The influence of nitric oxide donors on the responses to nitrergic nerve stimulation in the mouse duodenum

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Abstract

We investigated whether exogenous nitric oxide (NO) donors have a prejunctional and/or postjunctional inhibitory effect on the nitrergic responses and whether this inhibitory effect was mediated by NO itself and in part, by cyclic GMP in mouse duodenal strips. Nω-nitro-l-arginine inhibited relaxations induced by electrical field stimulation of nitrergic nerves, but not those with acidified NaNO2. Furthermore, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) inhibited both types of relaxations while 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT) and N-ethylmaleimide were ineffective. NO donors, nitroglycerin and sodium nitroprusside, inhibited relaxations induced by nitrergic nerve stimulation, but not those with acidified NaNO2. Hemoglobin, exogenous Cu2+/Zn2+ superoxide dismutase, diethyldithiocarbamic acid and pyrogallol did not influence the relaxation with nitrergic nerve stimulation. However, hemoglobin, diethyldithiocarbamic acid, pyrogallol and diethyldithiocarbamic acid plus pyrogallol attenuated the inhibitory effect of NO donors on relaxation with nitrergic nerve stimulation, and exogenous superoxide dismutase potentiated this inhibitory effect. Moreover, nitrergic nerve-mediated relaxations were inhibited by 8-bromo-cyclic GMP, but not by 8-bromo-cyclic AMP. These results suggest that exogenous NO donors have a prejunctional inhibitory effect on the nerve-mediated nitrergic relaxation and that the inhibitory effects of nitroglycerin and sodium nitroprusside are NO-dependent, but not related to NO metabolites such as peroxynitrite or a nitrosothiol intermediate. However, a contribution of S-nitrosothiol formed intracellularly cannot be entirely ruled out. Also, this prejunctional inhibition is mediated, at least in part, by the cyclic GMP, but not the cyclic AMP, pathway.

Introduction

Nitric oxide (NO) is formed via the enzymatic activity of NO synthase (NOS), which catalyzes the oxidation of l-arginine to NO plus l-citrulline. There are several different molecular forms of NOS, including neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS) Moncada et al., 1991, Brookes, 1993, Rand and Li, 1995, Kröncke et al., 1995. At the smooth muscle neuromuscular junction in the gut, nNOS has primarily been localized to nerve endings Murthy et al., 1993, Jarvinen et al., 1999. Recently, experimental results from molecular biological studies have indicated that NO may have an autoregulatory role via negative feedback on NOS activity (Griscavage et al., 1995). It was suggested that NO inhibits the activity of the constitutive isoforms of NOS from rat and bovine cerebellum or bovine aortic endothelial cells, as well as the inducible isoform of NOS from activated murine macrophages Rogers and Ignarro, 1992, Rengasamy and Johns, 1993, Buga et al., 1993, Assreuy et al., 1993, Griscavage et al., 1994 In addition, it has been shown that exogenous NO inhibited basal NO release from vascular endothelium both in vivo and in vitro (Ma et al., 1996). However, it is not completely established whether NO regulates its own synthase and release in the nerve terminals. De Man et al. (1995) demonstrated that prolonged exposure to NO donors inhibited electrically induced nerve-mediated nitrergic relaxations without affecting the postjunctional response to NO or vasoactive intestinal polypeptide, pointing to a prejunctional inhibitory effect on the nerve-mediated nitrergic responses in the rat gastric fundus. Similar findings were reported by Hosoda et al. (1998) for the rat gastric myenteric plexus, and it was proposed that NO synthesis and release were prejunctionaly inhibited by NO and that this inhibition was mediated, at least in part, through the cyclic GMP pathway. However, Lefèbvre and Vandekerckhove (1998) observed that nitroglycerin did inhibit both electrical field stimulation- and exogenous NO-induced relaxations and suggested that this effect was due to postjunctional tolerance to nitrergic stimuli in the pig gastric fundus. On the other hand, a recent study provided evidence that inhibition of enteric NOS by exogenous NO was mediated via the intermediate formation of nitrosothiol (Kurjak et al., 1999). However, the precise mechanism(s) by which NO inhibits NOS activity is still unknown. The first aim of the present study was to elucidate the role of NO in non-adrenergic non-cholinergic relaxations induced by electrical field stimulation in mouse duodenum. Secondly, we aimed to investigate whether exogenous NO donors have a prejunctional and/or postjunctional inhibitory effect on the nitrergic responses and, if this was the case, to investigate whether this effect was due to NO itself. It is known that superoxide anion generation is unavoidable in a variety of cell lines and NO reacts with superoxide anion to form peroxynitrite, therefore inhibition of NOS in response to NO donors may be mediated by peroxynitrite. Finally, we further studied whether cyclic GMP is involved in this inhibition.

Section snippets

Tissue preparation

Swiss albino mice of either sex, weighing 20–25 g, were used in these experiments. They were fasted for 24 h with free access to water. They were killed by stunning and cervical dislocation. Duodenal segments were rapidly removed and the proximal portion of duodenum (approximately 12–15-mm long) was mounted as a tube under 0.2-g tension in a 20-ml organ bath filled with Krebs solution (in mM: NaCl 117.9, KCl 4.7, CaCl2 2.5, KH2PO4 0.89, NaHCO3 25, glucose 10.1, Na2EDTA 0.05 and ascorbic acid

Relaxant effects of nitrergic nerve stimulation, acidified NaNO2, nitroglycerin and sodium nitroprusside in mouse duodenal strips

Short-term electrical field stimulation (1, 5 and 10 Hz, 25 V, 1 ms, pulse trains of 10 s) of nitrergic nerves elicited frequency-dependent transient and reproducible relaxations which were fast in onset in 0.1 μM serotonin-contracted tissues treated with atropine and guanethidine (n=8). Bolus injection of acidified NaNO2 (1, 10 and 100 μM) caused fast and transient relaxations in a concentration-dependent manner (n=8). Also, nitroglycerin (500 μM) and sodium nitroprusside (500 μM) induced fast

Discussion

Results of recent studies suggest that NO may have a prejunctional and/or postjunctional inhibitory effect on nitrergic relaxations and point to the presence of an autoregulatory mechanism for the nitrergic innervation. In the rat duodenum, NO has been shown to mediate non-adrenergic non-cholinergic relaxations in response to electrical field stimulation Martins et al., 1993, Postorino et al., 1995. Also, in the present study, relaxations induced by nitrergic nerve stimulation were completely

Acknowledgements

We are indebted to Dr. S. Cellek (The Wolfson Institue for Biomedical Research, University College London) for the gift of 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one. We thank Mr. Kenan Daǧlıoǧlu (Çukurova University Experimental Research Center) for the supply of mice. This work was supported by Çukurova University Research Foundation (TF.99.6) and parts of this work were presented at the annual meeting of the European Pharmacological Societies, Budapest, Hungary, 3–7 July 1999.

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