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/PACAP3 receptors coupled via Gs to adenylyl cyclase and with distinct receptors coupled via Gi1 and/or Gi2 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 125I-labeled atrial natriuretic peptide (125I-ANP) and 125I-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 Gi1 and 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 Gi1 and Gi2 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.
Murthy
K S
KS
Departments of Physiology and Medicine, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298-0711, USA.
Teng
B
B
Jin
J
J
Makhlouf
G M
GM
D000818:Animals; D009320:Atrial Natriuretic Factor / Q000378:metabolism / Q000494:pharmacology; D019556:COS Cells; D002417:Cattle; D019204:GTP-Binding Proteins / Q000502:physiology*; D005753:Gastric Mucosa / Q000378:metabolism; D006162:Guanylate Cyclase / Q000502:physiology*; D009130:Muscle, Smooth / Q000166:cytology / Q000201:enzymology*; D010446:Peptide Fragments / Q000494:pharmacology; D011817:Rabbits; D017461:Receptors, Atrial Natriuretic Factor / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology; D013270:Stomach / Q000166:cytology; D014660:Vasoactive Intestinal Peptide / Q000378:metabolism / Q000502:physiology; D000099242:Atrial Natriuretic Factor Receptor B
D009320:Atrial Natriuretic Factor; D019204:GTP-Binding Proteins; D006162:Guanylate Cyclase; D010446:Peptide Fragments; D017461:Receptors, Atrial Natriuretic Factor; D014660:Vasoactive Intestinal Peptide; D000099242:Atrial Natriuretic Factor Receptor B; C065367:atrial natriuretic factor (4-23)NH2, de-Gln(18)-de-Ser(19)-de-Gly(20,22)-de-Leu(21)-; C073616:atrial natriuretic factor receptor C