The accumulating evidence obtained from previous research supported by this NIH grant strongly indicate that the descending pain control systems activated by various opioid agonists for producing antinociception involve multiple descending pathways and different neurotransmitters. The descending pain control systems can be classified at least into three systems: 1) mu receptor mediated, 2) epsilon receptor mediated and 3)-kappa receptor mediated descending systems. The mu opioid system is activated by supraspinally-administered morphine, DAMGO and other mu opioid agonists and the antinociception is mediated by the activation of mu opioid receptors and subsequently induced the release of 5-HT and norepinephrine acting on serotonin receptors and alpha2-adrenoceptors, respectively, in the spinal cord. The epsilon system is activated by beta-endorphin and the antinociception is mediated by the release of Met-enkephalin and subsequent stimulation of opioid delta receptors in the spinal cord. The kappa system is activated by U50,488H or other kappa opioid agonists and the antinociception is mediated by the release of 5-HT and dynorphin(1-17) acting on 5-HT receptors and kappa-opioid receptors, respectively. Endomorphin-1 and endomorphin-2 are newly discovered endogenous ligands for mu opioid receptors. The present renewal grant application will focus on studying the neuronal mechanisms of antinociception induced by endomorphin-1 and endomorphin-2. Specific opioid receptor antagonists and antisense oligodeoxynucleotides (ODN) against mRNA of mu, delta and kappa opioid receptors will be used to identify the types of opioid receptors involved in endomorphin-induced antinociception. This research will also identify the midbrain and brainstem sites that are stimulated by endomorphins to cause antinociception and the release of biogenic amines, Met-enkephalin, dynorphins and other neurotransmitters for the spinal cord. The binding of (3H) DAMGO or (3H) endomorphins to mu opioid receptors in the midbrain, brainstem and spinal cord will be characterized. Additional experiments will ascertain the role of G-proteins in the antinociception induced by endomorphins. Antisense ODN to different types of G-alpha protein mRNA will be used to identify the types of G-proteins which are involved in mediating mu opioid-induced antinociception. The functional role of G-proteins in the antinociception evoked by endomorphins will be studied by measuring associated changes in GTPgammaS binding and GTPase activities stimulated by endomorphins. The tail-flick and hot-plate tests will be used to determine antinociceptive responsiveness of mice or/and rats in this research. These studies will provide valuable insight into the cellular mechanisms of mu opioid antinociception.
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