VIP-mediated G protein-coupled Ca2+ influx activates a constitutive NOS in dispersed gastric muscle cells.
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.
Murthy
K S
KS
Department of Physiology, Medical College of Virginia, Richmond 23298.
Zhang
K M
KM
Jin
J G
JG
Grider
J R
JR
Makhlouf
G M
GM
D000594:Amino Acid Oxidoreductases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000502:physiology*; D005753:Gastric Mucosa / Q000378:metabolism*; D066298:In Vitro Techniques; D009126:Muscle Relaxation; D009130:Muscle, Smooth / Q000166:cytology / Q000378:metabolism*; D019001:Nitric Oxide Synthase; D010451:Peptide PHI / Q000378:metabolism; D047428:Protein Kinase Inhibitors; D011817:Rabbits; D018000:Receptors, Peptide / Q000378:metabolism; D018005:Receptors, Vasoactive Intestinal Peptide / Q000378:metabolism; D015398:Signal Transduction; D013270:Stomach / Q000166:cytology; D014660:Vasoactive Intestinal Peptide / Q000502:physiology*
C042452:PHI peptide receptor; D010451:Peptide PHI; D047428:Protein Kinase Inhibitors; D018000:Receptors, Peptide; D018005:Receptors, Vasoactive Intestinal Peptide; D014660:Vasoactive Intestinal Peptide; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases; D019204:GTP-Binding Proteins; D002118:Calcium