vasoactive-intestinal-peptide and calmidazolium

In the goldfish pituitary, nerve fibers containing pituitary adenylate cyclase-activating polypeptide (PACAP) are located in close proximity to somatolactin (SL)-producing cells, and PACAP enhances SL release from cultured pituitary cells. However, there is little information about the mechanism of PACAP-induced SL release. In order to elucidate this issue, we used the cell immunoblot method. Treatment with PACAP at 10(-8) and 10(-7)M, but not with vasoactive intestinal polypeptide (VIP) at the same concentrations, increased the immunoblot area for SL-like immunoreactivity from dispersed pituitary cells, and PACAP-induced SL release was blocked by treatment with the PACAP selective receptor (PAC(1)R) antagonist, PACAP(6-38), at 10(-6)M, but not with the PACAP/VIP receptor antagonist, VIP(6-28). PACAP-induced SL release was also attenuated by treatment with the calmodulin inhibitor, calmidazolium at 10(-6)M. This led us to explore the signal transduction mechanism up to SL release, and we examined whether PACAP-induced SL release is mediated by the adenylate cyclase (AC)/cAMP/protein kinase A (PKA)- or the phospholipase C (PLC)/inositol 1,4,5-trisphosphate (IP(3))/protein kinase C (PKC)-signaling pathway. PACAP-induced SL release was attenuated by treatment with the AC inhibitor, MDL-12330A, at 10(-5)M or with the PKA inhibitor, H-89, at 10(-5)M. PACAP-induced SL release was suppressed by treatment with the PLC inhibitor, U-73122, at 3 x 10(-6)M or with the PKC inhibitor, GF109203X, at 10(-6)M. These results suggest that PACAP can potentially function as a hypophysiotropic factor mediating SL release via the PAC(1)R and subsequently through perhaps the AC/cAMP/PKA- and the PLC/IP(3)/PKC-signaling pathways in goldfish pituitary cells.

Topics: Adenylyl Cyclase Inhibitors; Adenylyl Cyclases; Animals; Calmodulin; Cells, Cultured; Enzyme Inhibitors; Estrenes; Female; Fish Proteins; Glycoproteins; Goldfish; Growth Substances; Imidazoles; Imines; Immunoblotting; Indoles; Intracellular Signaling Peptides and Proteins; Isoquinolines; Male; Maleimides; Phosphoinositide Phospholipase C; Pituitary Adenylate Cyclase-Activating Polypeptide; Pituitary Gland; Pituitary Hormones; Protein Kinase C; Pyrrolidinones; Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide; Signal Transduction; Sulfonamides; Vasoactive Intestinal Peptide

Understanding the mechanisms involved in the biogenesis of N-arachidonoylethanolamine (anandamide) and N-palmitoylethanolamine is important in view of the possible role of these lipids as endogenous cannabinoid substances. Anandamide (which activates cannabinoid CB1 receptors) and N-palmitoylethanolamine (which activates a CB2-like receptor subtype in mast cells) may both derive from cleavage of precursor phospholipid, N-acylphosphatidylethanolamine (NAPE), catalyzed by Ca(2+)-activated D-type phosphodiesterase activity. We report here that the de novo biosynthesis of NAPE is enhanced in a Ca(2+)-dependent manner when rat cortical neurons are stimulated with the Ca(2+)-ionophore ionomycin or with membrane-depolarizing agents such as veratridine and kainate. This reaction is likely to be mediated by a neuronal N-acyltransferase activity, which catalyzes the transfer of an acyl group from phosphatidylcholine to the ethanolamine moiety of phosphatidylethanolamine. In addition, we show that Ca2+-dependent NAPE biosynthesis is potentiated by agents that increase cAMP levels, including forskolin and vasoactive intestinal peptide. Our results thus indicate that NAPE levels in cortical neurons are controlled by Ca2+ ions and cAMP. Such regulatory effect may participate in maintaining a supply of cannabimimetic N-acylethanolamines during synaptic activity, and prime target neurons for release of these bioactive lipids.

Topics: Amides; Animals; Anti-Inflammatory Agents, Non-Steroidal; Arachidonic Acids; Arylamine N-Acetyltransferase; Astrocytes; Calcium; Calcium Channel Blockers; Calmodulin; Cannabinoids; Carbachol; Cyclic AMP; Endocannabinoids; Enzyme Inhibitors; Ethanolamine; Ethanolamines; Imidazoles; Ionomycin; Ionophores; Neurons; Nicotinic Agonists; Palmitic Acids; Phosphatidylethanolamines; Polyunsaturated Alkamides; Rats; Sodium Channel Agonists; Tritium; Vasoactive Intestinal Peptide; Veratridine

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.

Topics: Adenylate Cyclase Toxin; Amino Acid Oxidoreductases; Animals; Calcium; Calmodulin; Cell Membrane; Cell Separation; Enzyme Activation; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Imidazoles; Membrane Proteins; Muscle, Smooth; NADP; Neuropeptides; Nitric Oxide Synthase; Pertussis Toxin; Pituitary Adenylate Cyclase-Activating Polypeptide; Rabbits; Stomach; Vasoactive Intestinal Peptide; Virulence Factors, Bordetella