Investigation of a possible interaction between the heme oxygenase/biliverdin reductase and nitric oxide synthase pathway in murine gastric fundus and jejunum

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Abstract

This study investigated the possible interaction between the heme oxygenase (HO)/biliverdin reductase (BVR) and nitric oxide synthase (NOS) pathway in murine gastric fundus and jejunum, since previous studies have shown that both HO-2 and BVR are expressed in interstitial cells of Cajal (ICCs) and co-localized with neuronal NOS in a large proportion of myenteric neurons along the gastrointestinal tract. Neither HO inhibition by chromium mesoporphyrin (CrMP) nor co-incubation with CO or biliverdin/bilirubin affected nitrergic neurotransmission — i.e. relaxations induced by non-adrenergic non-cholinergic (NANC) nerve stimulation or exogenous NO — under normal physiological conditions. However, biliverdin/bilirubin reversed the inhibitory effect of the superoxide generator LY83583 on exogenous NO-induced relaxations in both tissues. When gastric fundus muscle strips were depleted of the endogenous antioxidant Cu/Zn superoxide dismutase (SOD) by the Cu-chelator DETCA, electrically induced NANC relaxations were also affected by LY82583; however, biliverdin/bilirubin could not substitute for the loss of Cu/Zn SOD when this specific antioxidant enzyme was depleted. In jejunal muscle strips, the combination DETCA plus LY83583 nearly abolished contractile phasic activity and, hence, did not allow studying nitrergic relaxation in these experimental conditions. In conclusion, this study does not establish a role for HO/CO in inhibitory NANC neurotransmission in murine gastric fundus and jejunum under normal physiological conditions. However, the antioxidants biliverdin/bilirubin might play an important role in the protection of the nitrergic neurotransmitter against oxidative stress.

Introduction

Heme oxygenase (HO—EC 1.14.99.3) is the rate-limiting enzyme in the degradation of heme, catalyzing the oxidative cleavage of heme to carbon monoxide (CO), biliverdin and ferrous iron. Biliverdin is subsequently reduced to bilirubin by biliverdin reductase (BVR—EC 1.3.1.24). The HO family consists of two distinct isozymes: the stress-inducible HO-1, which is upregulated by a variety of different stimuli; and the constitutive HO-2, which is expressed under basal conditions (Maines, 1997, Ryter et al., 2006, Wu and Wang, 2005).

HO-2 is widely expressed in the gastrointestinal tract and more specifically in myenteric and submucosal enteric neurons, interstitial cells of Cajal (ICCs), and mucosal epithelial cells (Gibbons and Farrugia, 2004). To a varying extent, this HO-2 isoform has been found to be co-localized with neuronal nitric oxide synthase (nNOS) in the enteric plexuses of pig gastric fundus and murine small intestine (100% co-localisation; Colpaert et al., 2002a, Zakhary et al., 1997), canine and feline lower oesophageal sphincter and ileum (50% co-localisation; Ny et al., 1996, Ny et al., 1997), and human gastric antrum and jejunum (10–40% co-localisation; Miller et al., 2001). Based on 1) the immunohistochemical co-localisation of HO-2 with nNOS in myenteric neurons; 2) the effects of some non-selective HO inhibitors on neurally-evoked non-adrenergic non-cholinergic (NANC) relaxations (Alcon et al., 2001, Grundemar and Ny, 1997, Rattan and Chakder, 1993, Zakhary et al., 1997); and 3) a study reporting reduced neurally-evoked ileal smooth muscle relaxations in HO-2−/− mice (Zakhary et al., 1997), the suggestion was made that NO and CO might function as co-neurotransmitters in the enteric nervous system. More recent studies, however, do not confirm a neural role for CO in enteric neurotransmission but indicate that CO - generated by HO-2 in the ICCs — more likely acts as an endogenous hyperpolarizing factor in the gastrointestinal tract (Farrugia et al., 2003, Sha et al., 2007).

As both HO-2 and BVR are expressed in ICCs and co-localized with nNOS in a large proportion of myenteric neurons (Colpaert et al., 2002a, Colpaert et al., 2002b, Miller et al., 1998, Miller et al., 2001, Ny et al., 1996, Ny et al., 1997, Zakhary et al., 1997), (a) possible interaction(s) between the HO/BVR and NOS pathway should be considered. Thus, the aim of our study was to investigate the role of the HO/BVR pathway in NANC neurotransmission and to elucidate the influence of CO and/or biliverdin–bilirubin on nitrergic neurotransmission in murine gastric fundus and jejunum.

Section snippets

Animals

Male Swiss (SPF Orl) mice (6–8 weeks, 30–35 g) were kept and cared for in standard cages in a 12 h–12 h light-darkness cycle with free access to water and pellets. All experimental procedures were approved by the Ethical Committee for Animal Experiments, Faculty of Medicine and Health Sciences, Ghent University, Belgium.

Tissue preparation and mounting

Mice were sacrificed by cervical dislocation and the stomach and a ± 8 cm long jejunal segment starting 5 cm distally from the ligament of Treitz were removed. Two full-thickness

Influence of l-NAME and CrMP on relaxations induced by EFS and NO

In gastric fundus muscle strips, EFS (0.5–8 Hz) induced transient and frequency-dependent NANC relaxations which were abolished by the NOS inhibitor l-NAME (300 µM; Fig. 1A). In jejunal muscle strips, EFS-evoked similar, transient relaxations which were abolished by l-NAME (300 µM) at low frequencies (≤ 2 Hz); however, residual acute twitch relaxations were still observed at higher frequencies (4–8 Hz; Fig. 1B). The combination of l-NAME (300 µM) with CrMP (10 µM) did not reduce jejunal NANC

Discussion

In the present study, we investigated the role of the HO/BVR pathway in NANC neurotransmission and its possible interaction with nitrergic neurotransmission in the murine gastric fundus and jejunum.

Over the past decade, confusing data have been published regarding a possible neural role for CO in the enteric nervous system. Based on the effects of some non-selective HO inhibitors—also affecting NOS activity (Grundemar, 1997)—on EFS-induced NANC relaxations (Alcon et al., 2001, Rattan and

Acknowledgements

O.D.B. is a research assistant of the Fund of Scientific Research Flanders. This study was financially supported by the Special Investigation Fund of Ghent University (GOA 1251004) and the Fund of Scientific Research Flanders (G.0053.02). The authors thank Mrs. Ellen Elinck and Mr. Valère Geers for technical assistance.

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