Effect Of Heroin On Purine Nucleotides Metabolism And The Therapeutical Effect Of Purine Nucleotides

Opiates, such as morphine and its chemical derivate heroin have caused serious medical and social problems. Heroin is a synthetic acetyl derivate of morphine. Heroin can pass the blood-cerebral barrier easier than morphine to play its pharmacological function, thus the addiction of heroin is stronger. Therefore, studies of the effects of repeated exposures to these compounds are important for the understanding of drug-dependent mechanisms. We have got some achievements in the realationship between morphine and heroin and purine nucleotide metabolism. The aims of the study were first to clarify the effects of heroin on purine nucleotide metabolism in vivo and in vitro, especially on purine nucleotide anabolism , second to investigate whether purine nucleotide administration affects heroin-dependent rats'behavior.The first part of this paper is the study on the tissue level. The rat model of heroin dependence was established. Adult male Wistar rats were randomly divided into 5 groups: The first group of rats was the saline group; The second The second group was the Heroin group, in which each rat was administered heroin for 9 days; The third group was the Heroin+AMP+GMP group, in which each rat was administered the same dose of heroin as the second group and purine nucleotides(AMP+GMP). The fourth group was the Heroin+AMP group, in which each rat was administered the same dose of heroin as the second group and AMP with a daily dose of 7.5 mg/kg. The fifth group was the Heroin+GMP group, in which each rat was administered the same dose of heroin as the second group and GMP.Tail flick latency, pressure pain and abstinent symptoms were evaluated to study the behavior of rats. The content of AMP and GMP in cortex was determined by HPLC. Adenosine deaminase (ADA) and xanthine oxidase(XO) are two of the key enzymes regulating the catabolism of purine nucleotides. The concentration of uric acid, creatinine and urea nitrogen in plasma and the ADA, XO activities in brain were measured. Real-time PCR was used to quantify the mRNA of ADA and XO in brain to investigate one of the possible mechanisms of heroin's effect on ADA and XO. Hypoxanthine-guaninephosphoribosyl transferase (HGPRT), adenine phosphoribosyl transferase (APRT) and adenosine kinase (AK) are the key enzymes of salvage biosynthesis. GTPCH is the key enzyme catalyzing the conversion of GTP to BH4. Thus, we examined the gene transcripts of HGPRT, APRT, AK and GTPCH by real-time PCR, to evaluate the purine nucleotide anabolism tendency of rat brain influenced by heroin solitarily or both of heroin and purine nucleotide compensation collectively.The second part of this paper is the study on the cell level. We determined the cell viability and cell survival rate by MTT colorimetry and trypanblue method respectively, and the effect of heroin and AMP and GMP on C6 glioma cell of the above ADA, XO, HGPRT, APRT, AK and GTPCH gene expression by Real Time PCR. The results are as follows:1. Establishment of heroin dependence modelThe abstinent symptoms, tail flick latency and pressure pain experiments were carried out to study whether purine nucleotide administration could affect the abstinent symptoms and possess antinoception in heroin-dependent rats.Naloxone induced abstinent symptoms were observed to assess physical dependence of the drug. The results showed that heroin plus nucleotides groups'counts of ptosis, tooth chattering and chewing were significantly lower than those in the heroin group,which suggests that nucleotide compensation may alleviate some of the physical dependence induced by heroin. The results mentioned above show that rats shaped physical dependence after exposure to heroin and the heroin-dependent model was established successfully. The pressure-pain threshold in heroin group was higher than that in saline group, and that in heroin plus purine nucleotide groups were higher than that in heroin group. In the tail flick test, tail flick latencies were significantly increased in heroin group and three heroin plus nucleotide group. Further more, tail flick latencies in three heroin plus nucleotide groups were much higher than that in heroin group. It is conceivable that purine nucleotides enhance heroin's antinociceptive analgesic effect. The mechanism of nucleotide effects needs to be studied further.Heroin treated rats had a lower increasing rate of body weight compared with those of the saline group. The purine nucleotide administration rats could alleviate the decreace of body weight compared with those of the heroin group.2. Effects of heroin and purine nucleotide administration on content of purine nuleotides in brain.HPLC method was used in this study to detect the absolute content of purine nucleotides in brain tissue. The content of AMP in heroin group was significantly decreased compared with that in saline group; purine nucleotide administration could alleviate the effect of heroin. Heroin could reduce the content of GMP in some degree, but with no significance.3. Effects of heroin and purine nucleotide administration on related indexes in plasma and brain tisuuse.Uric acid is the final oxidation product of purine metabolism in humans and higher primates. This study revealed a significant increase of plasma uric acid in heroin-administered rats compared with that of the Saline group, while purine nucleotides administration attenuated the increase of the uric acid level. Since uric acid level is a result of the balance between the breakdown of purine and the rate of uric acid excretion, we detected creatinine and urea nitrogen, which were sensitive indices reflecting the renal clearance rate, to assess the impact of heroin on renal function. The results showed that the concentrations of plasma creatinine and urea nitrogen did not significantly change in heroin group. These results indicated that the enhancement of plasma uric acid levels in heroin group rats was not associated with renal function, but mainly resulted from the increase of purine nucleotides catabolism.ADA and XO were key enzymes involved in purine catabolism. The results showed that heroin could increase the contents of ADA and XO in plasma. Purine nucleotides administration reduced the contents of ADA and XO dramatically, compared with that in the heroin administration group. This result suggested that the enhancement of purine nucleotide catabolism promoted by heroin could be restored by purine nucleotides administration.The results of ADA and XO contents in rats'brain tissue indicated that heroin can increase the ADA and XO contents in brain, which is believed to contribute to the enhancement of purine nucleotide catabolism and the high level of plasma uric acid. Purine nucleotides compensation decreased the ADA and XO contents dramatically compared with that in the heroin administration group, suggesting that heroin promoted rats'purine nucleotide catabolism, which could be restored by purine nucleotides compensation.4. Effects of heroin and purine nucleotide administration on gene expression of purine nucleotide metabolic key enzymes in rats'brainIn order to further investigate the mechanisms of heroin on ADA and XO levels, real-time PCR was used to quantify the mRNA of ADA and XO in brain tissue. The experiment data revealed significant increases of ADA and XO mRNA in the rat brains of heroin administration group, as compared with that of the Saline group rats. The increase also could be inhibited by purine nucleotide administration.Cellular purine nucleotides are derived from either de novo synthesis or salvage pathways. Nevertheless there is no de novo synthesis in brain and the anabolism of purine nucleotides mainly depends on the salvage pathway. HGPRT, APRT and AK are the key enzymes of the salvage pathway. We examined the gene transcript of HGPRT, APRT and AK by real-time PCR. Compared with the Saline group, the transcripts of HGPRT, APRT and AK gene decreased significantly in rats of heroin group, and these three transcripts of genes in heroin plus purine nucleotide groups were higher in some degree than that of the heroin group. These results indicate that heroin inhibited mRNA expression of HGPRT, APRT and AK, which could be restored by purine nucleotide administration. GTPCH gene expression was decreased dramatically in heroin group when compared with saline group (P<0.05); in heroin plus purine nucleotides groups ,GTPCH gene expression was increased with no significant differences.5. Effects of heroin and purine nucleotides on the morphologic changes of cortexWe observed the ultra-microstructure of cortex in saline, heroin and heroin plus purine nucleotides groups using the transmission electron microscope (TEM). The result of TEM showed that cortex of saline group was normal. Cortex of heroin group showed a great quantity of dark cells and retrogression change of the neuron and neurofibra in heroin group. The changes of ultra-microstructure in heroin and nucleotides administration group were much better than that in heroin group. Cortex of heroin plus nucleotides administration group presented normal neurons for the most part with occasionally emerged dark cells. We presume that purine nucleotide might improve the damage of cortex caused by heroin.6. Effect of heroin on the proliferation of C6 glioma cellC6 glioma cells has already been used as a model system to study the mechanism of opioid action. In this study, cell viability was determined with MTT colorimetry, and cell survival rate was detected by trypanblue method. The results showed that the higher heroin concentration was, the lower cell viability was. Cell survival was decreased and dead cell was increased in high content heroin. The results above indicate that heroin can inhibit C6 cell proliferation7. Effect of purine nucleotides administration on cell proliferation and purine nucleotide metabolic key enzymes gene expression in C6 glioma cellsIn this experiment, we determined the cell viability and the gene transcripts of ADA, XO, HGPRT, APRT, AK and GTPCH in C6 glioma cell treated with heroin and purine nucleotides.The results indicated that heroin can inhibit C6 cell proliferation. Purine nucleotide could alleviat this effect in some degree. Heroin could enhance the catabolism and inhibit the anabolism of purine nucleotide in cell level by regulating the gene expression of five key enzymes including ADA, XO, HGPRT, APRT and AK. Heroin can decreased GTPCH gene expression, which may affect the neurotransmitter production by reducing BH4. Purine nucleotide can restore this effect of heroin .These data were in accord with the results of animal experiments.In summary, heroin administration can promote the catabolism and inhibit the anabolism of purine nucleotide in rat brain, which imply that heroin may affect the nucleotide metabolism seriously and eventually lead to the lack of purine nucleotide. Purine nucleotide administration presented reversal effects on nucleotide metabolism changes induced by heroin in rat brains. Nucleotide administration could alleviate some of the withdrawal symptoms of the heroin-dependent rats. It seems that nucleotides administration can partially inhibit the development of physical dependence induced by heroin. Nucleotide administration may also increase the antinoceptive effects of heroin. Heroin can inhibit C6 cells proliferation, and decrease cell purine nucleotide anabolism and increase its catabolism. Exogenous purine nucleotides administration attenuated the effect of heroin on cell proliferation and nucleotide metabolism in C6 cells. The relationship between purine nucleotide metabolism and opioid addiction is worth to study further.