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Activation of Rho/Rho kinase signaling pathway by reactive oxygen species in rat aorta

Department of Physiology, Medical College of Georgia, Augusta, Georgia 30912-3000

Submitted 23 October 2003 ; accepted in final form 26 May 2004

	ABSTRACT

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Evidence indicates that both the Rho/Rho kinase signaling pathway and reactive oxygen species (ROS) such as superoxide and H₂O₂ are involved in the pathogenesis of hypertension. This study aimed to determine whether ROS-induced vascular contraction is mediated through activation of Rho/Rho kinase. Rat aortic rings (endothelium denuded) were isolated and placed in organ chambers for measurement of isometric force development. ROS were generated by a xanthine (X)-xanthine oxidase (XO) mixture. The antioxidants tempol (3 mM) and catalase (1,200 U/ml) or the XO inhibitor allopurinol (400 µM) significantly reduced X/XO-induced contraction. A Rho kinase inhibitor, (+)-(R)-trans-4-(1-aminoethyl-N-4-pyridil)cyclohexanecarboxamide dihydrochloride (Y-27632), decreased the contraction in a concentration-dependent manner; however, the Ca²⁺-independent protein kinase C inhibitor rottlerin did not have an effect on X/XO-induced contraction. Phosphorylation of the myosin light chain phosphatase target subunit (MYPT1) was increased by ROS, and preincubation with Y-27632 blocked this increased phosphorylation. Western blotting for cytosolic and membrane-bound fractions of Rho showed that Rho was increased in the membrane fraction by ROS, suggesting activation of Rho. These observations demonstrate that ROS-induced Ca²⁺ sensitization is through activation of Rho and a subsequent increase in Rho kinase activity but not Ca²⁺-independent PKC.

myosin light chain phosphatase; smooth muscle contraction; antioxidants

In recent years, studies have revealed that the small GTPase Rho and its downstream target Rho kinase mediate the Ca²⁺ sensitization of smooth muscle contraction (37). The exchange of bound GDP for GTP activates Rho and stimulates its translocation from cytosol to membrane. Rho-GTP phosphorylates Rho kinase, which inhibits MLC phosphatase activity by phosphorylation of the MLC phosphatase target subunit (MYPT1). A decrease in MLC phosphatase activity increases phosphorylation of myosin and therefore contributes to smooth muscle contraction at low levels of intracellular Ca²⁺ concentration. Strong evidence suggests that increased Rho/Rho kinase-dependent Ca²⁺ sensitization contributes to hypertension and inhibition of Rho or Rho kinase reduces blood pressure (33, 42). Some heterotrimetric G protein-coupled receptor (GPCR) agonists, including angiotensin II (ANG II) and endothelin (ET)-1, induce smooth muscle contraction through both intracellular Ca²⁺-dependent and Rho/Rho kinase-mediated Ca²⁺-independent signaling pathways (24, 26).

Another important factor that has been suggested to contribute to increased blood pressure in experimental hypertensive models is oxidative stress. Studies have shown that increased production of reactive oxygen species (ROS), most notably superoxide and H₂O₂, is linked to abnormal activities of NADPH oxidase, endothelial NO synthase, and/or xanthine oxidase (XO) in vascular cells. Administration of antioxidants or inhibition of ROS-generating enzymes normalizes or reduces elevated blood pressure and improves vascular function (10, 22, 28). In addition, recent studies have demonstrated that both blood pressure and superoxide production are significantly lower in knockout mice lacking selected NADPH oxidase subunits than in wild-type mice (21, 43).

ROS are important modulators of vascular tone. Many studies have shown that ROS increase the tone of arteries by reducing NO bioavailability and endothelium-dependent relaxation (5, 19). However, ROS also contract vessels after removal of endothelium, suggesting that ROS have direct effects on smooth muscle (3, 29, 48). Although accumulating evidence suggests that ROS can function as second messengers to activate multiple intracellular signal cascades, the mechanisms of ROS-induced vasoconstriction are still incompletely understood and the effects of ROS on the Rho/Rho kinase Ca²⁺-independent signaling pathway have not been investigated. Therefore, we report the results of studies designed to test the hypothesis that ROS induce vascular smooth muscle contraction via activation of Rho/Rho kinase.

	MATERIALS AND METHODS

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Tissue preparation and isometric force measurement. All animal procedures performed were in accordance with the "Guiding Principles for Research Involving Animals and Human Beings" of the American Physiological Society.

Male Sprague-Dawley rats (275–300 g) were anesthetized with pentobarbital sodium (50 mg/kg ip), and the thoracic aorta was quickly removed and cleaned in physiological salt solution (PSS) of the following composition (mM): 118 NaCl, 4.7 KCl, 1.18 KH₂PO₄, 1.6 CaCl₂·2H₂O, 1.6 MgSO₄·7H₂O, 25 NaHCO₃, 5.5 dextrose, and 0.03 EDTA. The aorta was cut into 2-mm rings, and the endothelium was gently removed. The aortic rings were mounted in a muscle bath containing PSS at 37°C and bubbled with 95% O₂-5% CO₂. Isometric force generation was recorded with a Multi Myograph System (Danish Myo Technology A/S). A resting tension of 3 g was imposed on each ring, and the rings were allowed to equilibrate for 90 min. The rings were contracted with phenylephrine (PE, 0.1 µM), and acetylcholine (1 µM) was added during the plateau phase to verify efficient removal of endothelium. During the experiments, the rings were contracted with xanthine (X) and XO with or without preincubation with tempol (3 mM), catalase (1,200 mU/ml), allopurinol (400 µM), (+)-(R)-trans-4-(1-aminoethyl-N-4-pyridil)cyclohexanecarboxamide dihydrochloride (Y-27632; 1 µM), or rottlerin (1 µM). In another set of experiments, the rings were contracted with phorbol 12,13-dibutyrate (PDBu) in the presence or absence of Y-27632 (1 µM). The contractions were compared with 10 µM PE-induced maximum contractions. The average maximal force generated in response to PE was 27.7 ± 2.2 mN.

Detection of phospho-MYPT1 levels. Aortic rings were maximally contracted with X/XO in the presence or absence of Y-27632 and immediately snap frozen in liquid nitrogen. The rings were solubilized in RIPA buffer (mM): 50 Tris·HCl (pH 7.4), 150 NaCl, 1 EDTA, 1 NaF, 1 Na₃VO₄, and 1 PMSF, with 0.25% Na-deoxycholate and 1.0% NP-40, and centrifuged at 10,000 g and 4°C for 30 min. The supernatant was collected, and the phosphorylation levels of MYPT1 at Thr⁶⁹⁶ were analyzed by Western blot.

Determination of Rho translocation by Western blot analysis. Aortic rings were subjected to a maximum contraction with X/XO, immediately snap frozen in liquid nitrogen, and homogenized in cold homogenization buffer (mM): 100 Tris·HCl (pH 7.4), 1 EGTA, 1 EDTA, 1 PMSF, and 1 Na₃VO₄. The homogenates were centrifuged at 100,000 g and 4°C for 20 min. The supernatant (cytosolic fraction) was collected, and the pellet (membrane fraction) was resuspended in homogenization buffer containing 1% Triton X-100. Protein concentrations were determined with a bicinchoninic acid kit (Pierce). Rho migration was determined by Western blot. Briefly, equal amounts of proteins were separated by SDS-PAGE and subsequently transferred to nitrocellulose membrane. The membrane was then incubated with monoclonal Rho antibody. Finally, the membranes were incubated with a horseradish peroxidase-linked secondary antibody and visualized with an enhanced chemiluminescence kit (Amersham).

Materials. Y-27632, PDBu, and rottlerin were purchased from Calbiochem (San Diego, CA). X, XO, Tempol, catalase, allopurinol, and -actin were purchased from Sigma (St. Louis, MO). Rho monoclonal antibody was obtained from BD Biosciences (San Diego, CA). Phospho-MYPT1 antibody was purchased from Upstate (Charlottesville, VA).

Statistics. Data are expressed as means ± SE. The unpaired Student's t-test was performed to determine the significance of differences between mean values. When P < 0.05, the differences were considered to be statistically significant.

	RESULTS

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X/XO-induced contraction is ROS dependent and mediated by Rho kinase. X and XO constitute a physiological superoxide generating system (25). It has been shown that superoxide is produced when X is converted to uric acid by XO. XO (5–40 mU/ml) and X (50–400 µM) induced concentration-dependent contractions in endothelium-denuded rat aortic rings. The maximum contraction reached was 64 ± 7% of the PE-induced maximum contraction (10 µM) (Fig. 1A). After the rings were washed to remove X/XO, the response of aortic rings to PE did not change (data not shown), suggesting that the contractile components of the smooth muscle cells were not altered by exposure to ROS. Twenty milliunits/milliliter of XO and two hundred micromolar X were chosen to perform further experiments because X/XO-induced contraction reached a plateau phase at these concentrations. To investigate the role of superoxide and H₂O₂ in X/XO-induced contraction, two antioxidants, tempol and catalase, were preincubated with aortic rings for 15 min. Tempol, the membrane-permeant SOD mimetic that converts superoxide to H₂O₂, reduced the effect of X/XO on smooth muscle from 52 ± 2% to 3 ± 0.6% of the PE-induced contraction (P < 0.01, Fig. 1B). Catalase, which catalyzes H₂O₂ to H₂O, decreased the X/XO-induced contraction to 39 ± 4% of the PE-induced contraction (P < 0.05; Fig. 1C). The inhibitory effect of antioxidants suggested that contraction induced by X/XO is mainly due to the generation of superoxide. This is supported by the fact that preincubation with allopurinol (400 µM), a XO inhibitor, diminished the contractions to X/XO (Fig. 1D). These observations indicate that ROS were produced by X/XO enzymatic reaction.

View larger version (16K): [in this window] [in a new window]   Fig. 1. Xanthine (X)/xanthine oxidase (XO)-induced smooth muscle contraction is mediated by ROS. A: rat aortic rings were incubated with different concentrations of X (5, 10, 20, and 40 mU/ml) and XO (50, 100, 200, and 400 µM). Smooth muscle contractility is expressed as the percentage of phenylephrine (PE; 10 µM)-induced maximum contraction. Values are means ± SE of 4 experiments. B: aortic rings were preincubated with Tempol (3 mM) for 15 min. Tempol inhibited X (200 µM)/XO (20 mU/ml)-induced contractions (control). Values are means ± SE of 5 experiments. **P < 0.01 vs. control. C: preincubation with catalase (1,200 U/ml) for 15 min reduced X (200 µM)/XO (20 mU/ml)-induced contractions (control). Values are means ± SE of 6 experiments. *P < 0.05 vs. control. D: preincubation with allopurinol (400 µM) for 15 min decreased X (200 µM)/XO (20 mU/ml)-induced contractions (control). Values are means ± SE of 6 experiments. **P < 0.01 vs. control.

View larger version (11K): [in this window] [in a new window]   Fig. 2. X/XO-induced smooth muscle contraction is mediated by Rho kinase. A: (+)-(R)-trans-4-(1-aminoethyl-N-4-pyridil)cyclohexanecarboxamide dihydrochloride (Y-27632) preincubated with rat aortic rings for 15 min inhibits X (200 µM)/XO (20 mU/ml)-induced contractions (control) in a concentration-dependent manner. Values are means ± SE of 6 experiments. **P < 0.01 vs. control. B: Y-27632 (1 µM) has no effect on phorbol 12,13-dibutyrate (PDBu)-induced contractions. Values are means ± SE of 4 experiments. C: rottlerin (1 µM) preincubated with the rings for 15 min has no effect on X (200 µM)/XO (20 mU/ml)-induced contractions (control). Values are means ± SE of 6 experiments.

Studies have suggested that Y-27632 is more selective for Ca²⁺-independent PKC- and PKC- than for Ca²⁺-dependent PKC isoforms such as PKC- and PKC- (8, 42). To determine whether PKC- and - are involved in X/XO-induced contraction, the aortic rings were preincubated with rottlerin (1 µM), a selective inhibitor for PKC- and -. The results show that rottlerin did not reduce the X/XO-induced contraction (Fig. 2C).

ROS increases phosphorylation levels of MYPT1. To further establish a role for Rho kinase in X/XO-induced smooth muscle contraction, phosphorylation levels of MYPT1 were examined. Previous studies showed that Rho kinase inhibits MLC phosphatase activity through phosphorylation of MYPT1, which promotes MLC phosphorylation, resulting in smooth muscle contraction (38). We examined the MYPT1 phosphorylation levels in X/XO-treated aortic rings in the presence and absence of Y-27632 by Western blot analysis. X/XO increased MYPT1 phosphorylation by 1.7 ± 0.2-fold (P < 0.05) compared with control. Preincubation with Y-27632 (1 µM) blocked this phosphorylation (Fig. 3), indicating that X/XO-increased Rho kinase activity was responsible for inactivation of MLC phosphatase.

View larger version (16K): [in this window] [in a new window]   Fig. 3. Rho kinase mediates X/XO-increased phosphorylation levels of the myosin light chain phosphatase target subunit (MYPT1). Rat aortic rings were preincubated with and without Y-27632 for 15 min and challenged with X (200 µM)/XO (20 mU/ml). Phosphorylation levels of MYPT1 were measured as described in MATERIALS AND METHODS. Top: representative immunoblot of phosphorylated (p) MYPT1. Bottom: densitometric analysis of immunoblots from 3 independent experiments. Values are means ± SE. *P < 0.05 vs. control.

View larger version (18K): [in this window] [in a new window]   Fig. 4. X/XO induces Rho translocation. Rat aortic rings were incubated with X (200 µM)/XO (20 mU/ml) and cytosol, and membrane-bound fractions of Rho were measured by Western blot analysis, as described in MATERIALS AND METHODS. Top: representative immunoblot of Rho translocation from cytosol to membrane. Bottom: densitometric analysis of membrane-bound fraction of Rho from 3 independent experiments. Values are means ± SE. *P < 0.05 vs. control.

	DISCUSSION

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Vascular reactivity and peripheral vascular resistance are both increased in hypertension (44). Increased ROS levels have been linked to hypertension in rat models such as spontaneously hypertensive rats, deoxycorticosterone acetate-salt hypertensive rats, Dahl salt-sensitive rats, ANG II-induced hypertensive rats, and renovascular hypertensive rats (22, 35, 41, 46, 47). Several studies have examined the contractile response of arteries to ROS. Superoxide and H₂O₂ induce vasoconstriction in rat aorta (3, 20, 29), canine basilar artery (48), and rat pulmonary artery (18). Additionally, our data show that the antioxidant Tempol significantly reduced X/XO-induced contractions and the antioxidant catalase decreased X/XO-induced contractions significantly but to a smaller extent. It is possible that a small amount of superoxide produced by X/XO is converted to H₂O₂ by SOD, which contributes to X/XO-induced contractions. The mechanism for the contractile response to ROS is not completely understood, and data from various studies are conflicting. It has been reported that ROS can affect cellular functions through modulation of intracellular Ca²⁺ levels. Bielefeldt et al. (4) demonstrated that both H₂O₂ and X/XO-generated superoxide increased intracellular Ca²⁺ concentration in smooth muscle cells and antioxidants abolished the changes in Ca²⁺ concentration. ROS increase the intracellular Ca²⁺ concentration via activation of voltage-sensitive Ca²⁺ channels, leading to an influx of extracellular Ca²⁺. Removal of extracellular Ca²⁺ or administration of Ca²⁺ channel blockers markedly reduces the contraction induced by exogenous ROS (3, 48). In addition, ROS release Ca²⁺ from intracellular stores by inhibiting Ca²⁺-ATPase activity and promoting inositol triphosphate-induced Ca²⁺ release (13, 14). Increases in intracellular Ca²⁺ levels result in activation of proteins including PKC and Ca²⁺-dependent calmodulin kinases, leading to smooth muscle contraction. In contrast, other studies suggest that ROS-induced contraction is independent of intracellular Ca²⁺ in pulmonary artery (17, 18, 27). Other possible mechanisms include activation of mitogen-activated protein kinase or tyrosine kinase (17, 27, 48). The effect of ROS on the Rho/Rho kinase Ca²⁺-independent pathway has not been investigated previously.

The state of MLC phosphorylation is the result of a balance between MLC kinase and MLC phosphatase activities and determines the contractile activity of smooth muscle (37, 38). MLC phosphatase consists of three subunits: a 38-kDa catalytic subunit of type 1 phosphatase, a 110-kDa MYPT1, and a small 20-kDa subunit whose function is not clear. Recent studies showed that inhibition of MLC phosphatase leads to increased Ca²⁺ sensitization and smooth muscle contraction (15, 36). One of the upstream proteins of MLC phosphatase is Rho kinase, which phosphorylates MYPT1 at Thr⁶⁹⁶ (9). Y-27632 is a selective inhibitor of Rho kinase. It has been reported that this pyridine derivative is 200–2,000 times more specific for Rho kinase than other protein kinases including Ca²⁺-dependent PKC, MLC kinase, and cAMP-dependent protein kinase that are the major players in regulation of smooth muscle tone (7, 16, 42). Sakamoto et al. (30) demonstrated that Y-27632 has no direct effects on intracellular Ca²⁺ levels or the activities of MLC phosphatase and MLC kinase at concentrations <100 µM. Studies using Y-27632 have shown Rho kinase to mediate the tonic component of vasoconstrictor responses to agonists including PE, serotonin, ET-1, prostaglandin F₂, and histamine (39, 40). In this study, we demonstrate that 1 µM Y-27632 abolishes X/XO-induced contractions but not PDBu-induced contractions, suggesting a role for Rho kinase as a mediator of the X/XO-induced contractions (Fig. 2). Eto et al. (8) reported that purified PKC- was inhibited by 10 µM Y-27632 and PDBu-induced contraction was reduced up to 65% in rabbit femoral artery with the same concentration of Y-27632. The difference between our results and theirs may be due to the higher concentration of Y-27632 used in their work and the species of the animals. Our results with a PKC-/-specific inhibitor show that PKC- and - are not involved in X/XO-induced contraction. Therefore, the inhibitory effect of 1 µM Y-27632 on X/XO-induced Ca²⁺ sensitization ascribes to the inhibition of Rho kinase. This is further supported by the observation that phosphorylation levels of MLC phosphatase are increased by ROS and Y-27632 prevents this increase.

Studies show that Rho kinase can be activated by Rho as well as by other second messengers such as arachidonic acid and sphingosylphosphorylcholine (2, 34). Therefore, we examined whether Rho is involved in activation of Rho kinase in ROS-treated aortic rings. To activate its target protein, Rho must exchange GDP for GTP and bind to the plasma membrane via a posttranslational COOH-terminal geranylgeranyl lipid modification (1). Our data show that ROS increase membrane-bound Rho in aortic rings during maximum contraction, indicating that ROS increase Rho kinase activity through activation of Rho.

Although increased activity of the Rho/Rho kinase signaling pathway contributes to hypertension, the regulation of the Rho/Rho kinase pathway is still not clear (23, 42, 45). One of the mechanisms of activation of Rho/Rho kinase is via GPCR, mainly G_12/13, on smooth muscle cells (11, 12). Regulation of the Rho/Rho kinase pathway can also occur at a nonreceptor level. Recent studies suggest that NO has an inhibitory effect on Rho/Rho kinase activities. Sauzeau et al. (31) observed that sodium nitroprusside reversed membrane translocation of Rho and induced actin disassembly. cGMP-dependent protein kinase (PKG) is thought to destabilize membrane binding of Rho through phosphorylation at Ser¹⁸⁸. Additionally, constitutively active PKG was demonstrated to inhibit the lysophosphatidic acid-induced translocation of Rho in HeLa and NIH 3T3 cells, indicative of the NO/PKG-mediated inactivation of Rho (32). Functional studies in the rat aorta have confirmed these observations (6). Our data suggest a role for ROS in the activation of the Rho/Rho kinase pathway and provide a new mechanism of regulation of the Ca²⁺-independent Rho/Rho kinase signaling pathway.

In conclusion, this novel study demonstrates the direct activation of the Rho/Rho kinase signaling pathway by ROS in rat aorta, suggesting an important role for ROS-mediated Rho/Rho kinase activation in vasoconstriction. The mechanism of ROS activation of the Rho/Rho kinase pathway remains to be examined.

	GRANTS

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This work is supported by National Heart, Lung, and Blood Institute Grants HL-18575 and HL-71138. L. Jin is a postdoctoral fellow supported by the American Foundation for Urological Diseases.

	ACKNOWLEDGMENTS

 
We are grateful to Drs. E. Inscho, R. White, A. Dorrance, C. Teixeira, and A. Linder for critical review.

	FOOTNOTES

 

Address for reprint requests and other correspondence: L. Jin, Dept. of Physiology, Medical College of Georgia, 1120 15th St., Augusta, GA 30912-3000 (E-mail: ljin{at}mcg.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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