The use and abuse of opiates and our incomplete understanding of how endogenous opioid peptides work is still a major health issue. We propose studying the effects of opiates on second messenger systems in dorsal root ganglion (DRG) cells, chosen because they express mu, delta and kappa opiate receptors, and in a neuroblastoma x DRG hybrid cell line (F-11), which is DRG-like and which we can grow in large quantities. We shall test the hypothesis that the acute affects of opiates in inhibiting neurotransmitter release are mediated by the inhibition of specific Ca2+ currents in these cells and are largely independent of cyclic AMP changes. We will do this by using a combination of electrophysiological, microfluorometric imaging and biochemical techniques. We will use a combined patch clamp/microfluorimetry technique to test the hypothesis that opiates inhibit [Ca2+]i transients and that these effects depend on the activation state of the DRG cell. Digital imaging techniques will be used to determine Ca2+ changes in soma versus processes and axon terminals, and to detect response (receptor) heterogeneity in the cell cultures. Pertussis toxin will be used to differentially inhibit these acute opioid effects and to distinguish them from the actions of other G-proteins. We shall also test the hypothesis that chronic effects of opiates are more related to effects on cyclic AMP levels and cross-talk between the inositol phosphate/calcium, cyclic AMP and other second messenger systems. Particular emphasis will be placed on the effect of elevated cAMP levels on the phospholipase C-mediated stimulus-secretion coupled pathway, the role of Ras-like G-proteins in this pathway and the mechanisms behind these effects. Our general strategy will be to make initial observations in DRG cells, and then try to reproduce them in F-11 cells where we have more material for biochemical studies such as phospholipid metabolism and protein kinase regulation.
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