Modulation of Atrial Natriuretic Peptide (ANP)-C Receptor and Associated Signaling by Vasoactive Peptides

Abstract

We have previously shown that pretreatment of A10 smooth muscle cells (SMC) with angiotensin II (Ang II) attenuated atrial natriuretic peptide (ANP) receptor-C (ANPC) mediated-inhibition of adenylyl cyclase without altering [125I]-ANP binding. In the present studies, we have investigated the modulation of ANP-C receptor signaling by C-ANP4–23, an analog of ANP that interacts specifically with ANP-C receptor and endothelin-1 (ET-1). Pretreatment of A10 SMC with C-ANP 4–23 for 24hrs resulted in the reduction in ANP receptor binding activity, whereas ET-1 treatment of the cells attenuated the expression of ANP-C receptor by about 60% as determined by immunoblotting. This decrease in receptor binding and receptor expression was reflected in attenuation of ANP-C receptor mediated-inhibition of adenylyl cyclase. C-ANP4–23 [des(Gln18,Ser19,Gln20,Leu21,Gly22) ANP4–23 NH2] a ring deleted peptide of ANP inhibited adenylyl cyclase activity in a concentration dependent manner with an apparent Ki of about 1 nM in control cells. The maximal inhibition observed was by about 30% which was almost completely attenuated by C-ANP4–23 and ET-1 treatments. In addition, Ang II-mediated inhibition of adenylyl cyclase was also attenuated by such treatments. The expression of Giα-2 and Giα-3 protein was decreased by C-ANP4–23 treatment, whereas it was augmented by ET-1 treatment. On the other hand, the expression of Gsα was augmented by C-ANP4–23and not by ET-treatment, whereas the expression of Gβ protein was unaltered by such treatments. The Gsα-mediated effects of some agonists on adenylyl cyclase activity were significantly increased by C-ANP4–23 treatment and was decreased by ET-1 treatment. These results suggest that both the vasoactive peptides down regulate ANP-C receptor in A10 SMC. The C-ANP4–23-induced down-regulation of ANP-C receptor and decreased expression of Giα proteins may be responsible for the attenuation of C-ANP4–23-mediated inhibition of adenylyl cyclase activity, whereas ET-induced attenuation of C-ANP4–23-mediated inhibition of adenylyl cyclase may be attributed to the decreased expression of ANP-C receptor and not to the overexpression of Giα proteins. From these studies it may be suggested that the desensitization of ANP-C receptors by ANP and endothelin in vivo may be one of the possible mechanisms for the pathophysiology of hypertension.