The specific aims of this proposal are to determine the mechanisms of hormone, autocoid and drug regulation of the isoenzyme forms of guanylate cyclase and cyclic GMP synthesis. The precise mechanisms involved in hormone receptor coupling to the different guanylate cyclase isoenzymes will be examined in considerable detail. Experiments will utilize intacts cell cultures and tissues as well as crude and purified components and their reconstitution in cell-free preparations. The effects of atrial natriuretic factor and E. coli heat-stable enterotoxin on cyclic GMP synthesis as well as other ligands and hormones will serve as models and prototypes for these studies. The effects of ATP and other nucleotides and a recently purified protein activator of guanylate cyclase will also be examined. The role of these materials as well as protein phosphorylation and dephosphorylation in guanylate cyclase regulation and hormone coupling to cyclic GMP synthesis will be examined. Methods will include the purification and characterization of coupling factors for hormonal regulation of guanylate cyclase and their functional reconstitution. These studies are expected to increase our understanding of the role of cyclic GMP as a fundamental second messenger to mediate hormone effects and, thus, should provide mechanisms to modify biochemical and physiological responses in normal and pathological states.