Original article
Nitrite confers protection against myocardial infarction: Role of xanthine oxidoreductase, NADPH oxidase and KATP channels

https://doi.org/10.1016/j.yjmcc.2007.07.057Get rights and content

Abstract

Reduction of nitrite to nitric oxide during ischemia protects the heart against injury from ischemia/reperfusion. However the optimal dose of nitrite and the mechanisms underlying nitrite-induced cardioprotection are not known. We determined the ability of nitrite and nitrate to confer protection against myocardial infarction in two rat models of ischemia/reperfusion injury and the role of xanthine oxidoreductase, NADPH oxidase, nitric oxide synthase and KATP channels in mediating nitrite-induced cardioprotection. In vivo and in vitro rat models of myocardial ischemia/reperfusion injury were used to cause infarction. Hearts (n = 6/group) were treated with nitrite or nitrate for 15 min prior to 30 min regional ischemia and 180 min reperfusion. Xanthine oxidoreductase activity was measured after 15 min aerobic perfusion and 30 min ischemia. Nitrite reduced myocardial necrosis and decline in ventricular function following ischemia/reperfusion in the intact and isolated rat heart in a dose- or concentration-dependent manner with an optimal dose of 4 mg/kg in vivo and concentration of 10 μM in vitro. Nitrate had no effect on protection. Reduction in infarction by nitrite was abolished by the inhibition of flavoprotein reductases and the molybdenum site of xanthine oxidoreductase and was associated with an increase in activity of xanthine dehydrogenase and xanthine oxidase during ischemia. Inhibition of nitric oxide synthase had no effect on nitrite-induced cardioprotection. Inhibition of NADPH oxidase and KATP channels abolished nitrite-induced cardioprotection. Nitrite but not nitrate protects against infarction by a mechanism involving xanthine oxidoreductase, NADPH oxidase and KATP channels.

Introduction

Until recently the nitrite anion (NO2) has been considered to be an oxidation product of nitric oxide, useful as an index of the catalytic activity of nitric oxide synthase [1]. Approximately 80–90% of plasma nitrite is derived from endothelial nitric oxidase synthase derived nitric oxide [2]. Recently it has been shown that plasma cerulloplasmin has a nitric oxide oxidase activity and may play a role in the conversion of nitric oxide to nitrite in vivo [3]. The nitrite anion, which is present between 0.2 and 10 μM in the blood and tissues [4], [5], may represent a storage form of nitric oxide that is made available under conditions of oxygen deprivation to maintain cell survival. Cardioprotective effects of nitrite have recently been demonstrated. Reduction of nitrite to nitric oxide during ischemia protects the heart against injury from ischemia–reperfusion [6], [7]. The underlying mechanisms by which nitrite is reduced to nitric oxide have not yet been fully defined. Of the candidates for nitrite reduction in vivo, deoxyhemoglobin, deoxymyoglobin and xanthine oxidoreductase have been shown to have an oxygen-sensitive nitrite reductase activity [8], [9], [10]. Data suggest that deoxyhemoglobin plays a role in nitrite-dependent control of vascular tone during hypoxia [11], while xanthine oxidoreductase has been implicated in the cardioprotective effects of nitrite under conditions of oxygen deprivation [7]. In mammals, xanthine oxidoreductase exists in two interconvertible forms, xanthine dehydrogenase (EC 1.1.1.204), which predominates in vivo, and xanthine oxidase (EC 1.1.3.22). The extent to which nitrite alters the activity of xanthine dehydrogenase and xanthine oxidase during myocardial ischemia is not known.

In this study we have selected in vitro and in vivo models of regional myocardial ischemia and reperfusion to determine the ability of nitrite to confer acute cardioprotection in both the presence and absence of hemoglobin. The objectives of our study were to determine (1) the ability of nitrite and nitrate to confer protection against injury from regional ischemia–reperfusion in vivo and in vitro and (2) to assess the contribution of xanthine oxidoreductase, nitric oxide synthase, NADPH oxidase and the sarcolemmal and mitochondrial ATP-sensitive potassium (KATP) channels to nitrite-induced cardioprotection.

Section snippets

Materials and methods

Male Sprague Dawley rats at 8 weeks of age used in this study received humane care in compliance with the Guide for the Care and Use of Laboratory Animals formulated by the National Research Council, 1996.

Nitrite, nitrate and cardioprotection in vivo

We determined the dose–response relationship for nitrite to confer protection against injury from myocardial infarction using an in vivo model of regional myocardial ischemia and reperfusion. Rats were treated with an I.V. bolus of sodium nitrite (0.04, 0.4, 1.0, 4.0, 7.0 or 10.0 mg/kg) 15 min prior to 30 min occlusion of the left anterior descending coronary artery and 2 h of reperfusion. The effects of increasing nitrite dosage on hemodynamic parameters in the in vivo rat model of ischemia

Discussion

The present data indicate that nitrite protects the heart against injury from infarction and decreased post-ischemic ventricular function in both in vivo and in vitro models of ischemia/reperfusion. This effect of nitrite is dose- and concentration-dependent with optimal protection occurring at 4 mg/kg I.V. in vivo and 10 μM in vitro. Nitrite conferred cardioprotection over the range of 10–100 μM in vitro. One hundred micromolar nitrite also vasodilates aortic ring preparations [21], with

Acknowledgments

This work was supported in part by grants HL54075 (JEB), GM55792 (NH) and HL08361 (GJG) from the National Institutes of Health.

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