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.