Flow- and acetylcholine-induced dilatation in small arteries from rats with renovascular hypertension — effect of tempol treatment

https://doi.org/10.1016/j.ejphar.2007.03.058Get rights and content

Abstract

We investigated whether renovascular hypertension alters vasodilatation mediated by nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF) and the influence of the superoxide dismutase mimetic tempol on vasodilatation. One-kidney one-clip hypertensive Sprague–Dawley rats, treated with either vehicle or tempol (from weeks 5 to 10 after placement of the clip), and uninephrectomized control rats were investigated. In renal hypertensive rats systolic blood pressure increased to 171 ± 6 mmHg (n = 10), while in tempol-treated rats systolic blood pressure remained normal (139 ± 7 mmHg, n = 5). In isolated pressurized mesenteric small arteries NO-mediated dilatation was obtained by increasing flow rate and EDHF-mediated dilatation by acetylcholine. In arteries from hypertensive rats, flow-induced dilatation was blunted, as compared to normotensive and tempol-treated rats, while acetylcholine-induced dilatation remained normal. Measured by dihydroethidium staining there was an increased amount of superoxide in arteries from vehicle-treated rats, but not from tempol-treated rats. Expression by immunoblotting of endothelial NO synthase and the NAD(P)H oxidase subunit p47phox remained unaffected by high blood pressure and tempol treatment. Simultaneous measurements of NO-concentration and relaxation were performed in isolated coronary arteries from the same animals. As compared to vehicle-treated rats, both acetylcholine-induced relaxation and NO-concentration increased in arteries from tempol-treated animals, while only the relaxation was improved by the NO donor, S-nitroso-N-acetylpenicillamine (SNAP). In conclusion renovascular hypertension selectively inhibits flow-induced NO-mediated vasodilatation, while EDHF-type vasodilatation remains unaffected, suggesting that high blood pressure leads to increased generation of superoxide contributing to decreased NO bioavailability. Furthermore, the abnormal endothelium function can be corrected by tempol treatment, but this seems to involve mechanisms partly independent of NO.

Introduction

Free oxygen radicals have attracted much attention as a contributing factor to the endothelial dysfunction associated with hypertension. One such free radical is superoxide (O2) which can be generated by the NAD(P)H oxidase system in the vascular wall. Superoxide is degraded to hydrogen peroxide (H2O2) by superoxide dismutase, but may also react with nitric oxide (NO) resulting in the production of peroxynitrite (ONOO) thus decreasing the bioavailability of NO. A consequence of this would be a reduced NO-dependent vasodilatation (McIntyre et al., 1999).

A short transient pressure increase in isolated arteries is associated with increased superoxide production and diminished flow-mediated dilatation (Huang et al., 1998, Ungvari et al., 2003, Christensen et al., 2007). This abnormality can be corrected by inhibition of the NAD(P)H oxidase with apocynin (Hamilton et al., 2002, Ungvari et al., 2003) and the superoxide dismutase mimetic tempol (Christensen et al., 2007). Also long term high blood pressure in vivo is related to increased vascular superoxide production and alterations in endothelial function. This has been demonstrated in various models of high blood pressure such as the spontaneously hypertensive rat (Zalba et al., 2000) and the DOCA salt sensitive rat model of hypertension (Ghosh et al., 2004). Also rats with renovascular hypertension show impairment of NO-dependent vasodilatation due to superoxide production (Heitzer et al., 1999). Similar abnormalities have been described in patients with essential hypertension (Taddei et al., 1998) as well as renovascular mediated hypertension (Higashi et al., 2002). Also in rats with hypertension due to aortic banding the superoxide generating NAD(P)H oxidase is upregulated (Vaziri and Ni, 2005). Thus, the ubiquitously finding of an activated NAD(P)H system during hypertension suggests this to be due to high blood pressure per se rather than related to the cause of hypertension.

Previous studies have demonstrated that flow-mediated dilatation of mesenteric small arteries from normotensive rats is almost entirely dependent on NO, whereas receptor-mediated dilatation to acetylcholine possess the characteristics of endothelial derived hyperpolarizing factor (EDHF) type and relies on activation of Ca2+-activated potassium channels (Edwards et al., 1998, Thorsgaard et al., 2003). This discrimination between flow- and acetylcholine-induced dilatation also seems to apply to human small arteries (Paniagua et al., 2001, Buus et al., 2000). The different mechanisms for flow- and acetylcholine-induced dilatation allowed us to investigate whether high blood pressure preferentially affects NO-dependent or EDHF-type vasodilatation. In contrast to the mesenteric small arteries, acetylcholine relaxation is NO-mediated in rat coronary small arteries (Symons et al., 2006), and intraluminal measurements of NO concentrations allow direct assessment of NO bioavailability (Simonsen et al., 1999).

Despite the well established role for superoxide as NO scavenger in endothelial dysfunction during high blood pressure conditions, it has not been studied whether receptor- and flow-mediated dilatation is affected in the same vascular preparation under similar experimental conditions. The primary aim of the present study was therefore to investigate to which extent high blood pressure in a rat model of renovascular hypertension influences NO-dependent flow-mediated dilatation or EDHF type acetylcholine-induced dilatation in isolated small arteries. Secondary, we tested whether scavenging of superoxide with tempol selectively affects either type of vasodilatation.

Section snippets

Animal model of hypertension

The investigation conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1996). Male Sprague–Dawley rats (7–8 weeks, 180–200 g) were obtained from Møllegaard Breeding Center, Skensved, Denmark. The rats were anaesthesized with intraperitoneal midazolam (0.413 mg/kg), fentanyl (0.026 mg/kg) and fluanisone (0.825 mg/kg) and supplied with extra fluanisone if necessary. The right kidney was removed in

Blood pressure measurements

Five and 10 weeks after placement of the clip systolic blood pressure was significantly elevated in one-kidney, one-clip (1K1C) rats compared to sham operated normotensive control rats (Fig. 1A). Tempol treatment from weeks 5 to 10 after placement of the clip lowered systolic blood pressure to the level of the normotensive group, while blood pressure in the group receiving vehicle remained elevated (Fig. 1A). There were no differences in heart rate among the three experimental groups (Fig. 1B)

Discussion

The present study is the first to directly compare NO- and non-NO-dependent dilatation in isolated arteries from hypertensive animals under similar experimental conditions. We confirm the finding from several previous studies that high blood pressure results in impaired flow-mediated vasodilatation (Huang et al., 1998, Ungvari et al., 2003), but also demonstrates that the EDHF type dilatation remains normal. Thus, the main findings of the present study are as follows: (1) Renovascular

Acknowledgements

We thank Helle Zibrandtsen for technical assistance. This study was supported by grants from the Danish Heart Association, the Danish Research Council, and Aarhus University Research Foundation.

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