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Heat shock augments angiotensin II-induced vascular contraction through increased production of reactive oxygen species
Biochemical and Biophysical Research Communications 399 (2010) 452–457
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Heat shock augments angiotensin II-induced vascular contraction through increased production of reactive oxygen species Jee In Kim a,b, Sang Won Jung a, Enyue Yang a, Kwon Moo Park b, Masumi Eto c, In Kyeom Kim a,* a
Department of Pharmacology and Cardiovascular Research Institute, Kyungpook National University School of Medicine, Daegu 700-422, Republic of Korea Department of Anatomy and BK21, Kyungpook National University School of Medicine, Daegu 700-422, Republic of Korea c Department of Molecular Physiology and Biophysics, Thomas Jefferson University School of Medicine, 1020 Locust Street, 436 JAH., Philadelphia, PA 19107, USA b
a r t i c l e
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Article history: Received 26 July 2010 Available online 3 August 2010 Keywords: Vascular smooth muscle Heat shock Angiotensin II Reactive oxygen species NADPH oxidase Hydrogen peroxide
a b s t r a c t A temporal increase in temperature triggers a series of stress responses and alters vascular smooth muscle (VSM) contraction induced by agonist stimulation. Here we examined the role of reactive oxygen species (ROS) in heat shock-dependent augmentation of angiotensin II (AngII)-induced VSM contraction. Endothelium-denuded rat aortic rings were treated with heat shock for 45 min at 42 °C and then subjected to assays for the production of force, ROS, and the expression of ROS-related enzymes. AngIIinduced contraction was enhanced in heat shock-treated aorta. AngII-induced production of hydrogen peroxide and superoxide were elevated in response to the heat shock treatment. Pre-treatment with superoxide dismutases (SOD) mimetic and inhibitors for glutathione peroxidase and NADPH oxidase but not for xanthine oxidase eliminated an increase in the AngII-induced contraction in the heat shock-treated aorta. Heat shock increased the expression of p47phox, a cytosolic subunit of NADPH oxidase, but not Cu–Zn–SOD and Mn–SOD. In addition, heat shock increased contraction that was evoked by hydrogen peroxide and pyrogallol. These results suggest that heat shock causes an elevation of ROS as well as a sensitization of ROS signal resulting in an augmentation of VSM contraction in response to agonist. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction A drastic change in temperature is a challenge for our body, and produces acute stress response signals for the maintenance of homeostasis. Temporal increase in temperature under extreme environment causes changes in the expression of a large group of proteins, including various heat-shock proteins (HSPs). Studies suggest an involvement of HSPs in the regulation of vascular smooth muscle (VSM) contraction and relaxation through various kinases and signals [1]. Independently, we have reported that a temporal hyperthermic treatment at 42 °C increases the contraction of aortic VSM induced by high-K+ and phenylephrine [2–4]. The VSM contraction is determined by phosphorylation of myosin light chain (MLC), which is balanced by activities of MLC kinase (MLCK) and phosphatase (MLCP) [5]. In our previous study we showed that heat shock changed signal transduction in the control of MLC phosphorylation and VSM contraction through the inhibition of MLCP [6]. However, the underlying mechanisms how heat shock augments VSM contraction is not fully understood. Reactive oxygen species (ROS), including superoxide, hydrogen peroxide, and hydroxyl anion, nitric oxide, and peroxynitrite are * Corresponding author. Fax: +82 53 426 7345. E-mail address: [email protected] (I.K. Kim). 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.07.115
increasingly recognized as important mediators that control VSM contraction [7]. VSM cells contain numerous sources of ROS, such as the NADPH oxidases as the most dominant players, in addition to xanthine oxidase, the mitochondrial respiratory chain, lipoxygenases and nitric oxide synthases [8]. While an overproduction of ROS causes disorders in vascular bed, normal level of ROS production is involved in the regulation of VSM contraction and relaxation, depending on the tissues and conditions. For example, hydrogen peroxide induces the vasodilatation of pulmonary artery and the vasoconstriction of aorta strips [9,10]. Effect of heat stress on ROS production is controversial [11–14] and the process how heat stress affects ROS production remains to be elucidated. Interestingly, ROS induces vasoconstriction through Rho-kinase pathway in rat aorta that transduces agonist stimulation into the inhibition of MLCP [15]. Therefore, we hypothesized that heat shock enhances ROS production that causes an augmentation of agonist-induced contraction of aorta smooth muscle (SM). Of the numerous vasoactive agents regulating vascular NAD(P)H oxidase, AngII appears to be one of the most important agonist [16,17]. In rat aorta, AngII stimulates vasoconstriction via ROS-dependent mechanisms [10]. Accordingly, oxidative stress has been demonstrated in various models of experimental hypertension, including AngII-induced hypertension [18,19]. Here we showed that heat shock increases AngII-induced contraction of
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aorta that is mediated by increased ROS production, as well as the hyper-responsiveness to ROS in aorta SM contraction. 2. Materials and methods 2.1. Tissue preparation The present investigation is in accordance with Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health [20]. Male Sprague–Dawley rats, weighing 320–350 g, were used. With animals under anesthesia with sodium pentobarbital (50 mg kg1 i.p.), the thoracic aorta was excised and immersed in cold modified Krebs’ solution composed of (in mM) NaCl, 115.0; KCl, 4.7; CaCl2, 2.5; MgCl2, 1.2; NaHCO3, 25.0; KH2PO4, 1.2; and dextrose, 10.0. The aorta was cleaned of all adherent connective tissue and cut into 4 mm long ring segments as described previously [6]. All of the rings were denuded of endothelium by gentle rubbing the internal surface using a forceps. 2.2. Tension studies Two stainless-steel triangles were inserted through each vessel ring. One triangle was anchored to a stationary support, and the other was connected to an isometric force transducer (Grass FT03C, Quincy, MA, USA) in a water-jacketed organ bath maintained at 37 °C and aerated with a mixture of 95% O2 and 5% CO2. The rings were stretched passively by imposing the optimal resting tension of 2.0 g, maintained throughout the experiment. Each ring was equilibrated before tissue viability was tested with 50 mM KCl. Only the aortas showing above 2.0 g of contractile responses were used for further experiments. Tension was recorded using a computerized data acquisition system (PowerLab/8SP, ADInstruments, Castle Hill, NSW, Australia).
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2.5. ROS assay Superoxide level was assayed using dihydroethidium (DHE) that reacts with superoxide to form the red fluorescent adducts ethidium and oxy-ethidium [21]. Isolated rat aortic rings were loaded with 0.1 lM DHE for 20 min. DHE-loaded aortas were stimulated with 1 lM AngII for 4 min. Aortas were embedded in O.C.T. compound (Tissue-Tek, Sakura Finetek USA, Inc., Torrance, CA) to be frozen. Serial cryostat sections (10 lm thick) were deposited onto glass slides. Samples were air dried at 4 °C. Fluorescence laser scanning microscopy was conducted on a Zeiss Axiovert 100 M coupled with a Zeiss LSM510 laser scanning system (Germany). Red fluorescence was measured using Adobe Photoshop imager (Adobe systems Inc., USA). Hydrogen peroxide level was assayed using a ferric oxide-sensitive dye, xylenol orange, as described previously [22]. Aortas were stimulated with 1 lM AngII for 30 s and frozen immediately by immersion in liquid nitrogen. Frozen aortic rings were homogenized in a sucrose buffer (pH 7.5) containing 210 mM mannitol, 70 mM sucrose, 1 mM EDTA, and 10 mM hepes. Each sample was centrifuged at 13,000 rpm for 20 min at 4 °C. The protein concentration in the supernatant was measured by Bradford method using Bio-Rad Protein Assay kit (Bio-Rad, Hercules, CA, USA). Equal amount of protein samples (100 ll) were added to FOX reagent (900 ll) (25 mM H2SO4, 100 mM sorbitol, 2.5 mM ferrous ammonium sulfate, 0.1 mM xylenol orange) and incubated for 30 min at room temperature prior to measurement at 560 nm. Hydrogen peroxide oxidizes iron (II) to iron (III) in the presence of sorbitol, iron (III) then forms a purple complex with xylenol orange, which absorbs at 560 nm. The absorbance was measured using Ultraspec 2100 pro (Amersham pharmacia biotech, Piscataway, NJ, USA). 2.6. Materials
2.3. Heat shock Half of the strips were exposed to 42 °C for 45 min (heat-shock group) followed by returning to 37 °C whereas the other half (control group) were remained at 37 °C. Both of the groups were further incubated for 4 h before study. 2.4. Western blot analysis Aortic rings with various treatments were quickly frozen at proper time points by immersion in LN2. Stored aortic rings were homogenized in a lysis buffer containing 320 mM sucrose, 50 mM Tris, 1 mM EDTA, 1% Triton X-100, 1 mM DTT, and protease inhibitors leupeptin (10 lg/ml), trypsin inhibitor (10 lg/ml), aprotinin (2 lg/ml), phenylmethylsulfonyl fluoride (PMSF; 100 lg/ml), and phosphatase inhibitor b-glycerophosphate (50 mM). Proteinmatched samples (Bradford assay) were electrophoresed on 10% polyacrylamide gel with 0.1% SDS and transferred to nitrocellulose membranes, and then subjected to an immunoblot with p47phox antibody (1:1000, Upstate Biotechnology, Lake Placid, NY, USA). For the SODs, samples were electrophoresed on 12% gel then subjected to immunoblot with anti-Cu–Zn–SOD antibody (1:5000, Chemicon, Temecula, CA, USA) and Mn–SOD antibody (1:1000, Calbiochem, Darmstadt, Germany). Horseradish peroxidase-conjugated anti-rabbit IgG or anti-sheep IgG were used as secondary antibodies respectively (1:5000, Sigma, St. Louis, MO, USA). For the loading control anti-b actin antibody (1:2000, Sigma, St. Louis, MO, USA) was used. The protein bands were exposed with enhanced chemiluminescence reagents, visualized on X-ray films and quantified with image acquisition and analysis software; Lab Works program (Ultra-Violet Product Ltd.).
Angiotensin II, apocynin, glutathione peroxidase, allopurinol; (4-hydroxypyrazolo[3,4-d]pyrymidine), MnTMPyP; [manganese (III) tetrakis (N-methyl-2-pyridyl) porphyrin], H2SO4, sorbitol, xylenol orange; [o-cresolsulfonephthalein-30 -300 -bis-(methyliminodiacetic acid sodium salt)], ferrous ammonium sulfate, dihydroethidium, hydrogen peroxide solution and pyrogallol were obtained from Sigma Chemical Company (St. Louis, MO, USA). KCl was obtained from Junsei chemicals Co., Ltd. (Tokyo, Japan). All other reagents were of analytical grade or better.
2.7. Statistical analysis The results are expressed as mean ± SE of the mean which were analyzed by Student’s t-test. P values < 0.05 were regarded as statistically significant. 3. Results 3.1. Heat shock-augmented aorta SM contraction evoked by angiotensin II Previous studies show that the contraction of rat aorta SM in response to the stimulation with high K+ or phenylephrine is enhanced after temporal heat shock treatment (42 °C for 45 min) [3,6]. We further examined whether the heat shock treatment causes an augmentation of AngII-induced aorta SM contraction. The maximum force of heat shock-untreated aorta SM evoked by 1 lM AngII was set as 100%. The contraction of the heat shocktreated aorta SM reached 200% of the contraction of the heat shock-untreated tissues (Fig. 1). Thus, the signals in the AngII-in-
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Fig. 1. Angiotensin-induced contraction after heat shock. (A) Time course of heat shock and representatives of myographs in response to AngII in control and heat shocktreated aorta. (B) Control (open bar) and heat shock-treated (closed bar) aorta were stimulated with 1 lM AngII. SM contractility is expressed as the percentage of 1 lM AngIIinduced contraction in control. Graph shows that heat shock significantly augmented contractile response to AngII. Values are means ± SEM of six independent experiments (**P < 0.01 vs. control, Student’s t-test).
Fig. 2. ROS production by angiotensin and ROS-related protein expression following heat shock in isolated rat aorta. (A) Both control (open bars) and heat shock-treated (closed bars) aorta were stimulated with 1 lM AngII for 30 s and subjected to determination of hydrogen peroxide generation. Graph shows that heat shock significantly increased production of hydrogen peroxide by AngII. Values are means ± SEM of three independent experiments. (*P < 0.05, **P < 0.01 vs. vehicle-treated control, ##P < 0.01 vs. AngII-treated control, Student’s t-test). (B) Dihydroethidium (DHE)-loaded aortas were pre-incubated for 30 min either in the absence or presence of apocynin, followed by stimulation with 1 lM AngII for 4 min. AngII-produced superoxide in both control and heat shock-treated aortas was determined by fluorescence laser scanning micrographs (400 objective). Heat shock significantly increased AngII-induced superoxide production. Values are means ± SEM of five independent experiments. (*P < 0.05, **P < 0.01 vs. superoxide production by vehicle (ethanol) in control and heat shock-treated aorta respectively, ##P < 0.01 vs. AngII -induced superoxide production in control, Student’s ttest). (C) Representative immunoblots showing expression of a cytosolic subunit of NADPH oxidase p47phox, superoxide dismutases Cu–Zn–SOD and Mn–SOD in control and heat shock-treated aorta. b-Actin was used as a loading control. p47phox expression significantly increased after heat shock whereas Cu–Zn–SOD and Mn–SOD did not change. Values are means ± SEM of six independent experiments. (*P < 0.05, vs. control, Student’s t-test).
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Fig. 3. Inhibition of angiotensin-induced contraction by various ROS inhibitors. Both control and heat shock-treated aorta were pretreated with SOD mimetic MnTMPyP (30 lM), Glutathione peroxidase (GPx) (2.2 U), NADPH oxidase inhibitor apocynin (50 lM), xanthine oxidase inhibitor allopurinol (400 lM), for 30 min followed by stimulation with 1 lM AngII. MnTMPyP, GPx, apocynin inhibited heat shock-augmented contraction to the control level whereas allopurinol did not. Values are means ± SEM of five independent experiments. (*P < 0.05, **P < 0.01 vs. without pre-treatment of inhibitors, Student’s t-test).
duced contraction of aorta SM tissue are sensitive to heat shock treatment. 3.2. Involvement of ROS in augmentation of aorta SM contraction after heat shock Zuo et al. reported that heat shock treatment of skeletal muscle increases production of ROS [11]. We assayed the level of hydrogen peroxide and superoxide in rat aorta tissues after heat shock with or without AngII stimulation. The level of hydrogen peroxide was increased in the tissue after heat shock treatment under un-stimulated condition (Fig. 2A and B, vehicle). The level of both ROS was elevated in response to AngII stimulation in the heat shock-untreated control (Fig. 2A and B, AngII), as reported previously [16,17]. Importantly, AngII-induced elevation in ROS level was further augmented in response to heat treatment (Fig. 2, AngII, closed bars). The heat-shock-induced elevation of ROS production was accompanied with up-regulation of NADH oxidase (Fig. 2C). Heat shock treatment induced a 3-fold increase in the expression of p47phox, a cytosolic subunit of NADPH oxidase. On the other hand,
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the expression of superoxide dismutases Cu–Zn–SOD and Mn–SOD was not changed after heat shock treatment, suggesting NADPH oxidase through the overexpression of p47phox is involved in the heat shock-induced elevation of ROS. Next we examined whether the elevated ROS is involved in the augmentation of aorta SM contraction induced by AngII stimulation, using cell permeable scavengers for ROS, such as SOD mimetic MnTMPyP (30 lM), Glutathione peroxidase GPx (2.2 U), inhibitors for NADPH oxidase (50 lM apocynin), and xanthine oxidase (400 lM allopurinol) (Fig. 3). A 30 min pre-treatment with MnTMPyP or GPx, eliminated the heat shock-induced augmentation of the contraction indicating that superoxide and/or hydrogen peroxide mediate heat shock-induced augmentation of SM contraction. In consistent to the up-regulation of p47phox, the augmented contraction was eliminated by the pre-treatment with apocynin, but not with allopurinol, indicating that increased NADPH expression is the major source of the heat shock-dependent production of ROS. 3.3. Sensitization of ROS-induced contraction of aorta SM in response to heat shock Yang et al. reported that hydrogen peroxide induces the contraction of endothelium-denuded aorta ring that is attenuated by the addition of a PKC inhibitor [23]. We examined whether heat shock alters the sensitivity in contraction of aorta SM in response to ROS. Cumulative addition of hydrogen peroxide (Fig. 4A) or pyrogallol that generates superoxide (Fig. 4B) induced contraction of aorta SM under low potassium (KCl 10 mM) pretreated condition. The extent of ROS-induced contraction was significantly greater in heat shock-treated aorta (Fig. 4). These results suggest that heat shock treatment increases not only production of ROS but also SM contractility induced by ROS signal. Thus the dual effects of heat shock on ROS signal caused the augmentation of AngII-induced contraction of aorta SM. 4. Discussion The major finding of the present study is that heat shock treatment causes NADPH up-regulation and consequencing augmentation of AngII-induced SM contraction through the increased ROS production. Heat shock also enhanced ROS-induced SM contractility resulting in a synergy that amplifies the ROS signal. This augmentation of AngII signal is consistent to our previous results showing that heat shock treatment augments the vasoconstriction
Fig. 4. Hydrogen peroxide- or pyrogallol-induced vascular contraction after heat shock. Cumulative contractile response curves. Aortic rings were incubated with 10 mM KCl for 10 min then stimulated with cumulative concentration (3, 30, 300 lM) of hydrogen peroxide (A) or superoxide-producing pyrogallol (B). SM contractility is expressed as the percentage of 50 mM KCl-induced maximum contraction. Graphs show that heat shock significantly augmented contractile response to hydrogen peroxide and pyrogallol. Values are means ± SEM of five independent experiments (*P < 0.05 vs. control, Student’s t-test).
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of rat aorta rings induced by various stimuli, such as phenylephrine and KCl [3,6]. It is well documented that ROS, particularly superoxide (O) and hydrogen peroxide (H2O2), are important in AngIImediated signal transduction in VSM cells [24,25]. Indeed, ROS level is higher after heat treatment, and depletion of ROS with MnTMPyP or GPx significantly inhibited heat shock-augmented contraction. Thus, ROS production is driven in VSM under hyperthermic condition. Under physiological conditions, ROS are produced in a controlled manner at low concentrations and function as signaling molecules regulating the contraction/relaxation and growth of VSM cells [17,26–28]. ROS production is increased in pathological samples, such as cultured VSMC and isolated arteries from hypertensive rats and humans [28–30], causing the increased contractility [26]. Importantly, Schnackenberg et al. reported that the suppression of superoxide production attenuates the development of hypertension [29]. We expect that the heat-shock model will provide further insights into the role of ROS in controlling blood pressure. Activation of AT1 receptors acutely produces ROS through NADPH oxidase and activation of c-Src [8]. A NADPH oxidase inhibitor apocynin that [31] blocks association of p47phox with membrane-associated subunits p22phox and gp91phox [32,33], inhibits heat shock-augmented SM contraction. In contrast, a xanthine oxidase inhibitor allopurinol had no effect on the contraction even at 400 lM. Furthermore, the heat shock treatment indeed upregulated NADPH oxidase via p47phox expression. Thus, it is evident that heat shock treatment selectively activates NADPH oxidase but not xanthine oxidase. We presume that a series of HSPs stabilizes p47phox and activates NADPH oxidase pathway as a part of heat shock response. NADPH oxidase activation has been linked to cardiovascular diseases [25,32,34]. For instance, genetic models of hypertension, such as SHR [35] and stroke-prone SHR [30] exhibited enhanced NAD(P)H oxidase-mediated superoxide generation in aorta and mesentery arteries. These processes were associated with increased expression of NAD(P)H oxidase subunits, particularly p22phox and p47phox [25]. Apparently, heat shock treatment temporally produces a similar environment to genetic hypertension via NADPH up-regulation. ROS is known to increase aortic SM contraction [15,31,36,37]. Endothelium is also involved in ROS-induced vasoconstriction, through the cyclooxygenase pathway and/or inhibition of nitric oxide [31]. In our study the vascular endothelium is removed from the tissue, so that the ROS effect is independent of endothelium. Shen et al. reported that ROS activates P2X ATP receptor and causes the contraction of denuded aorta tissues [36]. Although ATP is not intentionally added to the bath, we cannot rule out the possibility of ATP production from the cells causing an activation of P2X receptor. In addition, recent evidence suggests that ROS activates RhoA/ROCK signal in SM. For example, Denniss et al. reported that ROS activated RhoA-Rho kinase in VSM preparation to enhance endothelium-dependent contraction [38]. The finding is consistent to our previous works that heat shock treatment results in an inhibition of myosin phosphatase through RhoA/ROCK signal [6]. Therefore, it is possible that RhoA/ROCK activation transduces heat shock-induced ROS production into the vasoconstriction. Further investigation is needed to fully understand the novel mechanism underlying heat shock/ROS-induced vasoconstriction. In conclusion this study demonstrated that heat shock augments AngII signal through the up-regulation of NADPH oxidase and ROS production. The heat-shock model has potential to study the vasoconstriction related to hypertension. With greater insights and understanding of direct relationship between heat shock and NADPH oxidase-derived ROS production and the augmented vascular contraction which may cause vascular damage and leading to hypertension, it can be possible to make target therapies more effective so that detrimental actions of vascular oxygen free radicals can
be reduced. This also may present an intriguing avenue for future investigations on stress-associated cardiovascular condition. Acknowledgments This work was supported by a grant from the National Research Foundation (R01-2008-000-12198-0) and the Brain Korea 21 Project in 2010, Ministry of Education, Science and Technology, Republic of Korea, and NIH RO1 HL083261 grant to Dr. Masumi Eto. References [1] S. Salinthone, M. Tyagi, W.T. Gerthoffer, Small heat shock proteins in smooth muscle, Pharmacol. Ther. 119 (2008) 44–54. [2] Y. Kim, J. Kim, M. Kim, W. Baek, I. Kim, Effect of heat shock on the vascular contractility in isolated rat aorta, J. Pharmacol. Toxicol. Methods 42 (1999) 171–174. [3] I.K. Kim, T.G. Park, Y.H. Kim, J.W. Cho, B.S. Kang, C.Y. Kim, Heat-shock response is associated with enhanced contractility of vascular smooth muscle in isolated rat aorta, Naunyn Schmiedebergs Arch. Pharmacol. 369 (2004) 402–407. [4] Y. Seok, J.I. Kim, M. Ito, Y. Kureishi, T. Nakano, S.O. Kim, D.G. Lim, W.H. Park, I. Kim, Heat shock-induced augmentation of vascular contractility is independent of rho-kinase, Clin. Exp. Pharmacol. Physiol. 33 (2006) 264–268. [5] A.P. Somlyo, A.V. Somlyo, Ca2+ sensitivity of smooth muscle and nonmuscle myosin II: modulated by G proteins, kinases, and myosin phosphatase, Physiol. Rev. 83 (2003) 1325–1358. [6] J.I. Kim, S.B. Jeon, I. Baek, Y.M. Seok, H.M. Shin, I.K. Kim, Heat shock augments myosin phosphatase target-subunit phosphorylation, Biochem. Biophys. Res. Commun. 356 (2007) 718–722. [7] R.M. Touyz, E.L. Schiffrin, Reactive oxygen species in vascular biology: implications in hypertension, Histochem. Cell Biol. 122 (2004) 339–352. [8] R.E. Clempus, K.K. Griendling, Reactive oxygen species signaling in vascular smooth muscle cells, Cardiovasc. Res. 71 (2006) 216–225. [9] T.M. Burke, M.S. Wolin, Hydrogen peroxide elicits pulmonary arterial relaxation and guanylate cyclase activation, Am. J. Physiol. 252 (1987) H721–H732. [10] G. Torrecillas, M.C. Boyano-Adanez, J. Medina, T. Parra, M. Griera, S. LopezOngil, E. Arilla, M. Rodriguez-Puyol, D. Rodriguez-Puyol, The role of hydrogen peroxide in the contractile response to angiotensin II, Mol. Pharmacol. 59 (2001) 104–112. [11] L. Zuo, F.L. Christofi, V.P. Wright, C.Y. Liu, A.J. Merola, L.J. Berliner, T.L. Clanton, Intra- and extracellular measurement of reactive oxygen species produced during heat stress in diaphragm muscle, Am. J. Physiol. Cell Physiol. 279 (2000) C1058–C1066. [12] M. Sakatani, S. Kobayashi, M. Takahashi, Effects of heat shock on in vitro development and intracellular oxidative state of bovine preimplantation embryos, Mol. Reprod. Dev. 67 (2004) 77–82. [13] M. Okada, N. Hasebe, Y. Aizawa, K. Izawa, J. Kawabe, K. Kikuchi, Thermal treatment attenuates neointimal thickening with enhanced expression of heat-shock protein 72 and suppression of oxidative stress, Circulation 109 (2004) 1763–1768. [14] Y.J. Lee, D.H. Lee, C.K. Cho, H.Y. Chung, S. Bae, G.J. Jhon, J.W. Soh, D.I. Jeoung, S.J. Lee, Y.S. Lee, HSP25 inhibits radiation-induced apoptosis through reduction of PKCdelta-mediated ROS production, Oncogene 24 (2005) 3715–3725. [15] L. Jin, Z. Ying, R.C. Webb, Activation of Rho/Rho kinase signaling pathway by reactive oxygen species in rat aorta, Am. J. Physiol. Heart Circ. Physiol. 287 (2004) H1495–H1500. [16] P.N. Seshiah, D.S. Weber, P. Rocic, L. Valppu, Y. Taniyama, K.K. Griendling, Angiotensin II stimulation of NAD(P)H oxidase activity: upstream mediators, Circ. Res. 91 (2002) 406–413. [17] A.M. Zafari, M. Ushio-Fukai, M. Akers, Q. Yin, A. Shah, D.G. Harrison, W.R. Taylor, K.K. Griendling, Role of NADH/NADPH oxidase-derived H2O2 in angiotensin IIinduced vascular hypertrophy, Hypertension 32 (1998) 488–495. [18] J.B. Laursen, S. Rajagopalan, Z. Galis, M. Tarpey, B.A. Freeman, D.G. Harrison, Role of superoxide in angiotensin II-induced but not catecholamine-induced hypertension, Circulation 95 (1997) 588–593. [19] A. Virdis, M.F. Neves, F. Amiri, R.M. Touyz, E.L. Schiffrin, Role of NAD(P)H oxidase on vascular alterations in angiotensin II-infused mice, J. Hypertens. 22 (2004) 535–542. [20] N.I.O. Health, Guide for the Care and Use of Laboratory Animals, NIH publication No. 85-23, revised ed. NIH, Bethesda, MD, 1996. [21] H. Zhao, S. Kalivendi, H. Zhang, J. Joseph, K. Nithipatikom, J. Vasquez-Vivar, B. Kalyanaraman, Superoxide reacts with hydroethidine but forms a fluorescent product that is distinctly different from ethidium: potential implications in intracellular fluorescence detection of superoxide, Free Radic. Biol. Med. 34 (2003) 1359–1368. [22] Z.Y. Jiang, J.V. Hunt, S.P. Wolff, Ferrous ion oxidation in the presence of xylenol orange for detection of lipid hydroperoxide in low density lipoprotein, Anal. Biochem. 202 (1992) 384–389. [23] Z.W. Yang, T. Zheng, A. Zhang, B.T. Altura, B.M. Altura, Mechanisms of hydrogen peroxide-induced contraction of rat aorta, Eur. J. Pharmacol. 344 (1998) 169–181.
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Heat shock augments angiotensin II-induced vascular contraction through increased production of reactive oxygen species Jee In Kim a,b, Sang Won Jung a, Enyue Yang a, Kwon Moo Park b, Masumi Eto c, In Kyeom Kim a,* a
Department of Pharmacology and Cardiovascular Research Institute, Kyungpook National University School of Medicine, Daegu 700-422, Republic of Korea Department of Anatomy and BK21, Kyungpook National University School of Medicine, Daegu 700-422, Republic of Korea c Department of Molecular Physiology and Biophysics, Thomas Jefferson University School of Medicine, 1020 Locust Street, 436 JAH., Philadelphia, PA 19107, USA b
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Article history: Received 26 July 2010 Available online 3 August 2010 Keywords: Vascular smooth muscle Heat shock Angiotensin II Reactive oxygen species NADPH oxidase Hydrogen peroxide
a b s t r a c t A temporal increase in temperature triggers a series of stress responses and alters vascular smooth muscle (VSM) contraction induced by agonist stimulation. Here we examined the role of reactive oxygen species (ROS) in heat shock-dependent augmentation of angiotensin II (AngII)-induced VSM contraction. Endothelium-denuded rat aortic rings were treated with heat shock for 45 min at 42 °C and then subjected to assays for the production of force, ROS, and the expression of ROS-related enzymes. AngIIinduced contraction was enhanced in heat shock-treated aorta. AngII-induced production of hydrogen peroxide and superoxide were elevated in response to the heat shock treatment. Pre-treatment with superoxide dismutases (SOD) mimetic and inhibitors for glutathione peroxidase and NADPH oxidase but not for xanthine oxidase eliminated an increase in the AngII-induced contraction in the heat shock-treated aorta. Heat shock increased the expression of p47phox, a cytosolic subunit of NADPH oxidase, but not Cu–Zn–SOD and Mn–SOD. In addition, heat shock increased contraction that was evoked by hydrogen peroxide and pyrogallol. These results suggest that heat shock causes an elevation of ROS as well as a sensitization of ROS signal resulting in an augmentation of VSM contraction in response to agonist. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction A drastic change in temperature is a challenge for our body, and produces acute stress response signals for the maintenance of homeostasis. Temporal increase in temperature under extreme environment causes changes in the expression of a large group of proteins, including various heat-shock proteins (HSPs). Studies suggest an involvement of HSPs in the regulation of vascular smooth muscle (VSM) contraction and relaxation through various kinases and signals [1]. Independently, we have reported that a temporal hyperthermic treatment at 42 °C increases the contraction of aortic VSM induced by high-K+ and phenylephrine [2–4]. The VSM contraction is determined by phosphorylation of myosin light chain (MLC), which is balanced by activities of MLC kinase (MLCK) and phosphatase (MLCP) [5]. In our previous study we showed that heat shock changed signal transduction in the control of MLC phosphorylation and VSM contraction through the inhibition of MLCP [6]. However, the underlying mechanisms how heat shock augments VSM contraction is not fully understood. Reactive oxygen species (ROS), including superoxide, hydrogen peroxide, and hydroxyl anion, nitric oxide, and peroxynitrite are * Corresponding author. Fax: +82 53 426 7345. E-mail address: [email protected] (I.K. Kim). 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.07.115
increasingly recognized as important mediators that control VSM contraction [7]. VSM cells contain numerous sources of ROS, such as the NADPH oxidases as the most dominant players, in addition to xanthine oxidase, the mitochondrial respiratory chain, lipoxygenases and nitric oxide synthases [8]. While an overproduction of ROS causes disorders in vascular bed, normal level of ROS production is involved in the regulation of VSM contraction and relaxation, depending on the tissues and conditions. For example, hydrogen peroxide induces the vasodilatation of pulmonary artery and the vasoconstriction of aorta strips [9,10]. Effect of heat stress on ROS production is controversial [11–14] and the process how heat stress affects ROS production remains to be elucidated. Interestingly, ROS induces vasoconstriction through Rho-kinase pathway in rat aorta that transduces agonist stimulation into the inhibition of MLCP [15]. Therefore, we hypothesized that heat shock enhances ROS production that causes an augmentation of agonist-induced contraction of aorta smooth muscle (SM). Of the numerous vasoactive agents regulating vascular NAD(P)H oxidase, AngII appears to be one of the most important agonist [16,17]. In rat aorta, AngII stimulates vasoconstriction via ROS-dependent mechanisms [10]. Accordingly, oxidative stress has been demonstrated in various models of experimental hypertension, including AngII-induced hypertension [18,19]. Here we showed that heat shock increases AngII-induced contraction of
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aorta that is mediated by increased ROS production, as well as the hyper-responsiveness to ROS in aorta SM contraction. 2. Materials and methods 2.1. Tissue preparation The present investigation is in accordance with Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health [20]. Male Sprague–Dawley rats, weighing 320–350 g, were used. With animals under anesthesia with sodium pentobarbital (50 mg kg1 i.p.), the thoracic aorta was excised and immersed in cold modified Krebs’ solution composed of (in mM) NaCl, 115.0; KCl, 4.7; CaCl2, 2.5; MgCl2, 1.2; NaHCO3, 25.0; KH2PO4, 1.2; and dextrose, 10.0. The aorta was cleaned of all adherent connective tissue and cut into 4 mm long ring segments as described previously [6]. All of the rings were denuded of endothelium by gentle rubbing the internal surface using a forceps. 2.2. Tension studies Two stainless-steel triangles were inserted through each vessel ring. One triangle was anchored to a stationary support, and the other was connected to an isometric force transducer (Grass FT03C, Quincy, MA, USA) in a water-jacketed organ bath maintained at 37 °C and aerated with a mixture of 95% O2 and 5% CO2. The rings were stretched passively by imposing the optimal resting tension of 2.0 g, maintained throughout the experiment. Each ring was equilibrated before tissue viability was tested with 50 mM KCl. Only the aortas showing above 2.0 g of contractile responses were used for further experiments. Tension was recorded using a computerized data acquisition system (PowerLab/8SP, ADInstruments, Castle Hill, NSW, Australia).
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2.5. ROS assay Superoxide level was assayed using dihydroethidium (DHE) that reacts with superoxide to form the red fluorescent adducts ethidium and oxy-ethidium [21]. Isolated rat aortic rings were loaded with 0.1 lM DHE for 20 min. DHE-loaded aortas were stimulated with 1 lM AngII for 4 min. Aortas were embedded in O.C.T. compound (Tissue-Tek, Sakura Finetek USA, Inc., Torrance, CA) to be frozen. Serial cryostat sections (10 lm thick) were deposited onto glass slides. Samples were air dried at 4 °C. Fluorescence laser scanning microscopy was conducted on a Zeiss Axiovert 100 M coupled with a Zeiss LSM510 laser scanning system (Germany). Red fluorescence was measured using Adobe Photoshop imager (Adobe systems Inc., USA). Hydrogen peroxide level was assayed using a ferric oxide-sensitive dye, xylenol orange, as described previously [22]. Aortas were stimulated with 1 lM AngII for 30 s and frozen immediately by immersion in liquid nitrogen. Frozen aortic rings were homogenized in a sucrose buffer (pH 7.5) containing 210 mM mannitol, 70 mM sucrose, 1 mM EDTA, and 10 mM hepes. Each sample was centrifuged at 13,000 rpm for 20 min at 4 °C. The protein concentration in the supernatant was measured by Bradford method using Bio-Rad Protein Assay kit (Bio-Rad, Hercules, CA, USA). Equal amount of protein samples (100 ll) were added to FOX reagent (900 ll) (25 mM H2SO4, 100 mM sorbitol, 2.5 mM ferrous ammonium sulfate, 0.1 mM xylenol orange) and incubated for 30 min at room temperature prior to measurement at 560 nm. Hydrogen peroxide oxidizes iron (II) to iron (III) in the presence of sorbitol, iron (III) then forms a purple complex with xylenol orange, which absorbs at 560 nm. The absorbance was measured using Ultraspec 2100 pro (Amersham pharmacia biotech, Piscataway, NJ, USA). 2.6. Materials
2.3. Heat shock Half of the strips were exposed to 42 °C for 45 min (heat-shock group) followed by returning to 37 °C whereas the other half (control group) were remained at 37 °C. Both of the groups were further incubated for 4 h before study. 2.4. Western blot analysis Aortic rings with various treatments were quickly frozen at proper time points by immersion in LN2. Stored aortic rings were homogenized in a lysis buffer containing 320 mM sucrose, 50 mM Tris, 1 mM EDTA, 1% Triton X-100, 1 mM DTT, and protease inhibitors leupeptin (10 lg/ml), trypsin inhibitor (10 lg/ml), aprotinin (2 lg/ml), phenylmethylsulfonyl fluoride (PMSF; 100 lg/ml), and phosphatase inhibitor b-glycerophosphate (50 mM). Proteinmatched samples (Bradford assay) were electrophoresed on 10% polyacrylamide gel with 0.1% SDS and transferred to nitrocellulose membranes, and then subjected to an immunoblot with p47phox antibody (1:1000, Upstate Biotechnology, Lake Placid, NY, USA). For the SODs, samples were electrophoresed on 12% gel then subjected to immunoblot with anti-Cu–Zn–SOD antibody (1:5000, Chemicon, Temecula, CA, USA) and Mn–SOD antibody (1:1000, Calbiochem, Darmstadt, Germany). Horseradish peroxidase-conjugated anti-rabbit IgG or anti-sheep IgG were used as secondary antibodies respectively (1:5000, Sigma, St. Louis, MO, USA). For the loading control anti-b actin antibody (1:2000, Sigma, St. Louis, MO, USA) was used. The protein bands were exposed with enhanced chemiluminescence reagents, visualized on X-ray films and quantified with image acquisition and analysis software; Lab Works program (Ultra-Violet Product Ltd.).
Angiotensin II, apocynin, glutathione peroxidase, allopurinol; (4-hydroxypyrazolo[3,4-d]pyrymidine), MnTMPyP; [manganese (III) tetrakis (N-methyl-2-pyridyl) porphyrin], H2SO4, sorbitol, xylenol orange; [o-cresolsulfonephthalein-30 -300 -bis-(methyliminodiacetic acid sodium salt)], ferrous ammonium sulfate, dihydroethidium, hydrogen peroxide solution and pyrogallol were obtained from Sigma Chemical Company (St. Louis, MO, USA). KCl was obtained from Junsei chemicals Co., Ltd. (Tokyo, Japan). All other reagents were of analytical grade or better.
2.7. Statistical analysis The results are expressed as mean ± SE of the mean which were analyzed by Student’s t-test. P values < 0.05 were regarded as statistically significant. 3. Results 3.1. Heat shock-augmented aorta SM contraction evoked by angiotensin II Previous studies show that the contraction of rat aorta SM in response to the stimulation with high K+ or phenylephrine is enhanced after temporal heat shock treatment (42 °C for 45 min) [3,6]. We further examined whether the heat shock treatment causes an augmentation of AngII-induced aorta SM contraction. The maximum force of heat shock-untreated aorta SM evoked by 1 lM AngII was set as 100%. The contraction of the heat shocktreated aorta SM reached 200% of the contraction of the heat shock-untreated tissues (Fig. 1). Thus, the signals in the AngII-in-
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Fig. 1. Angiotensin-induced contraction after heat shock. (A) Time course of heat shock and representatives of myographs in response to AngII in control and heat shocktreated aorta. (B) Control (open bar) and heat shock-treated (closed bar) aorta were stimulated with 1 lM AngII. SM contractility is expressed as the percentage of 1 lM AngIIinduced contraction in control. Graph shows that heat shock significantly augmented contractile response to AngII. Values are means ± SEM of six independent experiments (**P < 0.01 vs. control, Student’s t-test).
Fig. 2. ROS production by angiotensin and ROS-related protein expression following heat shock in isolated rat aorta. (A) Both control (open bars) and heat shock-treated (closed bars) aorta were stimulated with 1 lM AngII for 30 s and subjected to determination of hydrogen peroxide generation. Graph shows that heat shock significantly increased production of hydrogen peroxide by AngII. Values are means ± SEM of three independent experiments. (*P < 0.05, **P < 0.01 vs. vehicle-treated control, ##P < 0.01 vs. AngII-treated control, Student’s t-test). (B) Dihydroethidium (DHE)-loaded aortas were pre-incubated for 30 min either in the absence or presence of apocynin, followed by stimulation with 1 lM AngII for 4 min. AngII-produced superoxide in both control and heat shock-treated aortas was determined by fluorescence laser scanning micrographs (400 objective). Heat shock significantly increased AngII-induced superoxide production. Values are means ± SEM of five independent experiments. (*P < 0.05, **P < 0.01 vs. superoxide production by vehicle (ethanol) in control and heat shock-treated aorta respectively, ##P < 0.01 vs. AngII -induced superoxide production in control, Student’s ttest). (C) Representative immunoblots showing expression of a cytosolic subunit of NADPH oxidase p47phox, superoxide dismutases Cu–Zn–SOD and Mn–SOD in control and heat shock-treated aorta. b-Actin was used as a loading control. p47phox expression significantly increased after heat shock whereas Cu–Zn–SOD and Mn–SOD did not change. Values are means ± SEM of six independent experiments. (*P < 0.05, vs. control, Student’s t-test).
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Fig. 3. Inhibition of angiotensin-induced contraction by various ROS inhibitors. Both control and heat shock-treated aorta were pretreated with SOD mimetic MnTMPyP (30 lM), Glutathione peroxidase (GPx) (2.2 U), NADPH oxidase inhibitor apocynin (50 lM), xanthine oxidase inhibitor allopurinol (400 lM), for 30 min followed by stimulation with 1 lM AngII. MnTMPyP, GPx, apocynin inhibited heat shock-augmented contraction to the control level whereas allopurinol did not. Values are means ± SEM of five independent experiments. (*P < 0.05, **P < 0.01 vs. without pre-treatment of inhibitors, Student’s t-test).
duced contraction of aorta SM tissue are sensitive to heat shock treatment. 3.2. Involvement of ROS in augmentation of aorta SM contraction after heat shock Zuo et al. reported that heat shock treatment of skeletal muscle increases production of ROS [11]. We assayed the level of hydrogen peroxide and superoxide in rat aorta tissues after heat shock with or without AngII stimulation. The level of hydrogen peroxide was increased in the tissue after heat shock treatment under un-stimulated condition (Fig. 2A and B, vehicle). The level of both ROS was elevated in response to AngII stimulation in the heat shock-untreated control (Fig. 2A and B, AngII), as reported previously [16,17]. Importantly, AngII-induced elevation in ROS level was further augmented in response to heat treatment (Fig. 2, AngII, closed bars). The heat-shock-induced elevation of ROS production was accompanied with up-regulation of NADH oxidase (Fig. 2C). Heat shock treatment induced a 3-fold increase in the expression of p47phox, a cytosolic subunit of NADPH oxidase. On the other hand,
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the expression of superoxide dismutases Cu–Zn–SOD and Mn–SOD was not changed after heat shock treatment, suggesting NADPH oxidase through the overexpression of p47phox is involved in the heat shock-induced elevation of ROS. Next we examined whether the elevated ROS is involved in the augmentation of aorta SM contraction induced by AngII stimulation, using cell permeable scavengers for ROS, such as SOD mimetic MnTMPyP (30 lM), Glutathione peroxidase GPx (2.2 U), inhibitors for NADPH oxidase (50 lM apocynin), and xanthine oxidase (400 lM allopurinol) (Fig. 3). A 30 min pre-treatment with MnTMPyP or GPx, eliminated the heat shock-induced augmentation of the contraction indicating that superoxide and/or hydrogen peroxide mediate heat shock-induced augmentation of SM contraction. In consistent to the up-regulation of p47phox, the augmented contraction was eliminated by the pre-treatment with apocynin, but not with allopurinol, indicating that increased NADPH expression is the major source of the heat shock-dependent production of ROS. 3.3. Sensitization of ROS-induced contraction of aorta SM in response to heat shock Yang et al. reported that hydrogen peroxide induces the contraction of endothelium-denuded aorta ring that is attenuated by the addition of a PKC inhibitor [23]. We examined whether heat shock alters the sensitivity in contraction of aorta SM in response to ROS. Cumulative addition of hydrogen peroxide (Fig. 4A) or pyrogallol that generates superoxide (Fig. 4B) induced contraction of aorta SM under low potassium (KCl 10 mM) pretreated condition. The extent of ROS-induced contraction was significantly greater in heat shock-treated aorta (Fig. 4). These results suggest that heat shock treatment increases not only production of ROS but also SM contractility induced by ROS signal. Thus the dual effects of heat shock on ROS signal caused the augmentation of AngII-induced contraction of aorta SM. 4. Discussion The major finding of the present study is that heat shock treatment causes NADPH up-regulation and consequencing augmentation of AngII-induced SM contraction through the increased ROS production. Heat shock also enhanced ROS-induced SM contractility resulting in a synergy that amplifies the ROS signal. This augmentation of AngII signal is consistent to our previous results showing that heat shock treatment augments the vasoconstriction
Fig. 4. Hydrogen peroxide- or pyrogallol-induced vascular contraction after heat shock. Cumulative contractile response curves. Aortic rings were incubated with 10 mM KCl for 10 min then stimulated with cumulative concentration (3, 30, 300 lM) of hydrogen peroxide (A) or superoxide-producing pyrogallol (B). SM contractility is expressed as the percentage of 50 mM KCl-induced maximum contraction. Graphs show that heat shock significantly augmented contractile response to hydrogen peroxide and pyrogallol. Values are means ± SEM of five independent experiments (*P < 0.05 vs. control, Student’s t-test).
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of rat aorta rings induced by various stimuli, such as phenylephrine and KCl [3,6]. It is well documented that ROS, particularly superoxide (O) and hydrogen peroxide (H2O2), are important in AngIImediated signal transduction in VSM cells [24,25]. Indeed, ROS level is higher after heat treatment, and depletion of ROS with MnTMPyP or GPx significantly inhibited heat shock-augmented contraction. Thus, ROS production is driven in VSM under hyperthermic condition. Under physiological conditions, ROS are produced in a controlled manner at low concentrations and function as signaling molecules regulating the contraction/relaxation and growth of VSM cells [17,26–28]. ROS production is increased in pathological samples, such as cultured VSMC and isolated arteries from hypertensive rats and humans [28–30], causing the increased contractility [26]. Importantly, Schnackenberg et al. reported that the suppression of superoxide production attenuates the development of hypertension [29]. We expect that the heat-shock model will provide further insights into the role of ROS in controlling blood pressure. Activation of AT1 receptors acutely produces ROS through NADPH oxidase and activation of c-Src [8]. A NADPH oxidase inhibitor apocynin that [31] blocks association of p47phox with membrane-associated subunits p22phox and gp91phox [32,33], inhibits heat shock-augmented SM contraction. In contrast, a xanthine oxidase inhibitor allopurinol had no effect on the contraction even at 400 lM. Furthermore, the heat shock treatment indeed upregulated NADPH oxidase via p47phox expression. Thus, it is evident that heat shock treatment selectively activates NADPH oxidase but not xanthine oxidase. We presume that a series of HSPs stabilizes p47phox and activates NADPH oxidase pathway as a part of heat shock response. NADPH oxidase activation has been linked to cardiovascular diseases [25,32,34]. For instance, genetic models of hypertension, such as SHR [35] and stroke-prone SHR [30] exhibited enhanced NAD(P)H oxidase-mediated superoxide generation in aorta and mesentery arteries. These processes were associated with increased expression of NAD(P)H oxidase subunits, particularly p22phox and p47phox [25]. Apparently, heat shock treatment temporally produces a similar environment to genetic hypertension via NADPH up-regulation. ROS is known to increase aortic SM contraction [15,31,36,37]. Endothelium is also involved in ROS-induced vasoconstriction, through the cyclooxygenase pathway and/or inhibition of nitric oxide [31]. In our study the vascular endothelium is removed from the tissue, so that the ROS effect is independent of endothelium. Shen et al. reported that ROS activates P2X ATP receptor and causes the contraction of denuded aorta tissues [36]. Although ATP is not intentionally added to the bath, we cannot rule out the possibility of ATP production from the cells causing an activation of P2X receptor. In addition, recent evidence suggests that ROS activates RhoA/ROCK signal in SM. For example, Denniss et al. reported that ROS activated RhoA-Rho kinase in VSM preparation to enhance endothelium-dependent contraction [38]. The finding is consistent to our previous works that heat shock treatment results in an inhibition of myosin phosphatase through RhoA/ROCK signal [6]. Therefore, it is possible that RhoA/ROCK activation transduces heat shock-induced ROS production into the vasoconstriction. Further investigation is needed to fully understand the novel mechanism underlying heat shock/ROS-induced vasoconstriction. In conclusion this study demonstrated that heat shock augments AngII signal through the up-regulation of NADPH oxidase and ROS production. The heat-shock model has potential to study the vasoconstriction related to hypertension. With greater insights and understanding of direct relationship between heat shock and NADPH oxidase-derived ROS production and the augmented vascular contraction which may cause vascular damage and leading to hypertension, it can be possible to make target therapies more effective so that detrimental actions of vascular oxygen free radicals can
be reduced. This also may present an intriguing avenue for future investigations on stress-associated cardiovascular condition. Acknowledgments This work was supported by a grant from the National Research Foundation (R01-2008-000-12198-0) and the Brain Korea 21 Project in 2010, Ministry of Education, Science and Technology, Republic of Korea, and NIH RO1 HL083261 grant to Dr. Masumi Eto. References [1] S. Salinthone, M. Tyagi, W.T. Gerthoffer, Small heat shock proteins in smooth muscle, Pharmacol. Ther. 119 (2008) 44–54. [2] Y. Kim, J. Kim, M. Kim, W. Baek, I. Kim, Effect of heat shock on the vascular contractility in isolated rat aorta, J. Pharmacol. Toxicol. Methods 42 (1999) 171–174. [3] I.K. Kim, T.G. Park, Y.H. Kim, J.W. Cho, B.S. Kang, C.Y. Kim, Heat-shock response is associated with enhanced contractility of vascular smooth muscle in isolated rat aorta, Naunyn Schmiedebergs Arch. Pharmacol. 369 (2004) 402–407. [4] Y. Seok, J.I. Kim, M. Ito, Y. Kureishi, T. Nakano, S.O. Kim, D.G. Lim, W.H. Park, I. Kim, Heat shock-induced augmentation of vascular contractility is independent of rho-kinase, Clin. Exp. Pharmacol. Physiol. 33 (2006) 264–268. [5] A.P. Somlyo, A.V. Somlyo, Ca2+ sensitivity of smooth muscle and nonmuscle myosin II: modulated by G proteins, kinases, and myosin phosphatase, Physiol. Rev. 83 (2003) 1325–1358. [6] J.I. Kim, S.B. Jeon, I. Baek, Y.M. Seok, H.M. Shin, I.K. Kim, Heat shock augments myosin phosphatase target-subunit phosphorylation, Biochem. Biophys. Res. Commun. 356 (2007) 718–722. [7] R.M. Touyz, E.L. Schiffrin, Reactive oxygen species in vascular biology: implications in hypertension, Histochem. Cell Biol. 122 (2004) 339–352. [8] R.E. Clempus, K.K. Griendling, Reactive oxygen species signaling in vascular smooth muscle cells, Cardiovasc. Res. 71 (2006) 216–225. [9] T.M. Burke, M.S. Wolin, Hydrogen peroxide elicits pulmonary arterial relaxation and guanylate cyclase activation, Am. J. Physiol. 252 (1987) H721–H732. [10] G. Torrecillas, M.C. Boyano-Adanez, J. Medina, T. Parra, M. Griera, S. LopezOngil, E. Arilla, M. Rodriguez-Puyol, D. Rodriguez-Puyol, The role of hydrogen peroxide in the contractile response to angiotensin II, Mol. Pharmacol. 59 (2001) 104–112. [11] L. Zuo, F.L. Christofi, V.P. Wright, C.Y. Liu, A.J. Merola, L.J. Berliner, T.L. Clanton, Intra- and extracellular measurement of reactive oxygen species produced during heat stress in diaphragm muscle, Am. J. Physiol. Cell Physiol. 279 (2000) C1058–C1066. [12] M. Sakatani, S. Kobayashi, M. Takahashi, Effects of heat shock on in vitro development and intracellular oxidative state of bovine preimplantation embryos, Mol. Reprod. Dev. 67 (2004) 77–82. [13] M. Okada, N. Hasebe, Y. Aizawa, K. Izawa, J. Kawabe, K. Kikuchi, Thermal treatment attenuates neointimal thickening with enhanced expression of heat-shock protein 72 and suppression of oxidative stress, Circulation 109 (2004) 1763–1768. [14] Y.J. Lee, D.H. Lee, C.K. Cho, H.Y. Chung, S. Bae, G.J. Jhon, J.W. Soh, D.I. Jeoung, S.J. Lee, Y.S. Lee, HSP25 inhibits radiation-induced apoptosis through reduction of PKCdelta-mediated ROS production, Oncogene 24 (2005) 3715–3725. [15] L. Jin, Z. Ying, R.C. Webb, Activation of Rho/Rho kinase signaling pathway by reactive oxygen species in rat aorta, Am. J. Physiol. Heart Circ. Physiol. 287 (2004) H1495–H1500. [16] P.N. Seshiah, D.S. Weber, P. Rocic, L. Valppu, Y. Taniyama, K.K. Griendling, Angiotensin II stimulation of NAD(P)H oxidase activity: upstream mediators, Circ. Res. 91 (2002) 406–413. [17] A.M. Zafari, M. Ushio-Fukai, M. Akers, Q. Yin, A. Shah, D.G. Harrison, W.R. Taylor, K.K. Griendling, Role of NADH/NADPH oxidase-derived H2O2 in angiotensin IIinduced vascular hypertrophy, Hypertension 32 (1998) 488–495. [18] J.B. Laursen, S. Rajagopalan, Z. Galis, M. Tarpey, B.A. Freeman, D.G. Harrison, Role of superoxide in angiotensin II-induced but not catecholamine-induced hypertension, Circulation 95 (1997) 588–593. [19] A. Virdis, M.F. Neves, F. Amiri, R.M. Touyz, E.L. Schiffrin, Role of NAD(P)H oxidase on vascular alterations in angiotensin II-infused mice, J. Hypertens. 22 (2004) 535–542. [20] N.I.O. Health, Guide for the Care and Use of Laboratory Animals, NIH publication No. 85-23, revised ed. NIH, Bethesda, MD, 1996. [21] H. Zhao, S. Kalivendi, H. Zhang, J. Joseph, K. Nithipatikom, J. Vasquez-Vivar, B. Kalyanaraman, Superoxide reacts with hydroethidine but forms a fluorescent product that is distinctly different from ethidium: potential implications in intracellular fluorescence detection of superoxide, Free Radic. Biol. Med. 34 (2003) 1359–1368. [22] Z.Y. Jiang, J.V. Hunt, S.P. Wolff, Ferrous ion oxidation in the presence of xylenol orange for detection of lipid hydroperoxide in low density lipoprotein, Anal. Biochem. 202 (1992) 384–389. [23] Z.W. Yang, T. Zheng, A. Zhang, B.T. Altura, B.M. Altura, Mechanisms of hydrogen peroxide-induced contraction of rat aorta, Eur. J. Pharmacol. 344 (1998) 169–181.
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