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

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Abstract
In gastrointestinal smooth muscle, the neuropeptides vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) induce relaxation by interacting with VIP2/PACAP3receptors coupled via Gsto adenylyl cyclase and with distinct receptors coupled via Gi1and/or Gi2to 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 using125I-labeled atrial natriuretic peptide (125I-ANP) and125I-VIP as radioligands, VIP, ANP, and 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 Gi1and Gi2, 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, NG-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 Gi1and Gi2antibodies (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.