Interactions Between the Opioid and Adenosine Systems in the CNS.

1. A brief overview of the adenosine and opioid systems and their role in analgesia has been presented. However, the evidence available until now concerning the nature and mechanism of the interaction between opioids and adenosine has been broadly reviewed and discussed with reference to contemporary and current literature. There is a large body of evidence indicating a role of adenosine in opioid-mediated antinociception as well as in the development of opioid tolerance, dependence and withdrawal. In order to investigate the possible interaction between the opioid and adenosine systems in the CNS, mice deficient in p opioid receptors, enkephalin peptides or A2A adenosine receptors were employed. Manipulation of the opioid system by chronic naltrexone treatment was also carried out. 2. Quantitative autoradiographic mapping of A1 and A2A adenosine receptors and nitrobenzylthioinosine (NBTI) sensitive adenosine transporters in the brains and spinal cords of wild type, heterozygous and homozygous p opioid receptor knockout mice revealed a small but significant reduction in A1 and NBTI-sensitive adenosine transporter binding in mutant mice brains but not in spinal cords. Regions of greatest decrease in A1 receptor binding correlated with regions of high p receptor expression. No significant change in A2A receptor binding was detected in homozygous p opioid receptor knockout mice brains whereas no A2A receptors were detected in the spinal cord. In contrast, quantitative autoradiographic mapping of A1 adenosine receptors and NBTI-sensitive adenosine transporters in the brains of wild type and homozygous enlephalin knockout mice revealed a small but significant increase in A1 and NBTI-sensitive adenosine transporter binding in mutant mice. Regions of greatest increase in A1 receptor binding correlated with regions of greatest upregulation of u receptor expression in enkephalin knockout mice. Again, no significant change in A2A receptor binding was detected in enkephalin knockout brains. The results suggest that there may be functional interactions between p receptors and A1 receptors as well as p receptors and NBTI-sensitive adenosine transporters in the brain but not in the spinal cord. 3. In order to investigate whether the manipulation of the opioid system by chronic administration of an opioid antagonist could lead to changes in adenosine receptor expression, quantitative autoradiographic maping of u, A1 and A2A receptors was carried out in the brains of chronic naltrexone-treated mice. The significant increase of p receptor binding was followed by a significant upregulation of A1 receptors, especially in regions of high p receptor expression. Again, no change in A2A receptor binding was observed in chronic naltrexone-treated mice. The results suggest that there may be functional interactions between p receptors and A1 receptors in the brain. 4. To investigate if there are any compensatory alterations in opioid systems in A2A receptor knockout mice, quantitative autoradiographic mapping of u, d, k and ORL1 opioid receptors was carried out in the brains and spinal cords of wild-type and homozygous A2A receptor knockout mice. In addition, u-, d -and K-mediated antinociception using the tail immersion test was tested in wild-type and homozygous A2A receptor knockout mice. A significant reduction in d receptor binding and a significant increase in k receptor binding were detected in the spinal cords but not in the brains of the knockout mice. u and ORL1 receptor expression were not significantly altered. Moreover, a significant reduction in d-mediated antinociception and a significant increase in K-mediated antinociception were detected in mutant mice whilst u-mediated antinociception was unaffected. Comparison of basal nociceptive latencies showed a significant hypoalgesia in knockout mice when tested at 55 C but not at 52 C. The results suggest a functional interaction between the spinal d and k opioid and the peripheral adenosine system in the control of pain pathways. 5. The role of A2A adenosine receptors in the expression of morphine withdrawal was investigated by examining naloxone-precipitated morphine withdrawal signs in wild type and A2A receptor knockout mice. To further investigate if the opioid system is altered in the absence of the A2A receptor gene under conditions of morphine withdrawal, quantitative autoradiography of p opioid receptors and p opioid receptor stimulated guanylyl 5’-[y-[35S]thio]-triphosphate ([35H]GTPyS) autoradiography were carried out in brain sections of morphine-withdrawn wild type and A2A receptor knockout mice. Moreover, to investigate the involvement of D2 dopamine receptors in modulating A2A receptor effects, quantitative autoradiography of D2 dopamine receptors was carried out in brain sections of morphine-withdrawn wild type and A2A receptor knockout mice. A significant enhancement of withdrawal jumping, paw tremor and writhing were observed in A2A receptor knockout mice. Moreover, no significant changes in D2 dopamine and p opioid receptor binding were observed in any of the brain regions of the naloxone-precipitated morphine-withdrawn A2A receptor knockout mice analysed. However, a significant increase in the level of p receptor stimulated [35S]GTPyS binding was observed in the nucleus accumbens of morphine-withdrawn A2A receptor knockout mice compared to wild type. The data indicate that adenosine, via its action on A2A receptors, exerts an important control on the expression of opioid withdrawal. Several mechanisms may be involved in the adenosinergic control of morphine withdrawal including possible inhibitory effects of A2A receptor stimulation on p receptor G protein activation. 6. Despite the limitations associated with the technology of homologous recombination in the production of knockout mice, these animals have proved to be an important tool for the study of the interaction of the opioid with the adenosine system. However, these studies have prompted more questions than they have actually answered