Characterization of nitric oxide synthase in the opossum esophagus

Background/Aims: Nitric oxide mediates the nonadrenergic, noncholinergic neural control of esophageal motor function. The purpose of this study was to characterize the NO synthase found in the muscularis propria of the opossum esophagus and determine its distribution along the esophagus. Methods: Esophageal muscle was homogenized in HEPES buffer and ultracentrifuged. The supernatant was exposed to [³H]l-arginine. The [³H]l-citrulline produced by NO synthase was separated from [³H]l-arginine with a Dowex AG 50 W-X8 column (Biorad, Hercules, CA). Assays were performed in the presence and absence of Ca²⁺ or reduced nicotinamide adenine dinucleotide phosphate (NADPH). The distribution of NO synthase activity along the esophagus was determined. Results: The apparent Michaelis constant and maximum velocity of NO synthase were 7.5 ± 1.4 μmol/L l-arginine and 76.0 ± 17.3 pmol·mg protein^-1 ·min^-1, respectively. The enzyme required both Ca²⁺ and NADPH for activity. Smooth muscle tissue from the lower esophageal sphincter and the esophageal body 1-2 cm or 5-6 cm above the lower esophageal sphincter differed little in enzymatic activity, ranging from 0.97 to 1.27 pmol·mg wet wt^-1·min^-1. Striated muscle had less activity with 0.40 pmol·mg wet wt^-1·min^-1. Conclusions: These data indicate the presence of a constitutive NO synthase in the esophagus of the opossum.