An expanding body of evidence indicates that opiates, opioid peptides, and opiate addiction modulate cells of the immune system. Amongst these cells, opioids have been shown to affect several crucial functions of human polymorphonuclear leukocytes (PMN) that are linked to the generation of the reactive oxygen metabolites superoxide and hydrogen peroxide. Utilizing lucigenin-enhanced chemiluminescence (CL) to detect superoxide, a recent study demonstrated that morphine, Beta-endorphin and dynorphin1-14 all stimulated production of superoxide by PMN. Significant CL responses occurred at opioid concentrations as low as 10-14M, and maximum responses were present at 10-12M. Our preliminary studies, utilizing the highly specific ferricytochrome C reduction assay to quantitate superoxide, have confirmed these observations, indicating that stimulation of human PMN is stereoselective and does not occur with N-acetyl-Beta-endorphin. This proposal is designed to characterize the opiate receptors mediating oxygen metabolism and to begin to explore the biological implications of PMN regulation by endogenous opioids and exogenous opiates. Several classes of highly selective opioid receptor agonists will be used to stimulate oxygen metabolism and for radioligand saturation and competition experiments. Since opiates often modulate either the release or the target response to another signal, the interaction between opiates and other well studied activators of PMN oxygen metabolism will be evaluated. Previous investigations have shown that certain stressors are associated with increased oxygen metabolism by PMN and with the release of endogenous opioid peptides. A rat model will be developed to study: (1) the opioid-mediated effects of stress and (2) the effects of chronic opiate exposure on PMN oxygen metabolism. Purified rat PMN will be tested in vitro to assess superoxide production and radioligand binding properties. To evaluate the effects of stress on PMN superoxide generation in response to in vitro opioids, rats will be subjected to various acute and chronic stress paradigms prior to harvesting PMN. In addition, opioid antagonists will be administered concommitant to stress, and the origin(s) of the opioid mediator(s) will be investigated using surgical ablation prior to stress. In summary, these studies will seek to advance new concepts pertinent to opioid-mediated neuroimmunomodulation in response to stress and chronic opiate exposure. Both a purified human cell preparation and a rat model that permits direct correlation of both the functional and radioligand binding characteristics of PMN opiate receptors will be used.
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