Effect Of Heroin On Purine Nucleotides Metabolism In Rats

The abuse and addiction of opiates has become an important social problem, and it has jeopardized the physical and mental health of human being seriously. It is still an important task to clarify the basis of tolerance and dependence. There are few reports about the relationship between the effect of opioid and the metabolism of nucleotides. Our previous experiment demonstrated that opiates affected anabolic metabolism of nucleinic acid and nucleotides in cells, especially in cellula nervosa. Whether opiates, such as heroin, can affect the metabolism of nucleinic acid and nucleotides in biosystem? The experiment on whole animal is more important than on cellular level, so the rat model of heroin administration and withdrawal was established. ADA and XOD are the key enzymes of purine nucleotides catabolism, while HGPRT and AK are the key enzymes of nucleotide salvage synthesis. To illuminate whether heroin affects the nucleic acid metabolism by regulateing the gene transcription of the key enzymes in nucleotides metabolism, the gene transcription of ADA and XOD and the corresponding biochemistry metabolism catalyzed by these two enzymes were detected, and the gene transcription of HGPRT and AK was also detected. We approached the substance foundation of herion dependence and addiction from the view of biochemistry metabolism, and we provided a new aspect for the study of heroin function.1. Establishment of heroin administration and withdrawal modelThe adult male Wister rats were used to establish heroin administration and withdrawal model. The body weight, conditioned place preference experiment(CPP), score of withdrawal syndromes, and tail flick latency were evaluated. The result showed that:①There was no significant variance in the body weight of rats during heroin administration and withdrawal.②The rats preferred to stay in the heroin-related-side, and it reflected that the mental dependence of rats had emerged. The craving for heroin was potentialized with the time prolongation of heroin administration, and the effcet could not vanishe in short time.③Naloxone precipitated withdrawal syndromes, such as rearing, jumping, wet dog shakes, dressing up, hypoblepharon, stretch, diarrhea, and piloerection appeared in the two heroin administration groups. The result showed that rats had developed physical dependence to heroin in the 3 day-heroin administration group, and it was more serious in the 9 day-heroin administration group. The results of conditioned place preference experiment and the observation of naloxone precipitated withdrawal syndromes showed that the rats had developed both psychological dependence and physical dependence to heroin, and the rat model of heroin administration and withdrawal was established successfully.④The sensitivity to ache decreased in rats after heroin administration, and it was significant as compared with the control group. The effect was reinforced by the increasement of heroin dosage, and sensitivity to ache recovered during withdrawal.2. Detection of indexes in plasma and homogenatesIt was detected that plasma uric acid contents and ADA and XOD concentrations in parietal lobe, lobus temporalis, lobus frontalis, brain stem, liver, small intestine, muscle, and plasma. The result indicated that ADA and XOD concentrations in brain and extraneuronal organs increased during heroin administration through some mechanism, thus the purine nucleotides catabolism was strengthened, and then the plasma uric acid contents was increased which is the dead end product of purine nucleotides catabolism. In different encephalic regions and extraneuronal organs, the effects of heroin on ADA and XOD were diverse in time or intension. On the whole, the effect of heroin on brain lasted for a long time, for the ADA and XOD concentrations in brain descended slowly during withdrawal, so the effect of heroin could not vanish soon. While the effect of heroin on extraneuronal organs lasted for a short time, for the ADA and XOD concentrations in extraneuronal organs decreased gradually during withdrawal and fell to the normal level. PRPP amidotransferase is a key enzyme of purine nucleotides anabolism in the liver, small intestine, and muscle. There is no PRPP amidotransferase in brain and the anabolism of nucleotides mainly depends on the salvage pathway, therefore the ability of nucleotide anabolism is inferior in brain. The experiment demonstrated that heroin caused the increasement of ADA and XOD concentrations in brain and the reinforcement of purine nucleotides catabolism induced by heroin could not disappear soon, therefore the effect of heroin on purine nucleotides catabolism in brain was more serious. The ADA and XOD in tissues released to plasma, thus the enzyme contents in plasma increased. With regard to the plasma ADA concentrations, it increased significantly in two heroin administration groups, but it reached to the peak in the 3-day withdrawal group. Although the plasma ADA concentration of the 8-day withdrawal group was still higher than that of the control group, it had decreased significantly as compared with that of the 3-day withdrawal group. As shown above, ADA is an intracellular enzyme and it takes a period of time to be released into the blood from tissues and it also needs some time to be cleared away from the blood. When the data obtained from plasma were used to reflect the variable of intracellular enzyme, the postponement of time parameter should be thought over. It might also be one of the reasons that brain ADA was still in a high level during withdrawal and kept being released into the blood. Furthermore, ADA in other tissues might also affect the ADA concentration of plasma. It requires further detailed investigations to be clarified.3. Effect of heroin on genetic transcription of key enzymes in purine nucleotides metabolism⑴RT-PCR and RT-PCR-Southern blot hybridization were used to detect the genetic transcription of ADA, XOD, AK, and HGPRT in parietal lobe, lobus temporalis, lobus frontalis, brain stem, liver, small intestine, and muscle of rats.⑵Effect of heroin on genetic transcription of key enzymes in purine nucleotides catabolism.①The gene transcription of ADA and XOD in brain and extraneuronal organs increased to different extents during heroin administration. The ADA and XOD mRNA concentrations enhanced, so the expression of ADA and XOD gene to protein strengthened, thus the enzyme contents increased and it induced sequentially the potentialization of purine nucleotides catabolism, furthermore the effect was reinforced with time elongation and dosage increment.②During herion withdrawal, ADA mRNA concentration in tissues decreased accompanied with the degrade of enzyme content, but the brain ADA mRNA concentrations of the withdrawal groups were still higher than that of the control group. The result demonstrated that the effect of herion on ADA gene transcription in brain could not vananish in short time and this might be concerned with the addiction of herion. With the prolongation of withdrawal time, ADA mRNA concentration in brain continued to decrease, while the ADA content increased a little. Heroin affected ADA gene expression in brain might not only on gene transcription level, but also on post-transcriptional processing or translational level, thus the variances of enzyme concentration and mRNA level were not with one accord . The result also proved that the effect of heroin on brain was complicated and serious.③During herion withdrawal, XOD mRNA concentrations in brain were still higher than that of the control group, while the XOD mRNA concentrations in extraneuronal organs decreased. The resut proved that the effect of heroin on XOD gene transcription in brain could not disappearance in short time. It was dirrerent in the variation tendency of XOD mRNA concentration in different encephalic regions, and the dependablity of XOD concent to XOD mRNA concentration was also distinct in different encephalic regions. The result demonstrated that the mechanisms of heroin affect XOD gene expression were distinct in different encephalic regions, and in some encephalic regions the effect might not only be in gene transcription level. In addition, there was disparity between XOD content and XOD mRNA concentration in muscle during withdrawal. The result proved that there was no completely parallelism between enzyme content and mRNA concentration. According to the quantization comparision between polygene cell mRNA and corresponding protein drawed by Anderson, the coefficient correlation was only 0.48 between them. We decomposed that the regulation of herion to ADA and XOD might occur on transcription level, and might also occur on post-transcriptional processing and translational levels. In a word, heroin strengthened purine nucleotides catabolism in brain and extraneuronal organs by enhancing ADA and XOD gene transcription.⑶Effect of heroin on genetic transcription of key enzymes in purine nucleotides anabolism.①Heroin depressed the gene transcription of AK and HGPRT in brain, thus AK and HGPRT mRNA concentrations decreased, and the effect was reinforced with time elongation and dosage increment. Heroin depressed the AK gene transcription in small intestine and muscle, while the effect of herion on AK gene transcription in liver and the HGPRT gene transcription in liver, small intestine, and muscle were not significant. AK and HGPRT are key enzymes in purine nucleotides salvage synthesis. There is only salvage synthesis in brain, while the de nove synthesis of purine nucleotides is the primary way in extraneuronal organs. Heroin depressed the gene transcription of AK and HGPRT in brain, and affected seriously the anabolic metabolism of purine nucleotides in brain, so this might be concerned with heroin addiction.②During herion withdrawal, the depressant effect of herion on AK and HGPRT genetic transcription lessened, AK and HGPRT mRNA concentrations increased in tissues. But the inhibited effects of heroin on AK and HGPRT gene transcription were different in different encephalic regions and different extraneuronal organs. During herion withdrawal, HGPRT mRNA concentration in tissues and AK mRNA concentration in muscle were lower than those of the control group, while the AK mRNA concentration in brain was highter than that of the control group. The inhibitory action of heroin on AK gene transcription in brain might reinforce the effect of some compensation mechanisms, and the effect of the compensation mechanisms could not vanish soon during withdrawal, thus the AK mRNA concentration was higher than that of the control group.4. Sequencing of ADA cDNA segmentThere was 99% homology between ADA cDNA segment and the ADA mRNA sequence in gene bank. There was 99% homology between XOD cDNA segment and the XOD mRNA sequence in gene bank. There was 98% homology between AK cDNA segment and the AK mRNA sequence in gene bank. There was 99% homology between HGPRT cDNA segment and the HGPRT mRNA sequence in gene bank. The result of sequencing demonstrated that the genes obtained by RT-PCR were ADA, XOD, AK, and HGPRT repectively and the consequence of RT-PCR in the experiment was confident.On the whole, the gene transcription of ADA and XOD which are key enzymes of purine nucleotides catabolism was increased during heroin administration, thus ADA and XOD contents increased in tissues and it induced sequentially the potentialization of purine nucleotides catabolism, so the plasma uric acid concentration increased. On the same time, heroin depressed the gene transcription of AK and HGPRT which are key enzymes of purine nucleotides salvage synthesis,so heroin affected purine nucleotides anabolism in the whole organism. The effect could not vanished soon in brain that heroin strengthened purine nucleotides catabolism and depressed purine nucleotides anabolism. There is no PRPP glutamyl amidotransferase in brain, so it only depends on purine nucleotides salvage synthesis. It might be the fundamental effect of heroin that it affacted purine nucleotides metabolism, and it might also be an important material foundation of opioid dependence. This study approached the material foundation of heroin dependence and addition from the point of biochemiscal metabolism, and provided some new cognition for the role of opioids.