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CARDIOVASCULAR

Cyclooxygenase, p38 Mitogen-Activated Protein Kinase (MAPK), Extracellular Signal-Regulated Kinase MAPK, Rho Kinase, and Src Mediate Hydrogen Peroxide-Induced Contraction of Rat Thoracic Aorta and Vena Cava

Department of Pharmacology and Toxicology, Michigan State University, East Lansing, Michigan

Received for publication July 12, 2006
Accepted September 25, 2006.

	Abstract

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In hypertension, blood vessels exhibit increased reactive oxygen species production that may alter vascular tone. We previously observed that H₂O₂ contracted rat thoracic vena cava under resting tone and aorta contracted with KCl. In arteries but not veins, H₂O₂-induced contraction required extracellular Ca²⁺ influx. Because of this difference in Ca²⁺ utilization, we hypothesized that signaling pathways mediating H₂O₂-induced contraction in vena cava under resting tone differed from those mediating H₂O₂-induced contraction in aorta contracted with KCl. Inhibitors of cyclooxygenase (COX) 1 and 2 (indomethacin, 10 µM), thromboxane A₂ (TXA₂) receptors [ICI185282 (2RS,4RS,5SR-4-o-hydroxyphenyl-2-trifluoromethyl-1,3-dioxan-5-yl heptenoic acid), 10 µM], p38 mitogen-activated protein kinase (MAPK) [SB203580 (4-[5-(4-fluorophenyl)-2-[4-(methylsulfonyl)phenyl]-1H-imidazol-4-yl]pyridine), 10 µM], extracellular signal-regulated kinase (Erk) [PD98059 (2'-amino-3'-methoxyflavone), 10 µM], src [PP1 (4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine, 10 µM], and rho kinase [Y27632 (trans-4-[(1R)-1-aminoethyl]-N-4-pyridinylcyclohexanecarboxamide dihydrochloride), 10 µM], significantly reduced H₂O₂-induced contraction in vena cava under resting tone and aorta after KCl (30 mM) contraction. In contrast, the phosphatidylinositol 3-kinase (PI3-K) inhibitor LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one, 20 µM] did not reduce aortic or venous H₂O₂-induced contraction. p38 MAPK, Erk MAPK, and src inhibition did not reduce aortic or venous contraction to the TXA₂ receptor agonist U46619 [GenBank] (9,11-dideoxy-9,11-methanoepoxy PGF₂, 1 µM), whereas rho kinase inhibition significantly reduced aortic and venous contraction to U46619 [GenBank] , and PI3-K inhibition reduced venous contraction to U46619. [GenBank] Our data suggest that, in rat thoracic aorta and vena cava, a COX-derived metabolite is one important mediator of H₂O₂ contraction, possibly via rho kinase activation, and that H₂O₂-induced contraction via p38 and Erk MAPK probably occurs independently of TXA₂ receptor activation.

H₂O₂ has been proposed to be the most likely reactive oxygen species (ROS) involved in signal transduction because it is not a free radical and is inherently more stable than other ROS (Wolin et al., 2002; Ardanaz and Pagano, 2006). H₂O₂ can also freely pass through membranes and has a longer diffusion distance than superoxide (Wolin et al., 2002; Ardanaz and Pagano, 2006). H₂O₂ modifies vascular smooth muscle tone, causing contraction and relaxation, depending on experimental conditions, vascular bed, and species (Lucchesi et al., 2005; Thakali et al., 2006). Multiple signal transduction pathways have been reported to mediate H₂O₂-induced contraction, including (but not limited to) COX-stimulated thromboxane A₂ (TXA₂) production (Rodriguez-Martinez et al., 1998; Gao and Lee, 2001), tyrosine kinases (Jin and Rhoades, 1997), mitogen-activated protein kinases (MAPKs), phospholipase C (Sheehan et al., 1993), and rho kinase (Thakali et al., 2005), although the progression of signaling has not been established. TXA₂ receptors signaling also occurs via the activation of many of the same pathways including Erk MAPK, p38 MAPK, PKC (Bos et al., 2004), tyrosine kinases, and rho kinase (Tazzeo et al., 2003). Thus, one can envision H₂O₂ stimulating COX to produce vasoactive eicosanoids, specifically the vasoconstrictor TXA₂. However, many of these signaling elements, including Erk MAPK and p38 MAPK, are also redox-sensitive such that oxidation of active site thiol residues can activate kinases and inactivate phosphatases (Rhee et al., 2000, 2005; Oeckler et al., 2005).

Presently, we are comparing H₂O₂-induced contraction in a model artery and vein, and the rat thoracic aorta and the thoracic vena cava, respectively. We previously reported that, under basal conditions, veins but not arteries robustly contract to exogenous H₂O₂. However, if arteries were either depolarized with KCl (30 mM) or K⁺ channels were inactivated (tetraethylammonium, 10 mM), H₂O₂-induced contraction was significantly potentiated (Thakali et al., 2006). We observed that, although extracellular Ca²⁺ influx was necessary for aortic (KCl-potentiated) H₂O₂-induced contraction, venous H₂O₂-induced contraction (under basal conditions) occurred in the absence of extracellular Ca²⁺ influx (Thakali et al., 2006), suggesting mechanistic differences in arterial and venous H₂O₂-induced contraction. The goal of this work was to determine whether the signal transduction pathways mediating venous H₂O₂-induced contraction under basal conditions were the same pathways mediating aortic H₂O₂-induced contraction after KCl (30 mM) contraction.

	Materials and Methods

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Isolated Tissue Bath Protocol. All animal studies were performed in accordance with the Guide for the Care and Use of Laboratory Animals at Michigan State University (East Lansing, MI). Thoracic aorta and vena cava were removed from anesthetized male Sprague-Dawley rats (200–250 g) (50 mg/kg i.p. pentobarbital) and placed in physiological salt solution containing 130 mM NaCl, 4.7 mM KCl, 1.18 mM KH₂PO₄, 1.17 mM MgSO₄ 7H₂O, 1.6 mM CaCl₂ 2H₂O, 14.9 mM NaHCO₃, 5.5 mM dextrose, and 0.03 mM CaNa₂EDTA, pH 7.2. Vessels were cleaned of fat, and rings (3–4 mm) of aorta and vena cava were prepared for measurement of isometric tension as described previously (Thakali et al., 2006). In brief, rings were placed between two wire hooks, one that was attached to a stationary glass rod and the other that was connected to a force transducer to measure isometric contraction. Passive tension was pulled (aorta, 4 g; vena cava, 1 g), and vessels were equilibrated for 1 h in warmed (37°C), aerated (95% O₂,5%CO₂) physiological salt solution, with frequent buffer changes. Tissue viability was assessed by contraction to an adrenergic agonist (aorta, phenylephrine, 10 µM; vena cava, norepinephrine, 10 µM). Norepinephrine was used to contract vena cava because phenylephrine did not reproducibly contract vena cava, and phenylephrine was used to contract aorta to compare results with past experiments.

Cumulative concentration response curves to H₂O₂ (1 µM–1 mM) were performed in aorta after KCl (30 mM) contraction [69 ± 8% phenylephrine (10 µM) contraction] and vena cava under quiescent conditions (under passive tension). Cumulative concentration response curves to U46619 [GenBank] (1 nM–1 µM) were performed in aorta and vena cava under passive tension. When inhibitors were used (10 µM indomethacin, 10 µM ICI185282, 10 µM PD98059, 10 µM SB203580, 10 µM PP1, 10 µM Y27632, and 20 µM LY294002), an inhibitor or vehicle (0.1% ethanol for indomethacin, 0.1% DMSO for ICI184282, PD98059, SB203580, and PP1; 0.2% DMSO for LY294002; and deionized water for Y27632) was added for 1 h before performing cumulative concentration response curves to agonists. Cumulative concentration response curves to KCl (6–100 mM) were also performed in aorta in the presence of the signaling inhibitors. We found that Y27632 (10 µM) significantly reduced aortic KCl-induced contraction; thus, in the presence of Y27632 (10 µM), aorta were contracted with 100 mM KCl to more closely match the contraction induced by 30 mM KCl in vehicle-incubated aorta. The signaling inhibitors and concentrations of inhibitors were chosen, because we have demonstrated previously specificity and effectiveness of these inhibitors in arterial smooth muscle cell cultures (Watts, 1996; Banes et al., 1999; Florian and Watts, 1999; Gao and Lee, 2001; Northcott et al., 2002).

Tissue Thromboxane B₂ Measurement. Thromboxane B₂ (TXB₂) levels were measured in rat thoracic aorta and vena cava using an enzyme immunoassay (EIA) kit purchased from Cayman Chemicals (Ann Arbor, MI). In brief, cleaned rat thoracic vena cava were cut in half and incubated in 150 µl of EIA buffer, with one-half incubated in H₂O₂ (1 mM) and the other with vehicle (water) for 10 min at 37°C. Cleaned rings (3–4 mm) of rat thoracic aorta were incubated in 150 µl of EIA buffer plus KCl (30 mM), and one ring was incubated with H₂O₂ (1 mM) and the other with vehicle (water) for 10 min at 37°C. Microcentrifuge tubes containing vessels and EIA buffer/H₂O₂ or vehicle were frozen at –80°C for 4 h. The tubes were thawed, sonicated for 3 s, and centrifuged (1 min, 14,000 rpm). Fifty microliters of the supernatant was added to a secondary antibody-coated 96-well plate to determine the amount of TXB₂ present in each sample. A standard TXB₂ curve was also performed and is described below in the data analysis section. Total protein was determined after dissolving vessel pellets in NaOH (1 N) and performing a Lowry's protein assay.

Data Analysis. Data are presented as mean ± S.E. of the percentage of the initial contraction to phenylephrine (aorta, 10 µM) or norepinephrine (vena cava, 10 µM) for n experiments, where n indicates the number of rats used. In aorta contracted with KCl (30 mM), contraction to H₂O₂ was calculated as the contraction above the maximal KCl response. When comparing multiple concentration response curves with H₂O₂ or U46619 [GenBank] , two-way analysis of variance with Bonferroni's post hoc test was performed. For quantification of TXB₂ EIA, a standard curve using TXB₂ was run along with aortic and venous samples. For the standard curve, the ratio of absorbance of bound ligand (TXB₂) to the absorbance of total binding was plotted against the log of the concentration of TXB₂, and linear regression was performed to determine TXB₂ levels in aortic and venous samples. TXB₂ levels were normalized to total protein in each sample and are reported as picograms of TXB₂ per milligram of total protein. In all cases, p 0.05 was considered statistically significant.

Chemicals. Acetylcholine, H₂O₂ (30%), norepinephrine, and phenylephrine were solubilized in water, and indomethacin was solubilized in ethanol and were purchased from Sigma Chemical Co. (St. Louis, MO). PD98059, SB203580, LY294002, PP1, ICI185282, and U46619 [GenBank] were solubilized in DMSO, and Y27632 was solubilized in water and purchased from Biomol (Plymouth Meeting, PA).

	Results

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COX-Derived TXA₂ Mediates Aortic H₂O₂-Induced Contraction after KCl (30 mM) and Basal Venous H₂O₂-Induced Contraction. The nonspecific COX inhibitor, indomethacin (10 µM; Kalgutkar et al., 2000), significantly reduced aortic H₂O₂-induced contraction after KCl (30 mM) and basal venous H₂O₂-induced contraction (Fig. 1A). TXA₂ has been reported to mediate H₂O₂-induced contraction in rat aorta (Rodriguez-Martinez et al., 1998; Gao and Lee, 2001); thus, we investigated the role of the TXA₂ receptor in aortic and venous H₂O₂-induced contraction using the TXA₂ receptor antagonist ICI185282 (Fig. 1B). ICI185282 (10 µM) significantly reduced aortic H₂O₂-induced contraction after KCl (30 mM) and basal venous H₂O₂-induced contraction (Fig. 1B). As expected, the same concentration of ICI185282 (10 µM) significantly inhibited U46619 [GenBank] -induced contraction (1 nM–1 µM) in rat thoracic aorta (not KCl-contracted) and vena cava (Fig. 1C).

View larger version (31K): [in this window] [in a new window]   Fig. 1. A, nonselective COX inhibitor indomethacin (10 µM) significantly reduced KCl-potentiated aortic and basal venous H2O2-induced contraction. B, TXA2 receptor antagonist ICI185282 (10 µM) significantly reduced KCl-potentiated aortic and basal venous H2O2-induced contraction. C, TXA2 receptor antagonist ICI185282 (10 µM) significantly reduced aortic and venous contraction to the TXA2 receptor agonist U46619. Data are represented as means ± S.E.M. for the number (N) of animals in parentheses. EtOH, ethanol, NE, norepinephrine, PE, phenylephrine. *, statistically significant difference from vehicle (p < 0.05).

MAPKs (p38 and Erk), Src, and Rho Kinase but Not PI3-K Mediate Aortic and Venous H₂O₂-Induced Contraction. The p38 MAPK inhibitor SB203580 (10 µM), Erk MAPK inhibitor PD98059 (10 µM), and src inhibitor PP1 (10 µM) significantly reduced KCl (30 mM)-potentiated aortic H₂O₂-induced contraction and basal venous H₂O₂-induced contraction (Fig. 2A). KCl (6–100 mM)-induced contraction of rat thoracic aorta was significantly reduced by Y27632 (10 µM) (Fig. 3C). Thus, Y27632 (10 µM)-incubated aorta were contracted with a higher concentration of KCl (100 mM) before performing H₂O₂ concentration response curves to match the contraction to KCl (30 mM) in vehicle-incubated aorta. Y27632 (10 µM), a rho kinase inhibitor, significantly reduced KCl (100 mM)-potentiated aortic H₂O₂-induced contraction (Fig. 3A), and we previously observed a similar inhibition in vena cava (Fig. 3A; reproduced with permission from Thakali et al., 2005). The PI3-K inhibitor LY294002 (20 µM) did not significantly alter aortic H₂O₂-induced contraction after KCl (30 mM) contraction or basal venous H₂O₂-induced contraction (Fig. 4A). Inhibition of p38 MAPK, Erk MAPK, and src had no effect on maximal U46619 [GenBank] (1 µM) contraction in aorta and vena cava (Fig. 2B). Similarly to H₂O₂-induced contraction, rho kinase inhibition reduced maximal aortic and venous U46619 [GenBank] -induced contraction (Fig. 3B). Unlike H₂O₂-induced contraction, PI3-K inhibition modestly reduced maximal venous but not aortic U46619 [GenBank] -induced contraction (Fig. 4B).

View larger version (40K): [in this window] [in a new window]   Fig. 2. A. Erk MAPK inhibition (PD98059, 10 µM), src inhibition (PP1, 10 µM), and p38 MAPK inhibition (SB203580, 10 µM) significantly reduced KCl-potentiated aortic and basal venous H2O2-induced contraction. B, Erk MAPK inhibition (PD98059, 10 µM), src inhibition (PP1, 10 µM), or p38 MAPK inhibition (SB203580, 10 µM) did not significantly reduce maximal aortic or venous U46619-induced contraction. Data are represented as means ± S.E.M. for the number (N) of animals in parentheses. EtOH, ethanol, NE, norepinephrine, PE, phenylephrine. *, statistically significant difference from vehicle (p < 0.05).

View larger version (28K): [in this window] [in a new window]   Fig. 3. A, rho kinase inhibition (Y27632, 10 µM) significantly reduced KCl (100 mM)-potentiated aortic H2O2-induced contraction and venous H2O2-induced contraction (used with permission from Thakali et al., 2005). B, rho kinase inhibition (Y27632, 10 µM) significantly reduced aortic and venous U46619-induced contraction. C, rho kinase inhibition (Y27632, 10 µM) significantly reduced maximal KCl-induced contraction. Data are represented as means ± S.E.M. for the number (N) of animals in parentheses. dH2O, deionized water; NE, norepinephrine; PE, phenylephrine. *, statistically significant difference from vehicle (p < 0.05).

View larger version (33K): [in this window] [in a new window]   Fig. 4. A, PI3-K inhibitor LY294002 (20 µM) did not alter KCl-potentiated aortic or basal venous H2O2-induced contraction. B, PI3-K inhibitor LY294002 (20 µM) did not reduce aortic contraction to U46619 but significantly reduced venous contraction to U46619. Data are represented as means ± S.E.M. for the number (N) of animals in parentheses. NE, norepinephrine; PE, phenylephrine. *, statistically significant difference from vehicle (p < 0.05).

View larger version (17K): [in this window] [in a new window]   Fig. 5. H2O2 (1 mM) significantly decreased venous TXB2 levels and did not alter arterial (+KCl, 30 mM) TXB2 levels. Data are represented as mean picograms of TXB2 per milligram of protein ± S.E.M. for the number (N) of animals in parentheses. *, statistically significant difference from –H2O2 (p < 0.05).

	Discussion

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Common Signal Transduction Pathways Mediate H₂O₂-Induced Contraction in Arteries and Veins. Multiple signaling pathways have been reported to mediate H₂O₂-induced contraction, and this current study affirms the idea that H₂O₂ is a physiologically relevant signaling molecule capable of activating multiple signal transduction pathways. We observed that Erk MAPK, p38 MAPK, src, rho kinase, and a COX-derived metabolite mediate H₂O₂-induced contraction in rat thoracic vena cava under basal conditions and in rat thoracic aorta after KCl contraction. The absolute magnitude of inhibition of H₂O₂-induced contraction seems to be different between arteries and veins. It possible that better penetration or a higher concentration of indomethacin, PD98059, PP1, or SB203580 is achieved in the smooth muscle of rat thoracic vena cava compared with aorta because of differences in wall structure between the two vessel types, although we have not tested this possibility experimentally. Nonetheless, the same concentrations of indomethacin, ICI185282, PD98059, PP1, SB203580, and Y27632 inhibited H₂O₂-induced contraction in both arteries and veins, suggesting that within the context of these signaling pathways, H₂O₂-induced contraction in arteries and veins occurs via similar signal transduction pathways.

Interestingly, the PI3-K pathway was not involved in mediating H₂O₂-induced contraction in vena cava under basal conditions or in KCl-contracted aorta. We have previously verified inhibition of PI3-K activity with LY294002 (20 µM) in cultured rat aortic smooth muscle cells (Northcott et al., 2002). H₂O₂ stimulation reportedly increases phosphorylation of Akt, a commonly evaluated downstream target of PI3-K in cultured rat aortic smooth muscle cells and perfused mouse mesenteric resistance arteries (Griendling et al., 2000; Lucchesi et al., 2005). From our experiments, we concluded that PI3-K is not involved in H₂O₂-induced contraction, but we cannot discount the possibility that acute H₂O₂ exposure in aorta and vena cava may activate Akt independently of PI3-K or may activate PI3-K to induce changes in other endpoints, such as cell proliferation, growth and apoptosis, or vascular remodeling.

In the current study, we compared H₂O₂ signaling in vena cava under quiescent conditions to aorta contracted with KCl. We previously observed that aorta under quiescent conditions contracted minimally to exogenous H₂O₂ and that depolarization of aorta (by increasing the concentration of extracellular K⁺ or nonspecific potassium channel blockade) potentiated the aortic contraction to H₂O₂ (Thakali et al., 2006). The magnitude of H₂O₂-induced contraction in vena cava under quiescent conditions (when normalized to contraction to an adrenergic agonist) is much larger than H₂O₂-induced contraction in aorta contracted with KCl. This could be due to differences in the efficacy of H₂O₂ to elicit contraction in aorta and vena cava or even differences in H₂O₂ penetration through the blood vessel wall. It is also possible that aortic KCl preconditioning may preferentially activate some signaling pathways or alter sensitivity to signaling inhibitors compared with U46619 [GenBank] -induced contraction in aorta under quiescent conditions. However, we previously observed that aortic KCl-induced contraction was not reduced by PD98059, SB203580, or PP1 (Watts, 1996; Banes et al., 1999; Florian and Watts, 1999; Northcott et al., 2002), suggesting that for p38 MAPK, Erk MAPK, and src activity, KCl preconditioning does not result in the activation of different contractile pathways compared with contraction induced by U46619 [GenBank] alone. We observed that maximal aortic contraction to KCl was significantly reduced by Y27632 (10 µM), a rho kinase inhibitor. Thus, Y27632 (10 µM)-incubated aorta were contracted with 100 mM KCl to reach a magnitude of contraction comparable with vehicle (30 mM KCl contracted)-incubated aorta.

Currently, there is no consensus on the signal transduction cascade mediating vascular H₂O₂-induced contraction. Our studies were performed with the intention of clarifying H₂O₂-mediated signaling but instead highlight the complexity of ROS signaling. Yang et al. (1998) reported that aortic H₂O₂-induced contraction was mediated by COX, PKC, and protein tyrosine kinases and was Ca²⁺-dependent. Wolin et al. (2002) observed that pulmonary arterial contraction to H₂O₂ was reduced by Erk MAPK inhibition, whereas Lucchesi et al. (2005) observed that mouse mesenteric artery contraction to H₂O₂ after KCl depolarization was mediated by p38 MAPK, but not Erk MAPK. In context of the above-mentioned signaling pathways, Pelaez et al. (2000a) reported that in the pulmonary vasculature of the rat, H₂O₂-induced contraction under basal conditions was independent of myosin light chain phosphorylation and extracellular Ca²⁺ influx, and in porcine pulmonary arteries, H₂O₂-induced contraction was independent of Erk MAPK and PKC activity (Pelaez et al., 2000b). We conclude that species, vascular bed, and experimental conditions probably dictate the contractile signaling pathways activated by H₂O₂.

Role of COX and TXA₂ in Mediating Aortic and Venous H₂O₂-Induced Contraction. One possible scheme of H₂O₂ signal transduction is that H₂O₂ activates COX to produce prostanoid metabolites including TXA₂. Cyclooxygenase, also known as prostaglandin endoperoxide H synthase, catalyzes two reactions, a peroxidase reaction and a cyclooxygenase reaction in the conversion of arachidonic acid into prostaglandin H₂ (PGH₂), the precursor to prostanoids. The cyclooxygenase reaction mediated by COX is peroxide-dependent; hydroperoxides such as t-butyl peroxide, peroxynitrite, and H₂O₂ increase cyclooxygenase activity (although not as efficiently as aliphatic hydroperoxides) (Smith et al., 2000; Kulmacz, 2005), whereas scavengers of peroxides like glutathione peroxidase but not scavengers of superoxide reduce cyclooxygenase activity (Kulmacz, 2005). Rodriguez-Martinez et al. (1998) observed that H₂O₂-induced contraction in aorta from normotensive Wistar Kyoto and spontaneously hypertensive rats was reduced by a PGH₂/TXA₂ receptor antagonist and by nonspecific COX inhibition. The hypothesis that H₂O₂ could stimulate TXA₂ synthesis and thus vascular contraction was confirmed by Gao and Lee (2001), where they demonstrated that nonspecific COX inhibitors, TXA₂ synthase inhibitors, and a phospholipase A₂ inhibitor reduced H₂O₂-stimulated TXB₂ production (a stable metabolite of TXA₂) and that these inhibitors and TXA₂ receptor antagonists reduced H₂O₂-induced contraction of rat mesenteric arteries. Through the use of a nonspecific COX inhibitor and a TXA₂ receptor antagonist, our contractility data suggest that in both rat thoracic aorta and vena cava, H₂O₂ stimulates the production of the vasoconstrictor TXA₂. However, when TXB₂ (a stable metabolite of TXA₂) levels were measured in aorta and vena cava, H₂O₂ did not increase TXB₂ levels and in fact decreased TXB₂ levels in vena cava, contrary to studies by Rodriguez-Martinez et al. (1998) and Gao and Lee (2001). One limitation of our TXB₂ EIA was that because the assay was incompatible with the physiological salt solution used in contractility experiments, H₂O₂ incubations were performed in EIA buffer from the kit, the composition of which is proprietary information. Thus, our experimental conditions may not have been optimal for detection of H₂O₂-stimulated TXB₂.

It is also possible that TXB₂ was the incorrect endpoint to measure because PGH₂, the actual product of COX activity, causes contraction via TXA₂ receptor binding (Davidge, 2001). We observed that U46619 [GenBank] , a TXA₂ receptor-specific agonist, induced concentration-dependent contraction of both rat thoracic aorta and vena cava, verifying that in rat thoracic aorta and vena cava, TXA₂ receptors couple to contraction. We observed that U46619 [GenBank] -induced contraction, although inhibited by a TXA₂ receptor antagonist, is not dependent on p38 MAPK, Erk MAPK, and src signaling pathways in a similar fashion to H₂O₂-induced contraction. U46619 [GenBank] -mediated contraction occurred via PI3-K and rho kinase activation in vena cava and rho kinase activation in aorta. It is possible that the dichotomy between the signaling pathways mediating H₂O₂-induced contraction and U46619 [GenBank] -induced contraction in aorta and vena cava occurs because although U46619 [GenBank] causes contraction via TXA₂ receptor activation, U46619 [GenBank] may induce TXA₂ receptor-dependent signaling in a manner different from TXA₂ or U46619 [GenBank] may have some unknown effects independent of TXA₂ receptor activation. It is also possible that the signaling inhibitors and concentrations used are nonspecific. Although we have previously demonstrated the specificity of the concentrations of inhibitors used in aortic smooth muscle cell cultures (Watts, 1996; Banes et al., 1999; Florian and Watts, 1999; Gao and Lee, 2001; Northcott et al., 2002), a study by Davies et al. (2000) suggests that many commonly used signaling inhibitors inhibit two or more kinases, depending on the assay used to determine inhibitor specificity. Our data suggest that COX activity is partially responsible for mediating H₂O₂-induced contraction in arteries and veins. From these current studies, we cannot determine whether H₂O₂-induced contraction via p38 MAPK, Erk MAPK, and src occurs independently of TXA₂ receptor activation.

Redox Signaling: A Possible Mechanism Mediating H₂O₂-Induced Contraction? It is likely that H₂O₂-stimulated TXA₂ receptor activation does not wholly account for H₂O₂-induced contraction, and other mechanisms may mediate aortic and venous H₂O₂-induced contraction. Increased intracellular Ca²⁺ is another possible mediator of H₂O₂-induced contraction, although the focus of the current study was not to investigate differences in Ca²⁺ handling between arteries and veins. Although H₂O₂ is not a free radical like many other reactive oxygen species, it is an oxidant and can participate in redox signaling (Wolin et al., 2002; Cai, 2005; Ardanaz and Pagano, 2006). Three potential mechanisms by which H₂O₂ could modify vascular tone via redox signaling are thiol oxidation and activation of kinases mediating vascular contraction, thiol oxidation and inhibition of phosphatases that normally inhibit contraction, and direct oxidation and activation of contractile proteins. Receptor tyrosine kinases (e.g., epidermal growth factor receptor), nonreceptor tyrosine kinases (e.g., Src, Lck, and Abl), and other kinases, including MAPKs, Akt, Janus kinase, and upstream regulators of these kinases (e.g., Ras) can be activated by oxidation of cysteine residues (Rhee et al., 2000, 2005; Oeckler et al., 2005). Besides activating kinases, H₂O₂ can also inhibit the activity of both protein tyrosine phosphatases and serine-threonine phosphates via thiol oxidation of active site cysteine residues (Rhee et al., 2000; Oeckler et al., 2005). The oxidative inactivation of phosphatases is kinetically favored over the activation of kinases (Oeckler et al., 2005); thus, redox inhibition of phosphatases regulating kinase activity may be another important mechanism of H₂O₂ signaling. The ATPase activity of myosin is redox-sensitive, such that thiol oxidation induces myosin ATPase activity independent of Ca²⁺ binding and phosphorylation of the regulatory myosin light chain₂₀ (Sparrow et al., 1970; Chandra et al., 1985; Ngai and Walsh, 1987). H₂O₂ can also alter gene transcription, although the acute contractile effects of H₂O₂ are not likely mediated by changes in gene transcription. To date, most of the studies regarding redox signaling have been performed in cell lines in the context of studying apoptosis, growth, and proliferation. Rogers et al. (2006) recently reported that in dog and rat coronary arterioles, H₂O₂-mediated vasodilation was reversed by dithiothreitol, a reducing agent, suggesting that H₂O₂ modification of vascular tone was redox-sensitive. Another possible explanation of our confusing and contradictory results could be due to concurrent TXA₂ receptor activation and redox signaling by H₂O₂.

Our studies underscore the complexity of H₂O₂ signaling and the difficulty in predicting the physiological actions of ROS. Of the pathways investigated, their involvement in H₂O₂-induced contraction does not differ between arteries and veins. Our data suggest that ROS can alter both venous and arterial tone. We speculate that in hypertension, a disease characterized by increased oxidative stress, both the arterial and venous circulation are likely targets of elevated ROS levels.

	Footnotes

 
This work was supported by the National Heart, Lung, and Blood Institute (Grant PO1 HL-70687 to S.W.W. and G.D.F.) and by a Ford Foundation Diversity Fellowship (to K.T.).

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.106.110650.

ABBREVIATIONS: ROS, reactive oxygen species; COX, cyclooxygenase; TXA₂, thromboxane A₂; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; PKC, protein kinase C; U46619 [GenBank] , 9,11-dideoxy-9,11-methanoepoxy PGF₂; ICI185282, 2RS,4RS,5SR-4-o-hydroxyphenyl-2-trifluoromethyl-1,3-dioxan-5-yl heptenoic acid; PD98059, 2'-amino-3'-methoxyflavone; SB203580, 4-[5-(4-fluorophenyl)-2-[4-(methylsulfonyl)phenyl]-1H-imidazol-4-yl]pyridine; PP1, 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine; Y27632, trans-4-[(1R)-1-aminoethyl]-N-4-pyridinylcyclohexanecarboxamide dihydrochloride; LY294002, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; DMSO, dimethylsulfoxide; TXB₂, thromboxane B₂; EIA, enzyme immunoassay; PI3-K, phosphatidylinositol 3-kinase; PGH₂, prostaglandin H₂.

Address correspondence to: Keshari Thakali, B445 Life Sciences Building, Department of Pharmacology and Toxicology, Michigan State University, East Lansing, MI 48824-1317. E-mail: thakalik{at}msu.edu

	References

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