Expression in Rat Coronary Endothelial Cells

ORIGINAL ARTICLE

Nitric Oxide Does Not Downregulate Rho-Kinase (ROCK-2) Expression in Rat Coronary Endothelial Cells R. Nalan Tiftik, MD,* Aysxe Erol, MD,† Mehtap G. Cxınar, MD,† Havva Kubat, MD,* Mustafa Ark, PhD,* Sibel U¨lker, MD,† and Kansu Buÿu¨kafsxar, PhD*

Abstract: Rho kinase (ROCK) and nitric oxide (NO) are important targets in cardiovascular diseases. Therefore, we investigated the possible influence of NO on Rho kinase (ROCK-2 isoform) expressions in cultured rat coronary microvascular endothelial cells. The cells were isolated from Wistar rats on a Langendorff system, and were incubated overnight (~16 h) with an NO generator, A-23187 (1027 to 10-6 M), NO donors, such as sodium nitroprusside (1027 to 1026 M), glyceryl trinitrate (1027 to 1026 M), 2,2#-(hydroxynitrosohydrazono)bis-ethanimine (1027 to 1026 M), and NaNO2 (1024 to 1023 M) or a nitric oxide synthase (NOS) inhibitor, NG-nitro-Larginine methylester (231024 M), or two ROCK inhibitors, (+)(R)-trans-4-(1-aminoethyl)- N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632, 10-5 M) and fasudil (10-5 M) in the absence or presence of thrombin (4 U/mL). ROCK-2 and endothelial NOS (eNOS) expressions were detected by Western blotting. Moreover, nitrite/nitrate levels were detected by Griess method in the presence of the ROCK inhibitors. The NO donors and the NO generator had no significant effects on ROCK-2 expression. Y-27632 and fasudil did not alter eNOS expression and NO production. Nitrite/nitrate levels were 4.4 6 0.32 mM in control and 4.0 6 0.93 mM and in Y-27632 group. These results demonstrate that prolong NO donation could not suppress the expression of ROCK-2 protein, and the ROCK inhibitor did not change e-NOS expression and NO production in the cultured rat coronary microvascular endothelial cells. Key Words: coronary microvascular endothelial cells, fasudil, nitric oxide, Rho-kinase, Y-27632 (J Cardiovasc Pharmacol TM 2008;51:140–147)

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ndothelial cells do not only form a semipermeable dynamic barrier between the vascular space of blood vessels and underlying tissues but also secrete many vasoactive compounds that regulate vascular resistance and blood

Received for publication August 8, 2007; accepted October 11, 2007. From the *Department of Pharmacology, Medical Faculty, Mersin University, Campus Yenisxehir, Mersin, Turkey; and the †Department of Pharmacol_ ogy, Medical Faculty, Ege University, Izmir, Turkey. Supported by grants from the Technical and Scientific Research Council _ ¨ BITAK-SBAG-2711) of Turkey (TU and Turkish Academy of Sciences _ ¨ BA-GEBIP/2002-1-5). (K.B./TU The authors report no conflicts of interest. Reprints: Dr. Kansu Buÿu¨kafsxar. PhD, Department of Pharmacology, Medical Faculty, Mersin University, Campus Yenisxehir 33169 Mersin, Turkey (e-mail: [email protected]). Copyright Ó 2008 by Lippincott Williams & Wilkins

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coagulation.1 Analogous to smooth muscle cell, endothelial cells are able to contract in response to appropriate stimuli, which may result in opening of the gap between two neighbour cells, and consequently an increased vascular permeability.2 The increased vascular permeability contributes to the development of pathological condition, such as edema and atherosclerosis.3,4 Similar to many nonmuscle cells, the endothelial cell contraction is regulated by phosphorylation of the myosin light chain (MLC) by nonmuscle myosin light chain kinase (MLCK) and Rho-kinase (ROCK), an effector of small G protein, Rho.5,6 MLC phosphorylation is a key regulatory event for force development of endothelial cells. In concert with the increase in MLC phosphorylation, two enzymes, namely MLCK and ROCK, cooperatively act. The former enzyme is activated by an increase in intracellular Ca2+ concentration, and the latter is by a small GTPase protein, Rho, which is activated by agonist stimulation.7–9 Consequently, these two pathways constitute the major regulatory event in endothelial contractility, which plays important role in microvascular permeability.10 It has been known for many years that particular antianginal drugs, such as nitrovasodilators, release nitric oxide (NO) within target cells. NO activates soluble guanylyl cyclase to form 3#,5#-cyclyc guanosine monophosphate (cGMP), which induces vascular smooth muscle cell relaxation.11 Recently, it has been proposed that there may be a crosstalk between these two counteracting pathways within cardiovascular system. For instances, endothelial NO synthase (eNOS) could be suppressed by Rho/ROCK pathway in human endothelial cells,12,13 and RhoA expression is controlled by NO through cGMP-dependent protein kinase activation.14 Furthermore, Rho activation could downregulate eNOS expression by destabilizing the half-life of eNOS mRNA.15 On the other hand, it has been suggested that NO may inhibit Rho-kinase activity in the intact aorta.16 Moreover, cGMPdependent protein kinase signaling pathway inhibits RhoAinduced Ca2+ sensitization of contraction in vascular smooth muscle.17 RhoA attenuation stimulates eNOS activity in human umbilical endothelial cells.18 However, removal of endothelium had no effect on the vasodilatory effects of the Rho-kinase inhibitors, Y-27632 and fasudil, in mesenteric arterial bed.19 Recently, it has been clearly demonstrated that the Rho kinase inhibitor Y-27632 produced substantial vasorelaxation in a mice model of genetically reduced endothelial NO production (eNOS2/2), which was indistinguishable from that in the eNOS+/+ aorta, indicating that inhibition of Rho kinase stimulates nitric oxide-independent J Cardiovasc Pharmacol ä Volume 51, Number 2, February 2008

J Cardiovasc Pharmacol ä Volume 51, Number 2, February 2008

vasorelaxation.20 Moreover, antagonism of Rho kinase stimulates rat penile erection via a nitric oxide-independent pathway.21 Furthermore, Noma et al22 reported that fasudilinduced vasodilatation may not be due to eNOS gene expression-related NO release in human forearm. Consequently, it is controversial whether there may be indeed a counteraction between these pathways. For that reason, we investigated effects of NO donors or NO generators on Rho-kinase expression in the cultured rat coronary microvascular endothelial cells. For this purpose, Rho-kinase expression was detected by Western blotting after exposure to NO-generating or -donating compounds. NO amount in the medium was determined as its stable metabolites, nitrite and nitrate, by Griess method. Furthermore, eNOS expression was also detected in the presence of Rho-kinase inhibitors, fasudil and Y-27632.

NO Does Not Downregulate Rho-Kinase (ROCK-2) Expression

the medium with 0.5% serum for 24 h. On the day of experimentation, the coronary microvascular endothelial cells were washed with HEPES-Krebs solution of the following composition in mM: NaCl 99, KCl 4.7, CaCl2 1.87, MgSO4 1.2, K2HPO4 1.2, Na-HEPES 20, and glucose 11 (pH = 7.4). The cells were incubated overnight (16 h) with each of the following drugs in the absence or presence of thrombin (4 U/mL): glyceryl trinitrate (GTN, 1027 to 1026 M), sodium nitroprusside (SNP, 1027 to 1026 M), 2,2#-(hydroxynitrosohydrazono)bis-ethanimine (DETA-NO, 1026 to 1025 M), sodium nitrite, (NaNO2, 1024 to 1023 M), A-23187 (1027 to 1026 M), NG-nitro-L-arginine methylester (L-NAME, 2 3 1024 M), (+)-(R)-trans-4-(1-aminoethyl)- N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632, 1025 M), or fasudil (10-5 M). Thrombin was chosen to promote endothelial cell activation through Rho signaling.7

Western Blot Analysis for ROCK-2 MATERIALS AND METHODS Isolation of Coronary Microvascular Endothelial Cells The protocol of this study was approved by the local ethics committee of the medical faculty at Mersin University. Wistar rats (male, 250–270 g) were used in the study. The rats were killed by a blow on the head and exsanguinated. Hearts were employed for coronary microvascular endothelial cells. The cells were isolated as was described previously elsewhere.23 Briefly, the hearts were mounted and perfused retrogradely on a constant-flow Langendorff system (5 mL/min) with 0.04% collagenase (type 2). The ventricles were chopped and collagenase digestion was quenched by the addition of bovine serum albumin to the perfusate. Coronary microvascular endothelial cells were obtained by sedimentation of myoctes and incubated in 0.01% trypsin at 37°C for the prevention of nonendothelial cell attachment. The cells were then activated by washing in calcium and suspended in medium 199 supplemented with L-glutamine (200 mM), fetal calf serum (10%), penicillin (250 IU/mL), streptomycin (250 mg/mL), and amphotericin (12.5 mg/mL). Cell suspensions were plated in 75-cm2 flasks and incubated in an incubator (Heal Force, Shengai Lishen Scientific Equipment, China) at 37°C under 5% CO2 + 95% air. After 1 h incubation, cells were washed with sterile saline solution to remove unattached cells, and remaining cells were cultured to confluence. Cultured cells formed confluent monolayer with typical ‘‘cobblestone’’ morphology within 3 to 5 days. For further culture, cells were trypsinized and subcultured. By this method, the cells were identified as endothelial cells by the uptake of fluorescently labelled acetylated LDL.24 The endothelial cells may include some endocardial endothelial cells, as well as some endothelial cells from larger coronary vessels. However, the statistical average (more than 90%) represents predominantly microvascular endothelial cells since the vast majority of cardiac endothelial cells are found in small vessels and capillary bed. Therefore, the number of endocardial cells would be negligible (0.0002%).24 Cells at the third passages were used in the study. After confluence, the cells were rendered quiescent by incubation in q 2008 Lippincott Williams & Wilkins

After incubation, the medium was removed for later nitrite/nitrate detection and the coronary microvascular endothelial cells were washed with cold physiological salt solution (0.9% NaCl). The cells were then harvested by scratching and then lysed with a boiled buffer containing 0.1% sodium dodecyl sulfate (SDS) in 10 mM Tris (pH 7.4). The homogenate was centrifuged at 13.000 3g for 10 min at 4 °C, and the supernatant was removed. Protein concentrations were estimated by Bradford assay using a protein assay kit (BioRad, Munich, Germany). Equal amounts of protein (50 mg) were loaded in wells, electrophoresed on 10% polyacrylamideSDS gels and then transferred to a nitrocellulose membrane overnight. The membrane was blocked with the blocking agent of the enhanced chemiluminescence kit (Amersham Biosciences, Freiburg, Germany) in Tris-buffered solution containing 0.05% Tween-20 for 1 h. It was then probed with a primary antibody raised against ROCK-2 (ROKa, polyclonal IgG, Santa Cruz Biotechnology; 1:200 dilution) or actin (Sigma Co., Germany, 1:1000) followed by horseradish peroxidase conjugated secondary antibodies (1:1000 for ROCK-2 and 1:2000 for actin). Protein blots were then detected with a chemiluminescence detection kit (ECL plus, Amersham Biosciences, Freiburg, Germany) and visualized on commercial X-ray films. ROCK and eNOS protein levels were measured relative to actin protein, a housekeeping protein.

Nitrite/Nitrate Determination NO released from coronary microvascular endothelial cells was determined as its stable metabolites, nitrite and nitrate, by Griess method in basal condition and in the presence of thrombin, fasudil, or Y-27632. Briefly, the supernatant from each flask was collected after the incubation period and centrifuged to remove cells and particles. Nitrate was reduced to nitrite by equilibrating of samples (supernatants and standards) in sodium phosphate buffer (pH 7.5) containing flavin adenine dinucleotid (FAD) (0.02 mM), reduced nicotinamide adenine dinucleotid phosphate (NADPH) (0.5 mM) and nitrate reductase (0.1 U/mL) at room temperature for 90 min. Sodium nitrite (2–50 mM) dissolved in KrebsHEPES solution was used as the standard. Total nitrite was then determined spectrophotometrically using Griess reaction.

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The amount of total nitrite in each sample was calculated by linear regression using the absorbance of the sodium nitrite standards each day. The amount of total cellular protein in the respective flask was determined by Bradford’s method after lyses with a buffer containing SDS 0.1% in 10 mmol Tris (pH 7.4). Total nitrite accumulated in each flask was defined as mM per total protein (mg).

Drugs and Reagents Used DETA-NO, sodium nitroprusside, A-23187, and LNAME were obtained from Sigma Chemical Co (St. Louis, MO). (+)-(R)-Trans-4-(1-aminoethyl) -N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632), and fasudil were obtained from Tocris Cookson (Bristol, UK). GTN was used as Perlinganite ampoule (Melusin, Istanbul, Turkey). Bovine thrombin was purchased from Calbiochem (Darmstadt, Germany). Primary antibody for ROCK-2 and HRP-conjugated secondary antibody were obtained from Santa Cruz Biotechnology and actin antibodies (primary and secondary) from Sigma Co. (Darmstadt, Germany). The ECL plus kit was purchased from Amersham Biosciences (Freiburg, Germany). The kit was used according to the manufacturer’s guide. All chemicals, except for A-23187, were dissolved in saline. A-23187 was dissolved in dimethyl sulfoxide, the concentration of which was 0.1% in the medium. All reagents and chemicals were of highest purity and quality obtainable from commercial sources.

Statistical Evaluations All data represent means 6 standard error of the mean (SEM) of n observations. ROCK and eNOS densities were measured relative to actin bands. For statistical comparison, one-way analysis of variance followed by the Dunnet post-hoc test or Student’s t test were used. A P value less than 0.05 was considered significant.

FIGURE 1. Effect of thrombin (4 U/mL) incubation on Rhokinase (ROCK-2) expression in the rat coronary microvascular endothelial cells in culture. Confluent cells were incubated with thrombin or its vehicle (saline, basal) overnight (16 h) and the cells were washed with ice-cold Krebs-HEPES solution. Thereafter, boiled lyses solution containing SDS 0.1% in 10 mM Tris (pH = 7.4, 250 mL for each flask) was applied into flasks and the cells were scraped. Following homogenization, Western blotting was performed for Rho kinase protein (ROCK-2, ROKa). Loading control was made by the antibody against actin, a housekeeping protein. ROCK bands were measured relative to actin bands. For statistical analysis, Student’s t test (for unpaired observation) was used. *P , 0.05.

RESULTS Expression of Rho kinase (ROCK-2) in Basal and Thrombin Stimulated Conditions Rat coronary microvascular endothelial cells in culture are able to express Rho-kinase (ROCK-2) protein. Prolonged exposure to thrombin (4 U/mL, 16 h), which activates endothelial cells through Rho/Rho-kinase pathway7 significantly upregulated ROCK-2 expression, which was measured relative to actin (Fig. 1).

Effects of GTN, SNP, DETA-NO, NaNO2, A-23187, and L-NAME on ROCK-2 Expression Under Basal and Thrombin-Stimulated Conditions NO donors, GTN (1027 to 1026 M, 16 h, Fig. 2), SNP (1027 to 1026 M, 16 h, Fig. 2), DETA-NO (1027 to 1026 M, 16 h, Fig. 3), and NaNO2 (1024 to 1023 M, 16 h, Fig. 3) had no significant effects on ROCK-2 expression in both basal (without thrombin) and thrombin-stimulated condition. Neither A-23187 (1027 to 1026 M, 16 h, Fig. 4), a NO generator, nor L-NAME (231024 M, 16 h, Fig. 4), a NOS inhibitor, considerably affected ROCK-2 expression in any condition.

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Effects of Fasudil and Y-27632 on e-NOS Expression, and Nitrite/Nitrate Formation Under Basal and Thrombin-Stimulated Condition Long-term exposure to the Rho-kinase inhibitors Y-27632 and fasudil (both 1025 M, 16 h) altered neither e-NOS expression (Fig. 5) nor the concentration of stable nitric oxide metabolites (NO22/NO23) in the medium incubating the cells in a significant manner (Table 1).

DISCUSSION In cultured rat coronary microvascular endothelial cells, we examined the effect of NO, which was generated by the Ca2+ ionophore, A-23187 and the NO donors, glyceryl trinitrate, DETA-NO, NaNO2, and sodium nitroprusside on the expression of Rho-kinase enzyme, which has an important role in the regulation of cytoskeletal organization of nonmuscle cells,6 and in some cardiovascular diseases, such as angina pectoris and hypertension.25 q 2008 Lippincott Williams & Wilkins



FIGURE 2. Effects of GTN (1027 to 1026 M, 16 h) and SNP (1027 to 1026 M, 16 h) on ROCK-2 expression in the rat coronary microvascular endothelial cells in culture. Confluent cells were coincubated with or without thrombin (4 U/mL) and ROCK-2 protein level was measured by Western blotting. Loading control was made by actin antibody. ROCK bands were measured relative to actin bands. For statistical analysis, one-way analysis of variance followed by Dunnet post-hoc test was used.

In preliminary experiments, we tried lower concentrations of GTN and SNP but we failed to obtain any marked effects of these NO donors on ROCK-2 protein (data not shown). Then, we increased the concentrations of the NO donors up to 1027 to 1026 M, which are more than usual experimental concentrations. Again, there was no downregulatory effect of the NO donors on ROCK-2 enzyme. In this case, one would consider that these NO donors may be broken down during such prolonged incubation (overnight, 16 h). However, we applied these donors twice (every 6 h for 12 h) and had no favorable results (data not shown). Furthermore, we also tested DETA-NO, which donates constant NO into the medium over hours,26 with effective concentrations, and there were still no effects. As for the other NO-related substance, NaNO2, it also failed to change ROCK-2 expression. q 2008 Lippincott Williams & Wilkins

Furthermore, we tested an NO-generating compound, A-23187, which can activate nitric oxide synthase (NOS) by elevating intracellular free Ca2+ concentration,27 and this calcium ionophore had no significant effect over ROCK-2 expression. In confirmation, the cells were incubated with a NOS inhibitor, L-NAME, and we did not demonstrate any upregulation in ROCK-2 expression, providing a circumstantial evidence that NO may not downregulate ROCK-2 expression. Because the NO donors had no effects on Rho-kinase expression in basal (not stimulated) condition, we reasoned to use NO donors as well as the NO-generating substance A-23187 and the NOS inhibitor L-NAME on the endothelial cells, which were stimulated by thrombin that activates them through Rho/Rho-kinase pathway.7,9 Thrombin is a serine

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FIGURE 3. Effects of DETA-NO (1026 to 1025 M, 16 h) and NaNO2 (1024 to 1023 M, 16 h) on ROCK-2 expression in the rat coronary microvascular endothelial cells in culture. Confluent cells were coincubated with or without thrombin (4 U/mL) and ROCK-2 protein level was measured by Western blotting. Loading control was made by actin antibody. ROCK bands were measured relative to actin bands. For statistical analysis, one-way analysis of variance followed by Dunnet post-hoc test was used.

protease that is involved in the coagulation cascade and hemostasis. Furthermore, it promotes endothelial cell contraction, capillary permeability increase, and consequently leads to vascular inflammation.7 In our study, this enzyme substantially upregulated ROCK-2 expression. Yet, NO donors had no marked effects on the ROCK-2 expression of thrombinstimulated cells. Furthermore, despite thrombin-stimulation and incubation of an NO synthase inhibitor, L-NAME with an effective concentration (2 3 1024 M) did not upregulate ROCK-2 expression, indicating that NO have indeed no effects on ROCK-2 expression in the rat coronary microvascular endothelial cells. This contradictory finding may be due to cell type, such that these cells are obtained from microvascular circulation where rather than NO, other vasodilatory

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substances such as endothelium-derived hyperpolarizing factor (EDHF) and/or cardiac metabolites, such as adenosine, have a more substantial role in the control of microvascular tone. In our previous study, successful removal of endothelium did not change fasudil and Y-27632-induced vasodilatation19 in the rat mesenteric vascular bed, representing microvascular circulation and indicating that the ROCK inhibitors may not release sufficient quantity of any vasodilatory agents from the endothelium. In the line with this, Lo¨hn et al20 have suggested that the inhibition of Rho-kinase induces a strong vasorelaxation and blood pressure reduction with eNOS-independent manner by using eNOS knockout mice. Moreover, inhibition of Rhokinase with Y-27632 had no effect on exogenously applied NO-elicited relaxation.28 On the other hand, we only detected q 2008 Lippincott Williams & Wilkins



FIGURE 4. Effects of the Ca2+ ionophore A-23187 (1027 to 1026 M, 16 h) and the NOS inhibitor L-NAME (2 3 1024 M, 16 h) on ROCK-2 expression in the rat coronary microvascular endothelial cells in culture. Confluent cells were coincubated with or without thrombin (4 U/mL) and ROCK-2 protein level was measured by Western blotting. Loading control was made by actin antibody. ROCK bands were measured relative to actin bands. For statistical analysis, one-way analysis of variance followed by Dunnet posthoc test was used.

expression of Rho-kinase rather than its activity, which has generally been measured in the studies in which the interaction between Rho-signaling and L-arginine:NO pathway is investigated. Nevertheless, our findings clearly show that in the expression level of ROCK, prolonged NO donation may have no downregulatory effect on its expression in the rat coronary microvascular endothelial cells. We tentatively investigated ROCK-1 expression and found that indeed the endothelial cells are able to express ROCK-1 isoform of Rho-kinase (data not shown). In a separate study, these experiments should be repeated with regard to ROCK-1 protein. Evidence showing that Rho kinase inhibition may not cause the release of NO from endothelium comes from our other series of experiments in which endothelial NOS expression was measured after overnight incubation of the cells with two ROCK inhibitors, Y-27632 and fasudil. These inhibitors failed to upregulate eNOS expression. In addition, q 2008 Lippincott Williams & Wilkins

the inhibitors did not increase stable NO metabolites (nitrite/nitrate) in the incubating medium. However, it has been reported that Rho/Rho-kinase pathway downregulates eNOS expression in the human umbilical vein of endothelial cells.13 This discrepancy may be due to the fact that NO may play a modest role in the regulation of coronary microvascular circulation, or indeed ROCK inhibition may not enhance L-arginine:NO pathway in the rat coronary microvascular endothelium. On the other hand, these findings could not sufficiently rule out the possibility that there may be a crosstalk between NO and Rho/Rho-kinase pathways because ROCK activity, the expression of another isoform of ROCK-1, and possible effects of different exposure duration (ie, shorter and longer exposure to the NO donors) were not investigated in this study. On the other hand, long-term exposure to NO may cause the inhibition of cell respiration29 and facilitate the formation of superoxide and peroxynitrite anions, which have

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FIGURE 5. Effects of the Rho-kinase inhibitors Y-27632 (1025 M, 16 h) and fasudil (1025 M, 16 h) on eNOS expression in the rat coronary microvascular endothelial cells in culture. Confluent cells were coincubated with or without thrombin (4 U/mL) and eNOS protein level was measured by Western blotting. Loading control was made by actin antibody. eNOS bands were measured relative to actin bands. For statistical analysis, Student’s t test (for unpaired observation) was used. TABLE 1. Concentrations of Stable NO Metabolites in the Medium of Rat Coronary Microvascular Endothelial Cells Incubated With Rho-kinase Inhibitors (Y-27632, Fasudil) Within 16 Hours NO-2/NO-3 Levels (mM/mg Protein) Basal Thrombin (4 U/mL) Y-27632 (1025 M) Fasudil (1025 M) Thrombin (4 U mL21) + Y-27632 (1025 M) Thrombin (4 U mL21) + Fasudil (1025 M)

4.4 4.3 4.0 5.7 3.6 4.9

6 6 6 6 6 6

0.32 0.29 0.93 0.80 0.24 0.20

Data are means 6 SEM. Nitrite/nitrate concentrations were detected by Griess method and were relative to total protein concentration of each flask. The flask numbers were 6–15.

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been demonstrated to activate Rho/Rho-kinase signaling.30,31 It can be postulated that superoxide and peroxynitrite formation might suppress possible downregulatory effects of NO on ROCK-2 expression in our experimental model. Further studies are needed to clarify this possibility. In conclusion, these findings may suggest that NO donation seems to have no downregulatory effects over the expression of ROCK-2 isoforms of Rho-kinase in the cultured rat coronary microvascular endothelial cells. ACKNOWLEDGMENTS The authors thank S. Sxahan-Fırat and H. Kurt for their help with cell cultures, nitrite/nitrate detection, and Western blotting. q 2008 Lippincott Williams & Wilkins



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