It is well known that the Enteric Nervous System (ENS) plays a predominant role in the control of three effector systems: smooth muscle (responsible for motility), the vasculature, and the intestinal mucosa (1). As for the latter, a large body of evidence from different animal species and experimental settings in fact supports a role of the ENS in the regulation of epithelial ion transport (2). The involvement of ENS seems to occur both in physiologic as well as in pathologic, mainly inflammatory, circumstances. Of the two neural networks of the ENS, the myenteric and the submucosal plexuses, the latter is primarily involved in the modulation of epithelial ion transport. The mucosal epithelium of the intestine is in fact predominantly innervated by secretomotor neurons with cell bodies in the ganglia of the submucous plexus. In the classical model, these secretomotor neurons are thought to evoke secretion by releasing their neurotransmitters (mostly acetylcholine and Vasoactive Intestinal Polypeptide - VIP-) which act at muscarinic or VIPergic receptors on the enterocytes. In recent times however the role of another neurotransmitter, the gas Nitric Oxide (NO) has become increasingly clear. This small molecule is now thought to be the main neurotransmitter of the so-called Non-Adrenergic, Non Cholinergic (NANC) branch of the ENS causing smooth muscle relaxation in many gastrointestinal tract locations (3). NO is generated from L-Arginine by several isoforms of NO synthase at least one of which reported to be present in the myenteric as well as in the submucosal plexuses and in enterocytes (4). This localization raises the possibility of a role for NO in modulating intestinal epithelial transport. Indeed, recent evidence suggests that NO may serve as a physiologic regulator of intestinal ion transport both as a pro-secretive or as a pro-absorptive agent. Most of the investigations on the role of NO in intestinal ion transport however have been done by employing inhibitors of NO synthesis or NO donors which would increase the NO concentration. Since these indirect approaches do not provide insights into the role of NO as a compound endogenously released following an increased availability of its natural precursor, in order to assess the role of NO on intestinal transport, we (5) determined the effects on ion transport of its biological precursor L-Arginine. Furthermore, to verify whether such effects were indeed due to the release of NO from its precursor, we assessed the effect of a known NO antagonist such as 2-2-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO) on L-Arginine induced changes in water transport. Finally, since we have previously shown that Cholera toxin (CT) is able to elicit secretion in a non-contiguous intestinal segment, we aimed at verifying if this effect could be attributed to the same neurotransmitter NO. To this purpose, we exposed the rat jejunal mucosa to CT and measured the changes induced by the toxin in the jejunum and in the simultaneously but separately perfused colon, both in the presence and in the absence of carboxy-PTIO. We have thus found that in vivo jejunal perfusion of L-Arginine, but not L-Lysine or D-Arginine, induced a dose-dependent pro-secretory effect on the jejunal transport of water, Na and Cl. The addition of the NO scavenger carboxy-PTIO significantly reversed the effect of L-Arginine. As for the role of NO as the agent responsible for the distant spreading of the Cholerz toxin secretory effect, we found that the presence of carboxy-PTIO in the colon abolished the distal secretory effect due to Cholera toxin addition to jejunum. In vitro, the pro-secretory activity of the pathway L-Arginine-NO was confirmed by the comparable decrease of net transport of both, Na and Cl. Also, in CaCo2-cells, mucosal addition of L-arginine raised both Potential Difference (PD) and Short circuit current (Isc), well known indexes of stimulated anion secretion, an effect not seen in Cl-free medium, thus indicating that the enterocytes have the biological machinery to release NO. Finally, L-Arginine significantly raised tissue levels of cGMP. Taken together, these observations, along with several other lines of evidence in the literature, show that the biological pathway L-Arginine-NO not only is operating in the small intestine with a significant and direct secretory effect on ion and water transport, but also that NO acts as a neurotransmitter within the ENS to spread the secretory effect due to Cholera toxin to non-contiguous intestinal segments.
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