Until recently, the most potent activators of soluble guanylate cyclase and stimulators of tissue cyclic GMP accumulation have been nitric oxide (NO) and certain nitroso compounds. Although studies with NO have greatly improved our understanding of guanylate cyclase activation and the possible role of cyclic GMP in certain cell functions, serious questions have arisen pertaining to the physiological relevance of such observations made with chemicals. The recent original findings in this laboratory that the naturally occurring porphyrin, protoporphyrin IX, is an extremely potent activator, whereas heme (ferroprotoporphyrin IX) is a potent inhibitor (competitive with protoporphyrin IX) of soluble guanylate cyclase, suggest that tissue cyclic GMP formation could be regulated by intracellular porphyrins and metalloporphyrins. Thus, the principal objective of this proposal is to characterize the regulation of cyclic GMP formation by protoporphyrin IX and heme. The specific aims are (1) to examine the kinetics of activation by protoporphyrin IX and structural analogs, and inhibition by heme and related metalloporphyrins of purified soluble guanylate cyclase, (2) to determine the mechanism of guanylate cyclase activation by phenylhydrazine and related aromatic hydrazino compounds, (3) to study the relationships among protoporphyrin IX, NO-heme, and iron-phenyl-heme with respect to a common mechanism of enzyme activation, (4) to study and compare the properties of heme-containing, heme-free, and heme-reconstituted guanylate cyclase, and to examine the exchange of heme and NO-heme between enzyme and other hemoproteins, (5) to determine the mechanism of activation of guanylate cyclase by arachidonic acid and related polyunsaturated fatty acids, and (6) to ascertain whether chemically induced alterations in the intracellular concentration ratio of protoporphyrin IX to heme cause concomitant alterations in tissue cyclic GMP levels. These studies will provide a broader understanding of guanylate cyclase, regulation of tissue cyclic GMP levels, and perhaps the physiological function of cyclic GMP. Moreover, such information could provide insight to the role of cyclic GMP in pharmacologically evoked vascular smooth muscle relaxation and inhibition of platelet aggregation.