Constitutive Nitric Oxide Synthases in Rat Gastric Mucosa: Subcellular Distribution, Relative Activity and Different Carboxyl-Terminal Antigenicity of the Neuronal Form Compared with Cerebellum

Abstract

Background/Aims: To investigate whether the neuronal form of nitric oxide (NO) synthase (nNOS) in gastric mucosa differed from nNOS in the brain in subcellular distribution or antigenicity, and to determine the relative activity of the endothelial form of NO synthase (eNOS) and nNOS in gastric mucosa. Methods: Subcellular fractionation, assay of enzyme activity by conversion of arginine to citrulline, and immunoblotting were employed. Results: 10% of NO synthase activity in gastric mucosa was particulate compared with 31% in the cerebellum. Immunoblotting demonstrated that gastric mucosal nNOS protein was essentially cytosolic and that eNOS was largely particulate. Combination of these results with measurements of enzyme activity suggested that 96% of gastric mucosal activity was contributed by nNOS. Experiments with an antiserum raised against the carboxyl-terminal hexadecapeptide of rat brain NO synthase suggested a difference in antigenicity between forms of nNOS from the cerebellum and gastric mucosa. This difference did not seem to result from alternative splicing of the primary transcript, and was not present after enrichment of nNOS by affinity chromatography using 2′,5′-ADP agarose. Conclusion: The form of nNOS in rat gastric mucosa is largely cytosolic and is the major constitutive activity. When first isolated gastric nNOS differs in antigenicity from the equivalent brain enzyme, probably as a result of an unstable post-translational modification, close to the carboxyl-terminus.