14 research outputs found
Lipoic acid, but not tempol, preserves vascular compliance and decreases medial calcification in a model of elastocalcinosis
Nitric oxide regulates angiotensin-I converting enzyme under static conditions but not under shear stress
Mechanical forces including pressure and shear stress play an important role in vascular homeostasis via the control of the production and release of a variety of vasoactive factors. An increase in vascular shear stress is accompanied by nitric oxide (NO) release and NO synthase activation. Previously, we have demonstrated that shear stress induces angiotensin-I converting enzyme (ACE) down-regulation in vivo and in vitro. In the present study, we determined whether NO participates in the shear stress-induced ACE suppression response. Rabbit aortic endothelial cells were evaluated using the NO synthase inhibitor L-NAME, and two NO donors, diethylamine NONOate (DEA/NO) and sodium nitroprusside (SNP). Under static conditions, incubation of endothelial cells with 1 mM L-NAME for 18 h increased ACE activity by 27% (from 1.000 ± 0.090 to 1.272 ± 0.182) while DEA/NO and SNP (0.1, 0.5 and 1 mM) caused no change in ACE activity. Interestingly, ACE activity was down-regulated similarly in the presence or absence of L-NAME (delta(0 mM) = 0.26 ± 0.055, delta(0.1 mM) = 0.21 ± 0.22, delta(1 mM) = 0.36 ± 0.13) upon 18 h shear stress activation (from static to 15 dyn/cm²). Taken together, these results indicate that NO can participate in the maintenance of basal ACE levels in the static condition but NO is not associated with the shear stress-induced inactivation of ACE
Lipoic acid, but not tempol, preserves vascular compliance and decreases medial calcification in a model of elastocalcinosis
Vascular calcification decreases compliance and increases morbidity. Mechanisms of this process are unclear. The role of oxidative stress and effects of antioxidants have been poorly explored. We investigated effects of the antioxidants lipoic acid (LA) and tempol in a model of atherosclerosis associated with elastocalcinosis. Male New Zealand white rabbits (2.5-3.0 kg) were fed regular chow (controls) or a 0.5% cholesterol (chol) diet+104 IU/day vitamin D2 (vitD) for 12 weeks, and assigned to treatment with water (vehicle, n=20), 0.12 mmol·kg-1·day-1 LA (n=11) or 0.1 mmol·kg-1·day-1 tempol (n=15). Chol+vitD-fed rabbits developed atherosclerotic plaques associated with expansive remodeling, elastic fiber disruption, medial calcification, and increased aortic stiffness. Histologically, LA prevented medial calcification by ∼60% and aortic stiffening by ∼60%. LA also preserved responsiveness to constrictor agents, while intima-media thickening was increased. In contrast to LA, tempol was associated with increased plaque collagen content, medial calcification and aortic stiffness, and produced differential changes in vasoactive responses in the chol+vitD group. Both LA and tempol prevented superoxide signals with chol+vitD. However, only LA prevented hydrogen peroxide-related signals with chol+vitD, while tempol enhanced them. These data suggest that LA, opposite to tempol, can minimize calcification and compliance loss in elastocalcionosis by inhibition of hydrogen peroxide generation
Chronic hyperhomocysteinemia impairs vascular function in ovariectomized rat carotid arteries
Homocysteine is an independent risk factor for coronary heart disease, as well as for cerebrovascular and peripheral vascular diseases. The purpose of this study was to investigate the effects of hyperhomocysteinemia (HHcy) on vascular reactivity within carotid artery segments isolated from ovariectomized female rats. Treatment with DL-Hcy thiolactone (1 g/kg body weight per day) reduced the phenylephrine-induced contraction of denuded rings. However, the treatment did not alter KCl-induced contractions, or relaxations induced by sodium nitroprusside or acetylcholine. We report elevated expressions of iNOS, eNOS, and nitrotyrosine in homocysteine-treated rat artery sections. Moreover, the inhibition of NOS by L-NAME, 1,400 W, or L-NNA restored phenylephrine- induced vasoconstriction in carotid artery segments from Hcy-treated rats. In conclusion, our findings show that severe HHCy can promote an acute decrease in the endothelium-independent contractile responses of carotid arteries to adrenergic agonists. This effect was restored by nitric oxide synthase inhibitors, which further supports the involvement of nitric oxide in HHcy-derived vascular dysfunction.3851515152
Nonspecific blockade of vascular free radical signals by methylated arginine analogues
Methylated arginine analogues are often used as probes of the effect of nitric oxide; however, their specificity is unclear and seems to be frequently overestimated. This study analyzed the effects of NG-methyl-L-arginine (L-NMMA) on the endothelium-dependent release of vascular superoxide radicals triggered by increased flow. Plasma ascorbyl radical signals measured by direct electron paramagnetic resonance spectroscopy in 25 rabbits increased by 3.8 ± 0.7 nmol/l vs baseline (28.7 ± 1.4 nmol/l, Pa-phenyl-N-tert-butylnitrone (N = 22). However, in both preparations, this complete blockade was not reversed by co-infusion of excess L-arginine and was also obtained by N-methyl-D-arginine, thus indicating that it is not related to nitric oxide synthase. L-arginine alone was ineffective, as previously demonstrated for NG-methyl-L-arginine ester (L-NAME). In vitro, neither L-arginine nor its analogues scavenged superoxide radicals. This nonspecific activity of methylated arginine analogues underscores the need for careful controls in order to assess nitric oxide effects, particularly those related to interactions with active oxygen species
Adult rats are more sensitive to the vascular effects induced by hyperhomocysteinemia than young rats
We aimed to investigate the vascular effects of hyperhomocysteinemia (HHcy) on carotid arteries from young and adult rats. With this purpose young and adult rats received a solution of DL-homocysteine-thiolactone (1g/kg body weight/day) in the drinking water for 7, 14 and 28days. Increase on plasma homocysteine occurred in young and adult rats treated with DL-homocysteine-thiolactone in all periods. Vascular reactivity experiments using standard muscle bath procedures showed that HHcy enhanced the contractile response of endothelium-intact, carotid rings to phenylephrine in both young and adult rats. However, in young rats, the increased phenylephrine-induced contraction was observed after hyperhomocysteinemia for 14 and 28days, whereas in adult rats this response was already apparent after 7day treatment. HHcy impaired acetylcholine-induced relaxation in arteries from adult but not young rats. The contraction induced by phenylephrine in carotid arteries in the presence of Y-27632 was reversed to control values in arteries from young but not adult rats with hyperhomocysteinemia. HHcy did not alter the contraction induced by CaCl2 in carotid arteries from young rats, but enhanced CaCl2-induced contraction in the arteries from adult rats. HHcy increased the basal levels of superoxide anion in arteries from both groups. Finally, HHcy decreased the basal levels of nitrite in arteries from adult but not young rats. The major new finding of the present work is that arteries from young rats are more resistant to vascular changes evoked by HHcy than arteries from adult rats. Also, we verified that the enhanced vascular response to phenylephrine observed in carotid arteries of DL-homocysteine thiolactone-treated rats is mediated by different mechanisms in young and adult rats.533-49910
Cardioprotective Mechanism Of S-nitroso-n-acetylcysteine Via S-nitrosated Betadrenoceptor-2 In The Ldlr-/- Mice
Previous studies from our group have demonstrated the protective effect of S-nitroso-N-acetylcysteine (SNAC) on the cardiovascular system in dyslipidemic LDLr-/- mice that develop atheroma and left ventricular hypertrophy after 15 days on a high fat diet. We have shown that SNAC treatment attenuates plaque development via the suppression of vascular oxidative stress and protects the heart from structural and functional myocardial alterations, such as heart arrhythmia, by reducing cardiomyocyte sensitivity to catecholamines. Here we investigate the ability of SNAC to modulate oxidative stress and cell survival in cardiomyocytes during remodeling and correlation with β2-AR signaling in mediating this protection. Ventricular superoxide (O2-) and hydrogen peroxide (H2O2) generation was measured by HPLC methods to allow quantification of dihydroethidium (DHE) products. Ventricular histological sections were stained using terminal dUTP nick-end labeling (TUNEL) to identify nuclei with DNA degradation (apoptosis) and this was confirmed by Western blot for cleaved caspase-3 and caspase-7 protein expression. The findings show that O2- and H2O2 production and also cell apoptosis were increased during left ventricular hypertrophy (LVH). SNAC treatment reduced oxidative stress during on cardiac remodeling, measured by decreased H2O2 and O2- production (65% and 52%, respectively), and a decrease in the ratio of p-Ser1177 eNOS/total eNOS. Left ventricle (LV) from SNAC-treated mice revealed a 4-fold increase in β2-AR expression associated with coupling change to Gi; β2-ARs-S-nitrosation (β2-AR-SNO) increased 61%, while apoptosis decreased by 70%. These results suggest that the cardio-protective effect of SNAC treatment is primarily through its anti-oxidant role and is associated with β2-ARs overexpression and β2-AR-SNO via an anti-apoptotic pathway. © 2013 Elsevier Inc. All rights reserved.365866Cohn, J.N., Abnormalities of peripheral sympathetic nervous system control in congestive heart failure (1990) Circulation, 82, pp. 59-167Bristow, M.R., Beta-adrenergic receptor blockade in chronic heart failure (2000) Circulation, 101, pp. 558-569Santos, I.N., Spadari-Bratfisch, R.C., Stress and cardiac beta adrenoceptors (2006) Stress, 9, pp. 69-84Tsutsui, H., Kinugawa, S., Matsushima, S., Mitochondrial oxidative stress and dysfunction in myocardial remodelling (2009) Cardiovasc. Res., 81, pp. 449-456Atar, S., Ye, Y., Lin, Y., Freeberg, S.Y., Nishi, S.P., Rosanio, S., Huang, M.H., Birnbaum, Y., Atorvastatin-induced cardioprotection is mediated by increasing inducible nitric oxide synthase and consequent S-nitrosylation of cyclooxygenase-2 (2006) Am. J. Physiol. Heart Circ. Physiol., 290, pp. 1960-H1968Spinale, F.G., Coker, M.L., Thomas, C.V., Walker, J.D., Mukherjee, R., Hebbar, L., Time-dependent changes in matrix metalloproteinase activity and expression during the progression of congestive heart failure: Relation to ventricular and myocyte function (1998) Circ. Res., 82, pp. 482-495Ding, B., Price, R.L., Goldsmith, E.C., Borg, T.K., Yan, X., Douglas, P.S., Weinberg, E.O., Lorell, B.H., Left ventricular hypertrophy in ascending aortic stenosis mice: Anoikis and the progression to early failure (2000) Circulation, 101, pp. 2854-2862Palojoki, E., Saraste, A., Eriksson, A., Pulkki, K., Kallajoki, M., Voipio-Pulkki, L.M., Tikkanen, I., Cardiomyocyte apoptosis and ventricular remodeling after myocardial infarction in rats (2001) Am. J. Physiol. Heart Circ. Physiol., 280, pp. 2726-H2731Teiger, E., Than, V.D., Richard, L., Wisnewsky, C., Tea, B.S., Gaboury, L., Tremblay, J., Hamet, P., Apoptosis in pressure overload-induced heart hypertrophy in the rat (1996) J. Clin. Invest., 97, pp. 2891-2897Anversa, P., Kajstura, J., Olivetti, G., Myocyte death in heart failure (1996) Curr. Opin. Cardiol., 11, pp. 245-251Barouch, L.A., Cappola, T.P., Harrison, R.W., Crone, J.K., Rodriguez, E.R., Burnett, A.L., Hare, J.M., Combined loss of neuronal and endothelial nitric oxide synthase causes premature mortality and age-related hypertrophic cardiac remodeling in mice (2003) J. Mol. Cell. Cardiol., 35, pp. 637-644Krieger, M.H., Santos, K.F., Shishido, S.M., Wanschel, A.C., Estrela, H.F., Santos, L., De Oliveira, M.G., Laurindo, F.R., Antiatherogenic effects of S-nitroso-N-acetylcysteine in hypercholesterolemic LDL receptor knockout mice (2006) Nitric Oxide, 14, pp. 12-20Garcia, J.A., Dos Santos, L., Moura, A.L., Ricardo, K.F., Wanschel, A.C., Shishido, S.M., Spadari-Bratfisch, R.C., Krieger, M.H., S-nitroso-N-acetylcysteine (SNAC) prevents myocardial alterations in hypercholesterolemic LDL receptor knockout mice by antiinflammatory action (2008) J. Cardiovasc. Pharmacol., 51, pp. 78-85Hess, D.T., Matsumoto, A., Kim, S.O., Marshall, H.E., Stamler, J.S., Protein S-nitrosylation: Purview and parameters (2005) Nat. Rev. Mol. Cell Biol., 6, pp. 150-166Whalen, E.J., Foster, M.W., Matsumoto, A., Ozawa, K., Violin, J.D., Que, L.G., Nelson, C.D., Stamler, J.S., Regulation of beta-adrenergic receptor signaling by S-nitrosylation of G-protein-coupled receptor kinase 2 (2007) Cell, 129, pp. 511-522Kokkola, T., Savinainen, J.R., Monkkonen, K.S., Retamal, M.D., Laitinen, J.T., S-nitrosothiols modulate G protein-coupled receptor signaling in a reversible and highly receptor-specific manner (2005) BMC Cell Biol., 6, p. 21Whalen, E.J., Johnson, A.K., Lewis, S.J., Beta-adrenoceptor dysfunction after inhibition of NO synthesis (2000) Hypertension, 36, pp. 376-382Ricardo, K.F., Shishido, S.M., De Oliveira, M.G., Krieger, M.H., Characterization of the hypotensive effect of S-nitroso-N-acetylcysteine in normotensive and hypertensive conscious rats (2002) Nitric Oxide, 7, pp. 57-66Santos, I.N., Sumitame, M., Caceres, V.M., Moreira, M.F., Krieger, M.H., Spadari-Bratfisch, R.C., Evidence for two atypical conformations of beta-adrenoceptors and their interaction with Gi proteins (2005) Eur. J. Pharmacol., 513, pp. 109-118Furchgott, R.F., The pharmacological differentiation of adrenergic receptors (1967) Ann. N.Y. Acad. Sci., 139, pp. 553-570Bilski, A.J., Halliday, S.E., Fitzgerald, J.D., Wale, J.L., The pharmacology of a beta 2-selective adrenoceptor antagonist (ICI 118,551) (1983) J. Cardiovasc. Pharmacol., 5, pp. 430-437Danson, E.J., Zhang, Y.H., Sears, C.E., Edwards, A.R., Casadei, B., Paterson, D.J., Disruption of inhibitory G-proteins mediates a reduction in atrial beta-adrenergic signaling by enhancing eNOS expression (2005) Cardiovasc. Res., 67, pp. 613-623Caulfield, M.P., Birdsall, N.J., International Union of Pharmacology. XVII. Classification of muscarinic acetylcholine receptors (1998) Pharmacol. Rev., 50, pp. 279-290Heubach, J.F., Rau, T., Eschenhagen, T., Ravens, U., Kaumann, A.J., Physiological antagonism between ventricular beta 1-adrenoceptors and alpha 1-adrenoceptors but no evidence for beta 2- and beta 3-adrenoceptor function in murine heart (2002) Br. J. Pharmacol., 136, pp. 217-229Jaffrey, S.R., Snyder, S.H., The biotin switch method for the detection of S-nitrosylated proteins (2001) Sci. STKE, 2001. , pl1Forrester, M.T., Foster, M.W., Benhar, M., Stamler, J.S., Detection of protein S-nitrosylation with the biotin-switch technique (2009) Free Radical Biol. Med., 46, pp. 119-126Turko, I.V., Murad, F., Protein nitration in cardiovascular diseases (2002) Pharmacol. Rev., 54, pp. 619-634Heymes, C., Bendall, J.K., Ratajczak, P., Cave, A.C., Samuel, J.L., Hasenfuss, G., Shah, A.M., Increased myocardial NADPH oxidase activity in human heart failure (2003) J. Am. Coll. Cardiol., 41, pp. 2164-2171Nakamura, K., Kusano, K., Nakamura, Y., Kakishita, M., Ohta, K., Nagase, S., Yamamoto, M., Ohe, T., Carvedilol decreases elevated oxidative stress in human failing myocardium (2002) Circulation, 105, pp. 2867-2871Landmesser, U., Spiekermann, S., Dikalov, S., Tatge, H., Wilke, R., Kohler, C., Harrison, D.G., Drexler, H., Vascular oxidative stress and endothelial dysfunction in patients with chronic heart failure: Role of xanthine-oxidase and extracellular superoxide dismutase (2002) Circulation, 106, pp. 3073-3078Farquharson, C.A., Butler, R., Hill, A., Belch, J.J., Struthers, A.D., Allopurinol improves endothelial dysfunction in chronic heart failure (2002) Circulation, 106, pp. 221-226Shizukuda, Y., Buttrick, P.M., Oxygen free radicals and heart failure: New insight into an old question (2002) Am. J. Physiol. Lung Cell. Mol. Physiol., 283, pp. 237-L238Papapostolou, I., Patsoukis, N., Georgiou, C.D., The fluorescence detection of superoxide radical using hydroethidine could be complicated by the presence of heme proteins (2004) Anal. Biochem., 332, pp. 290-298Zhao, H., Joseph, J., Fales, H.M., Sokoloski, E.A., Levine, R.L., Vasquez-Vivar, J., Kalyanaraman, B., Detection and characterization of the product of hydroethidine and intracellular superoxide by HPLC and limitations of fluorescence (2005) Proc. Natl. Acad. Sci. USA, 102, pp. 5727-5732Chess, D.J., Xu, W., Khairallah, R., O'Shea, K.M., Kop, W.J., Azimzadeh, A.M., Stanley, W.C., The antioxidant tempol attenuates pressure overload-induced cardiac hypertrophy and contractile dysfunction in mice fed a high-fructose diet (2008) Am. J. Physiol. Heart Circ. Physiol., 295, pp. 2223-H2230Date, M.O., Morita, T., Yamashita, N., Nishida, K., Yamaguchi, O., Higuchi, Y., Hirotani, S., Otsu, K., The antioxidant N-2-mercaptopropionyl glycine attenuates left ventricular hypertrophy in in vivo murine pressure-overload model (2002) J. Am. Coll. Cardiol., 39, pp. 907-912Aruoma, O.I., Halliwell, B., Hoey, B.M., Butler, J., The antioxidant action of N-acetylcysteine: Its reaction with hydrogen peroxide, hydroxyl radical, superoxide, and hypochlorous acid (1989) Free Radical Biol. Med., 6, pp. 593-597Thomas, S.R., Chen, K., Keaney, Jr.J.F., Hydrogen peroxide activates endothelial nitric-oxide synthase through coordinated phosphorylation and dephosphorylation via a phosphoinositide 3-kinase-dependent signaling pathway (2002) J. Biol. Chem., 277, pp. 6017-6024Drummond, G.R., Cai, H., Davis, M.E., Ramasamy, S., Harrison, D.G., Transcriptional and posttranscriptional regulation of endothelial nitric oxide synthase expression by hydrogen peroxide (2000) Circ. Res., 86, pp. 347-354Cai, H., Davis, M.E., Drummond, G.R., Harrison, D.G., Induction of endothelial NO synthase by hydrogen peroxide via a Ca(2+)/calmodulin-dependent protein kinase II/janus kinase 2-dependent pathway (2001) Arterioscler. Thromb. Vasc. Biol., 21, pp. 1571-1576Cai, H., Li, Z., Dikalov, S., Holland, S.M., Hwang, J., Jo, H., Dudley, Jr.S.C., Harrison, D.G., NAD(P)H oxidase-derived hydrogen peroxide mediates endothelial nitric oxide production in response to angiotensin II (2002) J. Biol. Chem., 277, pp. 48311-48317Fernandes, D.C., Wosniak, Jr.J., Pescatore, L.A., Bertoline, M.A., Liberman, M., Laurindo, F.R., Santos, C.X., Analysis of DHE-derived oxidation products by HPLC in the assessment of superoxide production and NADPH oxidase activity in vascular systems (2007) Am. J. Physiol. Cell. Physiol., 292, pp. 413-C422Adam, L., Bouvier, M., Jones, T.L., Nitric oxide modulates beta(2)-adrenergic receptor palmitoylation and signaling (1999) J. Biol. Chem., 274, pp. 26337-26343Chesley, A., Lundberg, M.S., Asai, T., Xiao, R.P., Ohtani, S., Lakatta, E.G., Crow, M.T., The beta(2)-adrenergic receptor delivers an antiapoptotic signal to cardiac myocytes through G(i)-dependent coupling to phosphatidylinositol 3′-kinase (2000) Circ. Res., 87, pp. 1172-1179Zhu, W.Z., Zheng, M., Koch, W.J., Lefkowitz, R.J., Kobilka, B.K., Xiao, R.P., Dual modulation of cell survival and cell death by beta(2)-adrenergic signaling in adult mouse cardiac myocytes (2001) Proc. Natl. Acad. Sci. USA, 98, pp. 1607-1612Communal, C., Singh, K., Sawyer, D.B., Colucci, W.S., Opposing effects of beta(1)- and beta(2)-adrenergic receptors on cardiac myocyte apoptosis: Role of a pertussis toxin-sensitive G protein (1999) Circulation, 100, pp. 2210-2212Grimm, M., Gsell, S., Mittmann, C., Nose, M., Scholz, H., Weil, J., Eschenhagen, T., Inactivation of (Gialpha) proteins increases arrhythmogenic effects of beta-adrenergic stimulation in the heart (1998) J. Mol. Cell. Cardiol., 30, pp. 1917-1928Xiao, R.P., Avdonin, P., Zhou, Y.Y., Cheng, H., Akhter, S.A., Eschenhagen, T., Lefkowitz, R.J., Lakatta, E.G., Coupling of beta2-adrenoceptor to Gi proteins and its physiological relevance in murine cardiac myocytes (1999) Circ. Res., 84, pp. 43-52Sabri, A., Pak, E., Alcott, S.A., Wilson, B.A., Steinberg, S.F., Coupling function of endogenous alpha(1)- and beta-adrenergic receptors in mouse cardiomyocytes (2000) Circ. Res., 86, pp. 1047-1053Brodde, O.E., Bruck, H., Leineweber, K., Cardiac adrenoceptors: Physiological and pathophysiological relevance (2006) J. Pharmacol. Sci., 100, pp. 323-337Sun, M., Chen, M., Dawood, F., Zurawska, U., Li, J.Y., Parker, T., Kassiri, Z., Liu, P.P., Tumor necrosis factor-alpha mediates cardiac remodeling and ventricular dysfunction after pressure overload state (2007) Circulation, 115, pp. 1398-1407Condorelli, G., Morisco, C., Stassi, G., Notte, A., Farina, F., Sgaramella, G., De Rienzo, A., Lembo, G., Increased cardiomyocyte apoptosis and changes in proapoptotic and antiapoptotic genes bax and bcl-2 during left ventricular adaptations to chronic pressure overload in the rat (1999) Circulation, 99, pp. 3071-3078Broillet, M.C., S-nitrosylation of proteins (1999) Cell. Mol. Life Sci., 55, pp. 1036-1042Stamler, J.S., Lamas, S., Fang, F.C., Nitrosylation. The prototypic redox-based signaling mechanism (2001) Cell, 106, pp. 675-683Lin, J., Steenbergen, C., Murphy, E., Sun, J., Estrogen receptor-beta activation results in S-nitrosylation of proteins involved in cardioprotection (2009) Circulation, 120, pp. 245-254Sun, J., Picht, E., Ginsburg, K.S., Bers, D.M., Steenbergen, C., Murphy, E., Hypercontractile female hearts exhibit increased S-nitrosylation of the L-type Ca2+ channel alpha1 subunit and reduced ischemia/reperfusion injury (2006) Circ. Res., 98, pp. 403-411Li, J., Billiar, T.R., Talanian, R.V., Kim, Y.M., Nitric oxide reversibly inhibits seven members of the caspase family via S-nitrosylation (1997) Biochem. Biophys. Res. Commun., 240, pp. 419-424Stamler, J.S., Redox signaling: Nitrosylation and related target interactions of nitric oxide (1994) Cell, 78, pp. 931-93
α1d-adrenoceptor-induced Relaxation On Rat Carotid Artery Is Impaired During The Endothelial Dysfunction Evoked In The Early Stages Of Hyperhomocysteinemia
Hyperhomocysteinemia is a known risk factor for cardiovascular diseases, but the underlying mechanisms of this pathology are complex. We aimed to evaluate the effect of hyperhomocysteinemia in vasorelaxations induced by α1D-adrenoceptor agonists. Vascular reactivity of rat carotid artery to the α-adrenoceptor agonist, phenylephrine, was enhanced in hyperhomocysteinemia. Mechanical removal of endothelium did not modify the carotid responsiveness to phenylephrine, compared to control. Phenylephrine induces endothelium-dependent relaxation, in the presence of 5-methyl urapidil (α1A-adrenoceptor antagonist). We hypothesised that endothelial-relaxant α1-adrenoceptors are impaired by hyperhomocysteinemia. Incubation with prazosin (selective α1-adrenoceptor antagonist) or BMY7378 (8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4,5]decane-7, 9-dione dihydrochloride) (selective α1D-adrenoceptor antagonist), similarly inhibited phenylephrine-induced relaxations in both control and hyperhomocysteinemic carotids. Immunohistochemistry showed enhanced immunoreactivity for eNOS and iNOS in hyperhomocysteinemic rats. In carotid arteries from hyperhomocysteinemic rats there was a decrease in superoxide dismutase activity and enhanced superoxide anion production. We conclude that α1D-adrenoceptors mediate endothelium-dependent relaxation triggered by phenylephrine in rat carotid artery and affect the final tone. Furthermore, the enhanced phenylephrine-induced contraction in carotid artery due to hyperhomocysteinemia is endothelium-dependent and involves a loss of the inhibitory effect of relaxant α1D-adrenoceptors by reducing NO biodisponibility. © 2006 Elsevier B.V. All rights reserved.54301/03/158391Amerini, S., Mantelli, L., Ledda, F., Enhancement of the vasoconstrictor response to KCl by nitric oxide synthesis inhibition: a comparison with noradrenaline (1995) Pharmacol. Res., 31, pp. 175-181Barbieri, A., Santagostino-Barbone, M.G., Zonta, F., Lucchelli, A., Pharmacological characterization of alpha-adrenoceptors that mediate contraction in splenic artery strips from the pig (1998) Naunyn-Schmiedeberg's Arch. Pharmacol., 357, pp. 654-661Bellamy, M.F., Mcdowell, I.F., Ramsey, M.W., Brownlee, M., Bones, C., Newcombe, R.G., Lewis, M.J., Hyperhomocysteinemia after an oral methionine load acutely impairs endothelial function in healthy adults (1998) Circulation, 98, pp. 1848-1852Benzuly, K.H., Padgett, R.C., Kaul, S., Piegors, D.J., Armstrong, M.L., Heistad, D.D., Functional improvement precedes structural regression of atherosclerosis (1994) Circulation, 89, pp. 1810-1818Boer, C., Scheffer, G.J., de Lange, J.J., Westerhof, N., Sipkema, P., Alpha1-adrenoceptor stimulation induces nitric oxide release in rat pulmonary arteries (1999) J. Vasc. Res., 36, pp. 79-81Brandes, R.P., Barton, M., Philippens, K.M., Schweitzer, G., Mugge, A., Endothelial-derived superoxide anion in pig coronary arteriesevidence from lucigenin chemiluminescence and histochemical techniques (1997) J. Physiol., 500, pp. 331-342Brizzolara, A.L., Stewart-Lee, A., Burnstock, G., Responses of rabbit basilar arteries to vasoconstrictor and vasodilator agents: the effects of atherosclerosis, age and sex (1994) J. Vasc. Res., 31, pp. 106-113Chen, C., Conklin, B.S., Ren, Z., Zhong, D.S., Homocysteine decreases endothelium-dependent vasorelaxation in porcine arteries (2002) J. Surg. Res., 102, pp. 22-30Daniel, E.E., Brown, R.D., Wang, Y.F., Low, A.M., Lu-Chao, H., Kwan, C.Y., Alpha-adrenoceptors in canine mesenteric artery are predominantly 1A subtype: pharmacological and immunochemical evidence (1999) J. Pharmacol. Exp. Ther., 291, pp. 671-679de Oliveira, A.M., Campos-Mello, C., Leitão, M.C., Corrêa, F.M.A., Maturation and aging-related differences in responsiveness of rat aorta and carotid arteries to alpha1-adrenoceptor stimulation (1998) Pharmacology, 57, pp. 305-313Desai, A., Lankford, H.A., Warren, J.S., Homocysteine augments cytokine-induced chemokine expression in human vascular smooth muscle cells: implications for atherogenesis (2001) Inflammation, 25, pp. 179-186Dudman, N.P., Wilcken, D.E., Homocysteine thiolactone and experimental homocysteinemia (1982) Biochem. Med., 27, pp. 244-253Filippi, S., Parenti, A., Donnini, S., Granger, H.J., Fazzini, A., Ledda, F., Alpha(1D)-adrenoceptors cause endothelium-dependent vasodilatation in the rat mesenteric vascular bed (2001) J. Pharmacol. Exp. Ther., 296, pp. 869-875Fujimoto, S., Itoh, T., Role of nitric oxide and nitric oxide-independent relaxing factor in contraction and relaxation of rabbit blood vessels (1997) Eur. J. Pharmacol., 330, pp. 177-184Futaki, N., Takahashi, S., Yokoyama, M., Arai, I., Higuchi, S., Otomo, S., NS-398, a new anti-inflammatory agent, selectively inhibits prostaglandin G/H synthase/cyclooxygenase (COX-2) activity in vitro (1994) Prostaglandins, 47, pp. 55-59Goetz, A.S., King, H.K., Ward, S.D., True, T.A., Rimele, T.J., Saussy Jr., D.L., BMY 7378 is a selective antagonist of the D subtype of alpha 1-adrenoceptors (1995) Eur. J. Pharmacol., 272, pp. R5-R6Greenberg, S., Diecke, F.P., Peevy, K., Tanaka, T.P., The endothelium modulates adrenergic neurotransmission to canine pulmonary arteries and veins (1989) Eur. J. Pharmacol., 162, pp. 67-80Hague, C., Gonzalez-Cabrera, P.J., Jeffries, W.B., Abel, P.W., Relationship between alpha(1)-adrenergic receptor-induced contraction and extracellular signal-regulated kinase activation in the bovine inferior alveolar artery (2002) J. Pharmacol. Exp. Ther., 303, pp. 403-411Harker, L.A., Ross, R., Slichter, S.J., Scott, C.R., Homocysteine-induced arteriosclerosis. The role of endothelial cell injury and platelet response in its genesis (1976) J. Clin. Invest., 58, pp. 731-741Hill, C.H., Mecham, R., Starcher, B., Fibrillin-2 defects impair elastic fiber assembly in a homocysteinemic chick model (2002) J. Nutr., 132, pp. 2143-2150Jahnichen, S., Eltze, M., Pertz, H.H., Evidence that alpha(1B)-adrenoceptors are involved in noradrenaline-induced contractions of rat tail artery (2004) Eur. J. Pharmacol., 488, pp. 157-167Jakubowski, H., Metabolism of homocysteine thiolactone in human cell cultures. Possible mechanism for pathological consequences of elevated homocysteine levels (1997) J. Biol. Chem., 272, pp. 1935-1942Jakubowski, H., Zhang, L., Bardeguez, A., Aviv, A., Homocysteine thiolactone and protein homocysteinylation in human endothelial cells. Implications for atherosclerosis (2000) Circ. Res., 87, pp. 45-51Kaley, G., Koller, A., Rodenburg, J.M., Messina, E.J., Wolin, M.S., Regulation of arteriolar tone and responses via L-arginine pathway in skeletal muscle (1992) Am. J. Physiol., 262, pp. H987-H992Lang, D., Kredan, M.B., Moat, S.J., Hussain, S.A., Powell, C.A., Bellamy, M.F., Powers, H.J., Lewis, M.J., Homocysteine-induced inhibition of endothelium-dependent relaxation in rabbit aorta: role for superoxide anions (2000) Arterioscler. Thromb. Vasc. Biol., 20, pp. 422-427Leite, P.F., Danilovic, A., Moriel, P., Dantas, K., Marklund, S., Dantas, A.P., Laurindo, F.R., Sustained decrease in superoxide dismutase activity underlies constrictive remodeling after balloon injury in rabbits (2003) Arterioscler. Thromb. Vasc. Biol., 23, pp. 2197-2202Li, Y., Zhu, H., Kuppusamy, P., Roubaud, V., Zweier, J.L., Trush, M.A., Validation of lucigenin (Bis-N-Methylacridinium) as a chemiluminescent probe for detecting superoxide anion radical for detecting superoxide anion radical production by enzymatic and cellular sources (1998) J. Biol. Chem., 273, pp. 2015-2023McCully, K.S., Carvalho, A.C., Homocysteine thiolactone, N-homocysteine thiolactonyl retinamide, and platelet aggregation (1987) Res. Commun. Chem. Pathol. Pharmacol., 56, pp. 349-360Miao, C.Y., Villeneuve, N., Brunel-Jacquemin, C., Petit, C., Guillaumin, J.P., Gransagne, D., Briant, C., Vanhoutte, P.M., Chronic mild hyperhomocysteinemia induces aortic endothelial dysfunction but does not elevate arterial pressure in rats (2005) J. Vasc. Res., 42, pp. 148-156Muramatsu, I., Ohmura, T., Kigoshi, S., Hashimoto, S., Oshita, M., Pharmacological subclassification of alpha 1-adrenoceptors in vascular smooth muscle (1990) Br. J. Pharmocol., 99, pp. 197-201Muramatsu, I., Murata, S., Isaka, M., Piao, H.L., Zhu, J., Suzuki, F., Miyamoto, S., Taniguchi, T., Alpha1-adrenoceptor subtypes and two receptor systems in vascular tissues (1998) Life Sci., 62, pp. 1461-1465Oliveira, C.P., Kassab, P., Lopasso, F.P., Souza, H.P., Janiszewski, M., Laurindo, F.R., Iriya, K., Laudanna, A.A., Protective effect of ascorbic acid in experimental gastric cancer: reduction of oxidative stress (2003) World J. Gastroenterol., 9, pp. 446-448Ossani, G.P., Fischer, P.A., Caram, S.G., Dominguez, G.N., Monserrat, A.J., Masnatta, L.D., Mild hyperhomocysteinemia promotes renal hemodynamic dysfunction without histopathologic changes in adult rats (2004) Kidney Int., 66, pp. 1866-1872Powers, R.W., Majors, A.K., Cerula, S.L., Huber, H.A., Schmidt, B.P., Roberts, J.M., Changes in markers of vascular injury in response to transient hyperhomocysteinemia (2003) Metabolism, 52, pp. 501-507Reddy, G.S., Wilcken, D.E., Experimental homocysteinemia in pigs: comparison with studies in sixteen homocystinuric patients (1982) Metabolism, 31, pp. 778-783Rees, D.D., Palmer, R.M., Schulz, R., Hodson, H.F., Moncada, S., Characterization of three inhibitors of endothelial nitric oxide synthase in vitro and in vivo (1990) Br. J. Pharmacol., 101, pp. 746-752Rodgers, G.M., Conn, M.T., Homocysteine, an atherogenic stimulus, reduces protein C activation by arterial and venous endothelial cells (1990) Blood, 75, pp. 895-901Ruffolo Jr., R.R., Nichols, A.J., Oriowo, M.A., Interaction of vascular alpha-1 adrenoceptors with multiple signal transduction pathways (1991) Blood Vessels, 28, pp. 122-128Satoh, M., Enomoto, K., Takayanagi, I., Koike, K., Differences of antagonism for a selective alpha1D-adrenoceptor antagonist BMY 7378 in the rabbit thoracic aorta and iliac artery (1998) J. Smooth Muscle Res., 34, pp. 151-158Stamler, J.S., Osborne, J.A., Jaraki, O., Rabbani, L.E., Mullins, M., Singel, D., Loscalzo, J., Adverse vascular effects of homocysteine are modulated by endothelium-derived relaxing factor and related oxides of nitrogen (1993) J. Clin. Invest., 91, pp. 308-318Stralin, P., Karlsson, K., Johansson, B.O., Marklund, S.L., The interstitium of the human arterial wall contains very large amounts of extracellular superoxide dismutase (1995) Arterioscler. Thromb. Vasc. Biol., 15, pp. 2032-2036Stuhlinger, M.C., Tsao, P.S., Her, J.H., Kimoto, M., Balint, R.F., Cooke, J.P., Homocysteine impairs the nitric oxide synthase pathway: role of asymmetric dimethylarginine (2001) Circulation, 104, pp. 2569-2575Theroux, T.L., Esbenshade, T.A., Peavy, R.D., Minneman, K.P., Coupling efficiencies of human alpha 1-adrenergic receptor subtypes: titration of receptor density and responsiveness with inducible and repressible expression vectors (1996) Mol. Pharmacol., 50, pp. 1376-1387Traupe, T., Ortmann, J., Munter, K., Barton, M., Endothelial therapy of atherosclerosis and its risk factors (2003) Curr. Vasc. Pharmacol., 1, pp. 111-121Ungvari, Z., Pacher, P., Rischak, K., Szollar, L., Koller, A., Dysfunction of nitric oxide mediation in isolated rat arterioles with methionine diet-induced hyperhomocysteinemia (1999) Arterioscler. Thromb. Vasc. Biol., 19, pp. 1899-1904Upchurch Jr., G.R., Welch, G.N., Fabian, A.J., Pigazzi, A., Keaney Jr., J.F., Loscalzo, J., Stimulation of endothelial nitric oxide production by homocyst(e)ine (1997) Atherosclerosis, 132, pp. 177-185Weiss, N., Mechanisms of increased vascular oxidant stress in hyperhomocys-teinemia and its impact on endothelial function (2005) Curr. Drug Metab., 6, pp. 27-36Welch, G.N., Loscalzo, J., Homocysteine and atherothrombosis. (1998) N. Engl. J. Med., 338, pp. 1042-1050Yousif, M., Kadavil, E.A., Oriowo, M.A., Heterogeneity of alpha 1-adrenoceptor subtypes mediating noradrenaline-induced contractions of the rat superior mesenteric artery (1998) Pharmacology, 56, pp. 196-206Zacharia, J., Hillier, C., Macdonald, A., Pharmacological characterization of alpha1-adrenoceptors in mouse isolated femoral small arteries (2004) Eur. J. Pharmacol., 503, pp. 155-163Zschauer, A.O., Sielczak, M.W., Smith, D.A., Wanner, A., Norepinephrine-induced contraction of isolated rabbit bronchial artery: role of alpha1- and alpha2-adrenoceptor activation (1997) J. Appl. Physiol., 82, pp. 1918-192