Examinando por Autor "Briones, AM"

Mostrando 1 - 9 de 9

Activation of BKCa channels by nitric oxide prevents coronary artery endothelial dysfunction in ouabain-induced hypertensive rats
(2009-01) Briones, AM; Padilha, A; Cogolludo, A; Alonso, MJ; Vassallo, DV; Pérez-Vizcaíno, F; Salaices, M
Objective Chronic-ouabain administration to rats induces hypertension and increases the endothelial modulation of vasoconstrictor responses. The aim of this study was to analyze whether ouabain-treatment affects the mechanisms involved in endothelium-dependent relaxation of coronary arteries. Methods Coronary arteries from control and ouabain-treated rats (aprox 8.0mg/day, 5 weeks) were used. Vascular reactivity was analyzed by isometric tension recording and membrane currents were measured using the whole-cell configuration of the patch-clamp technique. Results In 5-hydroxytryptamine (5-HT) precontracted arteries, acetylcholine (ACh, 1 nmol/l¿10mmol/l) induced a similar relaxant response in coronary arteries from both groups that was abolished by the nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester (100mmol/l). However, when arteries were contracted with high KCl (60 mmol/l) or preincubated with the large-conductance Ca2þ-activated Kþ (BKCa) channels-blocker iberiotoxin (0.1mmol/l), the relaxation elicited by ACh was more reduced in ouabain-treated than control rats. After iberiotoxin preincubation, the relaxant response of the nitric-oxide donor, DEA-NO (10 nmol/l¿100mmol/l) was significantly inhibited in ouabain-treated coronary arteries but not in control vessels. The soluble guanylyl cyclase activator BAY 41-2272 (10 nmol/l¿30mmol/l) induced relaxant responses that were inhibited by iberiotoxin. In coronary-artery myocytes isolated from ouabain-treated rats DEA-NO (1mmol/l) markedly increased the amplitude of the iberiotoxin-sensitive current in the whole range of test potentials, compared with nontreated rats. Conclusion Our results indicate that chronic ouabain treatment increases activation of BKCa currents by nitric oxide and this effect might contribute to preserve the endothelial function in coronary arteries in this hypertension model
Activation of p38 and ERK1/2 MAPK by superoxide anion participates in Angiotensin II-induced COX-2 expression in smooth muscle cells from resistance arteries
(2009-09) Hernanz, R; Beltrán, AE; Pérez-Girón, JV; Martín, A; Briones, AM; Palacios, R; Salaices, M; Alonso, MJ
Introduction: Angiotensin II (Ang II) regulates vascular smooth muscle cell (VSMC) function by activating signalling cascades that promote vasoconstriction, growth and inflammation. The mechanisms implicated in Ang II-induced pro-inflammatory actions include activation of several mitogen-activated protein kinases (MAPKs), reactive oxygen species generation and the modulation of prostaglandins production by regulating cyclooxygenase-2 (COX-2) expression. Aim: To investigate the effect of Ang II on COX-2 expression in VSMC derived from small resistance arteries and the mechanisms involved. Methods: VSMC derived from rat mesenteric resistance arteries were used. Protein expression was determined by Western Blot, mRNA levels by Q-RT-PCR and superoxide anion (O2-) production by dihydroethidine fluorescence. Results: Ang II (0.1 ¿M) time-dependently increased COX-2 protein expression (30 min - 8 h) and mRNA levels (15 min - 4 h), while COX- 1 expression remained unmodified. In addition, Ang II did not modify AT1 receptor expression. The AT1 antagonist losartan (10 ¿M), but not the AT2 antagonist PD 122319 (10 ¿M), abolished the increase in COX-2 expression induced by Ang II (2 h). COX-2 expression was also reduced by the respective NADPHox and xanthine oxidase inhibitors, apocynin (30 mM) and allopurinol (10 mM). Furthermore, Ang II (3- 30 min) increased O2- production; this effect was reduced by losartan, allopurinol and apocynin but not by PD 123319. Ang II (2-30 min) induced the phosphorylation of p38 and ERK1/2 MAPK; this effect was reduced by losartan, allopurinol and apocynin. In addition, the respective inhibitors of p38 and ERK1/2, SB 203580 (10 mM) and PD 98059 (25 mM), reduced the Ang II-induced COX-2 expression. Conclusions: The present results provide evidences that angiotensin II increases COX-2 expression in VSMC from resistance arteries, at least in part, through mechanisms that include O2- production and the subsequent activation of p38 and ERK1/2 MAPK
Atorvastatin prevents angiotensin II-induced vascular remodeling and oxidative stress
(2009-07) Briones, AM; Rodríguez-Criado, N; Hernanz, R; García-Redondo, AB; Rodrígues-Díez, RR; Alonso, MJ; Egido, J; Ruiz-Ortega, M; Salaices, M
Angiotensin II (Ang II) modulates vasomotor tone, cell growth, and extracellular matrix deposition. This study analyzed the effect of atorvastatin in the possible alterations induced by Ang II on structure and mechanics of mesenteric resistance arteries and the signaling mechanisms involved. Wistar rats were infused with Ang II (100 ng/kg per day, SC minipumps, 2 weeks) with or without atorvastatin (5 mg/kg per day). Ang II increased blood pressure and plasmatic malondialdehyde levels. Compared with controls, mesenteric resistance arteries from Ang II¿treated rats showed the following: (1) decreased lumen diameter; (2) increased wall/lumen; (3) decreased number of adventitial, smooth muscle, and endothelial cells; (4) increased stiffness; (5) increased collagen deposition; and (6) diminished fenestrae area and number in the internal elastic lamina. Atorvastatin did not alter blood pressure but reversed all of the structural and mechanical alterations of mesenteric arteries, including collagen and elastin alterations. In mesenteric resistance arteries, Ang II increased vascular O2.- production and diminished endothelial NO synthase and CuZn/superoxide dismutase but did not modify extracellular-superoxide dismutase expression. Atorvastatin improved plasmatic and vascular oxidative stress, normalized endothelial NO synthase and CuZn/superoxide dismutase expression, and increased extracellular superoxide dismutase expression, showing antioxidant properties. Atorvastatin also diminished extracellular signal¿ regulated kinase 1/2 activation caused by Ang II in these vessels, indicating an interaction with Ang II¿induced intracellular responses. In vascular smooth muscle cells, collagen type I release mediated by Ang II was reduced by different antioxidants and statins. Moreover, atorvastatin downregulated the Ang II¿induced NADPH oxidase subunit, Nox1, expression. Our results suggest that statins might exert beneficial effects on hypertension-induced vascular remodeling by improving vascular structure, extracellular matrix alterations, and vascular stiffness. These effects might be mediated by their antioxidant properties.
Hypertension increases contractile responses to hydrogen peroxide in resistance arteries through increased thromboxane A2, Ca2+, and superoxide anion levels
(2009-01) García-Redondo, AB; Briones, AM; Beltrán, A; Alonso, MJ; Simonsen, U; Salaices, M
This study investigated the mechanisms underlying the response to hydrogen peroxide (H2O2) in mesenteric resistance arteries from spontaneously hypertensive rats (SHRs) and normotensive Wistar Kyoto (WKY) rats. Arteries were mounted in microvascular myographs for isometric tension recording and for simultaneous measurements of intracellular Ca2+ concentration ([Ca2+]i), superoxide anion (O2.-) production was evaluated by dihydroethidium fluorescence and confocal microscopy, and thromboxane A2 (TXA2) production was evaluated by enzyme immunoassay. H2O2 (1¿100 microM) induced biphasic responses characterized by a transient endothelium-dependent contraction followed by relaxation. Simultaneous measurements of tension and Ca2+ showed a greater effect of H2O2 in arteries from hypertensive than normotensive rats. The cyclooxygenase (cox) inhibitor, indomethacin [1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1-H-indole-3-acetic acid] (1 microM); the COX-1 inhibitor, SC-58560 [5-(4-chlorophenyl)- 1-(4-methoxyphenyl)-3-trifluoromethyl pyrazole] (1 microM); the thromboxane (TXA2) synthase inhibitor, furegrelate [5-(3- pyridinylmethyl)-2-benzofurancarboxylic acid, sodium salt] (10 microM); and the TXA2/prostaglandin H2 receptor antagonist, SQ 29,548 ([1S-[1.alpha.,2.alpha.(Z),3.alpha.,4.alpha.]]-7-[3-[[2 [(phenylamino) carbonyl] hydrazino] methyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5- heptenoic acid)) (1 microM) abolished H2O2 contraction in arteries from WKY rats but only reduced it in SHRs. The O2.- scavenger, tiron (4,5-dihydroxy-1,3-benzenedisulfonic acid disodium salt) (1 mM), and the NADPH oxidase inhibitor, apocynin (4¿- hydroxy-3¿-methoxyacetophenone) (0.3 mM), decreased H2O2 contraction in arteries from SHRs but not in WKY rats. H2O2 induced TXA2 and O2.- production that was greater in SHRs than in WKY rats. The TXA2 analog, U46619 [9,11-di-deoxy-11alpha,9alpha-epoxymethano prostaglandin F2alpha (0.1 nM¿1 microM)], also increased O2.- production in SHR vessels. H2O2-induced TXA2 production was decreased by SC-58560. H2O2-induced O2.- production was decreased by tiron, apocynin, and SQ 29,548. In conclusion, the enhanced H2O2 contraction in resistance arteries from SHRs seems to be mediated by increased TXA2 release from COX-1 followed by elevations in vascular smooth muscle [Ca2+]i levels and O2.- production. This reveals a new mechanism of oxidative stress-induced vascular damage in hypertension.
Losartan and tempol treatments normalize the increased response to hydrogen peroxide in resistance arteries from hypertensive rats
(2009-09) García-Redondo, AB; Briones, AM; Avendaño, MS; Hernanz, R; Alonso, MJ; Salaices, M
Objective To analyse the role of angiotensin II, via AT1 receptors, and oxidative stress in the mechanisms underlying the increased response to hydrogen peroxide (H2O2) of mesenteric resistance arteries from spontaneously hypertensive rats (SHRs). Methods Arteries from normotensive and SHRs untreated or treated with the AT1 receptor antagonist, losartan (15mg/kg per day, 12 weeks), or with the superoxide dismutase analogue, tempol (1 mmol/l, 17 days), were used. Arteries were mounted in microvascular myographs for isometric tension recording; superoxide anion (O2 S) production was evaluated by dihydroethidium fluorescence, thromboxane A2 production by enzyme immunoassay and plasma nitrite levels by the Griess method. Results H2O2 (1¿100mmol/l) induced higher contractile responses in mesenteric resistance arteries from hypertensive than normotensive rats. In SHRs, losartan and tempol treatments induced the following effects: normalized the increased H2O2 contractile responses observed; modified neither the inhibitory effects of the cyclooxygenase inhibitor, indomethacin [1-(4- chlorobenzoyl)-5-methoxy-2-methyl-1-H-indole-3-acetic acid] (1mmol/l), and the thromboxane A2/prostaglandin H2 receptor antagonist, SQ 29 548 (1mmol/l), on H2O2 contraction, nor the increase in thromboxane A2 production in response to H2O2; abolished the increased vascular O2.- production; increased both the potentiatory effect of the nitric oxide inhibitor, N(G)-nitro-L-arginine methyl ester (100mmol/l), on H2O2 responses and the acetylcholineinduced relaxation. Moreover, losartan treatment abolished the effect of the O2.- scavenger, tiron (1 mmol/l), on H2O2 responses and increased plasma nitrite levels. Conclusion Nitric oxide removal by an excessive O2.- production, probably from an upregulated renin¿ angiotensin system, participates in the increased response to H2O2 in mesenteric resistance arteries from SHRs.
Ouabain treatment increases nitric oxide bioavailability and decreases superoxide anion production in cerebral vessels
(2008-10) Hernanz, R; Briones, AM; Martín, A; Beltrán, AE; Tejerina, T; Salaices, M; Alonso, MJ
Objective Chronic administration of ouabain induces hypertension and increases the contribution of nitric oxide to vasoconstrictor responses in peripheral arteries. The aim of this study was to analyse whether ouabain treatment alters the nitric oxide bioavailability in cerebral arteries. Methods Basilar arteries from control and ouabain-treated rats (approx. 8.0mg/day, 5 weeks) were used. Vascular reactivity was analysed by isometric tension recording, protein expression by western blot, nitric oxide levels by diaminofluorescein-induced fluorescence, superoxide anion (O2.-) production by ethidium fluorescence and lucigenin chemiluminescence and plasma total antioxidant status by a commercial kit. Results The relaxations induced by bradykinin (1 nmol/l¿10mmol/l) and L-arginine (0.01¿300mmol/l) and the contractile responses induced by both N-nitro-L-arginine methyl ester (0.1¿100mmol/l) and oxyhaemoglobin (0.01¿10mmol/l) were greater in arteries from ouabain-treated than control rats. However, the relaxation to diethylamine NONOate¿nitric oxide (0.1 nmol/l¿10mmol/l) and the contractions to KCl (7.5¿120 mmol/l) and 5-hydroxytryptamine (0.01¿10mmol/l) were similar in arteries from both groups. Ouabain treatment increased basal nitric oxide levels but did not modify endothelial and neuronal nitric oxide synthase protein expression. O2.- production was lower in cerebral arteries from ouabain-treated rats; however, plasma total antioxidant status and vascular protein expression of Cu/Zn-superoxide dismutase,Mn-superoxide dismutase and extracellular superoxide dismutase were similar in both groups. Conclusion Chronic ouabain treatment increased nitric oxide basal levels in basilar arteries probably due to the decreased O2.- levels. This might be an adaptive mechanism of the cerebral vasculature to the increase in blood pressure.
p38 MAPK contributes to angiotensin II-induced COX-2 expression in aortic fibroblasts from normotensive and hypertensive rats
(2009-01) Beltrán, A; Briones, AM; García-Redondo, AB; Rodríguez, C; Miguel, M; Alvarez, A; Alonso, MJ; Martínez-González, J; Salaices, M
Objective To investigate the effect of angiotensin II on cyclooxygenase-2 (COX-2) expression in aortic adventitial fibroblasts from normotensive [Wistar¿Kyoto (WKY)] rats and spontaneously hypertensive rats (SHRs). Methods Protein expression was determined by western blot, mRNA levels by real-time PCR, transcriptional activity by luciferase assays, superoxide anion (O2.-) production by dihydroethidine fluorescence and prostaglandin E2 by enzyme immunoassay. Results Angiotensin II (0.1mmol/l, 0.5¿6 h) time dependently induced COX-2 protein expression, this effect being transient in fibroblasts from WKY rats and maintained over time in SHRs. Angiotensin II effect was abolished by valsartan (1mmol/l), an angiotensin II type 1 receptor antagonist. Angiotensin II-induced prostaglandin E2 production was reduced by valsartan and the COX-2 inhibitor NS398 (1mmol/l). Angiotensin II increased O2.- production more in SHR than WKY rats. This increase was reduced by apocynin (30mmol/l) and allopurinol (10mmol/ l), respective nicotinamide adenine dinucleotide phosphate (NADPH) and xanthine oxidase inhibitors. However, angiotensin II-induced COX-2 expression was unaffected by apocynin, allopurinol, tempol (1 mmol/l) or catalase (1000 U/ml). Angiotensin II (2¿30 min) induced p38 mitogen-activated protein kinase (MAPK) phosphorylation, transiently in WKY rats but sustained in SHRs. The p38 inhibitor SB203580 (10mmol/l) reduced angiotensin II-induced COX-2 protein and mRNA levels. The angiotensin II effect was not prevented by inhibition of mRNA synthesis, and angiotensin II was unable to modulate COX-2 transcriptional activity. Conclusions Angiotensin II increases COX-2 expression in aortic fibroblasts through mechanisms including p38 MAPK pathway, independent of reactive oxygen species production and nonmediated by COX-2 transcriptional activity modulation. The sustained angiotensin-induced p38 MAPK activation in SHR cells might be related to the maintained COX-2 expression in this strain.
PPAR¿ activation improves oxidative stress and downregulates COX-2 expression in vascular cells
(2009-09) Palacios, R; Pérez-Girón, JV; Martín, A; Hernanz, R; Briones, AM; Salaices, M; Alonso, MJ
Introduction: The increased renin-angiotensin system (RAS) activity seems to contribute to the pathophysiology of hypertension by the increase in reactive oxygen species (ROS) levels and proinflammatory mediators. Endothelin-1 (ET-1) has been proposed to explain the cardiovascular damage induced by angiotensin II (AngII). Moreover, peroxisome proliferator activated receptor ¿ (PPAR¿) agonists have anti-inflammatory actions by interference with redox-sensitive transcription factors, such as NFkB or AP-1, involved in the transcription of proinflammatory genes including cyclooxygenase-2 (COX-2). Aim: To analyze if AngII contributes to the increased COX-2 levels in vascular smooth muscle cells (VSMC) from spontaneously hypertensive (SHR) rats by mechanisms dependent of ROS and ET-1 production and whether PPAR¿ activation regulates this effect. Methods: Aortic VSMC from SHR were stimulated with AngII in the absence and the presence of different drugs. mRNA levels were measured by qRT-PCR and protein expression by Western blot. Aortic segments from SHR and Wistar-Kyoto (WKY) rats untreated and treated with losartan (15 mg/Kg/day, 12 weeks) were also used. Results: COX-2 mRNA levels were greater in segments from SHR than WKY; the treatment with losartan reduced COX-2 levels in SHR. In VSMC from SHR, AngII (0.1 ¿M, 2 h) induced COX-2, ET-1 and NOX-1 mRNA levels; this effect was reduced by losartan (10 ¿M). AngII-induced COX-2 protein expression was also reduced by the NADPHox inhibitor apocynin (30 mM). The antagonist of the ETA receptor BQ 123 (1 ¿M), but not of the ETB receptor BQ 788 (1 ¿M), also reduced COX-2 and NOX-1 mRNA levels after AngII. The proteasome inhibitor lactacystin (20 ¿M) did not modify the ET-1 mRNA levels but inhibited those of NOX-1 and COX-2. AngII also increased c-jun expression; this expression was reduced by losartan but not by BQ 123. Moreover, the PPAR¿ activator pioglitazone (10 ¿M) decreased AngII-induced COX-2 and NOX-1 mRNA levels in VSMC from SHR. Conclusions: 1) The RAS activation contributes to the increased vascular COX-2 expression in hypertension. 2) AngII-induced COX-2 expression in VSMC is related with NOX-1 induction and NFkB and AP-1 activation. 3) AngII-induced ET-1 production and ETA activation contributes, at least partially, to the increased NOX-1 and COX-2 expression. 4) PPAR¿ activation inhibits AngII-induced COX-2 expression by reducing NOX-1 levels; we suggest that transrepression mechanisms on NFkB and/or AP-1 can play an important role in this inhibitory effect of PPAR¿ activation.
Toll-like receptor 4 contributes to vascular remodelling and endothelial dysfunction in angiotensin II-induced hypertension
(British Pharmacological Society, 2015-02-17) Hernanz, R; Martínez-Revelles, S; Palacios-Ramírez, R; Martín, A; Cachofeiro, V; Aguado, A; García-Redondo, L; Barrús, MT; de Batista, PR; Briones, AM; Salaices, M; Alonso, MJ
Toll-like receptor 4 (TLR4) signalling contributes to inflammatory cardiovascular diseases, but its role in hypertension and the associated vascular damage is not known. We investigated whether TLR4 activation contributed to angiotensin II (AngII)-induced hypertension and the associated vascular structural, mechanical and functional alterations. AngII was infused (1.44 mg·kg−1·day−1, s.c.) for 2 weeks in C57BL6 mice, treated with a neutralizing anti-TLR4 antibody or IgG (1 μg·day−1); systolic BP (SBP) and aortic cytokine levels were measured. Structural, mechanical and contractile properties of aortic and mesenteric arterial segments were measured with myography and histology. RT-PCR and Western blotting were used to analyse these tissues and cultured vascular smooth muscle cells (VSMC) from hypertensive rats (SHR). Aortic TLR4 mRNA levels were raised by AngII infusion. Anti-TLR4 antibody treatment of AngII-treated mice normalised: (i) increased SBP and TNF-α, IL-6 and CCL2 levels; (ii) vascular structural and mechanical changes; (iii) altered aortic phenylephrine- and ACh-induced responses; (iv) increased NOX-1 mRNA levels, superoxide anion production and NAD(P)H oxidase activity and effects of catalase, apocynin, ML-171 and Mito-TEMPO on vascular responses; and (v) reduced NO release and effects of L-NAME on phenylephrine-induced contraction. In VSMC, the MyD88 inhibitor ST-2825 reduced AngII-induced NAD(P)H oxidase activity. The TLR4 inhibitor CLI-095 reduced AngII-induced increased phospho-JNK1/2 and p65 NF-κB subunit nuclear protein expression. TLR4 up-regulation by AngII contributed to the inflammation, endothelial dysfunction, vascular remodelling and stiffness associated with hypertension by mechanisms involving oxidative stress. MyD88-dependent activation and JNK/NF-κB signalling pathways participated in these alterations.