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Published ahead of print on April 24, 2003, doi:10.1165/rcmb.2002-0157OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 29, pp. 465-471, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.2002-0157OC

Hypoxia Inhibits Myosin Phosphatase in Pulmonary Arterial Smooth Muscle Cells

Role of Rho-Kinase

Zhiqian Wang, M. Carita Lannér, Najia Jin, Darl Swartz, Liang Li and Rodney A. Rhoades

Department of Cellular/Integrative Physiology, and Department of Anatomy/Cell Biology, Indiana University School of Medicine, Indianapolis, Indiana

Address correspondence to: Dr. M. Carita Lannér, Department of Cellular and Integrative Physiology, Indiana University School of Medicine, Indianapolis, IN 46202. E-mail: clanner{at}iupui.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Rho-kinase was recently found to phosphorylate the myosin-binding subunit (MBS) of myosin phosphatase (MP) and to regulate MP activity. Although myosin light chain (MLC) phosphorylation in pulmonary arterial smooth muscle cells (PASMCs) is thought to be the cellular/molecular basis for hypoxic pulmonary vasoconstriction (HPV), very little is known about the role that Rho-kinase/MP plays in HPV. Rat PASMCs were cultured and made hypoxic (PO2 = 23 ± 2 mm Hg). Cells exposed to normoxia (PO2 ~ 148 mm Hg) served as controls. PASMCs exposed to hypoxia showed a significant increase in MLC and MBS phosphorylation, and a significant decrease in MP activity. Rho-kinase inhibitors (HA1077 or Y-27632) blocked hypoxia-induced MP inactivation and inhibited the hypoxia-induced MLC phosphorylation. Hypoxia was also found to induce stress fiber formation and actin polymerization in cultured PASMCs. In summary, these data show that MP inhibition in PASMCs is linked to activation of Rho-kinase, and that hypoxia inhibits the MP signaling pathway via Rho-kinase.

Abbreviations: Dulbecco's modified Eagle's medium, DMEM • dithiothreitol, DTT • fetal bovine serum, FBS • fluorescein isothiocyanate, FITC • hypoxic pulmonary vasoconstriction, HPV • immunoglobulin, IgG • lysophosphatidic acid, LPA • myosin-binding subunit, MBS • myosin light chain, MLC • MLC kinase, MLCK • myosin phosphatase, MP • okadaic acid, OA • pulmonary artery smooth muscle cell, PASMC • phosphate-buffered saline, PBS • Rho-associated serine/threonine kinase, Rho-kinase • sodium dodecyl sulfate, SDS • trichloroacetic acid, TCA


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Hypoxic pulmonary vasoconstriction (HPV) is a unique regulatory mechanism in the pulmonary circulation that functions to balance perfusion with ventilation. The Ca2+/calmodulin/myosin light chain (MLC) kinase (MLCK) pathway is required in the hypoxia-induced MLC phosphorylation/contraction seen in HPV (1, 2). This pathway is supported by several observations: (i) low O2 causes increase of intracellular calcium ([Ca2+]i) in pulmonary artery smooth muscle cells (PASMCs) (3, 4); (ii) Ca2+ channel blockers significantly attenuate HPV (5, 6); and (iii) hypoxic cell membrane depolarization causes influx of Ca2+ through activated voltage-gated Ca2+ channels (7). Although the Ca2+/calmodulin activation of MLCK is the dominant mechanism causing MLC phosphorylation in smooth muscle cells, another mechanism appears to be involved in the sustained phase of HPV. This is based on the observation that during the sustained phase of HPV, [Ca2+]i decreases while MLC phosphorylation remains elevated and a continuous force development is maintained (3, 4, 8).

MLC phosphorylation is regulated by a balance between myosin kinase and myosin phosphatase (MP) (9). Other studies have shown that the activity of MP is also under regulation (10, 11), and that the decrease of MP activity under a given stimulation is another important aspect of the functional regulation of smooth muscle contraction (9). Thus, the inhibition of MP under a specific physiologic condition is also important in the regulation of smooth muscle contraction.

MP is a holoenzyme composed of three subunits: a ~ 130-kD myosin-binding subunit (MBS), a 38-kD catalytic subunit (PP1c{delta}), and a small subunit of 20-kD of unknown function (12–14). The MBS is a critical subunit because it confers the specificity of the enzyme complex to myosin and regulates the activity of the complex (13). Phosphorylation of the MBS leads to inhibition of the MP, which has been demonstrated by in vitro (15) and in vivo (16–18) experiments. The phosphorylation of the MBS can be mediated by the Rho-associated kinase, Rho-kinase, which inhibits the MP (15). These studies have led to the recognition of Rho-kinase and MP as a fundamental signaling pathway in the regulation of smooth muscle contraction. Other mechanisms reported to regulate MP include arachidonic acid and PKC. Arachidonic acid binds to MP and caused dissociation of MBS from PP1C (15). PKC inhibits MP mainly through the phosphorylation of the inhibitor protein CPI17 (16, 17).

Our laboratory and others have recently shown that hypoxia significantly increases Rho-kinase activity in PASMCs, and that the Rho-kinase inhibitor (Y-27632) also significantly inhibits contraction during the sustained phase of HPV in an isolated pulmonary artery preparation (19, 20). These observations indicate the involvement of Rho-kinase in HPV. In the present study, we hypothesize that Rho-kinase/MP is a significant pathway in the sustained contraction of HPV. Data from the present study show that hypoxia inactivates MP in PASMCs, via activation of Rho-kinase and phosphorylation of the MBS. We conclude that the Rho-kinase/MP signaling pathway plays an important role in the sustained phase of the hypoxia-induced MLC phosphorylation of HPV.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Anti-MLC antibody (clone MY 21), anti-mouse immunoglobulin (IgG)–horseradish peroxidase, and fluorescein isothiocyanate (FITC)-conjugated anti–smooth muscle {alpha}-actin antibody (clone 14A) were purchased from Sigma (St. Louis, MO). Texas red–conjugated phalloidin was from Molecular Probes Inc. (Eugene, OR). Anti-MBS antibody was purchased from Berkeley Antibody Co. (Richmond, CA). Chemoluminescence reagents were from Amersham Biosciences UK (Buckinghamshire, UK) [{gamma}-32P]ATP was purchased from Dupont-New England Nuclear (Boston, MA). Protein A/G–conjugated sepharose beads were purchased from Santa Cruz (Santa Cruz, CA). HA1077 [1-(5-isoquinolinesulphonyl) homopiperazine dihydrochloride], protein phosphatase assay kit, and okadaic acid (OA) were purchased form Calbiochem (La Jolla, CA). Y-27632 [(+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride] was a kind gift from Yoshitomi Pharmaceutical Ind., Ltd, Osaka, Japan. Anti-phosphorylated MBS antibody (anti–P-{gamma}G-MBS667–1004) was a kind gift from Dr. Masaaki Ito (First Department of Medicine, Mie University, Tsu, Mie, Japan). Other chemicals and cell culture medium were purchased from Sigma.

PASMC Culture and Hypoxia Treatment
Cultured PASMCs from Sprague-Dawley rats were prepared using an explant method (20). The intrapulmonary arteries were excised and cleaned under sterile conditions. The endothelium of pulmonary artery was removed by gently rotating the vessel on a lightly sanded surgical steel rod. The vessels were cut into 2-mm pieces and incubated in Dulbecco's modified Eagle's medium (DMEM). The media was supplemented with 20% fetal bovine serum (FBS), 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 0.25 µg/ml amphotericin B. After 7 d, tissue explants were removed and fresh DMEM containing 20% FBS was added until cells reached confluence. Cells were subcultured after detachment with 0.05% trypsin in phosphate-buffered saline (PBS) and maintained in DMEM media containing 10% FBS. The cell culture medium was changed every 48 h, and cells were subcultured once weekly. Experiments were performed on cells from passages 2–5 after 24 h of serum starvation. To test the purity of smooth muscle cells, a sample was simultaneously stained with FITC-conjugated anti–smooth muscle {alpha}-actin antibody and 4,6-diamidino-2-phenylindole for smooth muscle {alpha}-actin and the nucleus, respectively. The purity was determined by fluorescence microscopy. Studies were performed on smooth muscle cells with purity higher than 90%. For the hypoxic studies, the cell culture medium was pre-bubbled with 5% CO2–95% N2. The cell medium was changed to the hypoxic medium and then cells were placed into a hypoxic incubator gassed with 3% O2–5% CO2 and balanced with N2 (PO2 23 ± 2 mm Hg) at 37°C for different time periods. Quick-freezing the cells in liquid nitrogen terminated the experiment.

Measurement of MLC Phosphorylation
Quick frozen PASMCs were denatured in cold 10% trichloroacetic acid (TCA), 10 mM dithiothreitol (DTT) in acetone, on ice. Pellets were harvested, washed with diethyl ether, and dissolved in 6 M urea, 20 mM Tris, 22 mM glycine, 10 mM DTT. Samples were applied to 10% acrylamide gel containing 40% glycerol for electrophoresis. Western blots were performed and MLC was detected with a monoclonal anti-MLC antibody. The MLC was visualized by blotting with a secondary antibody, anti-mouse IgG, conjugated to horseradish peroxidase, and detected via chemiluminescence. The amount of unphosphorylated MLC and phosphorylated MLC was determined by densitometry scanning (GS-670; Bio-Rad, Hercules, CA). The level of phosphorylated MLC was expressed as a percentage of phosphorylated MLC over total MLC (total MLC = phosphorylated MLC + unphosphorylated MLC).

Measurement of MBS Phosphorylation
Upon termination of the experiment, protein was denatured in cold 10% TCA, 10 mM DTT in acetone, on ice. Pellets were harvested by centrifugation at 15,000 x g for 10 min and washed with cold acetone three times. The dried cell powder was lysed in sodium dodecyl sulfate (SDS) sample buffer and analyzed by Western blot. Anti–phosphorylated-MBS antibody was used to detect phosphorylated MBS, and on the same blot anti-MBS antibody was used to detect total MBS.

Immunoprecipitation of MP and MP Assay
Confluent cultured PASMCs were lysed with a lysis buffer (1% Nonidet P-40, 20 mM Tris-HCl, pH 7.5, 0.15 M NaCl, 4 mM EDTA, 4 mM phenylmethylsulfonyl fluoride, 200 µg/ml leupeptin, 2 mM sodium orthovanadate). Cells were homogenized by passing the lysate through a 25-G needle, and cell debris was removed by centrifuging the solution at 16,000 x g for 10 min. Fifty microliters of prewashed protein A/G–conjugated sepharose beads were charged with 10 µl of anti-MBS antibody by mixing for 2 h at 4°C. Cell lysate was added to the charged sepharose beads and incubated for another 2 h at 4°C. After washing six times, the pellets were suspended in the protein phosphatase assay buffer containing OA for the phosphatase assay.

The phosphatase assay uses phosphorylated glycogen phosphorylase b as a substrate for the phosphatase. The assay measures two types of phosphatase, PP1 and PP2A, because glycogen phosphorylase is not a good substance for PP2B and PP2C. To exclusively measure PP1 activity, 10 nM OA was used to selectively inhibit PP2A (21). Therefore, we were able to measure PP1 activity in the MBS immunocomplex, which represents MP activity.

A Protein Phosphatase Assay Kit (Life Technologies, Gaithersburg, MD) was used to measure the phosphatase activity in the immunocomplex, and is based on the method described by Chisholm and Cohen (22). Briefly, phosphorylase b (0.1 mM) was phosphorylated in vitro by phosphorylase kinase (0.1 mg/ml) in the presence of [{gamma}-32P]ATP (5 mCi/ml) in phosphorylation buffer for 1 h at 30°C. The reaction was stopped with 90% ammoniumpersulfate solution (4°C), kept on ice for 1 h, and subsequently centrifuged at 12,000 x g for 10 min. The protein pellet was suspended and subsequently washed four times in ammoniumpersulfate solution (45% saturated). The labeled phosphorylase b protein was then concentrated to a final concentration of 3 mg/ml using Amicon Centricon-30R concentrators (Beverley, MA). Twenty microliters of the labeled substrate were mixed with the prepared immunocomplex in the presence of 10 nM OA. The reaction was allowed to proceed for 10 min at 30°C and then stopped with ice-cold 20% TCA. Samples were incubated on ice for 10 min and were centrifuged at 12,000 x g for 3 min. The radioactivity remaining in the supernatant, which reflects protein phosphatase activity, was measured with a liquid scintillation analyzer (Tri-Carb 2,300 TR; Packard; Global Medical Instrumentation, Inc., Albertville, MN).

Actin Staining
PASMCs were cultured sparsely in DMEM with 10% fetal calf serum on glass coverslips. The cells were then thoroughly washed and maintained in serum-free DMEM for 56 h. After treatment with hypoxia, cells were fixed in 4% formaldehyde and permeabilized in 0.2% Triton X-100 for 10 min. After three washes in PBS, the preparations were blocked against nonspecific binding of stain and antibody by incubation in 0.5% bovine serum albumin in PBS for 20 min at 37°C. The preparations were washed three times with warm PBS. For polymerized F-actin staining, cells were incubated with Texas red–conjugated phalloidin (100 nM) for 1.5 h at room temperature. To demonstrate expression of smooth muscle {alpha}-actin, cells were simultaneously stained with FITC-conjugated anti–smooth muscle {alpha}-actin antibody (1:250). Cells treated with 10 µM lysophosphatidic acid (LPA) for 20 min served as a positive control. Coverslips were mounted on a glass slide and examined with a fluorescence microscope (Diaphot; Nikon, Global Medical Instrumentation, Inc., Albertville, MN). Images were collected with a cool-SNAP camera HQ (Roper Scientific, Trenton, NJ) and stored and analyzed using IPLab software (Scanalytics, Fairfax, VA). Image capture times were optimally adjusted and kept constant.

Statistical Treatment of Data
Student's t test was used to compare results from the control and experimental groups. Results were expressed as mean values ± SE. A value of P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Hypoxia Inactivates MP in PASMCs
The expression of PP1, MBS, and Rho-kinase was confirmed in rat PASMCs by Western blotting (Figure 1). The effect of hypoxia on MP activity was measured in cultured PASMCs exposed for 20, 40, 60, and 90 min to normoxia and hypoxia (PO2 = 23 ± 2 mm Hg). MP activity was determined by measuring the amount of radioactively-labeled phosphorous removed by the MBS immunocomplex from phosphorylated glycogen phosphorylase b, in the presence of 10 nM OA. As shown in Figure 2A, hypoxia significantly decreased MP activity at all four time points observed (n = 3, P < 0.05). The decrease in MP activity under hypoxia was not due to reduced levels of the MBS or PP1, because the total protein levels of MBS and PP1 remained unchanged during 90 min of hypoxia (Figure 2B). However, Western blotting with anti–P-{gamma}G-MBS667–1004 showed an increase of MBS phosphorylation when PASMCs were exposed to hypoxia (Figure 3A), indicating that hypoxia causes MBS phosphorylation and inactivation of MP in PASMCs. Quantitation and the ratio of phosphorylated MBS to total MP is shown in Figure 3B. Importantly, a significant increase in the ratio of phosphorylated MBS is present at 20 min after exposure to hypoxia. The time of increase in phosphorylated MBS coincides with the time at which a significant decrease in MP activity in response to hypoxia is detected (Figure 2A).



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  		Figure 1. Expression of the components of the Rho-kinase/MP signaling pathway in PASMCs. Cultured PASMCs (passage 2) were serum-starved for 24 h before solubilization in lysis buffer. Denatured proteins were separated on a 10% SDS-PAGE, followed by immunoblotting with anti-Rho-kinase, anti-MBS, or anti-PP1C antibodies.

 


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  		Figure 2. Effect of hypoxia on MP activity and MBS phosphorylation in PASMCs. (A) PASMCs were treated with hypoxia (PO2 = 23 ± 2 mm Hg) for 20, 40, 60, and 90 min. PASMCs incubated under normoxic conditions are indicated as 0 min of hypoxia. Cells were homogenized, and MP was immunoprecipitated using anti-MBS antibody (anti–P-{gamma}G-MBS667–1004). Phosphatase activity, which is associated with the immunocomplex, was measured using 32P-labeled phosphorylase b as a substrate in the presence of 10 nM okadaic acid. Each value represents the mean ± SE from three separate experiments. In a set of preliminary experiments, immunocomplex obtained by using nonimmune IgG had a near-to-zero phosphatase activity. *P <= 0.05. (B) Representative Western blot showing the total content of MBS and the content of PP1C in PASMCs during 90 min of hypoxia. The protein concentrations of the cell lysates were measured in triplicate using the Bradford assay. Ten microgram of total protein was loaded in each lane (n = 3).

 


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  		Figure 3. Effect of hypoxia on content of MBS in PASMC and phosphorylation of MBS. (A) Representative Western blot showing MBS phosphorylation under hypoxia. LPA (10 µM, 15 min) stimulation served as a positive control. Protein denatured by ice-cold TCA was dissolved in SDS sample buffer. Anti-phosphorylated MBS antibody was used to detect phosphorylated MBS. After stripping, the same membrane was blotted with anti-MBS antibody to detect total MBS (n = 4). (B) The amount of phosphorylated MBS and total MBS was determined by densitometry scanning. The degree of phosphorylation of the MBS is expressed as the ratio of phosphorylated MBS over total MBS. Values represent the mean ± SE. *P <= 0.05

 
Inhibition of Rho-Kinase Blocks Hypoxia-Induced MP Inactivation
Cultured PASMCs were incubated with 5 µM Y-27632 or 10 µM HA1077 (two Rho-kinase inhibitors) for 30 min, followed by hypoxia for 60 min. The 5-µM concentration of Y-27632 was based on previous work in our laboratory indicating that maximal reduction of MLC20 phosphorylation occurred with this concentration (20). The 10-µM concentration of HA1077 was chosen based on work by Nagumo and coworkers, demonstrating complete inhibition of MLC20 in the presence of GTP-{gamma}-S (18). As shown in Figure 4, both HA1077 and Y-27632 significantly blocked the hypoxia-induced reduction of MP activity (n = 3, P < 0.05), suggesting that Rho-kinase is involved in the signaling pathway of hypoxia-induced inactivation of MP.



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  		Figure 4. The effect of Rho-kinase inhibition on hypoxia-induced MP inactivation in PASMCs. Myosin phosphatase activity was measured in PASMCs pretreated with vehicle (open bar), 5 µM Y-27632 (closed bar) or 10 µM HA1077 (striped bar) for 30 min, and then exposed to hypoxia (closed bar and striped bar) or normoxia (open bar) for 60 min. Phosphatase activity in the MBS immunocomplex was measured as described in MATERIALS AND METHODS. Values represent the mean ± SE from three separate experiments. *P <= 0.05.

 
Inhibition of Rho-Kinase Decreases Hypoxia-Induced MLC Phosphorylation
As seen in Figure 5, hypoxia caused a significant increase of MLC phosphorylation in PASMCs. Pretreatment of PASMCs with Y-27632 (5 µM) or HA1077 (10 µM) for 30 min blocked the hypoxia-induced MLC phosphorylation. The inhibitory effect of the Rho-kinase inhibitors on MLC phosphorylation was dose-dependent (data not shown). In addition, both HA1077 and Y-27632 significantly decreased the resting level of MLC phosphorylation in PASMC under normoxia.



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  		Figure 5. The effect of Rho-kinase inhibition on hypoxia-induced MLC phosphorylation in PASMCs. PASMCs were pretreated with vehicle, 5 µM Y-27632 or 10 µM HA1077 for 30 min, and then exposed to hypoxia (closed bar) or normoxia (open bar) for 60 min. Proteins were denatured with 10% TCA and separated using 10% acrylamide–40% glycerol gel. Phosphorylated and unphosphorylated MLC were detected using anti-MLC antibody by Western blot. The level of MLC phosphorylation was expressed as Phosphorylated MLC/(Phosphorylated MLC+ unphosphorylated MLC)x100%. Data are the summary of results from three separate experiments. Each value represents the mean ± SE. *P <= 0.05.

 
Hypoxia Induces Stress Fiber Formation in PASMCs
PASMCs were exposed to 60 min hypoxia to investigate whether hypoxia alone is sufficient to induce stress fiber formation. The formation of stress fibers under hypoxia indicates activation of Rho-kinase by hypoxia, because Rho-kinase activation is required to induce stress fiber formation (23).To minimize stress fiber formation in serum, PASMCs were serum-starved for 56–72 h before exposure to hypoxia. Figure 6 shows the representative responses of serum-starved PASMCs to hypoxia. Lysophosphatidic acid (LPA), a known agent for inducing stress fiber formation in cultured cells, served as a positive control. Cells pretreated with 10 µM LPA displayed a significant increase in stress fiber formation. The positive response is characterized by the formation of straight, parallel actin filaments from the woven, unorganized actin filaments in resting cells. Hypoxia caused a similar response in actin filament formation. Taken together, the hypoxia-induced stress fiber formation provides structure/function confirmation of Rho-kinase activation, and provides additional support to the idea that Rho kinase activation is functionally involved with hypoxia.



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  		Figure 6. The effect of hypoxia on stress fiber formation in PASMCs. Representative microscopic fields show serum-starved PASMCs, which were treated with hypoxia for 60 min or with 10 µM LPA for 20 min. The upper panels show phase-contrast images. The center panels show F-actin in stress fibers stained with Texas red–conjugated phalloidin. The lower panels show expression of smooth muscle {alpha}-actin detected with FITC-conjugated anti–smooth muscle {alpha}-actin monoclonal antibody.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Reversible phosphorylation of MLC at Ser-19 is the principal mechanism that induces activation of actomyosin ATPase and causes smooth muscle contraction (24). The regulation of MLC phosphorylation is the result of the balance between myosin kinase and MP. Ca2+/calmodulin-activated MLCK has been well established as the dominant kinase in causing MLC phosphorylation, which leads to contraction in smooth muscle. The primary contraction of HPV is manifested through the same basic mechanisms, requiring increased [Ca2+]i and activated MLCK (5, 6).

The purpose of the present study was to investigate the role of the Rho-kinase/MP pathway in the response of PASMC to hypoxia. Inhibition of MP under specific physiologic conditions is an important regulator of smooth muscle contraction (9). In other systems, Rho-kinase has been shown to phosphorylate MP, thereby inhibiting the phosphatase (15, 25, 26). We and others have demonstrated activation of Rho-kinase as an important regulator of hypoxia-induced smooth muscle contraction (19, 20). Therefore, we hypothesized the involvement of MP-inhibition by Rho-kinase in the hypoxic response of PASMCs.

The role of MP in the hypoxic response is indicated by the direct inhibition of MP by hypoxia (Figure 2A). Our study shows an ~ 25–45% decrease in MP activity measured using glycogen phosphorylase b as a substrate. Inhibition of MP activity by hypoxia was sustained for up to 90 min in our assay, with maximum inhibition attained at 20 min. Although glycogen phosphorylase b is a substrate for both type I and type II phosphatases, the inclusion of OA restricted the assay to type I phosphatases, which is the classification of the MP. The specificity of the assay is further assured by using the MBS immunocomplex to measure phosphatase activity, a method validated by other studies to assay MP activity (27, 28). These data demonstrate the inhibition of MP by hypoxia.

The inhibition of the MP by hypoxia was mediated by phosphorylation of the MBS. We observed that hypoxia generated an increase in the level of phosphorylation of the MBS, although the total amount of MBS present in the samples remained unchanged (Figures 2B, 3A and 3B). Phosphorylation of the MBS is a known mechanism of inhibition for this phosphatase (16). This suggests that the mechanism of inhibition by hypoxia is mediated by phosphorylation of the MBS. The concomitant increase in phosphorylation of the MBS and decrease in MP activity under hypoxia support hypoxic inhibition of MP through phosphorylation of the MBS.

In other tissues, Rho-kinase has been identified as the agent primarily responsible for phosphorylation and inactivation of MP (15, 17, 28, 29). In the present study, the Rho-kinase inhibitors HA1077 and Y-27632 blocked the hypoxia-induced inactivation of MP in PASMCs (Figure 4), suggesting that Rho-kinase is an upstream signal of MP inhibition. These data support the idea that hypoxia-induced activation of Rho-kinase contributes to the inhibition of MP in PASMCs. Our previous work, along with that of others, showed significant activation of Rho-kinase by hypoxia and the involvement of this phenomenon in the sustained phase of HPV in isolated rat pulmonary arteries (19, 20). In the present study, the activation of Rho-kinase by hypoxia is confirmed by the observation of actin stress fiber formation in the presence of hypoxia (Figure 6). The hypoxia-induced stress fiber formation occurs in the absence of other stimuli, such as growth factors, because the cells were serum-starved. The addition of Rho-kinase inhibitors (Y-27632 and HA1077) to cultured rat PASMC under normoxia blocked the formation of stress fibers, supporting the requirement for Rho-kinase in stress fiber formation (unpublished data). In addition, the reduction in stress fiber formation was pronounced enough to mask any hypoxia-induced formation of stress fibers. Because Rho-kinase activation has been shown to be required and sufficient for actin stress fiber formation (23), the induction of stress fibers in hypoxic PASMCs indicates Rho-kinase activation. The observation of Rho-kinase activation by hypoxia provides the basis for the involvement of Rho-kinase in MP inactivation by hypoxia.

Another aspect of Rho-kinase activity in PASMC is apparent in the reduction of basal MLC phosphorylation when PASMC were treated with the Rho-kinase inhibitors HA1077 or Y-27632 (Figure 5). However, the reduction in phosphorylation was not associated with an increase in MP activity (Figure 4). A similar observation has been reported in human platelets, where the Rho-kinase inhibitors HA1077 and Y-27632 caused a decrease in MLC phosphorylation without an increase in MP activity (28). These observations suggest that Rho-kinase may contribute to the basal level of MLC phosphorylation by directly phosphorylating MLC. Indeed, Rho-kinase has been shown to directly phosphorylate MLC in cell-free systems (30, 31). However, studies using smooth muscle tissue do not support the idea that Rho-kinase directly phosphorylates MLC (32, 33). A possible explanation for the observed differences seen between the in vitro and in situ studies may be spatial restriction of the Rho-kinase (34). This would allow very limited access to MLC and conditional access to MP. We speculate that under specific conditions, such as high serum levels, the restrictive limit for Rho-kinase access to MLC is relaxed, possibly due to upregulated expression of the Rho-kinase (35). The release of the restriction might be a mechanism for normal smooth muscle cells to migrate under certain pathophysiologic conditions.

In summary, the present study demonstrates that hypoxia causes inactivation of MP in PASMCs, via the activation of Rho-kinase. This mechanism, along with the Ca2+/calmodulin/MLCK pathway, contributes to hypoxia-induced MLC phosphorylation in PASMCs and HPV. A strategy of using a Ca2+ blocker and Rho-kinase inhibitor in combination may provide a more effective and safer approach in the treatment of hypoxic pulmonary hypertension.


   Acknowledgments
 
The authors thank Yoshitomi Pharmaceutical Ind., Ltd, Osaka, Japan for providing Y-27632. They thank Dr. Masaaki Ito for providing the anti-phosphorylated MBS antibody. Z.W. was supported by an American Heart Association Predoctoral Fellowship. The research was also supported by the Showalter foundation.

Received in original form August 16, 2002

Received in final form April 14, 2003


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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