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

In gastrointestinal smooth muscle, the neuropeptides vasoactiveintestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) induce relaxation by interacting withVIP₂/PACAP₃ receptors coupled via G_s toadenylyl cyclase and with distinct receptors coupled viaG_i1 and/orG_i2 to a smooth muscle endothelial nitric oxide synthase (eNOS). The present study identifies the receptoras the single-transmembrane natriuretic peptide clearance receptor(NPR-C). RT-PCR and Northern analysis demonstrated expression of thenatriuretic peptide receptors NPR-C and NPR-B but not NPR-A in rabbitgastric muscle cells. In binding studies using¹²⁵I-labeled atrial natriureticpeptide (¹²⁵I-ANP) and¹²⁵I-VIP as radioligands, VIP,ANP, and the selective NPR-C ligand cANP(4-23) bound with highaffinity to NPR-C. ANP, cANP-(4-23), and VIP initiated identicalsignaling cascades consisting ofCa²⁺ influx, activation of eNOSvia G_i1 andG_i2, stimulation of cGMP formation, and muscle relaxation. NOS activity and cGMP formation wereabolished (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 activitystimulated by all three ligands in muscle membranes was additivelyinhibited by G_i1 andG_i2 antibodies (82 ± 2 to 84 ± 1%). In reconstitution studies, VIP, cANP-(4-23), and guanosine 5'-O-(3-thiotriphosphate) stimulated NOS activity inmembranes of COS-1 cells cotransfected with NPR-C and eNOS. Theresults 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.