Cyclic GMP is an important second messenger in vertebrate and invertebrate muscle. Mammalian smooth muscle, mammalian cardiac muscle, and the skeletal muscles of invertebrates, like the lobster, are enriched for cyclic GMP dependent kinase and its substrates. Furthermore, these tissues increase cyclic GMP levels in response to hormonal stimuli. In mammals, vasodilating peptides, like Substance P, VIP, bradykinin, and ANP, raise cyclic GMP levels and thereby induce vascular and cardiac relaxation. In the lobster, we have shown that peptides extractable from lobster secretory organs elevate lobster muscle cyclic GMP levels (in some cases more than 200-fold), and subsequently relax depolarization-induced contractions. At least one of these peptides (peptide G1) has been purified to homogeneity. Because the lobster peptides are derived from endocrine structures, and because they have such powerful effects on cyclic GMP metabolism, it seems likely that they serve as members of a neurohormonal system that uses cyclic GMP as a second messenger. The experiments in this proposal analyze the mechanism(s) by which the peptides exert their effects on target tissues. Extensive use will be made of our recent observation that peptide G1 receptors remain coupled to the membrane form of guanylate cyclase in homogenates of lobster muscle, and that coupling can be preserved in detergent extracts. The nature of this coupling, and the structure of the membrane cyclase, will be investigated by biochemical and gene cloning techniques. Is this cyclase similar to peptide-sensitive vertebrate cyclases? Are additional components, such as a phosphodiesterase enzyme or cytoplasmic factors, also involved in mediating the cyclic GMP response? What role is played by low molecular weight regulatory agents like calcium ions? Although peptide G1 has no sequence homology with any of the vertebrate peptides that elevate cyclic GMP, it has many mechanistic features in common with the vertebrate ANP family. These include the prolonged time course and large magnitude of peptide effects in intact tissues, the calcium-independence of the response both in intact tissues and in homogenates, the ability to stimulate cyclase activity in broken cells (but with marked attenuation when compared to the response of intact cells), the integral association of the cyclase with the membrane, the ability to solubilize the cyclase in active form with low concentrations of nonionic detergents, the stimulatory effects of particular detergents on cyclase activity, and the characteristic positive cooperativity of the enzyme with respect to its substrate GTP. Thus, studies of cyclic GMP in the lobster may provide insight not only into the general mechanisms that regulate cyclic GMP metabolism, but may also enhance our understanding of human pathologies that have been linked to ANP. These include such disorders as hypertension, coronary vasospasm, and atherosclerosis.
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