Abstract
The mechanism by which atrial natriuretic peptide (ANP) acts on cells remains obscure. Using cultured vascular smooth muscle cells (VSMC) from rat aorta, we studied the structure-activity relationship of α-human(h) ANP and attempted to clarify its cellular mechanism of action .Binding studies using a variety of synthetic α-hANP7-28 analogs with deletion and substitution of amino-acid residue(s) within the ring structure and deamino-dicarba analogs with replacement of the disulfide bond with an ethylene linkage, suggested that the original cyclic structure is a minimum requirement for biological activity and the disulfide bond is not essential for receptor binding. The present study using VSMC loaded with a fluorescent Ca2+ indicator quin-2 revealed that α-rat(r) ANP has no effect on increases in cytosolic free Ca2+ concentration stimulated by angiotensin (A) II or arginine-vasopression (AVP). While both vasoconstrictive hormones rapidly stimulated phosphatidylinositol (PI) response in VSMC, pretreatment with rANP did not affect AII-or AVP-induced PI response. However, AII-stimulated phosphorylation level of 20 K-dalton (Da) myosin light chain (MLC), a regulatory contractile protein of VSMC, was attenuated by pretreatment with rANP. These data suggest that ANP may not act at site of either agonist-induced membrane PI hydrolysis or intracellular Ca2-signal system, but may partly be involved in phosphorylation/dephosphorylation of 20 K-Da MLC in cultured rat VSMC.
