The goal of this proposal is to investigate the actions of atrial natriuretic factor (ANF) on sympathetic neurotransmission in isolated neuronal cells and in isolated tissues. The proposed work will investigate the intracellular mechanism by which ANF suppresses catecholamine release in differentiated PC12 cells and rabbit isolated vasa deferentia and portal veins to determine if the neuromodulatory pathways defined in the PC12 cells occur in other sympathetically innervated organs. Specifically, the principal investigator will investigate whether ANF suppresses evoked neurotransmitter release is mediated through a G protein which 1) increases cyclic GMP accumulation; 2) alters cyclic AMP accumulation or 3) alters phospholipase products. Binding experiments will test for the presence of ANF receptors on PC12 cells. Pertussis toxin, and GTP and GDP analogues will be utilized to assess the role of inhibitory G proteins in mediating the neuromodulatory effects of ANF. It has been ascertained that pertussis toxin eliminates the suppression of adenylate cyclase and neurotransmission caused by ANF. ANF-induced alterations of second messenger synthesis and evoked catecholamine release will be correlated to test for potential cause and effect relationships. Cause and effect relationships among ANF effects on second messengers and neurotransmission will be tested further using pertussis toxin and ANF """"""""clearance receptor antagonists"""""""". The compound originally termed the ANF clearance receptor antagonist suppresses both adenylate cyclase activity and neurotransmission by a pertussis toxin-sensitive mechanism but does not activate guanylate cyclase. Potential interactions among the proposed second messengers also will be tested to discriminate between second messenger pathways which are in parallel or in series.
