Tempol, an antioxidant, restores endothelium-derived hyperpolarizing factor-mediated vasodilation during hypertension

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Abstract

Acetylcholine releases a non-prostanoid endothelium-derived hyperpolarizing factor (EDHF) and nitric oxide from physiological salt solution perfused rat mesenteric arteries. This study reports an impairment in EDHF-mediated vasodilation in deoxycorticosterone acetate (DOCA)-salt hypertensive versus control normotensive rats. Nitric oxide-mediated vasodilation to acetylcholine was not altered in the animals. We hypothesize that free radical species generated as by-products of arachidonic acid metabolism contribute to impaired EDHF-mediated dilation in DOCA-salt hypertension. With or without reduced nicotinamide adenine dinucleotide phosphate (NADPH) as co-factor, arterial microsomes generate free radical species upon incubation with arachidonic acid. The production of free radicals was significantly higher in DOCA-salt versus control rat microsomes, and was totally eliminated by addition of cyclooxygenase-2 inhibitors NS-398 or celecoxib at 30 μM. Treatment of DOCA-salt rats with tempol (an antioxidant; 15 mg/kg, i.p., 21 days) alleviates hypertension; improves acetylcholine-induced EDHF-mediated vasodilation in DOCA-salt rats, and decreases arachidonic acid-driven microsomal free radical production. Serum level of 8-isoprostanes is elevated in DOCA-salt hypertension versus control or sham-salt rats, and the increase was reversed by tempol treatment. These results show that EDHF-mediated dilation of rat mesenteric arteries is impaired in DOCA-salt induced hypertension. Our data also suggest that cyclooxygenase-2 mediates free radical production, and that free radicals modulate the EDHF-mediated vascular response in DOCA-salt induced hypertension.

Introduction

Endothelium-dependent vasodilation is impaired in human Miyoshi et al., 1997, Shimokawa, 1998, Lind et al., 2000 and animal forms of hypertension Fujii et al., 1992, Kimura and Nishio, 1999, including deoxycorticosterone acetate (DOCA)-salt-induced hypertension (Millette et al., 2000). The mechanisms proposed include: altered nitric oxide synthase activity (Sullivan et al., 2002); increased production of endothelium-derived prostanoid contracting factors and endothelins (Luscher, 1990); increased generation/availability of superoxide anions (Somers et al., 2000); and decreased influence of endothelium-derived hyperpolarizing factor (EDHF) (Fujii et al., 1992). These mechanisms vary in importance with different types of blood vessels and with different forms of hypertension. In the spontaneously hypertensive rats (SHR), for example, blunted endothelium-dependent relaxation found in the aorta is due to increased formation of contractile endoperoxide products Luscher, 1990, Cordellini, 1999, while in rat mesenteric arteries, blunted hyperpolarization (Fujii et al., 1992) and/or altered nitric oxide synthase 3 distribution (Sullivan et al., 2002) accounts for impaired endothelium-dependent vasodilation.

Acetylcholine elicits dilation of perfused rat mesenteric arterial bed by releasing two distinct, non-prostanoid relaxing factors (Adeagbo and Triggle, 1993). The first, described as endothelium-derived hyperpolarizing factor (EDHF), is insensitive to nitric oxide synthase inhibitors, and vasodilates by opening KCa channels. The second can be inhibited by nitric oxide synthase inhibitors when extracellular K+concentration is raised to about 25 mM and is characterized as endothelium-derived nitric oxide (EDNO). In the present study, we first determined the influence of DOCA-salt induced hypertension on EDHF- and EDNO-mediated components of acetylcholine-induced vasodilation.

Elevated production of free radical species contributes to the onset or progression of spontaneous (Kerr et al., 1999), Dahl salt-sensitive (Swei et al., 1999) and mineralocorticoid-salt-induced Somers et al., 2000, Beswick et al., 2001a, Beswick et al., 2001b hypertension in rats, as well as development of hypertension in humans Kumar and Das, 1993, Lacy et al., 2000). Free radical species are generated as by-products during the oxidative metabolism of either arachidonic acid by cyclooxygenases, lipoxygenases and cytochrome P450 enzymes (Puntarulo and Cederbaum, 1998), or l-arginine by nitric oxide synthases (Porasuphatana et al., 2003), and during activation of NADH/NADPH-dependent oxidases by hypertension Beswick et al., 2001a, Li et al., 2003. Since endothelium-dependent vasodilation of perfused rat mesenteric bed is accompanied by the release of arachidonic acid (Adeagbo et al., 2001), which we presume as the precursor for EDHF synthesis, we hypothesize that free radical by-products of cytochrome P450-mediated metabolism of arachidonic acid contribute to endothelium dysfunction during DOCA-salt-induced hypertension. We tested this hypothesis by investigating the effects of a broad spectrum antioxidant, tempol Mitchell et al., 1990, Laight et al., 1997, Zhang et al., 1998, on blood pressure and on EDHF-mediated vasodilation of DOCA-salt treated rats, and by measuring microsomal generation of free radical species from arachidonic acid under different experimental paradigms.

Section snippets

General animal care

Our experimental procedures conform to “Guiding Principles for Research Involving Animals and Human Beings” of the American Physiological Society (2002). Male Sprague–Dawley rats (Harlan, Indianapolis) were maintained on standard rat diet and water ad libitum for at least 1 week in Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC)-approved facilities before use for experiments. The project was approved by the Institutional Animal Care and Use Committee (IACUC) of

Results

The systolic as well as diastolic blood pressures were significantly (P<0.05) higher in uninephrectomized rats that received subcutaneous implants of DOCA (100 mg; 21 day-release) pellets and fed with potassium-supplemented high sodium chloride diet, and 1% saline drink. The age-matched control and sham-salt treated rats did not develop high blood pressure (Table 1). The body mass of DOCA-salt rats was significantly less than those of control and sham-salt rats; the heart mass and the heart

Discussion

The principal findings of the present study are threefold: (1) EDHF- but not EDNO-mediated dilation in response to acetylcholine is attenuated in vascular beds of DOCA-salt hypertensive rats compared to control and sham-salt rat vessels. Vasodilation elicited by KCa channel activators, 1-EBIO and NS-1619, is not altered. (2) Arterial microsomes incubated with arachidonic acid produce free radical species in the presence or absence of a co-factor, NADPH; microsomes of DOCA-salt hypertensive

Acknowledgements

The work was funded by an American Heart Association (Ohio Valley Affiliate) Grant-in-Aid #0051391B awarded to ASOA.

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