Oxidative stress induced by tert-butyl hydroperoxide causes vasoconstriction in the aorta from hypertensive and aged rats: role of cyclooxygenase-2 isoform.

We analyzed the mechanisms involved in the effect of tert-butyl hydroperoxide (t-BOOH) in isolated aortic rings with and without endothelium from normotensive Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) at 6, 18, and 24 months of age. t-BOOH (1 microM-10 mM) induced concentration-dependent contractions that were scarcely modified by aging and potentiated in SHR and by endothelium removal. The nitric oxide synthase and prostacyclin synthase inhibitors N(G)-nitro-L-arginine methyl ester (100 microM) and tranylcypromine (100 microM), respectively, increased both basal tone and the t-BOOH-induced contractions in intact segments from WKY, with these effects not observed in SHR. Indomethacin (10 microM), a nonspecific cyclooxygenase inhibitor, and SQ 29,548 (10 microM), a prostaglandin H(2)/thromboxane A(2) receptor blocker, abolished the t-BOOH-induced vasoconstriction, independent of age and hypertension. In both strains, these contractile responses were unaltered by the thromboxane synthase inhibitor imidazole (10 microM). The cyclooxygenase-2 inhibitor NS-398 (10 microM) abolished or markedly reduced the t-BOOH-induced contractions in segments with or without endothelium, respectively. In addition, expression of cyclooxygenase-2 protein was detected in aorta from WKY and SHR in either basal condition or after stimulation with t-BOOH. These results suggest that (1) t-BOOH-induced vasoconstriction in the aorta from WKY and SHR is essentially mediated by cyclooxygenase-2 metabolites, different from thromboxane-A(2), probably prostaglandin-H(2), and/or isoprostanes; (2) aging scarcely modifies, whereas endothelium negatively modulates, these contractions in both strains; and (3) nitric oxide and prostacyclin exert a negative modulator role on the t-BOOH-induced vasoconstriction in WKY, with this modulator role lost in SHR.

Changes in plasma oxidative state with age and their influence on contractions elicited by noradrenaline in the rat tail artery.

The present study analyzes the changes in plasma oxidative state with age and their influence on the contractions induced by noradrenaline (NA) in endothelium-denuded segments from the tail artery of 6- (young), 24- (old) and 30- (very old) month-old Sprague Dawley rats. The sensitivity (-log EC50) to NA increased with age, this increase being higher in old than in very old animals. Moreover, the maximum response (Emax) to NA did not change in old rats, whereas decreased in very old animals. We also found a progressive increase in the plasma oxidative state with age, measured as malondialdehyde (MDA) levels, that was accompanied by a decrease in the plasma antioxidative state, measured as glutathione peroxidase activity. In addition, MDA (0.5, 1 and 10 microM) potentiated the NA responses in 6-, 24- and 30-month-old rats, respectively, without affecting Emax. In young animals, catalase (1000 U/ml) or dimethylsulfoxide (7 mM), scavengers of hydrogen peroxide or hydroxyl radicals, respectively, did not modify either the contractions induced by NA in control situation or the potentiation of these responses caused by MDA. However, the superoxide anion scavenger, superoxide dismutase (SOD, 150 U/ml), completely reversed the increase in sensitivity to NA caused by MDA, without affecting NA responses in control situation. These results suggest that the increase in NA sensitivity with age could be due, at least in part, to the enhancement of plasma oxidative state during aging. In addition, in this alteration of the responses to NA caused by MDA, the generation of superoxide anions appears to be involved. This study supports the hypothesis that the enhancement of plasma oxidative state could play an important role in the increase of vascular resistance with age.

Mechanisms involved in relaxation induced by exogenous nitric oxide in pig coronary arteries.

The aim of the present study was to analyze the mechanisms involved in the vasodilator responses elicited by nitric oxide (NO) in segments of porcine posterior descending coronary artery. Exogenous NO (0.1-30 microM) induced concentration-dependent relaxations in segments precontracted with a concentration of the thromboxane A2 mimetic, U-46619 (30-300 nM) that produced a contraction 70% (submaximal contraction) of that elicited by 75 mM K+ (maximal contraction). The relaxations were almost abolished by 6-anilinoquinoline-5,8-quinone (LY-83583, 10 microM), an inhibitor of guanylate cyclase, and with precontraction with 40 or 60 mM K+. Relaxations were reduced by 5 mM tetraethylammonium (TEA), a blocker of Ca(2+)-activated K+ channels (Kca channels) and unaltered by ouabain (500 microM), 4-aminopyridine (1 mM), glibenclamide (10 microM), apamin (1 microM) and charybdotoxin (0.3 microM), inhibitors of sodium pump, voltage-sensitive, ATP-sensitive, small-conductance Kca and large-conductance KcaK+ channels, respectively. These results suggest that the relaxation caused by exogenous NO is mediated by guanylate cyclase activation, with only a slight participation of a hyperpolarizing mechanism mediated by activation of Kca channels.

Mechanisms of bradykinin-induced relaxation in pig coronary arteries.

Bradykinin (BK) induced endothelium- and concentration-dependent relaxations in segments of porcine posterior descending coronary arteries submaximally precontracted with the thromboxane A2 mimetic, U-46619. The effects of BK were reduced by L-NG-monomethylarginine (L-NMMA) and 6-anilinoquinoline-5,8-quinone (LY-83583), respective inhibitors of nitric oxide (NO) synthase and guanylate cyclase, but were unaffected by the cytochrome P450 monoxygenase blocker, thiopentone sodium; however, BK effects were slightly reduced by dimethyl sulfoxide (DMSO), an hydroxyl radical scavenger. Relaxant responses were reduced markedly by ouabain, a sodium pump inhibitor but only slightly by tetraethylammonium (TEA) and charybdotoxin, respective blockers of Ca-activated (KCa) and large-conductance (BKCa) K+ channels. However, BK responses were practically abolished by TEA + L-NMMA + ouabain while unaffected by apamin, 4-aminopyridine and glibenclamide, blockers of small-conductance KCa voltage-sensitive and ATP-sensitive K+ channels, respectively. In segments submaximally precontracted with K+, BK-induced relaxation was lower than that of those precontracted with U-46619, and was further reduced by L-NMMA, LY-83583 and especially, ouabain; L-NMMA + ouabain + TEA abolished the effect. Precontraction of segments with higher K+ concentrations almost abolished the relaxation. These results suggest that the relaxation to BK is mediated: 1) by endothelial NO release which activates guanylate cyclase of smooth muscle cells; 2) by hydroxyl radicals; and 3) by an endothelial hyperpolarizing factor, that does not seem to be a metabolite derived from cytochrome P450 monoxygenases and that relaxes activating K+ channels (mainly BKCa), and especially, the sodium pump.

Contractile responses elicited by hydrogen peroxide in aorta from normotensive and hypertensive rats. Endothelial modulation and mechanism involved.

The present study analyses the influence of hypertension and endothelium on the effect induced by hydrogen peroxide (H2O2) on basal tone in aortic segments from normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR) of 6-month-old, as well as the possible mechanisms involved. Single (1 mM) or cumulative (100 nM-10 mM) concentrations of H2O2 produced a transient contraction or a concentration-dependent increase of basal tone, respectively, in segments from WKY and SHR. In both cases, the contractions were higher in intact segments from hypertensive than from normotensive rats, and increased by endothelium removal in both strains. Catalase (1000 u ml(-1), a H2O2 scavenger) abolished the contraction elicited by 1 mM H2O2 in both strains. Superoxide dismutase (SOD, 150 u ml(-1)) and dimethylsulphoxide (DMSO, 7 mM), scavengers of superoxide anions and hydroxyl radicals, respectively, did not alter H2O2-induced contractions in intact segments from both strains. However, L-NG-nitroarginine methyl ester (L-NAME, 100 microM, a nitric oxide synthase inhibitor) increased the response to H2O2 in normotensive rats, although the increase was less than that produced by endothelium removal. Incubation of segments with 1 mM H2O2 for 15 min and subsequent washout reduced the contractile responses induced by 75 mM KCl in intact segments from SHR and in endothelium-denuded segments from both strains; this effect being prevented by catalase (1000 u ml(-1)). Indomethacin (10 microM, a cyclo-oxygenase inhibitor) and SQ 29,548 (10 microM, a prostaglandin H2/thromboxane A2 receptor antagonist) practically abolished the contractions elicited by H2O2 in normotensive and hypertensive rats. We conclude that: (1) the oxidant stress induced by H2O2 produces contractions mediated by generation of a product of the cyclo-oxygenase pathway, prostaglandin H2 or more probably thromboxane A2, in normotensive and hypertensive rats; (2) oxygen-derived free radicals are not involved in the effect of H2O2; (3) in normotensive rats, endothelium protects against H2O2-mediated injury to contractile machinery, determined by the impairment of KCl-induced contractions; and (4) endothelial nitric oxide has a protective role on the contractile effect induced by H2O2, that is lost in hypertension.