VIP-mediated G protein-coupled Ca2+ influx activates a constitutive NOS in dispersed gastric muscle cells

Abstract

Vasoactive intestinal peptide (VIP) and peptide histidine-isoleucine (PHI) receptors and the signaling pathways to which they are coupled were characterized in dispersed gastric smooth muscle cells. Radioligand binding using 125I-labeled VIP and PHI identified 4 classes of receptors: VIP-preferring and PHI-preferring receptors recognized by both ligands and readily desensitized by the preferred ligand, and VIP-specific and PHI-specific receptors recognized by only 1 ligand and resistant to desensitization. All except VIP-specific receptors were coupled to adenylate cyclase. VIP-specific receptors mediated a G protein-coupled Ca2+ influx that led to activation of NO synthase (NOS), NO-dependent activation of soluble guanylate cyclase, and activation of guanosine 3',5'-cyclic monophosphate (cGMP) kinase resulting in muscle relaxation. The entire cascade was blocked by Ca2+ channel and/or calmodulin antagonists. The NOS inhibitor NG-nitro-L-arginine abolished L-[3H]citrulline (coproduct of NO synthesis) and cGMP generation and partly inhibited (52 +/- 4%) relaxation. The components of response mediated by VIP-specific receptors (increase in [Ca2+]i, L-[3H]citrulline, and cGMP) were preserved after desensitization. Insertion of guanosine 5'-O-(beta-thio)diphosphate into reversibly permeabilized muscle cells abolished responses mediated by VIP-preferring and VIP-specific receptors. VIP stimulated both adenosine 3',5'-cyclic monophosphate (cAMP)-kinase and cGMP-kinase activities consistent with stimulation of cAMP and cGMP. Both kinases contributed to relaxation that was partly inhibited by cAMP-kinase [H-89 and (R)-p-adenosine 3',5'-cyclic monophosphorothioate] and cGMP-kinase (KT-5823) inhibitors and abolished by a combination of the 2 types of inhibitors. We conclude that VIP-specific receptors mediate a G protein-coupled Ca2+ influx leading to activation of a constitutive Ca2+/calmodulin-dependent NOS and generation of NO, which is partly responsible for relaxation in smooth muscle.

Distinct populations of sensory neurons mediate the peristaltic reflex elicited by muscle stretch and mucosal stimulation

“…3), a feature also observed in intact colonic segments (Grider, 1993). The relative potency of VIP 1 O-28 reflects the fact that VIP-induced NO production in muscle cells is the main source of NO Grider, 1993;Murthy et al, 1993). Addition of VIPlO-28 and L-NNA to the orad compartment had no effect on descending relaxation.…”

Neuronal constitutive nitric oxide synthase is involved in murine enteric inhibitory neurotransmission.

“…Note that VIP produces hyperpolarization in wild-type mice, but not in ncNOS(Ϫ) mice or tissues pre-treated with L-NA. Exogenously applied NO produces hyperpolarization in both wildtype and ncNOS(Ϫ) smooth muscle cells which is unaffected by VIP [10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28] . This demonstrates that VIP acts by releasing NO.…”

“…This calcium influx then activates a particulate, membrane-bound, calcium-and calmodulin-dependent constitutive isoform of NOS resembling ecNOS (10). The activation of ecNOS in the smooth muscle produces NO which causes elevation of intracellular cGMP and smooth muscle relaxation (11). By this formulation, VIP is the inhibitory neurotransmitter and NO acts as an intracellular mediator of VIP (20).…”

“…However, ncNOS is a soluble cytosolic enzyme whereas ecNOS is particulate and membrane bound. At the smooth muscle neuromuscular junction in the gut, ncNOS is primarily localized to nerve endings and ecNOS is primarily in the smooth muscle cells (11). These isoforms cannot be distinguished using pharmacologic agents, but they are products of distinct genes which can be selectively disrupted by homologous recombination (8,(11)(12)(13)(14)(15).…”

Involvement of Nitric Oxide and Vasoactive Intestinal Peptide in the Nonadrenergic-Noncholinergic Relaxation of the Porcine Retractor Penis Muscle

“…NO stimulates the presynaptic release of VIP in isolated guinea pig intestine ganglia (16) and in synaptosomal fraction of rat intestine (17). On the other hand, VIP stimulates the postsynaptic generation of NO in guinea pig stomach (18), ileum (19) and cecum (20). In rabbit corpus cavernosum, VIP induces relaxation partially by NO synthesis (21).…”