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Effect of oxidative stress on Rho kinase II and smooth muscle contraction in rat stomach Othman Al-Shboul and Ayman Mustafa
Abstract: Recent studies have shown that both Rho kinase signaling and oxidative stress are involved in the pathogenesis of a number of human diseases, such as diabetes mellitus, hypertension, and atherosclerosis. However, very little is known about the effect of oxidative stress on the gastrointestinal (GI) smooth muscle Rho kinase pathway. The aim of the current study was to investigate the effect of oxidative stress on Rho kinase II and muscle contraction in rat stomach. The peroxynitrite donor 3-morpholinosydnonimine (SIN-1), hydrogen peroxide (H2O2), and peroxynitrite were used to induce oxidative stress. Rho kinase II expression and ACh-induced activity were measured in control and oxidant-treated cells via specifically designed enzymelinked immunosorbent assay (ELISA) and activity assay kits, respectively. Single smooth muscle cell contraction was measured via scanning micrometry in the presence or absence of the Rho kinase blocker, Y-27632 dihydrochloride. All oxidant agents significantly increased ACh-induced Rho kinase II activity without affecting its expression level. Most important, oxidative stress induced by all three agents augmented ACh-stimulated muscle cell contraction, which was significantly inhibited by Y-27632. In conclusion, oxidative stress activates Rho kinase II and enhances contraction in rat gastric muscle, suggesting an important role in GI motility disorders associated with oxidative stress. Key words: smooth muscle, contraction, gastrointestinal, Rho kinase, oxidative stress. Résumé : Des études récentes ont montré que la signalisation par la Rho kinase et le stress oxydant sont impliqués dans la pathogenèse d’un certain nombre de maladies chez l’humain, comme le diabète sucré, l’hypertension et l’athérosclérose. Cependant, on en connaît peu sur l’effet du stress oxydant sur la voie de signalisation de la Rho kinase dans le muscle lisse gastro-intestinal (GI). Le but de cette étude était d’examiner l’effet du stress oxydant sur la Rho kinase II et la contraction musculaire dans l’estomac de rat. La 3-morpholinosydnonimine (SIN-1), un donneur de peroxynitrite, le peroxyde d’hydrogène (H2O2) et le peroxynitrite ont été utilisés pour induire un stress oxydant. L’expression de la Rho kinase II et son activité induite par l’ACh ont été mesurées dans ces cellules contrôles et des cellules soumises au stress oxydant a` l’aide d’un dosage ELISA spécifiquement conçu et d’une trousse de dosage d’activité, respectivement. La contraction de cellules musculaires uniques a été mesurée par micrométrie a` balayage en présence ou en absence d’un bloqueur de Rho kinase, le di-hydrochlorure de Y-27632. Tous les agents oxydants augmentaient l’activité de la Rho kinase induite par l’ACh, sans affecter son niveau d’expression. Qui plus est, le stress oxydant induit par les trois agents augmentait la contraction cellulaire musculaire stimulée par l’ACh, phénomène inhibé par le Y-27632 de manière significative. En conclusion, le stress oxydant active la Rho kinase II et accroit la contraction du muscle gastrique de rat, ce qui suggère qu’il jouerait un rôle important dans les troubles liés a` la motilité GI associés au stress oxydant. [Traduit par la Rédaction] Mots-clés : muscle lisse, contraction, gastro-intestinal, Rho kinase, stress oxidant.
Introduction Contraction of smooth muscle is regulated by both Ca2+dependent and Ca2+-independent (Ca2+ sensitization) mechanisms (Somlyo and Somlyo 1994). A rise in intracellular Ca2+ levels leads to myosin light chain (MLC) kinase activation, resulting in an increase in MLC phosphorylation. Importantly, MLC phosphorylation can also be increased through inhibition of MLC phosphatase, which augments smooth muscle force generation without a change in intracellular Ca2+ (Kitazawa et al. 1991). Recent studies have revealed that Rho kinase II, a serine/threonine kinase downstream of the small G protein RhoA, is important in developing smooth muscle tone. It inhibits MLC phosphatase activity by phosphorylation of the MLC phosphatase target subunit (MYPT1). A decrease in MLC phosphatase activity maintains phosphorylation of myosin and therefore contributes to smooth muscle contraction at low levels of intracellular Ca2+ concentration (Somlyo and Somlyo
2000). The importance of the RhoA/Rho kinase pathway has been demonstrated in the pathogenesis of cardiovascular disorders and as a target in the development of new drugs such as fasudil, a Rho kinase inhibitor (Hahmann and Schroeter 2010; Mills et al. 2001; Uehata et al. 1997). Several disease states or pathological situations in the GI tract are well-known to affect smooth muscle contractile function and many of these are characterized by the formation of reactive oxygen species (ROS). Free radical-induced oxidative stress has been involved in the pathogenesis of a number of human diseases such as diabetes mellitus, hypertension, and atherosclerosis (Buttery et al. 1996; Grunfeld et al. 1995; Kamata and Kobayashi 1996). Oxidative stress is associated with enhanced production of ROS, in particular superoxide anion that is normally converted to (H2O2) by the action of superoxide dismutase (SOD). H2O2 in turn is metabolized to H2O via the antioxidant enzymes; catalase and glutathione peroxidase. However, in pathological states the balance
Received 9 December 2014. Accepted 16 January 2015. O. Al-Shboul. Department of physiology and biochemistry, Faculty of Medicine, Jordan University of Science and Technology, Irbid 22110, Jordan. A. Mustafa. Department of anatomy, Faculty of Medicine, Jordan University of Science and Technology, Irbid 22110, Jordan. Corresponding author: Othman Abdullah Al-Shboul (e-mail: [email protected]). Can. J. Physiol. Pharmacol. 93: 405–411 (2015) dx.doi.org/10.1139/cjpp-2014-0505
Published at www.nrcresearchpress.com/cjpp on 28 January 2015.
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between synthesis and metabolism of reactive oxygen species (ROS) may be impaired, thus leading to generation of oxidative stress (Yu 1994). Superoxide anion can elicit direct vasoconstriction and scavenge nitric oxide, the most potent endogenous vasodilator, to produce peroxynitrite (·OONO–), which oxidizes proteins, breaks DNA strands, and reduces intracellular antioxidants such as glutathione and cysteine (Bayraktutan 2002; Gryglewski et al. 1986). Generation of superoxide anion as a mechanism of damage is well established in different models of acute and chronic vascular and pulmonary injuries, however very little is known about its effect on GI smooth muscle contractility (Farinati et al. 1996; Keshavarzian et al. 1992; Parks et al. 1982). This study will investigate the effect of oxidative stress induced by various oxidant agents on Rho kinase II, the smooth muscle predominant isoform (Murthy et al. 2001) and smooth muscle contraction in rat stomach. Insights into the molecular basis of abnormal smooth muscle function will prove invaluable in the treatment of the GI motility disorders associated with oxidative stress.
Materials and methods Preparation of dispersed gastric smooth muscle cells Smooth muscle cells were isolated from the circular muscle layer of the rat stomach by sequential enzymatic digestion, filtration, and centrifugation as described previously (Murthy et al. 1996; Murthy and Makhlouf 1995). Briefly, strips of circular muscle from the stomach were dissected and incubated at 31 °C for 30 min in HEPES medium containing 120 mmol/L NaCl, 4 mmol/L KCl, 2.0 mmol/L CaCl2, 2.6 mmol/L KH2PO4, 0.6 mmol/L MgCl2, 25 mmol/L HEPES, 14 mmol/L glucose, 2.1% Eagle’s essential amino acid mixture, 0.1% collagenase, and 0.01% soybean trypsin inhibitor. The tissue was continuously gassed with 100% oxygen during the entire isolation procedure. After the partly digested strips were washed twice with 50 mL of enzyme-free medium, the muscle cells were allowed to disperse spontaneously for 30 min. The cells were harvested by filtration through 500 m Nitex mesh and centrifuged twice at 350g for 10 min to eliminate broken cells and organelles. The cells were counted in a hemocytometer, and it is estimated that 95% of the cells excluded trypan blue. All the experiments were done within 2–3 h of cell dispersion. Identification of smooth muscle cells The smooth muscle identity of rat gastric muscle cells was verified by immunohistochemical staining of paraffin-embedded rat smooth muscle using anti-calponin antibody (ab46794) at 1/100 dilution. Oxidative stress treatment Aliquots (0.4 mL) of dispersed gastric smooth muscle cells were prepared. The aliquots were randomly distributed into either control or oxidant treatment groups. Aliquots designated for oxidant treatment were incubated with either SIN-1 (1 mmol/L) for 10 min, H2O2 (500 mol/L) for 10 min, or peroxynitrite (1 mmol/L) for 10 min. Samples nominated for antioxidant treatment were preincubated with tempol (1 mmol/L) 5 min prior to SIN-1 treatment. Cells were stimulated with ACh (1 mol/L) for 10 min in the presence or absence of oxidant treatment. Detection of nitrotyrosine levels in gastric smooth muscle cells SIN-1-treated dispersed gastric smooth muscle cells were collected to measure nitrotyrosine, a marker of peroxynitrite. The samples were homogenized on ice in 1× PBS, pH 7.4. The homogenates were centrifuged at 20 000g (4 °C for 10 min). Supernatants were used for the measurement of nitrotyrosine concentrations with a 3-Nitrotyrosine ELISA Kit from Abcam according to the manufacturer’s instructions. Nitrotyrosine concentrations were
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normalized per milligram of protein. Protein concentrations were measured using the Dc protein assay kit from Bio-Rad. Measurement of Rho kinase II activity Rho kinase II activity was analyzed by an enzyme immunoassay, using Cell Biolabs’ 96-well Rho kinase II activity assay kit. Experiments were done according to the manufacture’s protocol, using 10 L of protein lysate. The total starting protein concentration for every sample was 1 mg/mL. Measurement of Rho kinase II expression Muscle cells were solubilized in Triton X-100-based lysis buffer plus protease and phosphatase inhibitors (100 g/mL phenylmethanesulfonylfluoride (PMSF), 10 g/mL aprotinin, 10 g/mL leupeptin, 30 mmol/L sodium fluoride, and 3 mmol/L sodium vanadate). After centrifugation of the lysates at 20 000g for 10 min at 4 °C, the protein concentrations of the supernatant were determined with a Dc protein assay kit from Bio-Rad. Samples of equal amounts of proteins were quantitated by ELISA according to the manufacturers’ instructions. Measurement of contraction in dispersed smooth muscle cells Contraction in freshly dispersed gastric circular smooth muscle cells was determined by scanning micrometry (Huang et al. 2007). Aliquots (0.4 mL) of dispersed gastric smooth muscle cells containing approximately 104 cells/mL were prepared. The aliquots were randomly distributed into either control or oxidant treatment groups. Aliquots designated for oxidant treatment were incubated with ether SIN-1 (1 mmol/L) for 10 min, H2O2 (500 mol/L) for 10 min, or peroxynitrite (1 mmol/L) for 10 min. Cells were stimulated with ACh (1 mol/L) for 10 min in the presence or absence of oxidant treatment, and the reaction was terminated with 1% acrolein at a final concentration of 0.1%. Acrolein kills and fixes cells without affecting the cell length. The cells were viewed using a 20× or 40× objective of inverted Nikon TMS-f microscope, and cell images were acquired using a Canon digital camera and image acquisition software. The resting cell length was determined in a control group in which muscle cells were not treated with ACh. The mean length of at least 50 muscle cells was measured by image-J software from each group. The contractile response was defined as the decrease in the average length of at least 50 cells and was expressed as the percent change relative to control length. Materials Male Sprague Dawley (SD) rats were provided by the animal house of Jordan University of Science and Technology (JUST). Male SD rats (6 weeks of age, 200–250 g) were sacrificed using an overdose of ether. Assays were performed using a Rho kinase II assay kit (Cell Biolabs, INC., California, USA), a Rho kinase II ELISA kit (Cusabio Biotech, Newark, Delaware, USA), a 3-Nitrotyrosine ELISA Kit (ab116691) (Abcam Inc., Cambridge, Massachusetts, USA), and a Dc protein assay kit (Bio-Rad, Hercules, California, USA). The Anticalponin antibody (ab46794) was purchased from Abcam. The 500 m Nitex mesh was purchased from Amazon. Powdered tempol and Y-27632 dihydrochloride (Rho kinase inhibitor) were purchased from Santa Cruz Biotech(Santa Cruz, California, USA); sc-1616 and sc-281642 respectively. All remaining materials were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). Tempol was dissolved and diluted in 99% ethanol, Y-27632 dihydrochloride was dissolved in DMSO, while SIN–1 and ACh were dissolved in distilled water. Analysis of data Each experiment was performed on gastric smooth muscle cells that were harvested from 6 rats. Statistical analysis of all experiments was performed using Prism 5.0 software (GraphPad Software, San Diego, California, USA). Unpaired Student's t-test was used to reveal significant differences between the compared groups For the Rho kinase II activity experiments in SIN-1-treated Published by NRC Research Press
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Fig. 1. Identification of rat gastric smooth muscle cells. (A) Immunohistochemical staining of paraffin-embedded rat smooth muscle using ab46794 at 1/100 dilution. (B) Freshly isolated gastric smooth muscle cells appeared in spindle shape with diverse lengths under phase contrast microscopy.
samples pre-incubated with tempol, a one-way analysis of variance (ANOVA) was performed followed by Fisher’s post-hoc analysis to determine the significance of differences between experimental groups. A P < 0.05 was required for statistical significance in all the experiments. All data are shown as mean ± SEM.
Fig. 2. Effect of SIN-1 on nitrotyrosine levels in gastric smooth muscle cells. Levels of nitrotyrosine were measured in gastric smooth muscle cells using 3-nitrotyrosine assay kit and expressed as absorbance at 450 nm. SIN-1 treatment increased the levels of 3-nitrotyrosine. Values are means ± SEM of 4 experiments. (*P < 0.05 vs. control by unpaired t test). SIN-1, 3-morpholinosydnonimine.
Results Smooth muscle identity The smooth muscle identity of rat gastric muscle cells was verified by immunohistostaining with anti-calponin antibody. The results showed that greater than 95% of cells stained positive for calponin (Fig. 1A). Freshly isolated gastric smooth muscle cells appeared in spindle shape with diverse lengths under phase contrast microscopy (Fig. 1B). SIN-1-induced 3-nitrotyrosine formation To determine the effectiveness of SIN-1 treatment, we assessed the formation of peroxynitrite in both SIN-1-treated and control smooth muscle cells via quantitative measurement of 3-nitrotyrosine. SIN-1 significantly increased 3-nitrotyrosine levels in oxidant-treated cells compared to control (P < 0.05, n = 6) (Fig. 2). These results indicate peroxynitrite participation in nitrotyrosylation. Rho kinase II activity Treatment of freshly dispersed muscle cells with 1 mol/L ACh, a G␣q/13-coupled receptor agonist, for 10 min significantly increased Rho kinase II activity above basal level (P < 0.05, n = 6) (Fig. 3A, second column). Incubation of gastric smooth muscle cells with 1 mmol/L SIN-1, 500 mol/L H2O2, or 1 mmol/L peroxynitrite for 10 min significantly augmented ACh-stimulated Rho ki-
nase II activity (P < 0.05, n = 6) (Fig. 3A). The three oxidant agents had no effect on basal Rho kinase II activity (data not shown). To examine whether the enhanced ACh-stimulated Rho kinase II activity is indeed due to peroxynitrite generation in SIN-1-treated group, we pre-incubated gastric muscle cells with 1 mmol/L tempol, a peroxynitrite scavenger, 5 min prior to SIN-1 treatment. SIN-1-induced ACh-stimulated Rho kinase II activity was blocked by the presence of tempol (P < 0.05, n = 6) (Fig. 3B, fourth column). Published by NRC Research Press
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Fig. 3. Effect of SIN-1 (1 mmol/L for 10 min), H2O2 (500 mol/L for 10 min), and peroxynitrite (1 mmol/L for 10 min) incubation on ACh-induced (1 mol/L) of Rho kinase II activity in rat gastric smooth muscle. Rho kinase II activity is expressed as absorbance at 450 nm. (A) Note that the ACh-induced activation of Rho kinase II was significantly augmented by the 3 oxidant treatments. (B) Pre-treatment of cells with tempol (1 mmol/L) significantly blocked SIN-1-induced ACh-stimulated Rho kinase II activity (fourth column). Values are means ± SEM of 4 experiments. (*P < 0.05 vs. control basal; **P < 0.05 vs. control ACh; †P < 0.05 vs. SIN-1 + Ach (one way ANOVA followed by Bonferroni multiple comparison test). ACh, acetylcholine; H2O2, hydrogen peroxide; SIN-1, 3-morpholinosydnonimine.
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Fig. 4. Effect of SIN-1 (1 mmol/L for 10 min), (H2O2) (500 mol/L for 10 min), and peroxynitrite (1 mmol/L for 10 min) incubation on the expression levels of Rho kinase II protein in rat gastric smooth muscle. Rho kinase II protein expression level is expressed as absorbance at 450 nm. Note that all oxidant treatments didn’t affect the expression level of Rho kinase II protein. Values are means ± SEM of 4 experiments. (P > 0.05 by unpaired t test). SIN-1, 3-morpholinosydnonimine.
contraction in all oxidant treatment groups (Fig. 5), showing clearly that oxidative stress induced by SIN-1, H2O2, and peroxynitrite augments muscle contraction via pathways that are dependent on Rho kinase II activation.
Discussion
These results suggest that stimulation of Rho kinase II activity in stomach muscle cells is mediated by SIN-1-induced peroxynitrite formation. Rho kinase II expression To determine whether the effect of these various oxidant agents on Rho kinase II expression profile correlates with their effect on the kinase activity level, we compared the Rho kinase II protein level in SIN-1-, H2O2–, and peroxynitrite-treated versus control cells by ELISA. Despite the higher agonist-stimulated Rho kinase II activity in all oxidant-treated cells compared to control, the expression of Rho kinase II protein was not different in all groups of cells (P > 0.05) (Fig. 4). This raises the possibility that oxidative stress-induced Rho kinase II activity might be due to an effect on other upstream regulators of the enzyme such as RhoA. Smooth muscle contraction We further examined whether oxidant treatment was associated with changes in agonist-induced contraction and whether these changes, if any, are due to increased Rho kinase II activity level. For that reason, freshly dispersed gastric muscle cells from control and oxidant-treated groups were treated with 1 mol/L ACh and 1 mol/L Y-27632 (a highly potent Rho kinase inhibitor), and the decrease in muscle cell length was measured by scanning micrometry. Basal muscle cell lengths were similar in control and oxidant-treated groups. ACh caused contraction that was concentration-dependent in muscle cells from both control and oxidant-treated cells (Fig. 5). However, contraction in response to ACh was significantly higher in oxidant-treated cells compared to control cells (Fig. 5). Most importantly, pre-incubation with Y-27632, a selective inhibitor of Rho kinase that competes with ATP for its binding site on Rho kinase (Ishizaki et al. 2000), significantly inhibited ACh-stimulated muscle
Recent studies have suggested that both free radical-induced oxidative stress and Rho/Rho kinase signaling affect smooth muscle contractility and may contribute to the pathogenesis of a number of human diseases, such as diabetes mellitus, hypertension, and atherosclerosis (Buttery et al. 1996; Grunfeld et al. 1995; Kamata and Kobayashi 1996). Very little is known about the effect of oxidative stress on Rho kinase pathway and smooth muscle contraction in the GI tract. In the present study, we demonstrate that oxidative stress induced by the peroxynitrite-donor, SIN-1, H2O2, or peroxynitrite activates Rho kinase II and enhances agonistinduced contraction of dispersed gastric smooth muscle cells. There is an accumulating body of evidence that ROS-induced oxidative stress is involved in the increase of smooth muscle contractile function that might be associated with some disorders. For example, Gumusel et al. (1996) reported that in isolated rat aortic rings, the contractile response to phenylephrine was potentiated when the bathing solution was subjected to electrolysis. Moreover, previous studies in several species have demonstrated that exogenous application of H2O2 augments contraction in airway smooth muscle of humans (Rabe et al. 1995) as well as of rats (Schmidt 1990), dogs (Levin et al. 1981), cows, and other species, suggesting that H2O2 induces airway hyper-responsiveness and airflow limitation in patients with asthma or chronic obstructive pulmonary disease. In addition, Gao and Lee (2001) found that hydrogen peroxide induces a greater contraction in mesenteric arteries of spontaneously hypertensive rats through thromboxane A2 production. Our results are in support of these previous studies. We found that H2O2 significantly elevated ACh-induced gastric muscle contraction. Most importantly, H2O2-induced enhancement in muscle contraction was Rho kinase II-dependent, as it was inhibited by Y-27632, a Rho kinase II inhibitor. Other researchers found that administration of anti-oxidants or inhibition of ROS-generating enzymes normalizes or reduces elevated blood pressure and improves vascular function (Fukui et al. 1997; Laursen et al. 1997; Rajagopalan et al. 1996). Moreover, recent studies have shown that both blood pressure and superoxide production are significantly lower in knockout mice lacking selected NADPH oxidase subunits than in wild-type mice (Landmesser et al. 2002; Wang et al. 2001). Parallel to Published by NRC Research Press
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Fig. 5. Effect of SIN-1 (1 mmol/L for 10 min), (H2O2) (500 mol/L for 10 min), and peroxynitrite (1 mmol/L for 10 min) incubation on ACh-induced muscle contraction. Contraction of dispersed muscle cells from stomach was measured by scanning micrometry in response to 1 mol/L ACh. Cells were treated with ACh for 10 min and contraction was expressed as the percent change relative to control length. ACh lead to contraction in both control and treated groups. Contraction was significantly augmented in cells from the three oxidant-treated groups. ACh-induced contraction was significantly decreased by the Rho kinase II inhibitor Y27632. Values represent the means ± SEM. (*P < 0.05 vs. control ACh; †P < 0.05 vs. SIN-1 + ACh; €P < 0.05 vs. H2O2 + ACh; ¥P < 0.05 vs. peroxynitrite + ACh).
previous findings, free radicals were found to impair endotheliumdependent relaxation and increase the tone of arteries by reducing NO bioavailability (MacKenzie and Martin 1998). Interestingly, ROS also contract vessels after removal of endothelium, suggesting that ROS have direct effect on smooth muscle (Auch-Schwelk et al. 1989; Rodriguez-Martinez et al. 1998; Yang et al. 1999). Our findings of increased gastric muscle contraction via oxidative stress indeed reinforce ROS-induced elevation of muscle contraction reported in previous researches. In our research, we induced oxidative stress by incubating dispersed gastric smooth muscle cells with the peroxynitrite donor, SIN–1, H2O2, or peroxynitrite. It is well-known that during its decomposition, SIN-1 releases superoxide anion and nitric oxide, and these in turn spontaneously combine to form a highly reactive molecule, peroxynitrite (Beckman et al. 1990; Daiber et al. 2005). In physiological conditions, oxidative damage is minimized by endogenous anti-oxidant defenses (Forstermann 2010). However, in pathological conditions nitric oxide production drastically increases, and the produced nitric oxide competes with the SODs for superoxide anion to form peroxynitrite. Peroxynitrite can attack biological molecules such as lipids, DNA, and proteins via direct oxidative reactions or indirect radical-mediated mechanisms (Pacher et al. 2007). Interestingly, protein nitration is considered a characteristic of peroxynitrite generation and leads to inactivation of the tyrosine residue of a protein forming 3-nitrotyrosine. This process of protein modification has been detected in human atherosclerotic lesions using anti-nitrotyrosine antibodies (Luoma et al. 1998; Pennathur et al. 2004). In addition to nitration, peroxynitrite is implicated in tissue injury via lipid peroxidation (Szabo et al. 2007). We confirmed the effectiveness of SIN-1 treatment and demonstrated, using ELISA method, that SIN-1 indeed induced peroxynitrite generation by increasing 3-nitrotyrosine formation in gastric smooth muscle cells. Knowing the importance of Rho kinase II signaling pathway in increasing calcium sensitization and smooth muscle contraction, as being an upstream inhibitor of MLC phosphatase, our next aim was to explore the correlation between SIN-1-induced nitrotyrosylation, H2O2, and peroxynitrite treatment and Rho kinase II activity. Consistent with many previous studies (Fukata et al. 2001; Huang et al. 2005; Murthy et al. 2003), treatment of muscle cells with ACh significantly increased Rho kinase II activity. Most importantly, we found that pre-incubating gastric muscle cells with the oxidant agents used in this study, leads to significant enhancement in ACh-stimulated Rho kinase II activity and muscle contraction.
For the most part, this enhanced muscle contraction was partly dependent on Rho kinase II pathway, as it was significantly inhibited by Y27632, the Rho kinase II inhibitor. Although higher concentrations of Y27632 could also inhibit PKC, previous studies confirmed that Y27632 at 1 mol/L is highly selective for Rho kinase (Jin et al. 2004; Murthy et al. 2003). Current findings support those previously reported in the literature at various smooth muscle locations in the body. For example, Jin et al. (2004) found that ROS generated by a xanthine/xanthine oxidase mixture stimulates smooth muscle contraction in endothelium-denuded rat aorta. This reported vasoconstriction was mediated through activation of Rho and a subsequent increase in Rho kinase activity. Moreover, oxidative stress with H2O2 has been suggested to mediate airway smooth muscle contraction by increasing intracellular Ca2+ level and stimulating the Rho/Rho kinase pathway (Snetkov et al. 2011). Mishra et al. (2011) reported also that enhanced vascular reactivity of omental arteries in pre-eclampsia is attributed to ROS activation of the RhoA kinase pathway, and that the enhanced vascular reactivity is likely attributed to the infiltration of neutrophils. In addition, Shimokawa (2000) and colleagues revealed that RhoA and its downstream effector, Rho kinase, are involved in the hypercontraction of vascular smooth muscle of the coronary artery in vasospastic angina, which is probably mediated by oxidative stress. Very recently, one attractive study reported a SIN-1-induced enhancement of endothelial arginase activity. They concluded that oxidative species increase arginase activity through PKC-activated RhoA/Rho kinase pathway (Chandra et al. 2012). In regard to SIN-1 results, we aimed to further prove that the effect on Rho kinase was indeed due to nitration induced by SIN-1 treatment. So we pre-incubated gastric smooth muscle cells with the anti-oxidant tempol. Tempol is a well-known peroxynitrite scavenger that protects against oxidative stress (Bonini et al. 2002; Wilcox and Pearlman 2008). Moreover, tempol has been shown to effectively suppress pathological conditions associated with marked oxidative stress in vivo, such as hypertension (Elmarakby et al. 2007), experimental colitis (Karmeli et al. 1995), and cardiac reperfusion injury (Gelvan et al. 1991). Our data showed that tempol significantly reduced SIN-1-induced Rho kinase II activity. Interestingly, a group of researchers found that tempol decreases RhoA, the upstream activator of Rho kinase II, activation via reducing SIN-1-induced nitration of p190A in endothelial cells (Siddiqui et al. 2011). In addition, tempol was found to be efficient in reducing xanthine/xanthine oxidaseinduced contraction in rat aorta (Jin et al. 2004). Our findings with Published by NRC Research Press
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tempol strongly suggest that the increase in Rho kinase II activity is indeed due to SIN-1-induced peroxynitrite formation. Although pre-incubation of gastric smooth muscle cells with SIN-1, H2O2, or peroxynitrite enhanced ACh-stimulated Rho kinase II activity, protein expression level of Rho kinase II was not affected by various oxidant treatments. This raises the possibility that the increase in Rho kinase II activity associated with these different oxidant treatments may be caused by an effect of oxidative stress on other regulators of Rho kinase II such as Rho A, PKC, and other effectors. In conclusion, this study demonstrates the direct activation of Rho kinase II enzyme and thus contraction by ROS in gastric smooth muscle, suggesting an important role for ROS-mediated Rho/Rho kinase II pathway activation in GI motility disorders associated with oxidative stress. An understanding of the oxidative stress-induced smooth muscle changes at the intracellular level is important in the pathophysiology and may provide new insights for the development of therapeutic agents that should act on smooth muscle in the gut to treat motility disorders, as well as in other regions such as airways and vascular smooth muscle where similar intracellular mechanisms may prevail.
Acknowledgements This work was supported by Jordan University of Science & Technology, Irbid, Jordan (Grant No. 51/2012). The authors thank Dr. Nayef Al-Gharaibeh for help and providing laboratory facilities.
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ARTICLE
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Effect of oxidative stress on Rho kinase II and smooth muscle contraction in rat stomach Othman Al-Shboul and Ayman Mustafa
Abstract: Recent studies have shown that both Rho kinase signaling and oxidative stress are involved in the pathogenesis of a number of human diseases, such as diabetes mellitus, hypertension, and atherosclerosis. However, very little is known about the effect of oxidative stress on the gastrointestinal (GI) smooth muscle Rho kinase pathway. The aim of the current study was to investigate the effect of oxidative stress on Rho kinase II and muscle contraction in rat stomach. The peroxynitrite donor 3-morpholinosydnonimine (SIN-1), hydrogen peroxide (H2O2), and peroxynitrite were used to induce oxidative stress. Rho kinase II expression and ACh-induced activity were measured in control and oxidant-treated cells via specifically designed enzymelinked immunosorbent assay (ELISA) and activity assay kits, respectively. Single smooth muscle cell contraction was measured via scanning micrometry in the presence or absence of the Rho kinase blocker, Y-27632 dihydrochloride. All oxidant agents significantly increased ACh-induced Rho kinase II activity without affecting its expression level. Most important, oxidative stress induced by all three agents augmented ACh-stimulated muscle cell contraction, which was significantly inhibited by Y-27632. In conclusion, oxidative stress activates Rho kinase II and enhances contraction in rat gastric muscle, suggesting an important role in GI motility disorders associated with oxidative stress. Key words: smooth muscle, contraction, gastrointestinal, Rho kinase, oxidative stress. Résumé : Des études récentes ont montré que la signalisation par la Rho kinase et le stress oxydant sont impliqués dans la pathogenèse d’un certain nombre de maladies chez l’humain, comme le diabète sucré, l’hypertension et l’athérosclérose. Cependant, on en connaît peu sur l’effet du stress oxydant sur la voie de signalisation de la Rho kinase dans le muscle lisse gastro-intestinal (GI). Le but de cette étude était d’examiner l’effet du stress oxydant sur la Rho kinase II et la contraction musculaire dans l’estomac de rat. La 3-morpholinosydnonimine (SIN-1), un donneur de peroxynitrite, le peroxyde d’hydrogène (H2O2) et le peroxynitrite ont été utilisés pour induire un stress oxydant. L’expression de la Rho kinase II et son activité induite par l’ACh ont été mesurées dans ces cellules contrôles et des cellules soumises au stress oxydant a` l’aide d’un dosage ELISA spécifiquement conçu et d’une trousse de dosage d’activité, respectivement. La contraction de cellules musculaires uniques a été mesurée par micrométrie a` balayage en présence ou en absence d’un bloqueur de Rho kinase, le di-hydrochlorure de Y-27632. Tous les agents oxydants augmentaient l’activité de la Rho kinase induite par l’ACh, sans affecter son niveau d’expression. Qui plus est, le stress oxydant induit par les trois agents augmentait la contraction cellulaire musculaire stimulée par l’ACh, phénomène inhibé par le Y-27632 de manière significative. En conclusion, le stress oxydant active la Rho kinase II et accroit la contraction du muscle gastrique de rat, ce qui suggère qu’il jouerait un rôle important dans les troubles liés a` la motilité GI associés au stress oxydant. [Traduit par la Rédaction] Mots-clés : muscle lisse, contraction, gastro-intestinal, Rho kinase, stress oxidant.
Introduction Contraction of smooth muscle is regulated by both Ca2+dependent and Ca2+-independent (Ca2+ sensitization) mechanisms (Somlyo and Somlyo 1994). A rise in intracellular Ca2+ levels leads to myosin light chain (MLC) kinase activation, resulting in an increase in MLC phosphorylation. Importantly, MLC phosphorylation can also be increased through inhibition of MLC phosphatase, which augments smooth muscle force generation without a change in intracellular Ca2+ (Kitazawa et al. 1991). Recent studies have revealed that Rho kinase II, a serine/threonine kinase downstream of the small G protein RhoA, is important in developing smooth muscle tone. It inhibits MLC phosphatase activity by phosphorylation of the MLC phosphatase target subunit (MYPT1). A decrease in MLC phosphatase activity maintains phosphorylation of myosin and therefore contributes to smooth muscle contraction at low levels of intracellular Ca2+ concentration (Somlyo and Somlyo
2000). The importance of the RhoA/Rho kinase pathway has been demonstrated in the pathogenesis of cardiovascular disorders and as a target in the development of new drugs such as fasudil, a Rho kinase inhibitor (Hahmann and Schroeter 2010; Mills et al. 2001; Uehata et al. 1997). Several disease states or pathological situations in the GI tract are well-known to affect smooth muscle contractile function and many of these are characterized by the formation of reactive oxygen species (ROS). Free radical-induced oxidative stress has been involved in the pathogenesis of a number of human diseases such as diabetes mellitus, hypertension, and atherosclerosis (Buttery et al. 1996; Grunfeld et al. 1995; Kamata and Kobayashi 1996). Oxidative stress is associated with enhanced production of ROS, in particular superoxide anion that is normally converted to (H2O2) by the action of superoxide dismutase (SOD). H2O2 in turn is metabolized to H2O via the antioxidant enzymes; catalase and glutathione peroxidase. However, in pathological states the balance
Received 9 December 2014. Accepted 16 January 2015. O. Al-Shboul. Department of physiology and biochemistry, Faculty of Medicine, Jordan University of Science and Technology, Irbid 22110, Jordan. A. Mustafa. Department of anatomy, Faculty of Medicine, Jordan University of Science and Technology, Irbid 22110, Jordan. Corresponding author: Othman Abdullah Al-Shboul (e-mail: [email protected]). Can. J. Physiol. Pharmacol. 93: 405–411 (2015) dx.doi.org/10.1139/cjpp-2014-0505
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between synthesis and metabolism of reactive oxygen species (ROS) may be impaired, thus leading to generation of oxidative stress (Yu 1994). Superoxide anion can elicit direct vasoconstriction and scavenge nitric oxide, the most potent endogenous vasodilator, to produce peroxynitrite (·OONO–), which oxidizes proteins, breaks DNA strands, and reduces intracellular antioxidants such as glutathione and cysteine (Bayraktutan 2002; Gryglewski et al. 1986). Generation of superoxide anion as a mechanism of damage is well established in different models of acute and chronic vascular and pulmonary injuries, however very little is known about its effect on GI smooth muscle contractility (Farinati et al. 1996; Keshavarzian et al. 1992; Parks et al. 1982). This study will investigate the effect of oxidative stress induced by various oxidant agents on Rho kinase II, the smooth muscle predominant isoform (Murthy et al. 2001) and smooth muscle contraction in rat stomach. Insights into the molecular basis of abnormal smooth muscle function will prove invaluable in the treatment of the GI motility disorders associated with oxidative stress.
Materials and methods Preparation of dispersed gastric smooth muscle cells Smooth muscle cells were isolated from the circular muscle layer of the rat stomach by sequential enzymatic digestion, filtration, and centrifugation as described previously (Murthy et al. 1996; Murthy and Makhlouf 1995). Briefly, strips of circular muscle from the stomach were dissected and incubated at 31 °C for 30 min in HEPES medium containing 120 mmol/L NaCl, 4 mmol/L KCl, 2.0 mmol/L CaCl2, 2.6 mmol/L KH2PO4, 0.6 mmol/L MgCl2, 25 mmol/L HEPES, 14 mmol/L glucose, 2.1% Eagle’s essential amino acid mixture, 0.1% collagenase, and 0.01% soybean trypsin inhibitor. The tissue was continuously gassed with 100% oxygen during the entire isolation procedure. After the partly digested strips were washed twice with 50 mL of enzyme-free medium, the muscle cells were allowed to disperse spontaneously for 30 min. The cells were harvested by filtration through 500 m Nitex mesh and centrifuged twice at 350g for 10 min to eliminate broken cells and organelles. The cells were counted in a hemocytometer, and it is estimated that 95% of the cells excluded trypan blue. All the experiments were done within 2–3 h of cell dispersion. Identification of smooth muscle cells The smooth muscle identity of rat gastric muscle cells was verified by immunohistochemical staining of paraffin-embedded rat smooth muscle using anti-calponin antibody (ab46794) at 1/100 dilution. Oxidative stress treatment Aliquots (0.4 mL) of dispersed gastric smooth muscle cells were prepared. The aliquots were randomly distributed into either control or oxidant treatment groups. Aliquots designated for oxidant treatment were incubated with either SIN-1 (1 mmol/L) for 10 min, H2O2 (500 mol/L) for 10 min, or peroxynitrite (1 mmol/L) for 10 min. Samples nominated for antioxidant treatment were preincubated with tempol (1 mmol/L) 5 min prior to SIN-1 treatment. Cells were stimulated with ACh (1 mol/L) for 10 min in the presence or absence of oxidant treatment. Detection of nitrotyrosine levels in gastric smooth muscle cells SIN-1-treated dispersed gastric smooth muscle cells were collected to measure nitrotyrosine, a marker of peroxynitrite. The samples were homogenized on ice in 1× PBS, pH 7.4. The homogenates were centrifuged at 20 000g (4 °C for 10 min). Supernatants were used for the measurement of nitrotyrosine concentrations with a 3-Nitrotyrosine ELISA Kit from Abcam according to the manufacturer’s instructions. Nitrotyrosine concentrations were
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normalized per milligram of protein. Protein concentrations were measured using the Dc protein assay kit from Bio-Rad. Measurement of Rho kinase II activity Rho kinase II activity was analyzed by an enzyme immunoassay, using Cell Biolabs’ 96-well Rho kinase II activity assay kit. Experiments were done according to the manufacture’s protocol, using 10 L of protein lysate. The total starting protein concentration for every sample was 1 mg/mL. Measurement of Rho kinase II expression Muscle cells were solubilized in Triton X-100-based lysis buffer plus protease and phosphatase inhibitors (100 g/mL phenylmethanesulfonylfluoride (PMSF), 10 g/mL aprotinin, 10 g/mL leupeptin, 30 mmol/L sodium fluoride, and 3 mmol/L sodium vanadate). After centrifugation of the lysates at 20 000g for 10 min at 4 °C, the protein concentrations of the supernatant were determined with a Dc protein assay kit from Bio-Rad. Samples of equal amounts of proteins were quantitated by ELISA according to the manufacturers’ instructions. Measurement of contraction in dispersed smooth muscle cells Contraction in freshly dispersed gastric circular smooth muscle cells was determined by scanning micrometry (Huang et al. 2007). Aliquots (0.4 mL) of dispersed gastric smooth muscle cells containing approximately 104 cells/mL were prepared. The aliquots were randomly distributed into either control or oxidant treatment groups. Aliquots designated for oxidant treatment were incubated with ether SIN-1 (1 mmol/L) for 10 min, H2O2 (500 mol/L) for 10 min, or peroxynitrite (1 mmol/L) for 10 min. Cells were stimulated with ACh (1 mol/L) for 10 min in the presence or absence of oxidant treatment, and the reaction was terminated with 1% acrolein at a final concentration of 0.1%. Acrolein kills and fixes cells without affecting the cell length. The cells were viewed using a 20× or 40× objective of inverted Nikon TMS-f microscope, and cell images were acquired using a Canon digital camera and image acquisition software. The resting cell length was determined in a control group in which muscle cells were not treated with ACh. The mean length of at least 50 muscle cells was measured by image-J software from each group. The contractile response was defined as the decrease in the average length of at least 50 cells and was expressed as the percent change relative to control length. Materials Male Sprague Dawley (SD) rats were provided by the animal house of Jordan University of Science and Technology (JUST). Male SD rats (6 weeks of age, 200–250 g) were sacrificed using an overdose of ether. Assays were performed using a Rho kinase II assay kit (Cell Biolabs, INC., California, USA), a Rho kinase II ELISA kit (Cusabio Biotech, Newark, Delaware, USA), a 3-Nitrotyrosine ELISA Kit (ab116691) (Abcam Inc., Cambridge, Massachusetts, USA), and a Dc protein assay kit (Bio-Rad, Hercules, California, USA). The Anticalponin antibody (ab46794) was purchased from Abcam. The 500 m Nitex mesh was purchased from Amazon. Powdered tempol and Y-27632 dihydrochloride (Rho kinase inhibitor) were purchased from Santa Cruz Biotech(Santa Cruz, California, USA); sc-1616 and sc-281642 respectively. All remaining materials were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). Tempol was dissolved and diluted in 99% ethanol, Y-27632 dihydrochloride was dissolved in DMSO, while SIN–1 and ACh were dissolved in distilled water. Analysis of data Each experiment was performed on gastric smooth muscle cells that were harvested from 6 rats. Statistical analysis of all experiments was performed using Prism 5.0 software (GraphPad Software, San Diego, California, USA). Unpaired Student's t-test was used to reveal significant differences between the compared groups For the Rho kinase II activity experiments in SIN-1-treated Published by NRC Research Press
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Fig. 1. Identification of rat gastric smooth muscle cells. (A) Immunohistochemical staining of paraffin-embedded rat smooth muscle using ab46794 at 1/100 dilution. (B) Freshly isolated gastric smooth muscle cells appeared in spindle shape with diverse lengths under phase contrast microscopy.
samples pre-incubated with tempol, a one-way analysis of variance (ANOVA) was performed followed by Fisher’s post-hoc analysis to determine the significance of differences between experimental groups. A P < 0.05 was required for statistical significance in all the experiments. All data are shown as mean ± SEM.
Fig. 2. Effect of SIN-1 on nitrotyrosine levels in gastric smooth muscle cells. Levels of nitrotyrosine were measured in gastric smooth muscle cells using 3-nitrotyrosine assay kit and expressed as absorbance at 450 nm. SIN-1 treatment increased the levels of 3-nitrotyrosine. Values are means ± SEM of 4 experiments. (*P < 0.05 vs. control by unpaired t test). SIN-1, 3-morpholinosydnonimine.
Results Smooth muscle identity The smooth muscle identity of rat gastric muscle cells was verified by immunohistostaining with anti-calponin antibody. The results showed that greater than 95% of cells stained positive for calponin (Fig. 1A). Freshly isolated gastric smooth muscle cells appeared in spindle shape with diverse lengths under phase contrast microscopy (Fig. 1B). SIN-1-induced 3-nitrotyrosine formation To determine the effectiveness of SIN-1 treatment, we assessed the formation of peroxynitrite in both SIN-1-treated and control smooth muscle cells via quantitative measurement of 3-nitrotyrosine. SIN-1 significantly increased 3-nitrotyrosine levels in oxidant-treated cells compared to control (P < 0.05, n = 6) (Fig. 2). These results indicate peroxynitrite participation in nitrotyrosylation. Rho kinase II activity Treatment of freshly dispersed muscle cells with 1 mol/L ACh, a G␣q/13-coupled receptor agonist, for 10 min significantly increased Rho kinase II activity above basal level (P < 0.05, n = 6) (Fig. 3A, second column). Incubation of gastric smooth muscle cells with 1 mmol/L SIN-1, 500 mol/L H2O2, or 1 mmol/L peroxynitrite for 10 min significantly augmented ACh-stimulated Rho ki-
nase II activity (P < 0.05, n = 6) (Fig. 3A). The three oxidant agents had no effect on basal Rho kinase II activity (data not shown). To examine whether the enhanced ACh-stimulated Rho kinase II activity is indeed due to peroxynitrite generation in SIN-1-treated group, we pre-incubated gastric muscle cells with 1 mmol/L tempol, a peroxynitrite scavenger, 5 min prior to SIN-1 treatment. SIN-1-induced ACh-stimulated Rho kinase II activity was blocked by the presence of tempol (P < 0.05, n = 6) (Fig. 3B, fourth column). Published by NRC Research Press
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Fig. 3. Effect of SIN-1 (1 mmol/L for 10 min), H2O2 (500 mol/L for 10 min), and peroxynitrite (1 mmol/L for 10 min) incubation on ACh-induced (1 mol/L) of Rho kinase II activity in rat gastric smooth muscle. Rho kinase II activity is expressed as absorbance at 450 nm. (A) Note that the ACh-induced activation of Rho kinase II was significantly augmented by the 3 oxidant treatments. (B) Pre-treatment of cells with tempol (1 mmol/L) significantly blocked SIN-1-induced ACh-stimulated Rho kinase II activity (fourth column). Values are means ± SEM of 4 experiments. (*P < 0.05 vs. control basal; **P < 0.05 vs. control ACh; †P < 0.05 vs. SIN-1 + Ach (one way ANOVA followed by Bonferroni multiple comparison test). ACh, acetylcholine; H2O2, hydrogen peroxide; SIN-1, 3-morpholinosydnonimine.
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Fig. 4. Effect of SIN-1 (1 mmol/L for 10 min), (H2O2) (500 mol/L for 10 min), and peroxynitrite (1 mmol/L for 10 min) incubation on the expression levels of Rho kinase II protein in rat gastric smooth muscle. Rho kinase II protein expression level is expressed as absorbance at 450 nm. Note that all oxidant treatments didn’t affect the expression level of Rho kinase II protein. Values are means ± SEM of 4 experiments. (P > 0.05 by unpaired t test). SIN-1, 3-morpholinosydnonimine.
contraction in all oxidant treatment groups (Fig. 5), showing clearly that oxidative stress induced by SIN-1, H2O2, and peroxynitrite augments muscle contraction via pathways that are dependent on Rho kinase II activation.
Discussion
These results suggest that stimulation of Rho kinase II activity in stomach muscle cells is mediated by SIN-1-induced peroxynitrite formation. Rho kinase II expression To determine whether the effect of these various oxidant agents on Rho kinase II expression profile correlates with their effect on the kinase activity level, we compared the Rho kinase II protein level in SIN-1-, H2O2–, and peroxynitrite-treated versus control cells by ELISA. Despite the higher agonist-stimulated Rho kinase II activity in all oxidant-treated cells compared to control, the expression of Rho kinase II protein was not different in all groups of cells (P > 0.05) (Fig. 4). This raises the possibility that oxidative stress-induced Rho kinase II activity might be due to an effect on other upstream regulators of the enzyme such as RhoA. Smooth muscle contraction We further examined whether oxidant treatment was associated with changes in agonist-induced contraction and whether these changes, if any, are due to increased Rho kinase II activity level. For that reason, freshly dispersed gastric muscle cells from control and oxidant-treated groups were treated with 1 mol/L ACh and 1 mol/L Y-27632 (a highly potent Rho kinase inhibitor), and the decrease in muscle cell length was measured by scanning micrometry. Basal muscle cell lengths were similar in control and oxidant-treated groups. ACh caused contraction that was concentration-dependent in muscle cells from both control and oxidant-treated cells (Fig. 5). However, contraction in response to ACh was significantly higher in oxidant-treated cells compared to control cells (Fig. 5). Most importantly, pre-incubation with Y-27632, a selective inhibitor of Rho kinase that competes with ATP for its binding site on Rho kinase (Ishizaki et al. 2000), significantly inhibited ACh-stimulated muscle
Recent studies have suggested that both free radical-induced oxidative stress and Rho/Rho kinase signaling affect smooth muscle contractility and may contribute to the pathogenesis of a number of human diseases, such as diabetes mellitus, hypertension, and atherosclerosis (Buttery et al. 1996; Grunfeld et al. 1995; Kamata and Kobayashi 1996). Very little is known about the effect of oxidative stress on Rho kinase pathway and smooth muscle contraction in the GI tract. In the present study, we demonstrate that oxidative stress induced by the peroxynitrite-donor, SIN-1, H2O2, or peroxynitrite activates Rho kinase II and enhances agonistinduced contraction of dispersed gastric smooth muscle cells. There is an accumulating body of evidence that ROS-induced oxidative stress is involved in the increase of smooth muscle contractile function that might be associated with some disorders. For example, Gumusel et al. (1996) reported that in isolated rat aortic rings, the contractile response to phenylephrine was potentiated when the bathing solution was subjected to electrolysis. Moreover, previous studies in several species have demonstrated that exogenous application of H2O2 augments contraction in airway smooth muscle of humans (Rabe et al. 1995) as well as of rats (Schmidt 1990), dogs (Levin et al. 1981), cows, and other species, suggesting that H2O2 induces airway hyper-responsiveness and airflow limitation in patients with asthma or chronic obstructive pulmonary disease. In addition, Gao and Lee (2001) found that hydrogen peroxide induces a greater contraction in mesenteric arteries of spontaneously hypertensive rats through thromboxane A2 production. Our results are in support of these previous studies. We found that H2O2 significantly elevated ACh-induced gastric muscle contraction. Most importantly, H2O2-induced enhancement in muscle contraction was Rho kinase II-dependent, as it was inhibited by Y-27632, a Rho kinase II inhibitor. Other researchers found that administration of anti-oxidants or inhibition of ROS-generating enzymes normalizes or reduces elevated blood pressure and improves vascular function (Fukui et al. 1997; Laursen et al. 1997; Rajagopalan et al. 1996). Moreover, recent studies have shown that both blood pressure and superoxide production are significantly lower in knockout mice lacking selected NADPH oxidase subunits than in wild-type mice (Landmesser et al. 2002; Wang et al. 2001). Parallel to Published by NRC Research Press
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Fig. 5. Effect of SIN-1 (1 mmol/L for 10 min), (H2O2) (500 mol/L for 10 min), and peroxynitrite (1 mmol/L for 10 min) incubation on ACh-induced muscle contraction. Contraction of dispersed muscle cells from stomach was measured by scanning micrometry in response to 1 mol/L ACh. Cells were treated with ACh for 10 min and contraction was expressed as the percent change relative to control length. ACh lead to contraction in both control and treated groups. Contraction was significantly augmented in cells from the three oxidant-treated groups. ACh-induced contraction was significantly decreased by the Rho kinase II inhibitor Y27632. Values represent the means ± SEM. (*P < 0.05 vs. control ACh; †P < 0.05 vs. SIN-1 + ACh; €P < 0.05 vs. H2O2 + ACh; ¥P < 0.05 vs. peroxynitrite + ACh).
previous findings, free radicals were found to impair endotheliumdependent relaxation and increase the tone of arteries by reducing NO bioavailability (MacKenzie and Martin 1998). Interestingly, ROS also contract vessels after removal of endothelium, suggesting that ROS have direct effect on smooth muscle (Auch-Schwelk et al. 1989; Rodriguez-Martinez et al. 1998; Yang et al. 1999). Our findings of increased gastric muscle contraction via oxidative stress indeed reinforce ROS-induced elevation of muscle contraction reported in previous researches. In our research, we induced oxidative stress by incubating dispersed gastric smooth muscle cells with the peroxynitrite donor, SIN–1, H2O2, or peroxynitrite. It is well-known that during its decomposition, SIN-1 releases superoxide anion and nitric oxide, and these in turn spontaneously combine to form a highly reactive molecule, peroxynitrite (Beckman et al. 1990; Daiber et al. 2005). In physiological conditions, oxidative damage is minimized by endogenous anti-oxidant defenses (Forstermann 2010). However, in pathological conditions nitric oxide production drastically increases, and the produced nitric oxide competes with the SODs for superoxide anion to form peroxynitrite. Peroxynitrite can attack biological molecules such as lipids, DNA, and proteins via direct oxidative reactions or indirect radical-mediated mechanisms (Pacher et al. 2007). Interestingly, protein nitration is considered a characteristic of peroxynitrite generation and leads to inactivation of the tyrosine residue of a protein forming 3-nitrotyrosine. This process of protein modification has been detected in human atherosclerotic lesions using anti-nitrotyrosine antibodies (Luoma et al. 1998; Pennathur et al. 2004). In addition to nitration, peroxynitrite is implicated in tissue injury via lipid peroxidation (Szabo et al. 2007). We confirmed the effectiveness of SIN-1 treatment and demonstrated, using ELISA method, that SIN-1 indeed induced peroxynitrite generation by increasing 3-nitrotyrosine formation in gastric smooth muscle cells. Knowing the importance of Rho kinase II signaling pathway in increasing calcium sensitization and smooth muscle contraction, as being an upstream inhibitor of MLC phosphatase, our next aim was to explore the correlation between SIN-1-induced nitrotyrosylation, H2O2, and peroxynitrite treatment and Rho kinase II activity. Consistent with many previous studies (Fukata et al. 2001; Huang et al. 2005; Murthy et al. 2003), treatment of muscle cells with ACh significantly increased Rho kinase II activity. Most importantly, we found that pre-incubating gastric muscle cells with the oxidant agents used in this study, leads to significant enhancement in ACh-stimulated Rho kinase II activity and muscle contraction.
For the most part, this enhanced muscle contraction was partly dependent on Rho kinase II pathway, as it was significantly inhibited by Y27632, the Rho kinase II inhibitor. Although higher concentrations of Y27632 could also inhibit PKC, previous studies confirmed that Y27632 at 1 mol/L is highly selective for Rho kinase (Jin et al. 2004; Murthy et al. 2003). Current findings support those previously reported in the literature at various smooth muscle locations in the body. For example, Jin et al. (2004) found that ROS generated by a xanthine/xanthine oxidase mixture stimulates smooth muscle contraction in endothelium-denuded rat aorta. This reported vasoconstriction was mediated through activation of Rho and a subsequent increase in Rho kinase activity. Moreover, oxidative stress with H2O2 has been suggested to mediate airway smooth muscle contraction by increasing intracellular Ca2+ level and stimulating the Rho/Rho kinase pathway (Snetkov et al. 2011). Mishra et al. (2011) reported also that enhanced vascular reactivity of omental arteries in pre-eclampsia is attributed to ROS activation of the RhoA kinase pathway, and that the enhanced vascular reactivity is likely attributed to the infiltration of neutrophils. In addition, Shimokawa (2000) and colleagues revealed that RhoA and its downstream effector, Rho kinase, are involved in the hypercontraction of vascular smooth muscle of the coronary artery in vasospastic angina, which is probably mediated by oxidative stress. Very recently, one attractive study reported a SIN-1-induced enhancement of endothelial arginase activity. They concluded that oxidative species increase arginase activity through PKC-activated RhoA/Rho kinase pathway (Chandra et al. 2012). In regard to SIN-1 results, we aimed to further prove that the effect on Rho kinase was indeed due to nitration induced by SIN-1 treatment. So we pre-incubated gastric smooth muscle cells with the anti-oxidant tempol. Tempol is a well-known peroxynitrite scavenger that protects against oxidative stress (Bonini et al. 2002; Wilcox and Pearlman 2008). Moreover, tempol has been shown to effectively suppress pathological conditions associated with marked oxidative stress in vivo, such as hypertension (Elmarakby et al. 2007), experimental colitis (Karmeli et al. 1995), and cardiac reperfusion injury (Gelvan et al. 1991). Our data showed that tempol significantly reduced SIN-1-induced Rho kinase II activity. Interestingly, a group of researchers found that tempol decreases RhoA, the upstream activator of Rho kinase II, activation via reducing SIN-1-induced nitration of p190A in endothelial cells (Siddiqui et al. 2011). In addition, tempol was found to be efficient in reducing xanthine/xanthine oxidaseinduced contraction in rat aorta (Jin et al. 2004). Our findings with Published by NRC Research Press
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tempol strongly suggest that the increase in Rho kinase II activity is indeed due to SIN-1-induced peroxynitrite formation. Although pre-incubation of gastric smooth muscle cells with SIN-1, H2O2, or peroxynitrite enhanced ACh-stimulated Rho kinase II activity, protein expression level of Rho kinase II was not affected by various oxidant treatments. This raises the possibility that the increase in Rho kinase II activity associated with these different oxidant treatments may be caused by an effect of oxidative stress on other regulators of Rho kinase II such as Rho A, PKC, and other effectors. In conclusion, this study demonstrates the direct activation of Rho kinase II enzyme and thus contraction by ROS in gastric smooth muscle, suggesting an important role for ROS-mediated Rho/Rho kinase II pathway activation in GI motility disorders associated with oxidative stress. An understanding of the oxidative stress-induced smooth muscle changes at the intracellular level is important in the pathophysiology and may provide new insights for the development of therapeutic agents that should act on smooth muscle in the gut to treat motility disorders, as well as in other regions such as airways and vascular smooth muscle where similar intracellular mechanisms may prevail.
Acknowledgements This work was supported by Jordan University of Science & Technology, Irbid, Jordan (Grant No. 51/2012). The authors thank Dr. Nayef Al-Gharaibeh for help and providing laboratory facilities.
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