Vasoactive intestinal peptide/pituitary adenylate cyclase-activating peptide-dependent activation of membrane-bound NO synthase in smooth muscle mediated by pertussis toxin-sensitive Gi1-2.
Plasma membranes isolated from dispersed gastric muscle cells exhibited calmodulin-dependent NOS activity that was stimulated by Ca2+ in the range 0.1-1 mM (maximum 10 microM). Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP) (in the presence of GTP), and GTP gamma S (guanosine 5'-O-(gamma-thio)triphosphate) stimulated NOS activity in a concentration-dependent fashion above that maximally stimulated by Ca2+. The increase in NOS activity induced by VIP, PACAP, and GTP gamma S was abolished by GDP beta S (guanosine 5'-O-(beta-thio)diphosphate), which had no effect on NOS activity stimulated by Ca2+. The NOS inhibitor NG-nitro-L-arginine and the calmodulin antagonist calmidazolium abolished NOS activity stimulated by all agents including Ca2+. NOS activity stimulated by GTP gamma S, VIP, and PACAP was inhibited by Gi alpha 1-2 antibody but not by Gq alpha, Gs alpha, and Gi alpha 3 antibodies. NOS activity stimulated by VIP and PACAP was inhibited by 80-83% in membranes derived from pertussis toxin-treated cells. We conclude that a Ca2+/calmodulin-dependent NOS present in plasma membranes of gastric muscle cells is activated by two homologous peptide transmitters, VIP and PACAP, via a common receptor coupled to pertussis toxin (PTx)-sensitive Gi1-2. The study provides the first evidence of receptor-mediated G protein activation of NOS in smooth muscle cells.
Murthy
K S
KS
Department of Physiology, Medical College of Virginia, Richmond 23298-0711.
Makhlouf
G M
GM
D037361:Adenylate Cyclase Toxin; D000594:Amino Acid Oxidoreductases / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000037:antagonists & inhibitors / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism; D002469:Cell Separation; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D007093:Imidazoles / Q000494:pharmacology; D008565:Membrane Proteins / Q000378:metabolism; D009130:Muscle, Smooth / Q000201:enzymology*; D009249:NADP / Q000378:metabolism; D009479:Neuropeptides / Q000494:pharmacology*; D019001:Nitric Oxide Synthase; D037342:Pertussis Toxin; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D011817:Rabbits; D013270:Stomach / Q000166:cytology / Q000201:enzymology; D014660:Vasoactive Intestinal Peptide / Q000494:pharmacology*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D037361:Adenylate Cyclase Toxin; D002147:Calmodulin; D007093:Imidazoles; D008565:Membrane Proteins; D009479:Neuropeptides; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D010566:Virulence Factors, Bordetella; D014660:Vasoactive Intestinal Peptide; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C031938:calmidazolium; D009249:NADP; D006160:Guanosine Triphosphate; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D002118:Calcium