Study On Changes Of Heroin Addicted Rats' Purine Nucleotide And Glutamate Metabolism In Brain And Biological Effects Of Purine Nucleotide Compensation

Opioid like heroin abuse make the addicts shape psychological dependence and physical dependence to heroin and result in damage to multisystem.There is still no ideal drug for clinical therapy so far. Previous research work of our lab showed that both heroin and morphine promote purine nucleotide catabolism and inhibit purine nucleotide anabolism in several tissues. In this study, metabolism of brain purine nucleotide and glutamate, the most important excitory amino acid in brain, and biological effects of purine nucleotide compensation in addicted rats were investigated to discover the role of metabolism of brain purine nucleotide and glutamate in heroin addiction and withdrawal and to provide theoretical support to clinical detoxification therapy.1. Establishment of heroin addiction modelAdult male Wistar rats were randomly divided into control group (ip normal saline for 10 days), nucleotide group (ip AMP and GMP equimolar mixture for 10 days), heroin group (ip heroin for 10 days), heroin and nucleotide administration group (ip heroin and AMP and GMP mixture for 10days), withdrawal 3 days group and withdrawal 9 days group (withdraw for 3 days and 9 days respectively after 10 days heroin treatment), nucleotide 3 days group and nucleotide 9 days group (compensation nucleotide for 3 days and 9 days respectively following 10 days heroin treatment). Conditioned place preference (CPP) trial and abstinence symptom evoked by naloxone were done to test the establishment of heroin addition model. Antinociception was measured by changes in tail-flick latency.In the CPP trial, total time staying in the heroin-pairing side increases significantly after 9 days heroin administration which proves that the rats formed CPP to heroin. In contrast, control group and nucleotide groups showed obvious conditioned place aversion (CPA) because their total time staying in the drug-pairing side decreased dramatically after 5 days treatment and dropped even more after 9 days treatment. Heroin and nucleotide administration group's total time staying in the drug-pairing side changed little which confirm that purine nucleotide compensate with heroin inhibits rats'CPP to heroin. After 10 days heroin treatment, naloxone treatment evoked abstinent symptoms such as erection, wet dog shake, stretch, tooth chattering, ptosis and diarrhea.Above-mentioned results show that rats shaped psychological dependence and physical dependence after exposure to heroin for 10 days and the addiction model was successfully established and purine nucleotide compensate with heroin inhibits development of psychological dependence to heroin.In the tail-flick test, nociception of the rats subsided significantly after 6 days exposure to heroin and recovered after 2 days withdrawal. Purine nucleotide treatment showed no change in nociception while nucleotide compensate with heroin for 8 days significantly promote analgesia compared with merely heroin treated group, which suggest that purine nucleotide cooperate with heroin in antinociception.Heroin treated rats had a lower increasing rate of body weight than control ones and nucleotide and heroin administrated rats had a low increasing rate of body weight than control ones but a relative high rate than heroin treated rats.2. Effects of heroin and purine nucleotide compensation on related plasma indexesAdenosine deaminase (ADA) and xanthine oxidase (XOD) are key enzymes in purine nucleotide catabolism and uric acid (UA) is the deuto-end product of purine nucleotide in rodents. Activity of ADA and XOD and content of UA in heroin treated rats'plasma increased obviously compared with control ones while heroin and nucleotide administrated rats showed no difference in activity of ADA and XOD and content of UA in comparison with control ones, suggesting that heroin promotes rats'purine nucleotide catabolism in organism which could be inhibited by purine nucleotide compensation.Plasma creatinine (Cre) and urea nitrogen(UN), as index measuring renal function, increased simultaneously after 9 days withdrawal, suggesting that heroin affects renal function only after long term exposure and increased plasma UA result from purine nucleotide catabolism but not damaged renal function. 3. Effects of heroin and purine nucleotide compensation on ADA and XOD activity in rats'brainADA activity in midbrain, cortex, brain stem and cerebellum of heroin group increased significantly compared with control group, while these indexes of heroin and nucleotide administration group decreased in its degrees in comparison with heroin group.XOD activity in cortex, brain stem and cerebellum of heroin group increased dramatically compared with control group and those of heroin and nucleotide administration group decreased in various degrees too.These results suggest that heroin promotes purine nucleotide catabolism in above-mentioned brain region through enhances XOD and/or ADA activity and purine nucleotide compensation inhibits heroin induced ADA and XOD activity increase and then suppress over purine nucleotide decomposition.XOD activity in cortex, brain stem and cerebellum and ADA activity in cortex and brain stem showed no difference compare with control group, indicating that nucleotide may specifically inhibit or block heroin induced purine nucleotide decomposition rather than affect normal brain tissue directly and the detailed mechanism worth to be studied further.4. Effects of heroin and purine nucleotide compensation on mRNA level of purine nucleotide metabolism key enzymes in rats'brainSalvage synthesis is the main way of brain nucleotide anabolism. So adenosine kinase (AK) and hypoxanthine-guanosinephosphoribosyl tansferasel (HGPRT), key enzymes in nucleotide salvage synthesis, were chosen to evaluate purine nucleotide anabolism.AK and HGPRT mRNA had a lower expression in midbrain, cortex and brain stem of heroin group compared with control group while those of heroin and nucleotide administrated group were higher in different degree than heroin group. Brain stem AK mRNA of 3 days and 9 days withdrawal group decreased than control group and 9 days nucleotide group AK mRNA is higher than 9 days withdrawal group. Besides, AK mRNA in midbrain, cortex and cerebellum of 9 days nucleotide group is much higher than 9 days withdrawal group.ADA mRNA in midbrain, cortex, brain stem and cerebellum of heroin group increased significantly compared with control group while purine nucleotide administrated group decreased in different degree than heroin group. These results indicates that heroin inhibits mRNA expression of HGPRT and/or AK, the key enzymes in purine nucleotide salvage synthesis, in midbrain, cortex, brain stem and cerebellum and promotes mRNA expression of ADA, the key enzyme in purine nucleotide catabolism, in these region which could be inhibited by purine nucleotide compensation5. Effects of heroin and purine nucleotide compensation on Glu metabolism in rats'brainStudies focus on Glu receptors have showed that Glu, as a EAA, plays a role not only in development of opioid physical dependence but also in psychological dependence. However, the role of Glu metabolism changes remained unconcerned.Glutamate dehydrogenase(GDH) catalyzes deamination of Glu and glutamine synthetase(GS) catalyzes glutamine synthesis with Glu and ammonia. Both GDH and GS activity influence Glu concentration.In this study, heroin significantly suppressed GDH and GS activity in midbrain, cortex and cerebellum of rats and Glu concentration in these areas increased dramatically, suggesting that heroin increases midbrain, cortex and cerebellum Glu concentration through inhibits activity of GDH and GS.Midbrain GDH of heroin and nucleotide administration group was higher than heroin group and close to control group. Midbrain GDH of 3 days nucleotide group was higher than 3 days withdrawal group. Midbrain GS of 9 days nucleotide group was higher than 9 days withdrawal group. Correspondingly, Glu concentration of heroin and nucleotide administration group decreased significantly in comparison with heroin group. So, purine nucleotide compensation promotes midbrain GDH and GS activity and then enhances decomposition and utilization of GLu to inhibit heroin induced Glu concentration increase.No significant difference was found in cortex GDH and GS activity between heroin and nucleotide administration group and heroin group. But cortex GDH and GS of 3 days and 9 days nucleotide group were higher than 3 days and 9 days withdrawal group respectively. In consequence, Glu concentration of 3 days and 9 days nucleotide group decreased than corresponding withdrawal group.As far as brain stem is concerned, GDH of heroin and nucleotide administration group showed a significant increase compare with heroin group; GS of 3 days and 9 days nucleotide group were higher than 3 days and 9 days withdrawal group respectively; Glu of heroin and nucleotide administration group, 3 days and 9 days nucleotide group were all decreased in various degree than heroin group, 3 days and 9 days withdrawal group respectively; suggesting that purine nucleotide compensation decrease heroin induced Glu increase in brain stem.Different with above mentioned brain region, cerebellum GDH activity was enhanced by heroin treatment and cerebellum GS activity changed little but cerebellum Glu didn't change at all. The results indicate that Glu metabolism in brain is complicated and GDH and GS are important factors affecting Glu concentration but not unique one. Further study is needed to discover the mechanism.So, heroin increases Glu concentration in midbrain, cortex and brain stem through suppresses GDH and GS activity in these areas and then inhibits utilization and decomposition of Glu. The above effects could be blocked by purine nucleotide compensation, suggesting that purine nucleotide compensation may inhibit heroin dependence development or has therapeutic action.6. Effects of heroin and purine nucleotide compensation on morphology of midbrain and cortexMorphological observation showed that midbrain and cortex of control group were normal under light microscope and electron microscope.Cortex of heroin group showed little change under light microscope and a great quantity of dark cells under electron microscope.Midbrain of heroin group presented a great quantity of dark cells under electron microscope.Cortex and midbrain of heroin and nucleotide administration group showed little change under light microscope and presented normal neurons for the most part under electron microscope with occasionally emerged dark cells.In conclusion, heroin enhances purine nucleotide catabolism and inhibits their synthesis in rats'midbrain, cortex and brain stem through promotes activity of ADA and XOD, the key enzymes in purine nucleotide catabolism, and suppresses expression of AK and HGPRT, the key enzymes in purine nucleotide salvage synthesis. Heroin increases Glu concentration in midbrain, cortex and brain stem via suppresses GDH and GS activity. Purine nucleotide compensation inhibits the above mentioned heroin effects in different degrees and suppresses heroin induced CPP and pathological changes. All the results suggests that changes of purine nucleotide and Glu metabolism play a role in development of heroin dependence and purine nucleotide compensation may be potent in detoxification therapy for heroin daddicts.