ABSTRACT
Nitric oxide mediates neuromuscular events in the opossum esophagus. The NADPH diaphorase stain is used to localize nitric oxide synthase-containing enteric neurons. Cells stain by the NADPH diaphorase technique because they reduce nitroblue tetrazolium to the visible formazan. The effects of nitroblue tetrazolium on neuromuscular function and nitric oxide synthase of esophageal muscle were studied. The NADPH diaphorase stain was performed. Nitroblue tetrazolium inhibited lower esophageal sphincter relaxation, abolished the latency gradient of the off response, and inhibited nitric oxide synthase. The NADPH diaphorase technique stained myenteric plexus nerve cell bodies and nerve processes. Nitroblue tetrazolium is not a nonspecific muscle or nerve toxin, as nerve-mediated cholinergic responses, responses to exogenous nitric oxide, and responses to myogenic stimulation were maintained after nitroblue tetrazolium abolished the off response and lower esophageal sphincter relaxation. Nitroblue tetrazolium inhibits nitric oxide-mediated events and nitric oxide synthase. It stains neurons in the esophageal myenteric plexus.
Subject(s)
Esophagus/drug effects , Nitric Oxide Synthase/metabolism , Nitroblue Tetrazolium/pharmacology , Animals , Electric Stimulation , Esophagogastric Junction/drug effects , Esophagogastric Junction/physiology , Esophagus/enzymology , Esophagus/innervation , Esophagus/physiology , Histocytochemistry , In Vitro Techniques , Muscle Contraction/drug effects , Muscle Relaxation/drug effects , Myenteric Plexus/anatomy & histology , NADPH Dehydrogenase , Nitric Oxide Synthase/antagonists & inhibitors , Opossums , Staining and LabelingABSTRACT
Nitric oxide mediates nerve-induced hyperpolarization of circular smooth muscle of the esophagus. Two mechanisms are proposed to explain this hyperpolarization: an increase in K+ current or a decrease in Cl- current. These studies test the hypothesis that nitric oxide increases a K+ current in esophageal smooth muscle. Three outward K+ currents are present in circular smooth muscle cells from the opossum esophagus. One current is a Ca(2+)-activated K+ current (IKCa2+). This current is inhibited by charybdotoxin. Whole cell currents were recorded from isolated opossum esophageal smooth muscle cells using the whole cell patch-clamp technique. These studies showed that IKCa2+ is activated at potentials more positive than -30 mV. Bath application of S-nitroso-L-cysteine increased IKCa2+ by 50% above control levels throughout the entire activation range of potentials. The enhanced current was reversible on washout. Either charybdotoxin, an inhibitor of IKCa2+, or (R)-p-8-(4-chloropenylthio)-guanosine 3',5'-cyclic monophosphorothioate, an inhibitor of protein kinase G, antagonized the increase in outward current induced by S-nitroso-L-cysteine. These data suggest that nitric oxide activates IKCa2+ via the guanosine 3',5'-cyclic monophosphate-protein kinase G signal transduction pathway.
