervous system, which is involved in the formation of morphine dependence, and may be one of the important molecular mechanisms of morphine dependence. The concentration of extracellular glutamate depends mainly on the regulation of glutamate transporters; Glutamate Transporter-1(GLT-1) is the most widely distributed glutamate transporter. It is reported that GLT-1 is involved in the formation of morphine dependence. Aquaporin4(AQP4) is the most abundant AQPs in the central nervous system.Our previous experiments demonstrated that AQP4 is closely related with the addiction of morphine in morphine dependent formation, expression and function of AQP4 and GLT-1 in the process has a certain degree of positive correlation. It is reported that AQP4 and GLT-1 were specifically distributed in the astrocytes, which are involved in the process of morphine dependence, suggesting that AQP4 may regulate the expression and function of GLT-1 in astrocytes, and affect the synaptic glutamate levels to influence the development of morphine dependence. We hypothesized that AQP4 may form a macromolecular complex with GLT-1. At the same time, whether the specific expressed AQP4 and GLT-1 in astrocytes surface would form a complex with mu opioid receptor(MOR) and directly affect opioid receptor system, to participating inmorphine dependence. It is reported that during opioid administration the brain PKC activity is increased, which may be associated with opioid dependence and tolerance. At the same time, the expression of AQP4 was regulated by PKC; the expression of PKC can inhibit the activation of AQP4. Therefore, we hypothesized that in the formation process of morphine dependence, morphine activates PKC signal pathway though opioid receptor, the activation of PKC would regulate the expression of AQP4 and AQP4 related complexes, which would influence the formation of opioid dependence. This study intend to investing the function of AQP4 in morphine dependent process, and finding new areas of biological research for morphine dependence. Methods: 1) Male Rats were given for 5 days twice daily ascending doses of morphine(s.c.) at 8:00 and 20:00 according to the following schedule: day 1, 10 mg/kg; day 2, 20mg/kg; day 3, 30mg/kg; day 4, 40mg/kg; and day 5, 50 mg/kg to establish the morphine dependence model. 5 h after the last morphine or saline injection, naloxone was injected, and withdrawal symptoms were scored for 15 min to evalutate the model. To invest change of the expression of mRNA and protein level of AQP4,GLT-1,MOR in the prefrontal cortex, hippocampus, striatum and nucleus accumbensin by Real-time PCR and Western blot methods in rat morphine physical dependence model; 2) The method of culturing primary astrocytes It is modified by adding d Bc AMP. Astrocytes which were cultured for 15 d were treated with 1μM morphine for 72 h to establish morphine dependence model. Astrocytes were treated with 10μM naloxone after 72 h exposure,the occurrence of c AMP overshooting was observed to evaluate the model. To invest change of the expression of mRNA and protein level of AQP4,GLT-1,MOR by Real-time PCR and Western blot methods in astrocytes dependence model; 3) Co-location was observed by Immunofluorescence staining of AQP4, GLT-1, MOR on brain slice and astrocyte. The existence of large molecular complexes was proved by Co-immunoprecipitation(Co-IP) method and fluorescence resonance energy transfer(FRET) method; 4)Establish of the expression plasmid of AQP4 and GLT-1, in order to further validating the existence of complexes, and to seeking the possible interaction sites; 5) Observe the effect of PKC signaling pathway on expression of AQP4, GLT-1, MOR by administration of PKC agonist PMA and antagonist chelerythrine in astrocytes, to invest the relationship between the expression of these three molecules and morphine dependence in the process, and further elucidating the mechanism. Results: 1) The rat physical dependence model was successfully established. Morphine dependence rats showed obvious withdrawal symptoms including jumping, wet-dog shaking, writhing posture, paw tremors, genital grooming, teeth-chattering, chewing, salivation, rearing, ptosis, diarrhea, and irritability, while the group with the administration of naloxone did not appear obvious withdrawal symptoms. The mRNA and protein of AQP4, GLT-1 and MOR in four brain regions of the prefrontal cortex, hippocampus, striatum and nucleus accumbensin showed a downward trend in morphine dependence group, while the decrease of AQP4, GLT-1 and MOR were inhibited in naloxone group. 2) Method for primary culture of astrocytes was optimized, and the astrocytes morphine dependence model was successfully established. Morphine dependence group showed the phenomenon of naloxone overshooting. In morphine dependent cells, the mRNA and protein level of AQP4、GLT-1 and MOR showed a downward trend, while the down-regulation was inhibited in naloxone group. 3) It is demonstrated that in the rat cerebral cortex and astrocytes, AQP4, GLT-1 and MOR is co-localization by immunohistochemistry. Further evidence showed that endogenous AQP4 and GLT-1 in rat cerebral cortex and in vitro astrocytes could be reciprocal precipitation and showed FRET phenomenon by using the method of Co-immunoprecipitation and FRET. AQP4 and MOR could also be reciprocal precipitation and showed FRET phenomenon, while GLT-1 and MOR could not be reciprocal precipitation and showed no FRET phenomenon. 4) The vectors of AQP4, GLT-1 and AQP4 C terminal deletion were constructed, and wherein the expression in HEK293 cells was higher. AQP4-pEGFP-N1 and GLT-1-pDsRed were transferred to 293 cells, and exogenous expressed AQP4 and GLT-1 could also be co-immunoprecipitation, while there is no reciprocal precipitation for the expression of AQP4 C terminal deletion and GLT-1.5) Treated with PKC agonist PMA can reduce AQP4、GLT-1 and MOR expression. With PKC antagonist Chelerythrine before chronic morphine treatment, the down-regulation of AQP4, GLT-1 and MOR caused by chronic morphine treatment were inhibited. Conclusion: 1) mRNA and protein expression level of AQP4, GLT-1, MOR was found to be down regulated in rat morphine physical dependent model and astrocytes morphine dependent model. With early administration of naloxone, antagonist of MOR, the decline could be inhibited, which means the change of the three molecules in morphine dependence process is directly related to μopioid receptor. 2) In physiological condition, AQP4 and GLT-1 exist in a complex form, and at the same time, AQP4 and MOR was also a complex formation, which is involved in the regulation of morphine dependence. GLT-1 and MOR could not form complexes although the distribution is overlapping. 3) Validation of the AQP4 and GLT-1 complexes was made for expression AQP4 and GLT-1 in 293 cells. The interaction region of the complex may be related to the C terminal of AQP4. 4) The Mechanisms of AQP4 involving in morphine dependent is PKC signal pathway, blocking PKC signal pathway can inhibit the reduction of AQP4, GLT-1, MOR caused by chronic morphine treatment.