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CARDIOVASCULAR
Departamento de Farmacología, Facultad de Medicina, Universidad Autónoma de Madrid, Madrid, Spain (Y.A., R.H., A.M.B., A.G.-R., A.B., M.J.A., M.S.); and Departamento de Ciencias de la Salud III, Facultad de Ciencias de la Salud, Universidad Rey Juan Carlos, Alcorcón, Spain (J.V.P.-G., R.H., M.J.A.)
Received October 10, 2006; accepted January 19, 2007.
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Abstract |
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, 8-isoprostane, and total antioxidant status (TAS) by commercial kits; superoxide anion (
) by lucigenin chemiluminescence; and plasmatic malondialdehyde (MDA) by thiobarbituric acid assay. The COX-2 inhibitor N-[2-(cyclohexyloxyl)-4-nitrophenyl]-methane sulfonamide (NS-398) at 1 µM reduced phenylephrine responses more in SHR than in WKY rats. COX-2 protein and mRNA expressions, PGF2, PGI2, 8-isoprostane, and production, and MDA levels were higher in SHR, but TAS was similar in both strains. Losartan, but not hydralazine-hydrochlorothiazide treatment, reduced COX-2 expression and the effect of NS-398 on phenylephrine responses in SHR. Losartan also increased TAS and reduced PGF2, PGI2, 8-isoprostane, and production and MDA levels in SHR. Ang II (0.1 µM) induced COX-2 expression in VSMC from SHR that was reduced by 30 µM apocynin and 100 µM allopurinol, NADPH oxidase, and xanthine oxidase inhibitors, respectively. In conclusion, AT1 receptor activation by Ang II could be involved in the increased participation of COX-2-derived contractile prostanoids in vasoconstriction to phenylephrine with hypertension, probably through COX-2 expression regulation. The increased oxidative stress seems to be one of the mechanisms involved.
; Hernanz et al., 2003). Moreover, in the last years, it has become evident that prostanoid production from constitutively expressed COX-2 is also involved in the modulation of vascular responses (Henrion et al., 1997; García-Cohen et al., 2000; Adeagbo et al., 2005; Álvarez et al., 2005).
Hypertension, now considered a chronic inflammatory disease with elevated proinflammatory cytokine blood levels (Pauletto and Rattazzi, 2006; Ruiz-Ortega et al., 2006) and increased vascular COX-2 expression (Henrion et al., 1997; García-Cohen et al., 2000; Adeagbo et al., 2005; Álvarez et al., 2005), has been associated with changes in vascular responses such as impairment of endothelium-dependent vasodilator responses or enhancement of vasoconstrictor responses to different agonists (Schiffrin and Touyz, 2004). Angiotensin II (Ang II), the effector peptide of the renin-angiotensin system, physiologically regulates vascular tone and maintains normal vessel structure and function, although elevated levels of Ang II have been implicated in the pathophysiological processes that occur in hypertension. Recent studies have shown that Ang II has significant proinflammatory actions in the vascular wall, including the production of reactive oxygen species (ROS), inflammatory cytokines, and adhesion molecules (Schiffrin and Touyz, 2004; Cheng et al., 2005; Pauletto and Rattazzi, 2006), all of which are known to contribute to the inflammatory responses occurring in hypertension. Ang II also stimulates the release of prostaglandins in a variety of cells, including smooth muscle cells through the activation of phospholipase A2 (Freeman et al., 1998). In addition, Ang II regulates COX-2 expression and prostanoids production in vascular smooth muscle cells (VSMC) from normotensive rats through the activation of AT1 receptors (Ohnaka et al., 2000; Hu et al., 2002).
Increased oxidative stress has also been described in human and different models of hypertension, including spontaneously hypertensive rats (SHR) (McIntyre et al., 1999; Cai and Harrison, 2000; Griendling et al., 2000). Humoral factors, such as Ang II, may be responsible for the altered superoxide anion () generation in hypertension. Moreover, Ang II has been shown to stimulate superoxide generation by increasing the activity of NAD(P)H oxidase in cultured rat VSMC (Griendling et al., 1994), and this increase is higher in cells from SHR than in normotensive Wistar Kyoto (WKY) rats (Cruzado et al., 2005). In addition, increased vascular smooth muscle superoxide anion production via NADH/NADPH oxidase activation has been described in the hypertensive model induced by Ang II infusion (Rajagopalan et al., 1996). ROS such as superoxide anion or hydrogen peroxide also induce COX-2 (Feng et al., 1995; Kiritoshi et al., 2003), and these mediators have been implicated in the up-regulation of COX-2 expression and prostanoids release induced by Ang II in mesangial cells (Jaimes et al., 2005).
In a previous report, we have described that COX-2 expression is higher in aortic segments from SHR than in WKY rats; the increase in COX-2 levels raises contractile prostanoid production in SHR and thereby its participation in the vasoconstrictor response to phenylephrine (Álvarez et al., 2005). Similar results have been found by Adeagbo et al. (2005) using the deoxycorticosterone acetate-salt hypertensive model. The aim of the present study was to extend our previous results and to analyze whether Ang II, through the activation of AT1 receptors, is implicated in the increased participation of COX-2-derived contractile mediators in phenylephrine responses observed in aorta from hypertensive rats.
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Materials and Methods |
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Blood samples were collected in tubes containing 15% K3EDTA as anticoagulant (BD Vacutainer Systems, Preanalytical Solutions, Plymouth, UK) and placed in ice. Blood samples were centrifuged at 1500g for 15 min at 4°C. The obtained plasma was frozen at –20°C and kept at –80°C until used to determine malondialdehyde (MDA) concentration and total antioxidant status (TAS).
Reactivity Experiments. Vascular function was studied in aortic segments from WKY rats and SHR by isometric tension recording (Álvarez et al., 2005). The presence of endothelium was confirmed by the effect of 10 µM acetylcholine (Sigma-Aldrich) on segments contracted with phenylephrine at a concentration that produces close to 50% of the contraction induced by 75 mM KCl. Afterward, concentration-response curves to acetylcholine or phenylephrine were performed. The effect of the selective COX-2 inhibitor NS-398 at 1 µM (Calbiochem-Novabiochem GmbH, Bad Soden, Germany) in phenylephrine responses was investigated by its addition 30 min before phenylephrine. In some experiments, segments from WKY rats were incubated with 0.1 µM Ang II (Sigma-Aldrich), which was added from the time the aorta was removed from the animal and maintained through the experiment (6 h). In these vessels, the effect of NS-398 in phenylephrine responses was also evaluated.
Vasoconstrictor responses were expressed as a percentage of the tone generated by 75 mM KCl, and vasodilator responses were expressed as a percentage of the previous tone generated by phenylephrine. To compare the effect of NS-398 on the response to phenylephrine in segments from both strains and from all treatment groups, some results were expressed as a percentage of inhibition of the maximum response to phenylephrine induced by NS-398 in each artery.
Cell Cultures. VSMC were isolated from aortas of 5-month-old SHR. Arteries were treated during 20 min with 2 mg/ml collagenase (Worthington Biochemicals, Freehold, NJ), and adventitia was carefully removed. Then, VSMC were obtained from segments by the explant method. Cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. For experiments, cells from passages 3 to 8 were made quiescent by incubation in serum-free Dulbecco's modified Eagle's medium supplemented with 0.1% bovine serum albumin for 24 h. Cells were stimulated with Ang II for 2 h with or without pretreatment for 1 h with the specific inhibitors for xanthine oxidase (100 µM allopurinol) (Sigma/RBI, Natick, MA) and NADP(H) oxidase (30 µM apocynin) (Fluka-Sigma Chemical, Seelze, Germany).
Western Blot Analysis. COX-2 protein expression was determined in cellular lysates (20 µg of protein) and in homogenates from the aortic segments (30 µg of protein) used for reactivity experiments (approximately 6 h after extraction from the animal) and from segments obtained immediately upon removal from the animal (basal conditions), as described previously (Álvarez et al., 2005). In brief, proteins were separated by 7.5% SDS-polyacrylamide gel electrophoresis and then transferred to polyvinyl difluoride membranes overnight. Membranes were incubated with rabbit polyclonal antibody for COX-2 (1:1,000; Cayman Chemical, Ann Arbor, MI). After washing, membranes were incubated with anti-rabbit IgG antibody conjugated to horseradish peroxidase (1:2000; Bio-Rad, Hercules, CA). The immunocomplexes were detected using an enhanced horseradish peroxidase-luminol chemiluminescence system (ECL Plus; GE Healthcare, Little Chalfont, Buckinghamshire, UK) and subjected to autoradiography (Hyperfilm ECL; GE Healthcare). Signals on the immunoblot were quantified using the NIH Image computer program, version 1.56 (http://rsb.info.nih.gov/nih.image/). To compare the results for protein expression within the same experiment and with other experiments, we assigned a value of 1 in each gel to the expression of VSMC stimulated with Ang II or of arteries from losartan-untreated WKY rats in basal conditions.
Reverse Transcription-Polymerase Chain Reaction Assay. COX-2 mRNA was determined in aortic segments in similar conditions to those used for protein expression determination. Total RNA was obtained by using TRIzol (Invitrogen, Carlsbad, CA). In total, 1 µg of DNase I-treated RNA was reverse-transcribed into cDNA using the High-Capacity cDNA Archive kit (Applied Biosystems, Foster City, CA) in a 50-µl reaction. Polymerase chain reaction was performed in duplicate for each sample using 0.5 µl of cDNA as template for COX-2, 1x TaqMan Universal PCR Master Mix (Applied Biosystems), and 10x TaqMan Gene Expression Assays (Rn00568225_m1; Applied Biosystems) in a 20-µl reaction. For quantification, quantitative RT-PCR was carried out in an ABI Prism 7000 sequence detection system (Applied Biosystems) using the following conditions: 2 min 50°C and 10 min 95°C followed by 40 cycles of 15 s 95°C and 1 min 60°C. As a normalizing internal control, we amplified 
2 microglobulin (Rn00560865_m1). To calculate the relative index of gene expression, we used the 2–
Ct method (Livak and Schmittgen, 2001) with the untreated samples as calibrator.
Measurement of Prostaglandin F2, Prostacyclin, and 8-Isoprostane Production. The measurements of the metabolite of PGF2, 13,14-dihydro-15-keto-PGF2; the metabolite of prostacyclin (PGI2), 6-keto-PGF1; and 8-isoprostane were determined in the incubation medium after completion of the phenylephrine concentration-response curves, using enzyme immunoassay commercial kits (Cayman Chemical for PGF2 and 8-isoprostane and R&D Systems Europe for PGI2). The medium was frozen in liquid nitrogen, kept at –70°C until analysis, and processed following the manufacturers' instructions.
Measurement of MDA Production. Plasmatic MDA levels were measured by a modified thiobarbituric acid assay (Rodríguez-Martínez and Ruiz-Torres, 1992). Plasma was mixed with 20% trichloroacetic acid in 0.6 M HCl (1:1, v/v), and the tubes were kept in ice for 20 min to precipitate plasma components to avoid possible interference. Samples were centrifuged at 1500g for 15 min before adding 120 mM thiobarbituric acid in 260 mM Tris, pH 7, to the supernatant in a proportion of 1:5 (v/v). Then, the mixture was boiled at 97°C for 30 min. Spectrophotometric measurements at 535 nm were made at 20°C.
Plasmatic TAS Measurement. TAS was measured using the Calbiochem total antioxidant status assay kit (Calbiochem-Novabiochem), according to the manufacturer's instructions.
Detection of Superoxide Anion. Superoxide anion production in aortic segments was determined with lucigenin chemiluminescence. In brief, thoracic aortic rings (10–15 mm) were cleaned and placed in modified Krebs-HEPES (119 mM NaCl, 20 mM HEPES, 1 mM MgSO4, 4.6 mM KCl, 0.4 mM KH2PO4, 5 mM NaHCO3, 1.2 mM CaCl2, and 11.1 mM glucose, pH 7.4) for 30 min at 37°C. Aortic rings were placed in Krebs-HEPES buffer containing 20 µM lucigenin. The chemiluminescence was then recorded every 30 s during 10 min with a luminometer (Optocom I BG-1; MGM Instruments Inc., Hamden, CT). The differences between the values obtained before and after adding the rings to the buffer medium were calculated.
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The contractile response to phenylephrine (1 nM–30 µM) was slightly greater in segments from SHR than WKY rats, as reported previously (Álvarez et al., 2005). Losartan treatment did not affect contractile responses to phenylephrine both in WKY rats and SHR (Fig. 1). Thus, maximal response (WKY control: 110.9 ± 3.8, n = 32; WKY losartan: 102.8 ± 6.6, n = 16; SHR control: 122.2 ± 4.1, n = 36; and SHR losartan: 135.3 ± 6.5, n = 21) and pD2 values (WKY control: 6.84 ± 0.06; WKY losartan: 6.84 ± 0.08; SHR control: 7.04 ± 0.07; and SHR losartan: 7.21 ± 0.11) were similar in control and losartan-treated rats.
The selective COX-2 inhibitor NS-398 at 1 µM inhibited the phenylephrine response to a greater extent in segments from SHR than from WKY rats (Table 1), as described previously (Álvarez et al., 2005). Losartan treatment decreased, but it did not abolish, the inhibitory effect of this drug only in segments from SHR (Fig. 2; Table 1). To evaluate the effect of lowering blood pressure versus AT1 blockade, SHR were treated with a combination of hydralazine plus hydrochlorothiazide. This treatment did not modify the inhibitory effect of NS-398 in segments from SHR (Fig. 2; Table 1).
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To further analyze the modulatory effect of Ang II on COX-2-derived prostanoids, vessels from WKY rats were preincubated with 0.1 µM Ang II from the dissection of the artery. As shown in Table 1, NS-398 induced an inhibitory effect of phenylephrine response that was greater in Ang II-incubated segments than in the untreated vessels.
COX-2 Expression. In freshly isolated segments (basal) COX-2 protein expression was greater in SHR than WKY rats (Fig. 3); this higher expression was accompanied by increased mRNA expression in SHR (Fig. 4A). At the end of the reactivity experiments (6 h), COX-2 protein (Fig. 3) and mRNA expressions showed increased levels in both strains (relative mRNA expression 6 h versus basal: 14.4 ± 4.4, n = 8 for WKY rats and 24.1 ± 6.3, n = 9 for SHR; p < 0.05). In these conditions, the expression of COX-2 protein (Fig. 3) and mRNA (Fig. 4A) was also greater in SHR than WKY rats.
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Losartan treatment did not affect the expression of either COX-2 protein (Fig. 3) or mRNA (data not shown) in WKY rats, and it reduced the increase in COX-2 protein and mRNA expression observed in SHR both in basal conditions and after 6-h incubation (Figs. 3 and 4B). However, HH treatment did not affect COX-2 mRNA expression in aortic segments from SHR either in basal conditions or after 6-h incubation (Fig. 4B). These results suggest that the effect of losartan in COX-2 expression is independent of its effect on blood pressure.
Prostaglandin F2, PGI2, and 8-Isoprostanes Production. At the end of the reactivity experiments, aortic segments from both strains released detectable amounts of the metabolite of PGF2, 13,14-dihydro-15-keto-PGF2; of the metabolite of PGI2, 6-keto-PGF1; and 8-isoprostanes to the incubation medium (Fig. 5); the levels of the three compounds were higher in samples from SHR (Fig. 5). NS-398 at 1 µM reduced 13,14-dihydro-15-keto-PGF2, 6-keto-PGF1, and 8-isoprostanes levels only in the medium with SHR segments. Losartan treatment decreased the release of these compounds into the incubation medium from SHR, but it did not modify these levels in WKY rats (Fig. 5). After losartan treatment, 1 µM NS-398 did not further inhibit the release of 13,14-dihydro-15-keto-PGF2, 6-keto-PGF1, and 8-isoprostanes by SHR segments (Fig. 5). These results suggest that AT1 receptors activation participates in the increased prostanoids release from COX-2 in SHR.
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Vascular Superoxide Anion Production and Plasmatic MDA Levels and TAS. Basal superoxide anion production was higher in segments from SHR than from WKY rats. Losartan treatment prevented the rise in superoxide production in aorta from SHR (Fig. 6). Plasmatic MDA levels were also higher in SHR than in WKY rats, whereas no differences in TAS were observed in plasma samples from WKY rats and SHR. Losartan treatment reduced MDA levels in both strains, abolishing the differences between strains, and increased TAS only in SHR (Fig. 6). These results suggest that AT1 receptors activation participates in the increased oxidative stress observed in SHR.
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Discussion |
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The renin angiotensin system plays an important role in the pathogenesis of several cardiovascular diseases, including hypertension. In the present study, in keeping with findings obtained elsewhere, the AT1 receptor antagonist losartan significantly lowered blood pressure in hypertensive rats. Several mechanisms have been proposed as responsible for the hypertensinogenic effect of Ang II. Thus, Ang II has been shown to stimulate both hyperplasia and hypertrophy in VSMC (Touyz et al., 1999), and it plays a role in the profibrotic processes (Laviades et al., 1998) as well as in the altered vasoconstrictor or vasodilator responses described in hypertension (Schiffrin and Touyz, 2004). In addition, it is now accepted that an inflammatory reaction may be recorded in association with the hypertensive process, and the role of endogenous Ang II in vascular inflammation has also been suggested (Schiffrin and Touyz, 2004).
The present and previous results (Álvarez et al., 2005) demonstrate that the participation of contractile prostanoids from COX-2 in vasoactive responses is increased with hypertension. This is supported by the fact that phenylephrine-induced responses were inhibited by the selective COX-2 inhibitor NS-398 as well as by the protein synthesis inhibitor dexamethasone and the thromboxane A2/PGH2 receptor antagonist SQ 29,548 to a greater extent in aorta from SHR (Álvarez et al., 2005). In different hypertension models, other investigators have also described increased vascular contractility associated with increased production of contractile prostanoids from COX-2 (Zerrouk et al., 1998; Adeagbo et al., 2005). We have found that basal COX-2 mRNA and protein expressions were greater in aortic segments from hypertensive rats; such expressions were increased during the course of the experiment, being also greater in SHR than WKY rats. In addition, the NS-398-sensitive release of PGF2, and 8-isoprostane was greater in the hypertensive animals. In different models of hypertension, increased vascular basal COX-2 protein expression (Henrion et al., 1997; García-Cohen et al., 2000; Adeagbo et al., 2005; Álvarez et al., 2005) and prostanoids production from COX-2 (Zerrouk et al., 1998; Adeagbo et al., 2005; Álvarez et al., 2005) as well as serum and urinary 8-isoprostane levels have been described previously (Schnackenberg and Wilcox, 1999; Adeagbo et al., 2005).
It has been suggested that although COX-1 primarily contributes to basal prostanoids production in the kidney and aorta, Ang II increases prostanoids via COX-2 activity (Qi et al., 2006) and/or expression, but different results have been reported depending on the cell type or stimulus. Thus, in VSMC (Ohnaka et al., 2000; Hu et al., 2002), ventricular cardiomyocytes (Rebsamen et al., 2003), coronary vascular wall (Rocha et al., 2002), and mesangial cells and glomerulus (Jaimes et al., 2005) from normotensive rats, Ang II up-regulates COX-2 mRNA expression through the activation of AT1 receptors (Ohnaka et al., 2000; Hu et al., 2002). However, there is also evidence showing that Ang II attenuates COX-2 expression in kidney (Harris et al., 2004) and VSMC stimulated with interleukin-1 (Jiang et al., 2004). To our knowledge, there are no reports analyzing the influence of hypertension in Ang II-induced prostaglandin production from COX-2. Our results suggest that AT1 receptors activation by Ang II plays a role in the increased participation of contractile prostanoids in phenylephrine responses in aorta from hypertensive rats through the up-regulation of COX-2 expression and the subsequent increase of contractile prostanoids production. This is based on several observations. 1) In VSMC from SHR, Ang II increased COX-2 expression. 2) Losartan treatment reduced the increased COX-2 protein and mRNA expressions and the increased release of PGF2 and 8-isoprostane found in aortic segments from SHR, without further effects by the COX-2 inhibitor NS-398 in this release. In this line of evidence, losartan treatment is reported to decrease the increased inducible nitric-oxide synthase and COX-2 expression observed after experimental renal interstitial fibrosis (Manucha et al., 2004). However, the AT1 receptor blocker candesartan has also been reported to increase the renocortical COX-2 mRNA expression in 5-week-old SHR, but it has no effect in older rats (Hocherl et al., 2001). 3) Losartan treatment reduced the inhibitory effects of NS-398 in phenylephrine responses in aortic segments from SHR but not in WKY rats. 4) The incubation of aortic segments from WKY rats with Ang II increased the inhibitory effect of NS-398 in phenylephrine responses. 5) The treatment with hydralazine plus hydrochlorothiazide diminished blood pressure, but it did not modify the increase in both COX-2 mRNA expression and the inhibitory effect of NS-398 observed in segments from hypertensive rats without treatment, suggesting that the observed effects of losartan treatment were probably independent of its blood pressure-lowering properties.
After losartan treatment, no change in phenylephrine response in SHR was observed, despite down-regulation of the COX-2 pathway. As mentioned, losartan treatment decreased the production of the contractile prostanoid PGF2 and 8-isoprostane as well as the vasodilator PGI2. Therefore, it is possible that the final effect of these mediators in phenylephrine contraction could be counterbalanced, although other contractile mediators or signaling pathways might be up-regulated after AT1 receptors blockade to maintain contractile responses. Furthermore, Cediel et al. (2003) demonstrated improvement of smooth muscle cell function by AT1 receptor antagonists associated to the reversion of medial hypertrophy that could improve contractile machinery of smooth muscle cells.
Ang II mediates many of its cellular actions by stimulating formation of intracellular ROS, thereby participating in the increased oxidative stress observed in hypertension (Schiffrin and Touyz, 2004; Cheng et al., 2005). Ang II stimulates superoxide anion generation in vascular rings through AT1 receptor activation (Rajagopalan et al., 1996). Likewise, in cultured rat VSMC, Ang II stimulates superoxide anion production by increasing the activity of NAD(P)H oxidase (Griendling et al., 1994) greatly in SHR (Cruzado et al., 2005). Losartan treatment abolished the increased production observed in aorta from SHR. Moreover, losartan normalized the increased plasmatic MDA levels and increased TAS, suggesting that losartan may prevent oxidative stress in hypertension by inhibiting lipid peroxidation. Accordingly, losartan improved the impaired endothelium-dependent relaxation to acetylcholine observed in aorta from SHR, as reported previously (Schiffrin and Touyz, 2004). These results confirm the participation of the renin-angiotensin system in the endothelial dysfunction associated to hypertension, probably through its ability to generate ROS. Other investigators have also found reduction of malondialdehyde levels and plasmatic and urinary 8-isoprostrane levels as well as increase of antioxidant systems, after inhibition of the renin-angiotensin system (Mervaala et al., 2001; Donmez et al., 2002; Chamorro et al., 2004).
Induction of COX-2 expression after oxidative stress stimulus in mesangial cells (Feng et al., 1995; Kiritoshi et al., 2003) and reduction of COX-2 expression in renal cortex after antioxidant treatments have been reported previously (Li et al., 2005). Our group has described that oxidative stress induces higher COX-2 expression in aorta from hypertensive than from normotensive rats (García-Cohen et al., 2000). In agreement, we observed here that the increased COX-2 expression found in VSMC from SHR after Ang II stimulation was decreased by apocynin and allopurinol, inhibitors of superoxide anion production by NADPH oxidase and xanthine oxidase, respectively. As discussed above, since losartan is diminishing the oxidative stress both at vascular and systemic levels, together these findings point to the excess of ROS as one possible mechanism for the increased COX-2 expression and activity observed in SHR. In agreement, in deoxycorticosterone acetate-salt hypertension (Adeagbo et al., 2005) and in diabetic rats (Li et al., 2005), it has also been suggested that oxidative stress is a component of the mechanism involved in the production of contractile prostanoids from COX-2 that are responsible for the increased vascular reactivity to vasoconstrictors in aorta and kidney. In addition, ROS have been implicated in the up-regulation of COX-2 expression and prostanoids release induced by Ang II in mesangial cells (Jaimes et al., 2005).
In conclusion, endogenous Ang II activation of AT1 receptors seems to participate in the increased COX-2 expression and contractile prostanoids production from this isoform that are involved in phenylephrine responses with hypertension. Our findings point to oxidative stress as one of the mechanisms involved in these effects.
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Acknowledgements |
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: COX, cyclooxygenase; Ang II, angiotensin II; ROS, reactive oxygen species; VSMC, vascular smooth muscle cell(s); SHR, spontaneously hypertensive rat(s); WKY, Wistar Kyoto; HH, hydralazine-hydrochlorothiazide; MDA, malondialdehyde; TAS, total antioxidant status; NS-398, N-[2-(cyclohexyloxyl)-4-nitrophenyl]-methane sulfonamide; RT-PCR, reverse transcription-polymerase chain reaction; PG, prostaglandin; ANOVA, analysis of variance; SQ 29,548, [1S-[1,2(5Z)3,4]-7-[3-[[2-[(phenylamino)carbonyl]hydrazino]methyl]-7-oxobicyclo-[2.2.1]hept-2-yl]-5-heptenoic acid.
Address correspondence to: Dr. Mercedes Salaices, Departamento de Farmacología y Terapéutica, Facultad de Medicina, Universidad Autónoma de Madrid; 28029 Madrid, Spain. E-mail: mercedes.salaices{at}uam.es
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