Effect Of Morphine On Purine Nucleotide Catabolism

Opiates are among the most commonly abused illegal drugs all over the world. Chronic exposure to opiates, such as morphine and heroin, can cause serious physical dependence and psychological dependence. It is still an important task to investigate the basis of tolerance and dependence. The relationship between opiate addiction and changes in nucleotide catabolism remains poorly understood. In this study, we establish the rat model of morphine dependence and withdrawal and choose rat C6 glioma cells to try to clarify whether morphine affects the catabolism of purine nucleotides by regulating gene expression of adenosine deaminase (ADA) and xanthine oxidase(XO) which are key enzymes of purine nucleotide catabolism, to probe the substantial basis of morphine dependence and tolerance and provide a new way for our research. The first part of this paper deals with the whole animal level. A rat model of morphine addiction and withdrawal was established.Acute withdrawal syndromes were observed after naloxone administration and the weight of rats were measured before and after morphine administration. Concentrations of uric acid in plasma were measured with an autoanalyzer, and ADA and XO in plasma were measured with the test kit. Samples of the brain parietal lobe, liver, small intestine, and muscle were homogenized, and the concentrations of ADA and XO of the tissues were also measured by test kit. RT-PCR and RT-PCR-Southern blot were used to examine the gene transcripts of ADA and XO in these tissues. 1. Establishment of morphine addiction and withdrawal model: Adult, female Wistar rats were randomly divided into 6 groups for experiments. Each group of rats represented at least ten animals which were treated with①saline ②mophine for 3 days ③morphine for 7 days ④⑤morphine for 7 days and then killed on 11th and 15th day respectively ⑥morphine for 7 days and then followed by naloxone induced withdrawl syndrome on 8th day. The amount of HCl morphine was increased every day to set up morphine addiction model. In experiment, natural withdrawal 2 group was selected to observe the change of weight,and the weight of natural withdrawal 2 group decreased gradually during morphine administration. 2. Uric acid and enzymes detection in plasma of morphine dependent rats: As uric acid is the end product of purine nucleotide catabolism, determination of plasma uric acid concentration may reflect the catabolism state of purine nucleotides. In this study, we found that there was a significant increase in plasma uric acid of morphine treated rats as compared with controls(P<0.05). This result suggests that morphine addiction may cause the enhancement of purine nucleotide catabolism. While during natural withdrawal and naloxone precipitated withdrawal , concentrations of plasma uric acid in rats became decreased as compared with the morphine treated group. We consider that naloxone precipitated acute withdrawal caused a kind of stress state in rat body, so that the chemical modification may occur on xanthine oxidase, causing decrease of the enzyme activity of xanthine oxidase which may result in less amount of uric acid production, thus the level of uric acid drop down to about the same as that in control. Therefore , the question arises: What is mechanism of morphine's effects on purine nucleotide catabolism? Continued study showed that the morphine induced increase in uric acid concentration is highly correlated with that of ADA and XO. During morphine administration , the levels of plasma ADA and XO increased significantly(P<0.05), and after morphine abstinence the concentration of ADA and XO still kept high level. 3. Tissues ADA and XO detection of morphine dependent rats: ADA and XO is an intracellular enzyme, and the high concentration of plasma ADA and XO may be released from one or more tissues into blood; therefore, we determined the ADA and XO levels of several important tissues, such as parietal lobe, liver, small intestine and muscle during morphine administration and withdrawal. The ADA activities showed that there were significant increases of parietal lobe, liver and muscle ADA in morphine treated rats (P<0.05), and the ADA level in those tissues of the natural withdrawal groups recovered to the normal level gradually. While ADA levels in those tissues were still higher in naloxone precipitated withdrawal rats than in controls. The XO activities showed that there were significant increases of parietal lobe, small intestine and muscle XO in morphine treated rats (P<0.05),and the XO level in those tissues of the natural withdrawal groups recovered to the normal level gradually. While XO levels in parietal lobe and small intestine were still higher in naloxone precipitated withdrawal rats than in controls(P<0.05). The results indicated that morphine can act on theses tissues to induce the increased ADA and XO concentrations during morphine addiction, which is believed to contribute to the enhancement of purine nucleotide catabolism and the high level of plasma uric acid. 4.Tissues ADA and XO mRNA detection of morphine dependent rats: In order to investigate the mechanism of morphine's effect on ADA and XO, RT-PCR and RT-PCR-Southern blot was used to examine the gene transcripts of ADA and XO in these tissues. Compared with that in the control group, the transcripts of ADA mRNA were significantly higher in rats treated with morphine. During morphine abstinence and naloxone precipitated withdrawal, with the exception of muscle, ADA mRNA levels gradually recovered to normal. RT-PCR-Southern blot showed that morphine administration caused a significant increase of XO mRNA levels in these tissues. Compared with the morphine treatment groups, the effect of morphine decreased gradually and the XO mRNA levels of parietal lobe, liver and muscle recovered to the nomal level in the natural withdrawal groups. While XO mRNA levels of these tissues were down regulated in rats after treatment with naloxone precipitated withdrawal. In summary, acute and chronic morphine administration can increase the catabolism of purine nucleotide in brain and extraneuronal tissues by regulating the gene expression of key enzymes of ADA and XO, and the regulation maybe mediated by mu receptor. The second part of this paper consists of determination of effect of morphine on C6 glioma cell ADA and XO gene expression by RT-PCR and RT-PCR-Southern blot hybridization. In this experiment , exposure of morphine at all time point to C6 cells caused an enhancement of ADA gene expression, while XO transcripts increased respectively in 12h ,24h, 48h and 72h after exposure of morphine as compared with control in corresponding time point. The up-reagulation of morphine on ADA and XO can be reversed by naloxone. The results indicate that morphine can enhance the catabolism of purine nucleotide in cell level by regulating the gene expression of two key enzymes of ADA and XO, and the regulation maybe mediated by mu receptor. These data were in accord with the results of animal experiment. In summary, morphine administration can increase the catabolism of purine nucleotide by regulating two key enzymes ─ADA and XO in vivo and in...