Interplay between nitric oxide and vasoactive intestinal polypeptide in the pig gastric fundus smooth muscle

Abstract

The aim of this study was to investigate the exact mechanism of interaction between nitric oxide (NO) and vasoactive intestinal polypeptide (VIP) as inhibitory non-adrenergic non-cholinergic (NANC) neurotransmitters in isolated smooth muscle cells and smooth muscle strips of the pig gastric fundus. In isolated smooth muscle cells, the maximal relaxant effect of VIP (10⁻⁹ M) was inhibited by 94% by the NO synthase (NOS) inhibitor N^G-nitro-l-arginine (l-NA, 10⁻⁴ M) and by 85% by the inducible NOS (iNOS)-selective inhibitor N-(3-(aminomethyl)-benzyl)acetamide (1400W; 10⁻⁶ M). The relaxant effect of VIP was reduced by more than 70% by the guanylyl cyclase inhibitor 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ; 10⁻⁶ M), the glucocorticoid dexamethasone (10⁻⁵ M) and three protein kinase A inhibitors: (R)-p-cyclic adenosine-3′,5′-monophosphothioate ((R)-p-cAMPS; 10⁻⁶ M), {(8R,9S,11S)-(−)-9-hydroxy-9-n-hexylester-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H,11H-2,7b,11a-triazadibenzo[a,g]cycloocta[cde]-trin-den-1-one} (KT5720; 10⁻⁶ M) and N-(2-(p-bromo-cinnamylamino)ethyl))-5-isoquinoline sulfonamide dihydrochloride (H-89; 10⁻⁵ M). In contrast, no influence of the NOS inhibitors, ODQ, dexamethasone, nor the protein kinase A inhibitors could be observed on the relaxant effect of VIP in smooth muscle strips. These data demonstrate that the experimental method completely changes the influence of NOS inhibitors on the relaxant effect of VIP in the pig gastric fundus. The isolation procedure of the smooth muscle cells might induce iNOS that can be activated by VIP.

Introduction

It has been well recognized that both nitric oxide (NO) and vasoactive intestinal polypeptide (VIP) serve as inhibitory non-adrenergic non-cholinergic (NANC) mediators in the gastrointestinal tract D'Amato et al., 1992, Sanders and Ward, 1992, Brookes, 1993, Shuttleworth and Keef, 1995, Rand and Li, 1995. Still, there is a lot of controversy about the interaction between NO and VIP in the gastrointestinal tract. In many neurons of the myenteric plexus from different gastrointestinal tissues, including the pig gastric fundus, immunoreactivity revealed colocalization of VIP and neuronal NO synthase (nNOS, Furness et al., 1992, Berezin et al., 1994, Lefebvre et al., 1995) suggesting cotransmission of VIP and NO. Hereby NO is thought to induce relaxation via a guanosine 3′5′ cyclic monophosphate (cyclic GMP)-dependent pathway and VIP via an adenosine 3′5′ cyclic monophosphate (cyclic AMP)-dependent pathway. In line with the model of cotransmission of VIP and NO is the observation that the relaxation by VIP in many gastrointestinal tissues is not influenced by NOS inhibitors Tøttrup et al., 1991, Boeckxstaens et al., 1992, Barbier and Lefebvre, 1993, Keef et al., 1994. In contrast, the group of Makhlouf reported that in both isolated smooth muscle cells and smooth muscle strips of the guinea-pig gastric fundus and rat colon, the VIP-induced relaxation is inhibited by NOS inhibitors. A sequential link between NO and VIP is proposed whereby VIP seems to be the primary neurotransmitter, inducing relaxation partially via activation of adenylate cyclase and partially via stimulation of NO production in the smooth muscle cells Grider et al., 1992, Grider, 1993, Jin et al., 1993. The enzyme involved in the muscular production of NO would be endothelial NO synthase (eNOS) as measured by reverse transcription-polymerase chain reaction and Southern blot analysis in rabbit gastric and human intestinal smooth muscle cells (Teng et al., 1998).

Recent results obtained in our laboratory showed a completely different influence of NOS inhibitors in smooth muscle cells versus smooth muscle strips of the guinea-pig gastric fundus (Dick et al., 2000). In isolated smooth muscle cells, the relaxation by VIP is inhibited by NOS inhibitors, including the inducible NO synthase (iNOS)-selective inhibitor 1400W, whereas in isolated smooth muscle strips it is not. These results suggest that the experimental method determines the influence of NOS inhibitors on the relaxant effect of VIP in the guinea-pig gastric fundus. An NOS isoform with properties of iNOS, probably induced by the isolation procedure of the smooth muscle cells, might be involved in the relaxation induced by VIP in the isolated smooth muscle cells.

Among the non-primate mammalian species, the pig has been proposed as one of the best models for the study of nutritional issues in man, due to the similarity of the morphology and physiology of the gastrointestinal tracts (Miller and Ullrey, 1987). It has been shown before in the pig gastric fundus that NO and to a smaller extent VIP are involved in NANC relaxation (Lefebvre et al., 1995). The aim of this study was to investigate whether the differential effect of NOS inhibitors in gastric smooth muscle cells and strips, as observed in a rodent, could be reproduced in a non-rodent higher mammalian species, the pig.