H3 receptor-mediated inhibition of intestinal acetylcholine release:: pharmacological characterization of signal transduction pathways

The present study investigates the mechanisms through which prejunctional histamine Hg receptors modulate intestinal cholinergic neurotransmission. The experiments were performed on longitudinal muscle-myenteric plexus preparations of guinea pig ileum, preincubated with [H-3]choline, superfused with physiological salt solution containing hemicholinium-3, and subjected to electrical field stimulation. The stimulation-induced outflow of radioactivity was taken as an index of endogenous acetylcholine release. The electrically induced [H-3]acetylcholine release was inhibited by histamine (EC50=33.5 nM) or the H, receptor agonist R-alpha -methylhistamine (EC50=41.6 nM), whereas it was not affected by pyridylethylamine (H-1 agonist), impromidine (H-2 agonist), pyrilamine (H-1 antagonist), cimetidine (H-2 antagonist), thioperamide or clobenpropit (H-2 antagonists). The inhibitory effects of histamine or R-alpha -methylhistamine were antagonized by thioperamide (pK(d)= 8.31 and 8.53, respectively) or clobenpropit (pKd=9.44 and 9.32, respectively), but not by pyrilamine or cimetidine. The modulatory action of histamine on the evoked tritium outflow was attenuated by pertussis toxin and abolished by N-ethylmaleimide, two selective blockers of G(i)/G(o) proteins. Tetraethylammonium or 4-aminopyridine, acting as inhibitors of voltage-dependent K+ channels, enhanced the evoked tritium outflow when tested alone, and apparently counteracted the inhibitory effect of histamine. However, the blocking actions of tetraethylammonium and 4-aminopyridine were no longer evident when their enhancing actions were compensated by appropriate reductions of Ca2+ concentration in the superfusion medium. Histamine-induced inhibition of evoked tritium output was enhanced by omega -conotoxin, a selective blocker of N-type Ca2+ channels, or low Ca2+ concentration, whereas it was not modified by nifedipine, an antagonist of L-type Ca2+ channels. In addition, the inhibitory effect of histamine was not significantly affected by forskolin (activator of adenylyl cyclase), 8-bromo-cyclic AMP (a stable analog of cyclic AMP), rolipram (a selective blocker of type IV phosphodiesterase), phorbol myristate acetate (activator of protein kinase C), H-89 (N-(2-[p-bromocinnamyl amino]ethyl)-5-isoquinolinesulfonamide, inhibitor of protein kinase A), Ro-31-8220 (2-{1-[3-(amidinothio)propyl]1H-indol-3-yl}-3-(1-methylindol-3-yl)-maleimide, inhibitor of protein kinase C), KT5823 (N-methyl-(8R*,9S*,11S*)(-)-9-methoxy-9 -methoxycarbonyl-8-methyl-2, 3,9,10-tetrahydro-8,11-epoxy-1H,8H,11H-2,7b,11a-triazadibenzo [a,g]cycloocta[c,d,e]-trinden-1-one, inhibitor of protein kinase G), or lavendustin A (inhibitor of tyrosine kinase). The present results indicate that histamine inhibits intestinal cholinergic neurotransmission through presynaptic H-3 receptors coupled to G(i)/G(o), proteins. It is suggested that adenylyl cyclase, serine-threonine protein kinase and tyrosine kinase pathways are not implicated in this regulatory action, and that G(i)/G(o), proteins modulate the activity of N-type Ca2+ channels through a direct link, thus causing a reduced availability of extracellular Ca2+ at the level of ileal cholinergic nerve terminals.