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Am J Physiol Lung Cell Mol Physiol 292: L678-L684, 2007. First published November 3, 2006; doi:10.1152/ajplung.00178.2006
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Role of Rho kinases in PKG-mediated relaxation of pulmonary arteries of fetal lambs exposed to chronic high altitude hypoxia

Yuansheng Gao,1,2 Ada D. Portugal,1 Sewite Negash,1 Weilin Zhou,1 Lawrence D. Longo,3 and J. Usha Raj1

1Division of Neonatology, Harbor-UCLA Medical Center, Geffen School of Medicine at University of California, and Los Angeles Biomedical Research Institute, Los Angeles, California; 2Key Laboratory of Molecular Cardiovascular Sciences, Peking University, Ministry of Education, Beijing, China; and 3Center for Perinatal Biology, Loma Linda University, School of Medicine, Loma Linda, California

Submitted 16 May 2006 ; accepted in final form 30 October 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
An increase in Rho kinase (ROCK) activity is implicated in chronic hypoxia-induced pulmonary hypertension. In the present study, we determined the role of ROCKs in cGMP-dependent protein kinase (PKG)-mediated pulmonary vasodilation of fetal lambs exposed to chronic hypoxia. Fourth generation pulmonary arteries were isolated from near-term fetuses (~140 days of gestation) delivered from ewes exposed to chronic high altitude hypoxia for ~110 days and from control ewes. In vessels constricted to endothelin-1, 8-bromoguanosine-cGMP (8-Br-cGMP) caused a smaller relaxation in chronically hypoxic (CH) vessels compared with controls. Rp-8-Br-PET-cGMPS, a PKG inhibitor, attenuated relaxation to 8-Br-cGMP in control vessels to a greater extent than in CH vessels. Y-27632, a ROCK inhibitor, significantly potentiated 8-Br-cGMP-induced relaxation of CH vessels and had only a minor effect in control vessels. The expression of PKG was increased but was not accompanied with an increase in the activity of the enzyme in CH vessels. The expression of type II ROCK and activity of ROCKs were increased in CH vessels. The phosphorylation of threonine (Thr)696 and Thr850 of the regulatory subunit MYPT1 of myosin light chain phosphatase was inhibited by 8-Br-cGMP to a lesser extent in CH vessels than in controls. The difference was eliminated by Y-27632. These results suggest that chronic hypoxia in utero attenuates PKG-mediated relaxation in pulmonary arteries, partly due to inhibition of PKG activity and partly due to enhanced ROCK activity. Increased ROCK activity may inhibit PKG action through increased phosphorylation of MYPT1 at Thr696 and Thr850.

cGMP; myosin light chain phosphatases; vascular smooth muscle; cGMP-dependent protein kinase; Rho kinase

RHO KINASE (ROCK) is a serine/threonine protein kinase with a molecular mass of ~160 kDa. It is present in two isoforms, the type I and II. These are encoded by different genes and are expressed ubiquitously. Within the cells, ROCKs are distributed in the cytoplasm but can be translocated to the peripheral membrane on RhoA activation. Currently, more than 15 ROCK substrates have been identified. The phosphorylation of these proteins by ROCKs is related to a variety of functions such as contraction, migration, proliferation, apoptosis/survival, gene expression, and differentiation (33, 38).

ROCK-mediated phosphorylation of MYPT1, the regulatory subunit of myosin light chain phosphatase (MLCP), is recognized as a critical mechanism in the regulation of contractility of vascular smooth muscle cells. When stimulated with various constrictors, the activated ROCKs may inhibit MLCP and cause increased Ca2+ sensitivity of the contractile filaments through phosphorylation of MYPT1 at threonine (Thr)696 and Thr853 (human sequence) and through phosphorylation of PKC-potentiated inhibitor protein of 17 kDa (CPI-17) at Thr38 (43). ROCK activity may be preferentially augmented in a number of vascular diseases such as hypertension, atherosclerosis, and pulmonary hypertension (28, 42). In lungs, increased ROCK expression and/or activity is associated with chronic hypoxia-induced pulmonary hypertension (14, 16, 22, 37). Furthermore, ROCK inhibitors have been found to be effective in preventing and reversing chronic hypoxia-induced pulmonary hypertension in animal models and seem to have some therapeutic benefit in treatment of pulmonary hypertension in humans (7, 11, 14, 16, 36–38).

cGMP-dependent protein kinase (PKG) is the primary enzyme involved in mediating relaxation of smooth muscle induced by nitric oxide and cGMP (9, 15, 27). Activation of PKG results in reduction of Ca2+ sensitization of the contractile filaments by stimulation of MLCP activity through interaction between its leucine zipper motifs and MYPT1 and by the phosphorylation of MYPT1 by PKG at Serine (Ser)695 and Ser852. Ser695 and Ser852 are immediately adjacent to Thr696 and Thr853(human sequence), respectively. The latter two sites can be phosphorylated by ROCKs and are associated with inhibition of MLCP (27, 43, 44, 51). The opposing actions of PKG and ROCK at MYPT1 prompted us to speculate that, in chronically hypoxic lungs, ROCKs may attenuate PKG-induced vasodilation by their counteracting effects on MYPT1. The present study was designed to test this hypothesis. Our results suggest that chronic hypoxia-induced suppression of PKG-mediated relaxation of fetal ovine pulmonary arteries may result partially from inhibition of PKG activity and partially from enhanced ROCK activity. Furthermore, the effect of ROCKs may be due to an increased phosphorylation of MYPT1 at Thr696 and Thr850 (corresponding to Thr853 in human sequence).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. Pregnant ewes carrying single or twin fetuses (~140 days of gestation; term being 147 days, either sex) were obtained from Nebeker Ranch, Lancaster, CA (altitude: ~300 m; PaO2, 102 ± 2 mmHg). To induce chronic hypoxia in the fetus, some pregnant sheep were transported to Barcroft Laboratory, White Mountain Research Station, Bishop, CA (3,801 m altitude; PaO2, 60 ± 2 mmHg) at 30 days of gestation and kept there for ~110 days. The ewes were brought down to sea level immediately before delivery. They were anesthetized with thiamylal (10 mg/kg iv), and anesthesia was maintained on 1.5–2.0% halothane in oxygen throughout surgery. The fetus was delivered by cesarean section and killed by a lethal dose of pentobarbital (100 mg/kg) via the umbilical vein. After the fetuses were delivered, the ewe was then euthanized with T-61 (euthanasia solution; Hoechst-Roussel, Somerville, NJ). All procedures and protocols used in the present study were approved by the Animal Research Committees of Loma Linda University (29) and Los Angeles Biomedical Research Institute at Harbor-UCLA.

Tissue preparation. Fourth generation pulmonary arteries (outside diameter: 1.5–2.0 mm) were dissected free of parenchyma and cut into rings (length: 3 mm) in ice-cold modified Krebs-Ringer bicarbonate buffer [composition (in mM): 118.3 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 25 NaHCO3, 11.1 glucose].

Organ chamber study. Rings of pulmonary arteries were suspended in organ chambers filled with 10 ml of modified Krebs-Ringer bicarbonate solution maintained at 37°C and aerated with 95% O2-5% CO2 (pH 7.4). Each ring was suspended by two stirrups passed through the lumen. One stirrup was anchored to the bottom of the organ chamber, and the other one connected to a strain gauge (model FT03C; Grass Instrument, Quincy, MA) for the measurement of isometric force (13).

At the beginning of each experiment, vessel rings were brought to their optimal tension by stretching the vessels progressively until the contractile responses to 100 mM potassium chloride were maximal. The optimal resting tension was 0.80 ± 0.08 g (n = 6) and 0.67 ± 0.09 g (n = 6) for the control and chronically hypoxic vessels, respectively (P > 0.05). One hour of equilibration was allowed after the vessels were brought to their optimal tension. When contraction became stable, the effects of 8-bromoguanosine-cGMP (8-Br-cGMP) [a cell-permeable cGMP analog] were determined under control conditions or treated with Rp-8-Br-PET-cGMPS [a PKG inhibitor (4)] or Y-27632 [an inhibitor of Rho kinase (1, 10)]. The inhibitors were administrated at least 30 min before testing their effects.

PKG activity assay. Isolated pulmonary arteries of fetal lambs were homogenized in a buffer containing 50 mM Tris HCl (pH 7.4), 10 mM EDTA, 2 mM dithiothreitol, 1 mM IBMX, 100 µM nitro-L-arginine, and 10 µM indomethacin. The homogenate was sonicated and centrifuged at 13,000 g for 10 min at 4°C. Supernatants were assayed for protein kinase G activity by measuring the incorporation of 32P from [{gamma}-32P]ATP into a specific PKG substrate, BPDEtide (Biomol Research Laboratories, Plymouth Meeting, PA). Aliquots (20 µl) of supernatant were added to a mixture (total volume, 50 µl) containing 50 mM Tris HCl (pH 7.4), 20 mM MgCl2, 0.1 mM IBMX, 10 µM indomethacin, 100 µM nitro-L-arginine, 150 µM BPDEtide, 1 µM PKI (a synthetic PKA inhibitor; Peninsula Laboratories, Belmont, CA), and 0.2 mM [{gamma}-32P]ATP (specific activity 3,000 Ci/mmol). The mixture was incubated at 30°C for 10 min in the presence or absence of 3 µM exogenous cGMP. Reaction was terminated by spotting 40-µl aliquots of mixture onto phosphocellulose papers (2 cm x 2 cm; P81 Whatman) and placing them in ice-cold 75 mM phosphoric acid. The filter papers were washed, dried, and counted in a liquid scintillation counter. Assays were performed in triplicate with appropriate controls. After subtracting control counts, counts obtained indicate PKG activity, which is expressed as picomoles of 32P incorporated into PKG substrate per minute per milligram of protein. Protein content in supernatant was measured by the Bradford procedure using bovine serum albumin as a standard (3). Preliminary experiments confirmed the linearity of PKG activity at the protein concentration used within the incubation time (6, 12).

ROCK assay. Tissues were homogenized in a buffer containing 50 mM Tris HCl (pH 7.5), 100 µM EGTA, 1% 2-mercaptoethanol, 100 µM nitro-L-arginine, and 10 µM indomethacin. The homogenate was sonicated and centrifuged at 1,000 g for 10 min at 4°C. Aliquots (25 µl) of supernatant were added to a mixture (total volume, 50 µl) containing 50 mM Tris HCl (pH 7.5), 100 µM EGTA, 1% 2-mercaptoethanol, 10 mM MgCl2, 100 µM ATP, 10 µM indomethacin, 100 µM nitro-L-arginine, and 0.5 ng MYPT1 (the regulating subunit of myosin light chain phosphatase). The mixture was incubated at 30°C for 10 min in the presence of solvent, 30 or 50 µM arachidonic acid, and 10 µM Y-27632. The reaction was terminated by adding 50 µl 2x Laemmli sample buffer and boiled for 5 min after addition of Laemmli sample buffer. Samples were then subjected to Western blot analysis using a rabbit polyclonal antibody against phospho-MYPT1 at Thr696 (cat. no. 07-251, polyclonal; dilution, 1:1,000; Upstate Biotechnology, Lake Placid, NY) as the primary antibody (1, 19).

Western blots for PKG I, ROCK isoforms, MYPT1, and phospho-MYPT1. Tissue lysates prepared from whole tissue homogenate of fetal ovine pulmonary arteries were solubilized in 2x Laemmli sample buffer and clarified (800 g, 10 min) before SDS-PAGE. The lysates, each containing 20 µg of protein, were subjected to SDS-PAGE and electrotransferred to nitrocellulose. Nonspecific binding of antibody was blocked by washing with Tris-buffered saline containing 10% milk for 1 h. The blot was then subjected to two brief washes with TBS plus 0.5% Tween 20 and incubated in TBS plus 0.1% Tween 20 and the primary antibody with appropriate dilution for overnight. After two more washes in TBS plus 0.1% Tween 20, the blot was incubated for 40 min in secondary antibody, washed, and developed using the chemiluminescent detection method (SuperSignal West Pico Chemiluminescent Substrate; Pierce, Rockford, IL). The amount of the specific protein present in blots was quantified by densitometry using an Eagle Eye II Still Video System (Stratagene, La Jolla, CA) and normalized to scanning signals of actin (Oncogene, La Jolla, CA) (12). The primary antibodies used were anti-PKG type I (cat. no. KAS-PK005, polyclonal, detects an ~75-kDa protein, corresponding to PKG type I; dilution, 1:5,000; Stressgen, Victoria, BC, Canada); anti-ROCK-I (cat. no. 611137, monoclonal, detects an ~160-kDa protein, corresponding to ROCK-I; dilution, 1:1,000; BD Biosciences, San Jose, CA); anti-ROCK-II (cat. no. 610624, monoclonal, detects an ~180-kDa protein, corresponding to ROCK-II; dilution, 1:1,000; BD Biosciences); anti-MYPT1 (cat. no. 612165, monoclonal, detects an ~130-kDa protein, corresponding to MYPT1; dilution, 1:1,000; BD Biosciences); anti-phospho-MYPT1 at Thr696 (cat. no. 07-251, polyclonal, detects an ~130-kDa protein, corresponding to phospho-MYPT1 at Thr696; dilution, 1:1,000; Upstate Biotechnology); and anti-phospho-MYPT1 at Thr850 (cat. no. 36-003, polyclonal, detects an ~130-kDa protein, corresponding to phospho-MYPT1 at Thr853 in human MYPT1; dilution, 1:1,000; Upstate Biotechnology).

For Western analysis of MYPT1 and phospho-MYPT1 at Thr696 and Thr850, pulmonary arteries were first incubated in the modified Krebs-Ringer bicarbonate solution (37°C, 95% O2-5% CO2, pH 7.4) in the presence of solvent, endothelin-1 (6 x 10–9 M) for 30 min, followed by 8-Br-cGMP (10–4 M) for another 30 min. In some vessels, Rp-8-Br-PET-cGMPS (3 x 10–5 M) or Y-27632 (10–5 M) was included in the incubation buffer. They were administrated at least 30 min before the addition of endothelin-1. For all vessels, nitro-L-arginine (10–4 M) and indomethacin (10–5 M) were administered to exclude the involvement of endogenous nitric oxide and cyclooxygenase products (12, 34). At the end of incubation, the tissues were rapidly frozen with liquid nitrogen and homogenated in a buffer containing 50 mM Tris HCl (pH 7.4), 100 µM EGTA, 1 mM sodium orthovanadate, 1% 2-mercaptoethanol, and 10% SDS. The homogenate was sonicated (5 s for 3 times, 4°C), centrifuged (1,000 g, 10 min, 4°C), and then solubilized in 2x Laemmli sample buffer before SDS-PAGE.

Drugs. The following drugs were used (unless otherwise specified, all were obtained from Sigma, St. Louis, MO): 8-Br-cGMP, Rp-8-Br-PET-cGMPS (Rp isomer; Biolog Life Science Institute, La Jolla, CA), endothelin-1 (American Peptide, Sunnyvale, CA), indomethacin, nitro-L-arginine, and Y-27632 (Biomol, CA).

Indomethacin (10–5 M) was prepared in equimolar Na2CO3. This concentration of Na2CO3 did not significantly affect the pH of the solution in the organ chamber. The other drugs were prepared using distilled water.

Data analyses. Contractions are expressed in grams. Relaxations are expressed as percent of contraction of vessels to endothelin-1. Data are shown as means ± SE. When mean values of two groups were compared, Student's t-test for unpaired observations was used. When the mean values of the same group before and after stimulation were compared, Student's t-test for paired observations was used. Comparison of mean values of more than two groups was made with one-way ANOVA test with Student-Newman-Keuls test for post hoc testing of multiple comparisons. All these analyses were performed using a commercially available statistics package (SigmaStat; Jandel Scientific, San Rafael, CA). Statistical significance was accepted when the P value (2-tailed) was less than 0.05. In all experiments, n represents the number of lambs.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Vessel tension study. To exclude the involvement of endogenous nitric oxide and cyclooxygenase products, nitro-L-arginine (10–4 M) and indomethacin (10–5 M) were administered at the beginning of the experiments. These inhibitors had no effect on the resting tension of the vessels (data not shown, n = 6, P > 0.05).

Relaxation of pulmonary arteries induced by 8-Br-cGMP [a cell-permeable analog of cGMP (32)] was determined after the vessel tension was raised with endothelin-1 (6 x 10–9 M to 2 x 10–8 M) to a similar level (Table 1). The analog of cGMP caused a smaller relaxation of vessels obtained from the chronically hypoxic fetal lambs than from control lambs. Rp-8-Br-PET-cGMPS (3 x 10–5 M), a selective inhibitor of PKG (4, 6, 12), caused a greater attenuation of the relaxation to 8-Br-cGMP in control vessels than in chronically hypoxic vessels. In the presence of the PKG inhibitor, the difference in the relaxation response to 8-Br-cGMP between the control and chronically hypoxic vessels was abolished (Fig. 1).


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  		Table 1. Vessel tension of pulmonary arteries of fetal lambs raised by endothelin-1

 

Figure 1
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  		Fig. 1. Relaxation of pulmonary arteries of normoxic and chronically hypoxic fetal lambs induced by 8-bromoguanosine-cGMP (8-Br-cGMP) in vessels preconstricted with endothelin-1 (6 x 10–9 M). PKG-I (Rp-8-Br-PET-cGMPS) is at 3 x 10–5 M. Data are shown as means ± SE; n = 5–6 for each group. *Significantly different from chronically hypoxic vessels and from vessels treated with PKG-I; {dagger}significantly different from vessels treated with PKG-I (P < 0.05).

 
Y-27632 (10–5 M), a specific inhibitor of Rho kinase (1, 10), showed no effect on relaxation of control vessels to 8-Br-cGMP at concentrations ranging from 10–7 M to 3 x 10–4 M with the exception that relaxation caused by the cGMP analog at 3 x 10–5 M was moderately augmented. For chronically hypoxic vessels, the relaxation to 8-Br-cGMP was significantly potentiated. The relaxation of chronically hypoxic vessels treated with Y-27632 was similar to that of control vessels not treated with the inhibitor of Rho kinase (Fig. 2).


Figure 2
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  		Fig. 2. Relaxation of pulmonary arteries of normoxic and hypoxic fetal lambs induced by 8-Br-cGMP in vessels preconstricted with endothelin-1 (6 x 10–9 M). Y-27632 is at 10–5 M. Data are shown as means ± SE; n = 5–6 for each group. *Significantly different from normoxic vessels and from vessels treated with Y-27632; {dagger}significantly different from chronically hypoxic vessels treated with Y-27632 (P < 0.05).

 
PKG. The expression of PKG protein in pulmonary arteries from fetal lambs exposed to chronic hypoxia was significantly increased (Fig. 3). However, the PKG activity in chronically hypoxic arteries, both under basal conditions and when stimulated with cGMP (3 x 10–6 M), was not significantly different from that in control vessels. In all vessels, the increases in PKG activity caused by cGMP were abolished by Rp-8-Br-PET-cGMPS (3 x 10–5 M) (Fig. 4).


Figure 3
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  		Fig. 3. Effect of chronic hypoxia on cGMP-dependent protein kinase (PKG) protein expression in pulmonary arteries of fetal lambs. Top: Western blots. Bottom: densitometric scanning of PKG protein normalized to actin and expressed relative to that of normoxic vessels as 1. Data are shown as means ± SE; n = 6 for each group. *Significantly different from normoxia (P < 0.05).

 

Figure 4
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  		Fig. 4. PKG activity in pulmonary arteries of normoxic and chronically hypoxic fetal lambs. cGMP is at 3 x 10–6 M; PKG-I (Rp-8-Br-PET-cGMPS) is at 3 x 10–5 M. Data are shown as means ± SE; n = 6 for each group. *Significantly different from basal (P < 0.05).

 
Rho kinase. There was no significant difference in the level of expression of Rho kinase type I protein between the control and chronically hypoxic vessels. The expression of Rho kinase type II protein in chronically hypoxic vessels was increased compared with that in control vessels (Fig. 5). The basal level of Rho kinase activity was similar among different vessel groups. When stimulated with arachidonic acid, which can directly activate Rho kinase (1, 10), the increase in activity of Rho kinase was greater in chronically hypoxic vessels compared with that in controls. In the presence of Y-27632 (10–5 M), the activity of Rho kinase in all vessels was reduced below basal levels (Fig. 6).


Figure 5
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  		Fig. 5. Effect of chronic hypoxia on Rho kinase (ROCK) expression. Top: Western blots. Bottom: densitometric scanning of ROCK normalized to actin and expressed relative to that of normoxic vessels as 1. Data are shown as means ± SE; n = 6 for each group. *Significantly different from normoxia (P < 0.05). N, normoxia; H, hypoxia.

 

Figure 6
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  		Fig. 6. Effect of chronic hypoxia on ROCK activity of tissue homogenate of fetal ovine pulmonary arteries. Assay was conducted using MYPT1 (654-880) as the substrate and arachidonic acid (AA) as the stimulant for ROCK. The ROCK activity was determined by Western blotting using antibody against phosphorylated MYPT1 at Thr696. Top: Western blots. Bottom: densitometric scanning of phosphorylated MYPT1 at Thr696 normalized to actin and expressed relative to that of normoxic vessels as 1. Y-27632 is at 10–5 M. Data are shown as means ± SE; n = 5–6 for each group. *Significantly different from control; {dagger}significantly different from normoxia (P < 0.05).

 
Phosphorylation of MYPT1 at Thr696 and Thr850. The phosphorylation of MYPT1, the regulatory subunit of MLCP, at Thr696 and Thr850 by Rho kinase may suppress the activity of MLCP and thus result in vasoconstriction (43, 51). The basal levels of phosphorylation of MYPT1 at Thr696 and Thr850 of chronically hypoxic pulmonary arteries were similar to those of control vessels. Endothelin-1 (6 x 10–9 M) caused a greater phosphorylation of MYPT1 at Thr696 and Thr850 in chronically hypoxic vessels compared with that in controls. In the presence of endothelin-1, 8-Br-cGMP (10–4 M) suppressed the phosphorylation of MYPT1 in chronically hypoxic vessels to a lesser extent than in control vessels; this difference was eliminated when Y-27632 (10–5 M) was present. The effect of the cyclic nucleotide was blocked by Rp-8-Br-PET-cGMPS (3 x 10–5 M) (Fig. 7). When vessels were treated with endothelin plus Y-27632 (10–5 M), the phosphorylation of MYPT1 in all vessels was reduced to a similar level and was below basal values (0.26 ± 0.06 and 0.32 ± 0.09 for control and hypoxic vessels, respectively). Data are normalized to non-phosphorylated MYPT1 and expressed relative to values in normoxic vessels (n = 6, P > 0.05).


Figure 7
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  		Fig. 7. Effect of chronic hypoxia on ROCK-induced phosphorylation of MYPT1 at Thr696 and Thr850. Top: Western blots. Bottom: densitometric scanning of phosphorylated MYPT1 at Thr696 and Thr850 normalized to non-phosphorylated MYPT1 and expressed relative to that of normoxic vessels as 1. ET-1 is at 6 x 10–9 M; 8-Br-cGMP, 10–4 M; PKG-I (Rp-8-Br-PET-cGMPS) is at 3 x 10–5 M; Y-27632 is at 10–5 M. Data are shown as means ± SE; n = 6 for each group. *Significantly different from control; {dagger}significantly different from normoxia (P < 0.05).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the present study, relaxation of pulmonary arteries of fetal lambs induced by 8-Br-cGMP was markedly attenuated by Rp-8-Br-PET-cGMPS, a specific inhibitor of PKG (4). This is consistent with our previous studies and those by others (6, 11, 27), indicating an important role for PKG in cGMP-induced pulmonary vasodilation. The relaxation induced by 8-Br-cGMP in chronically hypoxic vessels was significantly lower than that in control vessels. However, following inhibition of PKG, the differential response to cGMP analog between the control and chronically hypoxic vessels was eliminated. This suggests that the PKG-dependent component of cGMP-induced relaxation was preferentially suppressed by chronic hypoxia.

We found that PKG protein expression was increased in the chronically hypoxic vessels compared with that in normoxic vessels. However, the activity of the enzyme in the hypoxic vessels was not increased proportionately. This would indicate that the intrinsic kinase activity of PKG protein was partially inhibited by chronic hypoxia and may in part contribute to attenuation of cGMP-mediated relaxation. However, the underlying mechanism of this inhibition is not clear. Studies show that the production of reactive oxygen species (ROS) is enhanced in chronically hypoxic pulmonary arteries (23) and that ROS inhibits cGMP-mediated relaxation of pulmonary arteries (5).

MLCP is a critical target for regulating the Ca2+ sensitivity of contractile filaments of smooth muscle cells. The activity of MLCP can be stimulated by PKG and inhibited by ROCKs through their action on MYPT1, the regulatory subunit of MLCP (28, 33, 39, 43, 51). Therefore, it would be logical to suspect that an increased ROCK activity may attenuate PKG-mediated relaxation through its antagonistic action on MYPT1. In our study, arachidonic acid-stimulated ROCK activity, which represents direct stimulation of ROCK activity (1, 10), was significantly greater in chronically hypoxic pulmonary arteries than in control vessels. This arachidonic acid-stimulated ROCK activity was blocked by Y-27632, a specific inhibitor of ROCKs. Increased ROCK activity has also been reported in pulmonary arteries of rats with chronic hypoxia-induced pulmonary hypertension (14, 22). ROCKs exist in two isoforms, type I and type II. Both are present in vascular smooth muscle. Currently, the functional distinctions between these isoforms are not clear (33). We found that the protein level of type II, but not type I, ROCK was greater in the chronically hypoxic vessels compared with control vessels. The increased stimulated ROCK activity that was measured may represent increased activity of the type II isoform.

In our studies, although ROCK II protein expression was increased in chronically hypoxic pulmonary arteries, basal ROCK activity was not increased. In pulmonary arteries of rats with chronic hypoxia-induced pulmonary hypertension, ROCK II expression and stimulated ROCK activity were increased. However, the basal activity of ROCK was unchanged (22). It is not clear why the basal activity of ROCK was not increased even though ROCK protein expression and stimulated ROCK activity were increased in the hypoxic pulmonary arteries. This may be due to the fact that ROCK activity depends not only on the protein level of the enzyme but also on the subcellular location of the protein (cytoplasmic vs. membrane localization) (28).

Thr695 and Thr850 are identified as the major inhibitory phosphorylation sites of MYPT1 by ROCKs. Phosphorylation of Thr696 has been associated with inhibition of the catalytic activity of MLCP, and the phosphorylation of Thr850 is associated with inhibition of the holoenzyme binding to myosin (43). Studies show that ROCK may inhibit MLCP activity through the phosphorylation of MYPT1 at either Thr695 (8, 17, 18, 40), Thr850 (24, 35, 50), or both (31, 48). In the present study, we found that the phosphorylation of MYPT1 at both Thr696 and Thr850 stimulated by endothelin-1, which indirectly stimulates ROCK through G protein-dependent activating RhoA/ROCK pathway (49), was greater in chronically hypoxic pulmonary arteries than in control vessels. This phosphorylation was inhibited by Y-27532, confirming that it is mediated by ROCKs. The finding that the phosphorylation was increased in hypoxic pulmonary arteries is consistent with the increased ROCK II protein expression and increased stimulated ROCK activity in the hypoxic vessels. These results are also in agreement with other studies that have reported increased ROCK signaling in pulmonary vessels following exposure to chronic hypoxia (14, 16, 22, 37).

The phosphorylation of MYPT1 at Thr696 and Thr850 by ROCKs could counteract the stimulatory action of PKG on MLCP activity and reduce the vasodilation induced by cGMP (43, 44, 51). PKG may stimulate MLCP activity through interaction between its leucine zipper motifs and MYPT1 (43). PKG may also antagonize phosphorylation of MYPT1 induced by ROCKs by phosphorylating MYPT1 at Ser695 and Ser852, sites that are immediately adjacent to Thr696 and Thr853 (human sequence), respectively (51). In our study, phosphorylation of Thr696 and Thr850 by ROCKs was reduced by 8-Br-cGMP to a lesser extent in hypoxic vessels than in controls. The differential inhibitory effect of the cGMP analog in hypoxic and control vessels was eliminated by Y-27632. These results are consistent with the results from the vessel tension study, which showed that the reduced relaxation of chronically hypoxic vessels induced by 8-Br-cGMP was reversed by inhibition of ROCKs with Y-27632.

In fetal and newborn lungs, PKG is the primary pathway for vasodilation induced by agents that elevate cGMP such as endothelium-derived nitric oxide and exogenous nitrovasodilators (6, 12, 25, 26). cGMP-mediated responses have been found to be impaired in hypoxia-induced pulmonary hypertension (2, 20, 21, 41, 45). As shown in the overall schema of Fig. 8, our results suggest that impaired relaxation of the hypoxic pulmonary arteries to cGMP may be caused in part by a suppression of intrinsic PKG activity and in part by an increased stimulated activity of ROCKs. Moreover, the enhanced ROCK activity in hypoxic vessels may attenuate PKG effect by an increased phosphorylation of the regulatory subunit MYPT1 of MLCP at Thr696 and Thr850. It should be noted that the alternations in signaling mechanisms in vascular smooth muscle following exposure to chronic hypoxia are very complex (30). Chronic hypoxia may increase, whereas nitric oxide, probably mediated by PKG, may reduce the activation of RhoA of pulmonary arteries (22, 37, 46). Regarding the opposing actions of PKG and ROCKs on MYPT1, in addition to the stimulatory sites of phosphorylation by PKG and the inhibitory sites of phosphorylation by ROCKs, PKG can stimulate MLCP activity by the interaction between its leucine zipper motifs and MYPT1 while ROCKs can inhibit MLCP activity by the phosphorylation of CPI-17 at Thr38. The phosphorylation of CPI-17 enhances its potency for inhibiting the catalytic subunit of MLCP (PP1c{delta}) (Fig. 8) (30, 41, 46, 47, 49). Despite this complexity of signaling in pulmonary vascular smooth muscle, it is noteworthy that a recent study in patients with severe pulmonary hypertension showed that fasudil, a specific inhibitor of ROCKs, is more potent than oxygen inhalation, nitric oxide inhalation, or nifedipine in reducing pulmonary vascular resistance (11). This suggests that ROCKs may be involved in the pathogenesis of pulmonary hypertension in humans. On the basis of our findings, the therapeutic benefits of ROCK inhibitors may be in part due to the restoration of PKG-mediated vasodilation.


Figure 8
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  		Fig. 8. Possible signal transduction pathways in pulmonary arteries for the opposing actions of PKG and RhoA/ROCK and the effects of chronic hypoxia. Chronic hypoxia may increase the Ca2+ sensitivity of the contractile filaments of smooth muscle through the inhibition of myosin light chain phosphatase (MLCP) via RhoA/ROCK pathway. ROCKs inhibit MLCP by phosphorylating the regulatory subunit of MLCP (MYPT1) at Thr696 and Thr853 (human sequence). ROCK may also inhibit the activity of MLCP through phosphorylation of PKC-potentiated inhibitor protein of 17 kDa (CPI-17). Activation of PKG by nitric oxide (NO) and cGMP may interfere with the activation of RhoA. PKG may phosphorylate MYPT1 at Ser695 and Ser852, which results in a decreased ROCK-mediated phosphorylation of MYPT1 at Thr696 and Thr853. This antagonizing effect of PKG may be impaired by chronic hypoxia. PKG can also directly stimulate MLCP activity through interaction between the leucine zipper (LZ) motives of PKG and MYPT1. GPCR, G protein-coupled receptors; G, G protein; DAG, diacylglycerol; RhoA, a small monomeric GTPase; M20, a 20-kDa subunit of MLCP with unknown function. The dashed line indicates inhibitory action.

 

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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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This study was supported in part by National Institutes of Health Grants HL-059435, HL-075187, and P01-HD-31226, the National Natural Science Foundation of China Grants 30370523 and 30470629 Program for Changjiang Scholars and Innovative Research Team in University, and Major National Basic Research Program of P. R. China (No. 2006CB503802).


   FOOTNOTES
 

Address for reprint requests and other correspondence: Y. Gao, Los Angeles Biomedical Institute, Harbor-UCLA Medical Center, 1124 W. Carson St., RB-1, Torrance, CA 90502 (e-mail: ysgao{at}labiomed.org)

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


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