Mechanism of neurogenic relaxation and modification of the response by enteric substances in isolated dog colon

Abstract

The mechanisms of neurogenic relaxation in the longitudinal muscle of the isolated canine colon and its modification by enteric substances were investigated. Relaxations induced by transmural electrical stimulation with electrical pulses, nicotine or K⁺ in the muscle strips contracted with bradykinin and treated with atropine were attenuated but not abolished by N^G-nitro-l-arginine (l-NA), and the inhibition was reversed by l-arginine. Oxyhemoglobin and ouabain inhibited the response, whereas K⁺ channel inhibitors, such as glibenclamide, tetraethylammonium, apamin and charybdotoxin, were without effect. In l-NA-treated strips, stimulation-induced relaxations were reduced by ouabain but not by oxyhemoglobin. Among substances tested, only norepinephrine, ATP, vasoactive intestinal peptide (VIP) and galanin produced relaxations. However, α- and β-adrenoceptor antagonists and aminophylline did not alter the response to nerve stimulation. In the strips made unresponsive to VIP and galanin, stimulation-induced relaxations were not influenced. Indomethacin, calcitonin gene-related peptide, cholecystokinin, peptide YY, substance P and serotonin did not modulate the neurogenic response. It is concluded that the relaxation associated with nerve stimulation is mediated by nitric oxide (NO) synthesized from l-arginine and also by substance(s) activating the electrogenic Na⁺ pump but not that opening K⁺ channels. Norepinephrine, ATP, VIP and galanin can be excluded as candidate inhibitory neurotransmitters, and the substances used so far are unlikely to modulate inhibitory nerve function.

Introduction

Mechanisms underlying non-adrenergic, non-cholinergic inhibitory neurotransmission in the gastrointestinal tract have been analyzed by electrophysiological, pharmacological and histological techniques with regard to the role of nitric oxide (NO) and neuropeptides (Lundberg, 1996). A contribution of NO to the inhibitory response to nerve stimulation was first defined using NO synthase inhibitors in the canine duodenum (Toda et al., 1990), together with a NO bioassay method for the canine ileocolonic junction (Bult et al., 1990). Accumulated data demonstrate that the involvement of neurogenic NO and peptides in the regulation of gastroenteric motility and tone differs according to location in the digestive tract and also the type of mammals concerned. In the rat stomach, relaxation caused by inhibitory nerve stimulation is associated with NO and vasoactive intestinal peptide (VIP) (Li and Rand, 1990; Lefebvre, 1993), while in the rat ileum, the electrical stimulation-induced contraction is blunted by NO synthase inhibitors, suggesting that NO derived from nerves mediates contraction or changes the release of other neurotransmitters (Bartho et al., 1992). NO is mainly involved in the neurogenic relaxation of longitudinal muscle of the proximal segment of the rat colon, whereas VIP is a likely neurotransmitter in the distal colon (Suthamnatpong et al., 1993). Nitroxidergic nerves may be involved only partially in the stimulation-induced relaxation in the circular muscle of the human ileum (Maggi et al., 1991).

We have reported that neurogenic relaxation of the canine duodenum (Toda et al., 1990, Toda et al., 1991, Toda et al., 1992) and the sphincter of Oddi (Tanobe et al., 1995) is mediated exclusively by NO or its stable analog, such as S-nitroso thiol, that possibly activates guanylate cyclase in smooth muscle and increases the production of cyclic GMP (Toda et al., 1992). However, our preliminary study of the isolated canine colon suggested that relaxations induced by nerve stimulation are mediated not only by NO but also by other inhibitory factors.

The present study was therefore undertaken to analyze the mechanism of the relaxation induced by nerve stimulation with electrical pulses, nicotine and K⁺ in longitudinal muscles of the proximal colon from dogs, in comparison with mechanisms underlying the neurogenic response of the canine duodenum and sphincter of Oddi. We also sought whether enteric substances, such as VIP, cholecystokinin (CCK), calcitonin gene-related peptide (CGRP), peptide YY, galanin, norepinephrine and serotonin, participate in and/or modulate the inhibitory response to nerve stimulation.