Nitric Oxide Produced by the Enterocyte Is Involved in the Cellular Regulation of Ion Transport

Abstract

The role of nitric oxide (NO) in the intestinal basal ion transport and under conditions of enterotoxin-induced ion secretion is controversial. Namely it is not clear whether NO enhances or counteracts intestinal ion secretion and whether the effects on transport result from a direct interaction with the enterocyte. The cell origin of NO is also unclear. We have tested the hypothesis that NO produced by the enterocyte directly regulates ion transport processes either in basal condition or in response to cholera toxin-induced secretion. Electrical variables reflecting transepithelial ion transport were measured in Caco-2 cell monolayers mounted in Ussing chambers exposed to the NO synthase inhibitor N-nitro-L-arginine methyl ester, in the presence or absence of cholera toxin. cAMP concentrations were also measured. NO release was determined by nitrite-nitrate concentration. NO synthase activities were assayed by Western blot analysis. N-nitro-L-arginine methyl ester had a secretory effect, as judged by increased basal short-circuit current and cAMP concentration. It also increased cholera toxin-induced electrical response and cAMP production. Either cholera toxin or the cAMP analog 8-bromo-cAMP induced a rapidly progressive and Ca 2ϩ -dependent increase in NO concentration, suggesting a homeostatic up-regulation of the constitutive form of NO synthase.Western blot analysis showed an increase in constitutive NO synthase enzyme isoform. These results indicate that the enterocyte regulates its own ion transport processes, either in basal condition or in the presence of active secretion, through the activation of a constitutive NO synthase-NO pathway, functioning as a braking force of cAMP-induced ion secretion. Intestinal water and electrolyte transepithelial transport is under a complex control by several agents including neurotransmitters, hormones, or paracrine agents (1). There is evidence that NO takes part in the intestinal ion transport processes with an effect that involves the enteric nervous system, the suppression of prostaglandin formation, and the opening of K ϩ channels (2, 3). NO is a gas with a half-life of less than 5 s, rapidly degrading to nitrite and nitrate in the presence of oxygen and water. NO is produced by a number of cells, including neuronal cells, and released in various organs. Being soluble in both water and lipids, it freely passes into adjacent target cells (2,4). NO is generated from L-arginine by a family of enzymes, NOS, whose activity can be stereospecifically inhibited by L-but not by D-arginine analogs (5). Two NOS are constantly active in the intestine and are termed cNOS. They include the neuronal NOS (NOS 1) and the endothelial NOS (NOS 3). Both are Ca 2ϩ /calmodulin-dependent, produce small amounts of NO in short burst, and are involved in several homeostatic processes. A third NOS form, which is Ca 2ϩ /calmodulin-independent, is activated by various signal molecules, including proinflammatory cytokines. This latter form, termed iNOS (NOS 2), requires at least 2 h to be act...

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