Increased cardiovascular risk after mercury exposure has been described, but the underlying mechanisms are not well explored. We analyzed the effects of chronic exposure to low mercury concentrations on endothelium-dependent responses in aorta and mesenteric resistance arteries (MRA). Wistar rats were treated with mercury chloride (1st dose 4.6 microg/kg, subsequent dose 0.07 microg.kg(-1).day(-1) im, 30 days) or vehicle. Blood levels at the end of treatment were 7.97 +/- 0.59 ng/ml. Mercury treatment: 1) did not affect systolic blood pressure; 2) increased phenylephrine-induced vasoconstriction; 3) reduced acetylcholine-induced vasodilatation; and 4) reduced in aorta and abolished in MRA the increased phenylephrine responses induced by either endothelium removal or the nitric oxide synthase (NOS) inhibitor N(G)-nitro-l-arginine methyl ester (l-NAME, 100 microM). Superoxide dismutase (SOD, 150 U/ml) and the NADPH oxidase inhibitor apocynin (0.3 mM) decreased the phenylephrine-induced contraction in aorta more in mercury-treated rats than controls. In MRA, SOD did not affect phenylephrine responses; however, when coincubated with l-NAME, the l-NAME effect on phenylephrine response was restored in mercury-treated rats. Both apocynin and SOD restored the impaired acetylcholine-induced vasodilatation in vessels from treated rats. Endothelial NOS expression did not change in aorta but was increased in MRA from mercury-treated rats. Vascular O2(-) production, plasmatic malondialdehyde levels, and total antioxidant status increased with the mercury treatment. In conclusion, chronic exposure to low concentrations of mercury promotes endothelial dysfunction as a result of the decreased NO bioavailability induced by increases in oxidative stress. These findings offer further evidence that mercury, even at low concentrations, is an environmental risk factor for cardiovascular disease.

BACKGROUND AND PURPOSERegular physical activity is an effective non-pharmacological therapy for prevention and control of hypertension. We investigated the effects of aerobic exercise training in vascular remodelling and in the mechanical and functional alterations of coronary and small mesenteric arteries from spontaneously hypertensive rats (SHR). EXPERIMENTAL APPROACHNormotensive Wistar Kyoto (WKY), SHR and SHR trained on a treadmill for 12 weeks were used to evaluate vascular structural, mechanical and functional properties. KEY RESULTSExercise did not affect lumen diameter, wall thickness and wall/lumen ratio but reduced vascular stiffness of coronary and mesenteric arteries from SHR. Exercise also reduced collagen deposition and normalized altered internal elastic lamina organization and expression of MMP-9 in mesenteric arteries from SHR. Exercise did not affect contractile responses of coronary arteries but improved the endothelium-dependent relaxation in SHR. In mesenteric arteries, training normalized the increased contractile responses induced by U46619 and by high concentrations of acetylcholine. In vessels from SHR, exercise normalized the effects of the NADPH oxidase inhibitor apocynin and the NOS inhibitor L-NAME in vasodilator or vasoconstrictor responses, normalized the increased O2 -production and the reduced Cu/Zn superoxide dismutase expression and increased NO production. CONCLUSIONS AND IMPLICATIONSExercise training of SHR improves endothelial function and vascular stiffness in coronary and small mesenteric arteries. This might be related to the concomitant decrease of oxidative stress and increase of NO bioavailability. Such effects demonstrate the beneficial effects of exercise on the vascular system and could contribute to a reduction in blood pressure.

1 Hypertension development, phenylephrine-induced contraction and Na + ,K + -ATPase functional activity and protein expression in aorta (AO), tail (TA) and superior mesenteric (SMA) arteries from ouabain-(25 mg day 71, s.c., 5 weeks) and vehicle-treated rats were evaluated. 2 Ouabain treatment increased systolic blood pressure (127+1 vs 160+2 mmHg, n=24, 35; P50.001) while the maximum response to phenylephrine was reduced (P50.01) in AO (102.8+3.9 vs 67.1+10.1% of KCl response, n=12, 9) and SMA (82.5+7.5 vs 52.2+5.8%, n=12, 9). 3 Endothelium removal potentiated the phenylephrine response to a greater extent in segments from ouabain-treated rats. Thus, dierences of area under the concentration-response curves (dAUC) in endothelium-denuded and intact segments for control and ouabain-treated rats were, respectively: AO, 56.6+9.6 vs 198.3+18.3 (n=9, 7); SMA, 85.5+15.4 vs 165.4+24.8 (n=6, 6); TA, 13.0+6.1 vs 39.5+10.4% of the corresponding control AUC (n=6, 6); P50.05. 4 The relaxation to KCl (1 ± 10 mM) was similar in segments from both groups. Compared to controls, the inhibition of 0.1 mM ouabain on KCl relaxation was greater in AO (dAUC: 64.8+4.6 vs 84.0+5.1%, n=11, 14; P50.05), similar in SMA (dAUC: 39.1+3.9 vs 43.3+7.8%, n=6, 7; P40.05) and smaller in TA (dAUC: 62.1+5.5 vs 41.4+8.2%, n=12, 13; P50.05) in ouabaintreated rats. 5 Protein expression of both a 1 and a 2 isoforms of Na + ,K + -ATPase was augmented in AO, unmodi®ed in SMA and reduced in TA from ouabain-treated rats. 6 These results suggest that chronic administration of ouabain induces hypertension and regional vascular alterations, the latter possibly as a consequence of the hypertension.

Hypertension is considered as a low-grade inflammatory disease, with adaptive immunity being an important mediator of this pathology. TLR4 may have a role in the development of several cardiovascular diseases; however, little is known about its participation in hypertension. We aimed to investigate whether TLR4 activation due to increased activity of the renin-angiotensin system (RAS) contributes to hypertension and its associated endothelial dysfunction. For this, we used aortic segments from Wistar rats treated with a non-specific IgG (1 µg/day) and SHRs treated with losartan (15 mg/kg·day), the non-specific IgG or the neutralizing antibody anti-TLR4 (1 µg/day), as well as cultured vascular smooth muscle cells (VSMC) from Wistar and SHRs. TLR4 mRNA levels were greater in the VSMC and aortas from SHRs compared with Wistar rats; losartan treatment reduced those levels in the SHRs. Treatment of the SHRs with the anti-TLR4 antibody: 1) reduced the increased blood pressure, heart rate and phenylephrine-induced contraction while it improved the impaired acetylcholine-induced relaxation; 2) increased the potentiation of phenylephrine contraction after endothelium removal; and 3) abolished the inhibitory effects of tiron, apocynin and catalase on the phenylephrine-induced response as well as its enhancing effect of acetylcholine-induced relaxation. In SHR VSMCs, angiotensin II increased TLR4 mRNA levels, and losartan reduced that increase. CLI-095, a TLR4 inhibitor, mitigated the increases in NAD(P)H oxidase activity, superoxide anion production, migration and proliferation that were induced by angiotensin II. In conclusion, TLR4 pathway activation due to increased RAS activity is involved in hypertension, and by inducing oxidative stress, this pathway contributes to the endothelial dysfunction associated with this pathology. These results suggest that TLR4 and innate immunity may play a role in hypertension and its associated end-organ damage.

1 This study compares the role of endothelial factors in a-adrenoceptor contractile responses in mesenteric resistance (MRA) and superior (SMA) mesenteric arteries from ouabain-treated (8.0 mg day À1 , 5 weeks) and untreated rats. The role of the renin-angiotensin system was also evaluated. 2 Ouabain treatment increased systolic blood pressure. In addition, ouabain reduced the phenylephrine response in SMA but did not alter noradrenaline responses in MRA. 3 Endothelium removal or the nitric oxide synthase (NOS) inhibitor (L-NAME, 100 mM) increased the responses to a-adrenergic agonists in both vessels. After ouabain treatment, both endothelial modulation and the L-NAME effect were increased in SMA, while only the L-NAME effect was increased in MRA. Endothelial NOS expression remained unaltered after ouabain treatment. 4 Indomethacin (10 mM) similarly reduced the noradrenaline contraction in MRA from both groups; in contrast, in SMA, indomethacin only reduced phenylephrine-induced contractions in segments from untreated rats. Co-incubation of L-NAME and indomethacin leftward shifted the concentration-response curves for noradrenaline more in MRA from ouabain-treated rats; tetraethylammonium (2 mM) shifted the noradrenaline curves further leftward only in MRA from untreated rats. 5 Losartan treatment prevents the development of hypertension but not all vascular changes observed after ouabain treatment. 6 In conclusion, a rise in endothelial NO and impaired prostanoid participation might explain the reduction in phenylephrine-induced contraction in SMA after ouabain treatment. An increase in the modulatory effect of endothelial NO and impairment of endothelium-dependent hyperpolarizing factor effect might explain why the ouabain treatment had no effect on noradrenaline responses in MRA.

Eugenol is a natural pungent substance and the main component of clove oil, with vasorelaxant action. To elucidate some of the possible mechanisms involved in this action isometric tension was measured in aortic rings from male Wistar rats precontracted with phenylephrine (PHE, 10 À7 M) or KCl (75 mM). Responses to increasing concentrations of eugenol (10 À6 -10 À2 M) were obtained in the presence and absence of endothelium. In the presence of eugenol, dose -response curves to PHE (10 À9 to 10 À4 M) and KCl (5 -125 mM) were displaced downwards. Concentration-dependent relaxation was observed in rings precontracted with PHE (10 À7 M) and KCl (75 mM). The tension increment produced by increasing external calcium concentration (0.25 -3 mM) was also reduced by eugenol (300 mM) treatment. The inhibitory effects of eugenol (300 mM) were compared to those induced by nifedipine (0.01 mM), a selective Ca 2+ channel blocker, producing similar relaxant effects. Two other protocols were performed. After precontraction with PHE (10 À7 M), increasing concentrations of eugenol (10 À6 -10 À2 M) were used before and after N w -nitro-L-arginine (L-NAME, 10 À4 M) and methylene blue (10 À5 M) treatment. Eugenolinduced relaxation was reduced by endothelial damage (rubbing), L-NAME and methylene blue treatments. Results suggested that eugenol produces smooth muscle relaxation resulting from the blockade of both voltage-sensitive and receptor-operated channels that are modulated by endothelial-generated nitric oxide. D

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1 The aim of this study was to assess the effects of treatment with isoproterenol (ISO, 0.3 mg kg À1 day À1 , s.c.) for 7 days on the vascular reactivity of rat-isolated aortic rings. Additionally, potential mechanisms underlying the changes that involved the endothelial modulation of contractility were investigated. 2 Treatment with ISO induced cardiac hypertrophy without changes in haemodynamic parameters. Aortic rings from ISO-treated rats showed an increase in the contraction response to phenylephrine (PHE) and serotonin, but did not change relaxations produced by acetylcholine or isoproterenol. Removal of the endothelium increased the responses to PHE in both groups. However, this procedure was less effective in ISO-treated as compared with control rats. Endothelial cell removal abolished the increase in the response to PHE in ISO-treated rats. The presence of N o -nitro-L-arginine methyl ester shifted the concentration-response curve to PHE to the left in both groups of rats. However, this effect was more pronounced in the ISO group. In addition, aminoguanidine (50 mM) potentiated the actions of PHE only in the ISO group. ISO treatment increased nitric oxide synthase (NOS) activity and neuronal NOS and endothelial NOS protein expression in the aorta. 3 Neither losartan (10 mM) nor indomethacin (10 mM) abolished the effects of ISO on the actions of PHE. Superoxide dismutase (SOD, 150 U ml À1 ) and L-arginine (5 mM), but neither catalase (300 U ml À1 ) nor apocynin (100 mM), blocked the effect of ISO treatment. In addition, we observed an increase in superoxide anion levels as measured by ethidium bromide fluorescence and of copper and zinc superoxide dismutase protein expression in ISO-treated rats. 4 In conclusion, our data suggest that ISO treatment alters the endothelial cell-mediated modulation of the contraction to PHE in rat aorta. The increased maximal response of PHE seems to be due to an increase in superoxide anion generation, which inactivates some of the basal NO produced and counteracts NO-mediated negative modulation even in the presence of high NO production and antioxidant defence.