G protein-dependent activation of smooth muscle eNOS via natriuretic peptide clearance receptor

In gastrointestinal smooth muscle, the neuropeptides vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) induce relaxation by interacting with VIP2/PACAP(3) receptors coupled via G(s) to adenylyl cyclase and with distinct receptors coupled via G(i1) and/or G(i2) to a smooth muscle endothelial nitric oxide synthase (eNOS). The present study identifies the receptor as the single-transmembrane natriuretic peptide clearance receptor (NPR-C). RT-PCR and Northern analysis demonstrated expression of the natriuretic peptide receptors NPR-C and NPR-B but not NPR-A in rabbit gastric muscle cells. In binding studies using I-125-labeled atrial natriuretic peptide (I-125-ANP) and I-125-VIP as radioligands, VIP, ANP, aha the selective NPR-C ligand cANP-(4-23) bound with high affinity to NPR-C. ANP, cANP-(4-23), and VIP initiated identical signaling cascades consisting of Ca2+ influx, activation of eNOS via G(i1) and G(i2), stimulation of cGMP formation, and muscle relaxation. NOS activity and cGMP formation were abolished (93 +/- 3 to 96 +/- 2% inhibition) by nifedipine, pertussis toxin, the NOS inhibitor, N-G-nitro-L-arginine, and the antagonists ANP-(1-11) and VIP-(10-28). NOS activity stimulated by all three ligands in muscle membranes was additively inhibited by G(i1) and G(i2) antibodies (82 +/- 2 to 84 +/- 1%). In reconstitution studies, VIP, cANP-(4-23), and guanosine 5'-O-(3-thiotriphosphate) stimulated NOS activity in membranes of COS-1 cells cotransfected with NPR-C and eNOS. The results establish a unique mechanism for G protein-dependent activation of a constitutive NOS expressed in gastrointestinal smooth muscle involving interaction of the relaxant neuropeptides VIP and PACAP with a single-transmembrane natriuretic peptide receptor, NPR-C.