The proposed studies will expand upon new data from the Principal Investigator's laboratory which demonstrate that in vivo morphine administration produces robust reductions in T-cell function which appear to be mediated by macrophage-derived nitric oxide. Since both macrophages and T-cells are key immunoregulatory cells, and the target of HIV-1 in humans, understanding the mechanisms by which opioids exert these actions has important human health implications.
Specific Aim 1 will test the hypothesis that morphine, acting at the level of the central nervous system, induces pharmacologically-specific alterations in nitric oxide production by splenic macrophages. It is proposed to compare the dose-dependent effects of morphine administered systemically, intracerebroventrically (ICV), and in vitro on the expression of nitric oxide by splenic macrophages. Antagonism studies also will be performed. Tests will be conducted on splenocytes to identify the subtype and number of macrophages expressing inducible nitric oxide synthase (iNOS). The level of nitric oxide activity produced by splenocytes will be assessed by measuring iNOS protein using western blotting and by measuring the concentration of nitrate/nitrite in vitro. The relationship between morphine-induced alterations in nitric oxide production and changes in the function of lymphocytes will be established.
Specific Aim 2 will determine the subtype(s) of opioid receptors involve in opioid-induced alterations of nitric oxide production. To distinguish the role of mu, kappa, and delta opioid receptor subtypes, the investigators will compare the effects of ICV and in vitro administration of selective opioid agonists and antagonists on splenic nitric oxide production.
Specific Aim 3 will characterize the effect of opioid administration on nitric oxide production in vivo. These experiments will assess the dose-dependent effects of morphine, and selective opioid agonists and antagonists. To measure alterations of nitric oxide production in vivo, a complementary approach which uses RT-PCR and western blotting to directly measure alterations of splenic iNOS mRNA and protein expression will be applied. Serum nitrite/nitrate levels will also be measured. Collectively, these investigations could provide important new information concerning the site of action and the subtype of receptor involved in opioid-induced modulation of nitric oxide production.
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