Nitric oxide has been implicated in the regulation of blood flow, mucosal integrity and mucus secretion in the gastric mucosa. An antiserum directed against the C-terminal hexadecapeptide of rat brain nitric oxide synthase (NOS) and monoclonal antibodies to the neuronal and endothelial forms of NOS were used to establish the location of isoforms of NOS in rat gastric glandular mucosa. Antibodies to the neuronal form of NOS reacted with a band of 160 kDa on immunoblots of brain and gastric mucosa, and the addition of the hexadecapeptide inhibited recognition by the antipeptide antiserum. The antibody to endothelial NOS detected a band of 140 kDa on protein blots of samples of intestinal mesentery and gastric mucosa. Immunohistochemistry using these antibodies demonstrated that material related to neuronal NOS was present in surface cells of the gastric mucosa, and showed a similar localization to intense NADPH diaphorase activity. The antibody to endothelial NOS did not stain the surface of the gastric mucosa but recognized blood vessels in the lower region of the gastric glands and in the sub-mucosa. This study suggests that nitric oxide might act both as an intra- and inter-cellular messenger to regulate mucus release, and that the NOS present in surface cells is related more closely to the neuronal than to the endothelial isoform.

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Nitric oxide (NO) synthase activity, which converts arginine to citrulline and NO, is present in homogenates of rat gastric mucosal cells. The aims of this study were to identify the form of NO synthase expressed in gastric cells isolated from fed rats, and to investigate the metabolism of arginine by suspensions of intact mucosal cells. Antibodies directed against the neuronal form of NO synthase recognised a protein of 160 kDa on immunoblots of extracts of gastric cells, and stained isolated cells of approx. 8 microm in diameter. NO synthase was enriched in a cell fraction which banded at high-density in a Percoll gradient, and was inhibited (IC50) by N(G)-nitro-L-arginine (0.8 microM), N(G)-monomethyl-L-arginine (12.6 microM), L-canavanine (147 microM), trifluoperazine (140 microM) and by phosphorylation involving protein kinase C. Intact gastric cells converted exogenous arginine to ornithine and citrulline. Arginase was present in the cells, and was predominantly responsible for arginine metabolism because formation of ornithine and citrulline was reduced by the arginase inhibitors, N(G)-hydroxy-L-arginine and L-ornithine, but not by NO synthase inhibitors such as N(G)-nitro-L-arginine. In conclusion, NO synthase that resembles the neuronal isoform is present in gastric mucosal cells, but a pathway involving arginase seems to be largely responsible for citrulline formation from exogenous arginine in intact mucosal cells.

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