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J. Biol. Chem., Vol. 275, Issue 28, 21722-21729, July 14, 2000

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Cyclic GMP-dependent Protein Kinase Signaling Pathway Inhibits RhoA-induced Ca²⁺ Sensitization of Contraction in Vascular Smooth Muscle^*

Vincent Sauzeau, Hélène Le Jeune, Chrystelle Cario-Toumaniantz, Albert Smolenski, Suzanne M. Lohmann, Jacques Bertoglio^§, Pierre Chardin^¶, Pierre Pacaud, and Gervaise Loirand

From the Laboratoire de Physiologie Cellulaire et Moléculaire, INSERM U-533, Faculté des Sciences, 44322 Nantes, France,  Institute of Clinical Biochemistry and Pathobiochemistry, Wuerzburg, Germany, ^§ INSERM U-461, 92296 Chatenay-Malabry, France, and ^¶ IPMC-CNRS UPR 411, 06560 Sophia-Antipolis, France

Received for publication, February 1, 2000, and in revised form, April 21, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The potent vasodilator action of cyclic GMP-dependent protein kinase (cGK) involves decreasing the Ca²⁺ sensitivity of contraction of smooth muscle via stimulation of myosin light chain phosphatase through unknown mechanisms (Wu, X., Somlyo, A. V., and Somlyo, A. P. (1996) Biochem. Biophys. Res. Commun. 220, 658-663). Myosin light chain phosphatase activity is controlled by the small GTPase RhoA and its target Rho kinase. Here we demonstrate cGMP effects mediated by cGK that inhibit RhoA-dependent Ca²⁺ sensitization of contraction of blood vessels and actin cytoskeleton organization in cultured vascular myocytes. Ca²⁺ sensitization and actin organization were inhibited by both 8-bromo-cGMP and sodium nitroprusside (SNP). SNP also caused translocation of activated RhoA from the membrane to the cytosol. SNP-induced actin disassembly was lost in vascular myocytes in culture after successive passages but was restored by transfection of cells with cGK I. Furthermore, cGK phosphorylated RhoA in vitro, and addition of cGK I inhibited RhoA-induced Ca²⁺ sensitization in permeabilized smooth muscle. 8-Bromo-cGMP-induced actin disassembly was inhibited in vascular myocytes expressing RhoA^Ala-188, a mutant that could not be phosphorylated. Collectively, these results indicate that cGK phosphorylates and inhibits RhoA and suggest that the consequent inhibition of RhoA-induced Ca²⁺ sensitization and actin cytoskeleton organization contributes to the vasodilator action of nitric oxide.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The contractile state of vascular smooth muscle controls the vessel lumen size, and abnormal increase in vascular smooth muscle tone is involved in the pathogenesis of vascular diseases such as hypertension and atherosclerosis (1). The major regulatory mechanism of smooth muscle contraction is phosphorylation/dephosphorylation of the 20-kDa myosin light chain (MLC)¹ (2). MLC is phosphorylated by the Ca²⁺-calmodulin-activated myosin light chain kinase (MLCK) and dephosphorylated by the Ca²⁺-independent myosin light chain phosphatase (MLCP). Thus, a rise in cytosolic Ca²⁺ concentration produces smooth muscle contraction by activation of MLCK and consequent phosphorylation of MLC. However, it is now well established that MLC phosphorylation and tension can be induced independently of change in cytosolic Ca²⁺ concentration (1, 2). Agonists (noradrenaline, endothelin, thromboxane, etc.) that bind to G-protein-coupled receptors produced contraction by increasing both the cytosolic Ca²⁺ concentration and the Ca²⁺ sensitivity of the contractile apparatus. The increased sensitivity of vascular smooth muscle toward Ca²⁺ results from inhibition of MLCP activity leading to increased MLC phosphorylation and tension at a constant Ca²⁺ concentration. The Ca²⁺-sensitizing effect of vasoconstrictors is ascribed to the activation of the small 22-26-kDa GTPase RhoA that activates Rho kinase which, in turn, phosphorylates the regulatory subunit of MLCP and inhibits its activity (3-6).

RhoA-dependent Ca²⁺-sensitization constitutes a major component of the sustained rise in tension induced by vasoconstrictors in various vascular beds including pulmonary artery, mesenteric artery, and portal vein (3, 6, 7). Rho kinase-dependent MLCP inhibition is responsible not only for the RhoA-dependent Ca²⁺ sensitization in smooth muscle but also for agonist-induced stimulation of actomyosin-based cytoskeleton organization (actin stress fiber formation) in cultured smooth muscle cells (8, 9).

Conversely, relaxation of vascular smooth muscle results from a decrease in cytosolic Ca²⁺ concentration and/or reduced Ca²⁺ sensitivity of the contractile apparatus. Physiologically released endothelial nitric oxide (NO) elevates cGMP, the second messenger responsible for relaxation of vascular smooth muscle and consequent enlargement of the vessel lumen (10). cGMP-induced relaxation involves activation of the cGMP-dependent protein kinase (cGK) (11, 12). The potent vasodilator action of the cGMP/cGK pathway has been ascribed to a decrease in cytosolic Ca²⁺ through activation of multiple Ca²⁺ lowering mechanisms (13), and "Ca²⁺ desensitization" by stimulation of MLCP activity through unknown mechanisms (14, 15). Also, most recently cGK was shown to bind directly to MLCP by a leucine zipper interaction and phosphorylate in vitro the myosin-binding subunit of MLCP (16). However, phosphorylation of the regulatory subunit of MLCP was reported not to affect the phosphatase activity toward MLC, suggesting that indirect mechanisms are involved in the Ca²⁺-desensitizing effect of cGMP (17). Such an indirect mechanism involving telokin, a low molecular weight protein expressed in phasic smooth muscle, has been proposed (18). Telokin is phosphorylated in smooth muscle cells relaxed by application of 8-Br-cGMP. Telokin accelerates dephosphorylation of MLC and relaxation at constant Ca²⁺ concentration, and the relaxing effect of telokin and 8-Br-cGMP in permeabilized smooth muscle are synergistic. However, a truncated form of telokin that does not contain the phosphorylation site for cGK is also able to relax permeabilized smooth muscle (18). In addition, cGMP/cGK pathway induces Ca²⁺ desensitization in tonic smooth muscles that do not express telokin, suggesting that other mechanisms are involved in the cGMP-dependent stimulation of MLCP activity.

In the present study, we have analyzed the role of cGMP/cGK pathway on RhoA-dependent Ca²⁺ sensitization and actin stress fiber organization in vascular smooth muscle since both processes depend on MLCP inhibition (19). We show that both RhoA-dependent Ca²⁺ sensitization of the contractile apparatus and actin cytoskeleton organization in vascular smooth muscle are inhibited by cGMP through cGK phosphorylation of RhoA Ser-188 that causes subsequent translocation of membrane-bound activated RhoA to the cytosol. Inhibition of RhoA-induced Ca²⁺ sensitization of the contractile apparatus by cGMP/cGK is thus identified as a new signaling pathway that contributes to the vasodilator action of NO. A short report of our work has been recently published in abstract form (20).

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Tension Measurements in Intact Fibers-- Wistar rats and guinea pigs were stunned and then killed by cervical dislocation. Rabbits were killed with sodium pentobarbital (100 mg/kg intravenously). The aorta, pulmonary artery, and portal vein were collected in physiological saline solution (PSS, in mM; 130 NaCl, 5.6 KCl, 1 MgCl₂, 2 CaCl₂, 11 glucose, 10 Tris, pH 7.4, with HCl) cleaned of fat and adherent connective tissue, and cut in rings or strips. The endothelium was carefully removed by gently rubbing the intimal surface with the tip of small forceps. Smooth muscle strips or rings were then suspended under isometric conditions and connected to a force transducer (Pioden Controls Ltd., Canterbury, UK) in organ baths filled with Krebs-Henseleit solution (in mM: 118.4 NaCl, 4.7 KCl, 2 CaCl₂, 1.2 MgSO₄, 1.2 KH₂PO₄, 25 NaHCO₃, 11 glucose) maintained at 37 °C, and equilibrated with 95% O₂, 5% CO₂. The preparations were initially placed under a resting tension of 1500 mg, left to equilibrate for 1 h and washed at 20-min intervals. The absence of endothelium was confirmed in each ring by the inability of carbachol (10 µM) to relax phenylephrine (PE, 1 µM)-induced contraction.

Isometric Tension Measurement in Skinned Fibers-- Guinea pigs were stunned and then killed by cervical dislocation. The portal vein was collected in PSS, cleaned of fat and adherent connective tissue, and longitudinally opened. The endothelium was carefully removed by gently rubbing the intimal surface with the tip of small forceps. Small muscle strips (approximately 200 µm wide and 4 mm long) were isolated from the media and tied at each end with a single silk thread to the tips of two needles, one of which was connected to a force transducer (AE 801, SensoNor, Horten, Norway). Strips were placed in a well on a bubble plate filled with PSS (21) and stretched to about 1.3 resting length. The solution was rapidly changed by sliding the plate to an adjacent well. After measuring contraction evoked by high K⁺ solution, the strips were incubated in the normal relaxing solution (in mM: 85 KCl, 5 MgCl₂, 5 Na₂ATP, 5 creatine phosphate, 2 EGTA, and 20 Tris maleate, brought to pH 7.1 at 25 °C with KOH) for few minutes, followed by treatment with -escin (50-70 µM) in the relaxing solution for 35 min at 25 °C as described previously (22). The skinned muscle strip was then washed several times with fresh relaxing solution containing 10 mM EGTA. Calmodulin (1.5 µM) was added to the bathing solutions throughout the experiments. Tension developed by permeabilized muscle strips were measured in activating solutions, containing 10 mM EGTA and a specified amount of CaCl₂ to give a desired concentration of free Ca²⁺ (22).

Smooth Muscle Cell Culture-- Smooth muscle cells from young rat (45 g) aorta were isolated by enzymatic dissociation as described previously (23). Cells were cultured in DMEM with 10% fetal calf serum (FCS), 100 units/ml penicillin, and 100 µg/ml streptomycin. Secondary cultures were obtained by serial passages after the cells were harvested with 0.5 g/liter trypsin and 0.2 g/liter EDTA (trypsin/EDTA) and reseeded in fresh DMEM containing 10% FCS and antibiotics.

Western Blot Analysis-- For measurements of Rho distribution, strips of endothelium-denuded aortic muscle were washed twice with PSS at 37 °C and changed to PSS with or without PE (10 µM) for 1 h in the absence or in the presence of sodium nitroprusside (SNP, 10 µM). The tissues were then rapidly frozen in liquid nitrogen and homogenized in lysis buffer containing (in mM) 20 Hepes-NaOH, 10 KCl, 10 NaCl, 5 MgCl₂, 1 dithiothreitol, and Complete (Roche Molecular Biochemicals, 1 tablet/50 ml). Nuclei and unlysed cells were removed by low speed centrifugation. The supernatant was then centrifuged at 100,000 × g for 30 min to generate membrane and cytosolic fractions. The membrane pellet was resuspended in the same buffer. Protein concentration of fractions was measured and adjusted and then Laemmli sample buffer was added, and equal amounts of protein from membrane and cytosolic fractions were loaded in each lane of SDS-12% polyacrylamide gels, which were then electrophoresed and transferred to nitrocellulose. The amounts of proteins were checked by staining with Ponceau Red. Before immunoblotting, the membrane was blocked with 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Tween 20, 5% non-fat milk for 1 h at room temperature and then probed with a mouse monoclonal anti-RhoA antibody (2 µg/ml) for 3 h at room temperature. After three washes, membranes were incubated for 1 h at room temperature with horseradish peroxidase-conjugated goat anti-mouse antibody (16 ng/ml). The signal from immunoreactive bands was detected by ECL.

For cGK I expression analysis, lysates were prepared from aortic smooth muscle cells at different passages. Samples were then analyzed by Western blot using rabbit cGK I antiserum diluted 1:1000. The immunoreactive bands were detected by ECL, and quantified using ImageQuant (Molecular Dynamics, Sunnyvale, CA).

Actin Staining-- After dissociation, aortic myocytes were cultured in DMEM with 10% FCS on glass coverslips for 2 days. The cells were then washed and maintained in serum-free DMEM in the absence or in the presence of 10 µM SNP for 1 h or 100 µM 8-Br-cGMP for 40 min. When Rp-8-Br-cGMPS was used, it was added 2 h before other treatment. Cells were then fixed for 30 min in 4% paraformaldehyde, permeabilized in 0.5% Triton X-100, and then rinsed in phosphate-buffered saline. For polymerized F-actin staining, cells were incubated with FITC-conjugated phalloidin (5 µg/ml) for 45 min at room temperature and then washed with phosphate-buffered saline. Actin staining was also performed with a monoclonal anti--smooth muscle actin antibody followed by FITC-conjugated anti-mouse antibody which gave results similar to those obtained with FITC-conjugated phalloidin. When dual labeling was performed, cells were simultaneously stained with FITC-conjugated phalloidin and Texas Red-labeled DNase I (10 µg/ml) to localize monomeric G-actin (24) and then washed in phosphate-buffered saline. Coverslips were mounted on a glass slide and examined with a fluorescence microscope (Eclipse E-600, Nikon, Champigny-sur-Marne, France). The background fluorescence signal was estimated by collecting planes from areas of the slide without cells and was electronically subtracted before analysis. Images were collected with a cool-SNAP camera (Princeton Instruments, Evry, France) and stored and analyzed using Metamorph software (Universal Imaging, West Chester, PA). For each area examined, images of FITC-phalloidin and Texas Red-DNase I fluorescence were collected. The time of measurements and image capturing and the image intensity gain at both wavelengths were optimally adjusted and kept constant. The ratio of fluorescence of FITC-phalloidin and Texas Red-DNase I (F- to G-actin ratio), used to quantify actin cytoskeleton organization was calculated for at least 20 cells in each experimental condition and expressed as percentage of the ratio obtained under control condition. A decrease in the F- to G-actin ratio was assumed to represent depolymerization of actin filaments.

Recombinant Protein Expression-- RhoA and RhoA^Ala-188 were expressed in Escherichia coli, purified, then geranylgeranylated in vitro by type 1 geranylgeranyltransferase and loaded with GTPS as described previously (22, 25, 26).

In Vitro Kinase Assay-- Phosphorylation of recombinant RhoA and geranylgeranylated (GG) RhoA was determined in a kinase assay system using cGK I (10,000 units/reaction; Calbiochem, France; Biochem, Meudon, France) according to protocol provided by the manufacturer. The reaction was carried out in a phosphorylation buffer (50 mM Tris, 10 mM MgCl₂, 1 mM dithiothreitol, 0.1 µM cGMP, 20 µM ATP, and 10 µCi of [-³²P]ATP) and with 500 ng of RhoA substrate for 30 min at 30 °C. The reaction was stopped by addition of cold phosphorylation buffer, and samples were boiled in Laemmli buffer. Proteins were separated by in SDS-PAGE and visualized by autoradiography.

Expression of RhoA Mutants-- Full-length RhoA^WT, RhoA^Ala-188, RhoA^Val-14, and RhoA^{Val-14,Ala-188} were cloned in pSG5 vector (Stratagene, La Jolla, CA), and full-length cGK I was cloned in pcDNA3 (Invitrogen, Groninger, The Netherlands). RhoA or cGKI plasmids were transiently transfected, together with the CD8 plasmid, into aortic myocytes grown on coverslips by using using Fugene reagent (Roche Molecular Biochemicals). Forty eight hours after transfection, cells were washed in FCS-free DMEM then maintained in serum-free DMEM in the absence or in the presence of 10 µM SNP for 1 h or 100 µM 8-Br-cGMP for 40 min. Anti-CD8 antibody-coated beads were added just prior to fixation to visualize transfected cells (27). Cells were then fixed and stained as described above.

Statistics-- All results are expressed as the mean ± S.E. of sample size n. Significance was tested by means of Student's t test. Probabilities less than 5% (p < 0.05) were considered significant.

Chemicals and Drugs-- Texas Red-DNase I was obtained from Molecular Probe (Leiden, The Netherland). Anti-CD8 antibody-coated beads were purchased from Dynal (Compiègne, France), and mouse monoclonal RhoA antibody (26C4) was purchased from Santa Cruz Biotechnology. The RhoA inhibitor C3 exoenzyme was kindly provided by Dr. P. Boquet (Inserm U452, Nice University Medical School, Nice, France). The Rho kinase inhibitor Y-27632 was a gift from Yoshitomi Pharmaceutical Industries, Ltd (Saitama, Japan). R_p-8-Br-cGMPS was from Calbiochem. All other reagents were purchased from Sigma.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

cGMP/cGK Pathway Inhibits the RhoA/Rho Kinase-dependent Ca²⁺ Sensitization in Intact Smooth Muscle-- The relaxing effect of cGMP/cGK pathway on agonist-induced Ca²⁺ sensitization of the contractile apparatus was first examined in intact endothelium-denuded vessels (rat aorta, rabbit pulmonary artery, and guinea pig portal vein) stimulated with PE (1 µM). In all vessels, the PE-induced contraction was strongly inhibited by the Rho kinase inhibitor Y-27632 (6) attesting to involvement of RhoA/Rho kinase-dependent Ca²⁺ sensitization in the contraction mechanism. Y-27632 (10 µM) inhibited PE-induced tension by 98 ± 2, 95 ± 3, and 94 ± 4%, in intact rat aorta, rabbit pulmonary artery, and guinea pig portal vein, respectively (n = 6). Fig. 1A shows typical traces illustrating the classical dose-dependent relaxation of PE-induced contraction of rat aorta under control conditions. The concentration of 8-Br-cGMP which gave half-maximal relaxation (IC₅₀) corresponded to 80 µM (Fig. 1B and Table I). Arterial rings were then maintained in the presence of the voltage-gated Ca²⁺ channel inhibitor methoxyverapamil (D600, 20 µM) and the Ca²⁺ store-depleting agent thapsigargin (TSG, 2 µM) to inhibit agonist-induced change in cytosolic Ca²⁺ (28, 29). Under these conditions, the rate of rise of the PE-induced contraction was not modified in all vessels tested. However, measurements of intracellular Ca²⁺ in freshly isolated aortic cells maintained in similar conditions indicated that PE did not produce any rise in [Ca²⁺]_i (not shown). This suggests that the basal Ca²⁺ concentration in the presence of D600/TSG allowed the development of Ca²⁺-sensitizing mechanisms responsible for the D600/TSG-resistant component of the PE-induced contraction. The maximal rise in tension induced by PE in the presence of D600 and TSG was reduced to 72 ± 9% (n = 6) of the control responses (Fig. 1A), and the tension was concentration-dependently inhibited by the Rho kinase inhibitor Y-27632 with an IC₅₀ of 1 µM (not shown). The TSG/D600-resistant component of the PE-induced contraction was also concentration-dependently inhibited by 8-Br-cGMP (Fig. 1, A and B). In the presence of D600/TSG, the concentration-response curve to 8-Br-cGMP was shifted to the left and the IC₅₀ was decreased to 18 µM (Fig. 1B and Table I). Similar results were obtained with SNP (Fig. 1C and Table I) for which the IC₅₀ was 6.2 nM under control conditions and to 3.1 nM in the presence of D600/TSG. Results similar to those obtained with rat aorta were also obtained with rabbit pulmonary artery and guinea pig portal vein (Table I). These results indicate that RhoA/Rho kinase-dependent Ca²⁺ sensitization strongly contributed to PE-induced contraction and that its inhibition is implicated in the mechanism of cGMP/cGK relaxation of smooth muscle. To analyze further the inhibitory effect of 8-Br-cGMP, we next used -escin-permeabilized smooth muscle strips.

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Relaxing effect of 8-Br-cGMP and SNP on the sustained rise in tension induced by PE in intact denuded muscle rings of aorta. A, typical traces showing the relaxing effect of increasing concentrations of 8-Br-cGMP on the sustained tension induced by PE (1 µM) under control conditions (left, filled circle) and in the presence of TSG (2 µM) and D600 (20 µM) (right, open circle). Concentration-response curve of the relaxing effect of 8-Br-cGMP (B) and SNP (C) under control conditions (filled circles) and in the presence of TSG and D600 (open circles). Results are expressed as percentage of the maximal PE-induced tension measured before the application of the first concentration of 8-Br-cGMP or SNP. Each point represents mean ± S.E. of 5-6 experiments.

cGMP/cGK Pathway Relaxes Permeabilized Smooth Muscle-- Ca²⁺-dependent contractions and Ca²⁺ sensitization of contractile proteins could be independently evoked in -escin-permeabilized smooth muscle strips. Ca²⁺-dependent contractions were induced by an increase in Ca²⁺ concentration (submaximal pCa (log [Ca²⁺]) 6.2 or 6 or maximal pCa 4.5), and Ca²⁺ sensitization was evoked by addition of GTPS at pCa 6.3 in guinea pig portal vein (Fig. 2A). Contractions evoked at submaximal Ca²⁺ concentration (pCa 6.2 or 6) were only slightly inhibited by 8-Br-cGMP and were not sensitive to either C3 exoenzyme or the Rho kinase inhibitor Y-27632 (Fig. 2B). This indicates that Rho/Rho kinase pathway does not participate to the Ca²⁺-induced contraction in -escin-permeabilized muscle, in contrast to previous observations in -toxin-permeabilized muscle (7, 14). The relaxing effect of 8-Br-cGMP on the contraction induced by GTPS at pCa 6.3 (43.3 ± 4.6%, n = 7) was ~4 times higher than that obtained at the same tension level reached at pCa 6 (10.9 ± 2.4%, n = 7; p < 0.0001) (Fig. 2, A and B). The GTPS-induced Ca²⁺ sensitization was inhibited by C3 and Y-27632 (Fig. 2B), indicating that it depends on RhoA/Rho kinase activation. Similar results were obtained in -escin-permeabilized strips from rabbit pulmonary artery in which 8-Br-cGMP relaxed GTPS-induced contraction 2.5 times more effectively than Ca²⁺-dependent contraction. These results confirm that RhoA-dependent signaling pathway is a target for the cGMP-induced Ca²⁺ desensitization.

View larger version (24K): [in this window] [in a new window]   Fig. 2.   Effect of 8-Br-cGMP on GTPS- and Ca2+-induced contraction in -escin-permeabilized smooth muscle strips. A, typical traces showing the relaxing effect of 10 µM 8-Br-cGMP on the pCa 6-induced contraction and on the GTPS (10 µM)-induced tension in portal vein-permeabilized muscle strips. pCa 4.5 was used to evoke maximal contraction. B, relaxing effects of C3 exoenzyme (1.5 µg/ml; white bar), Y-27632 (10 µM; gray bar), and 8-Br-cGMP (10 µM; black bar) on the tension measured either at pCa 6.2, pCa 6, and in the presence of 10 µM GTPS. Relaxation is shown as a percentage of the maximal tension (100%) obtained in the absence of inhibitors. Comparisons were made between relaxation obtained in the absence (0%) versus presence of inhibitor (*, p < 0.05). C3 and Y-27632 had no inhibitory effect at pCa 6.2 and pCa 6 but completely inhibited the tension induced by GTPS. 8-Br-cGMP had only a slight relaxing effect at pCa 6.2 and pCa 6 but strongly inhibited the tension induced by GTPS.

cGMP/cGK Pathway Inhibits Actin Stress Fiber Organization in Aortic Smooth Muscle Cells-- Staining of aortic smooth muscle cell actin cytoskeleton with FITC-phalloidin revealed a dense and organized network of actin stress fibers (Fig. 3A) that is inhibited by the RhoA-inactivating exoenzyme C3 or the Rho kinase inhibitor Y-27632 (Fig. 3C). Incubation of cells with 8-Br-cGMP (100 µM) or SNP (10 µM) decreased the extent of phalloidin staining of stress fibers (Fig. 3, B and C). This effect was inhibited by the cGK inhibitor R_p-8-Br-cGMPS (100 µM). Similar results were obtained using a monoclonal anti--smooth muscle actin antibody to image stress fibers (not shown). A gradual decrease of the SNP-induced actin disassembly was observed with successive passages of aortic smooth muscle cells in culture, 75% of the response being lost between passages 1 and 8 (Fig. 4A). Western blot examination of cGK expression indicated that the loss of SNP-induced actin disassembly in cultured cells was associated with a decrease in endogenous cGK I (Fig. 4B). Such down-regulation of cGK I has previously been reported to occur upon smooth muscle cell passaging (30). When normalized to its value at passage 1, endogenous cGK was decreased to 0.6 and 0.1 in cells at passage 4 and 6, respectively. To analyze a causal relationship between down-expression of cGK and loss of the SNP effect, cGK I was expressed in aortic smooth muscle cells that have lost endogenous cGK. cGK I transfection of passage 8 aortic smooth muscle cells caused full restoration of the inhibitory action of 8-Br-cGMP (not shown) or SNP (Fig. 4, A and C) on actin cytoskeleton organization. cGK I was co-expressed with CD8 for identification of transfected cells with anti-CD8 antibody-coated beads. All transfected cells showed a strong disassembly of actin fibers in response to 8-Br-cGMP (not shown) or SNP, whereas actin cytoskeleton of non-transfected cells (not labeled by beads) was almost not affected (Fig. 4C). Similar results were obtained with transfection of cGK I (not shown). Therefore, these results suggest that inhibition of RhoA/Rho kinase-dependent modulation of MLCP by 8-Br-cGMP or SNP resulted from the activation of cGK. Since potential sites for phosphorylation by cGK were not found in the amino acid sequence of Rho kinase (Rock I and Rock II), we investigated the possibility that cGMP/cGK could inhibit RhoA-dependent pathway by phosphorylating RhoA.

View larger version (58K): [in this window] [in a new window]   Fig. 3.   Inhibitory effects of 8-Br-cGMP on actin cytoskeleton organization in aortic smooth muscle cells. F-actin staining using FITC-phalloidin in aortic smooth muscle cells under control conditions (A) and in the presence of 8-Br-cGMP (100 µM, B) showing the inhibitory effect of 8-Br-cGMP on actin stress fiber formation (magnification × 600). C, effects of C3 exoenzyme (15 µg/ml), Y-27632 (10 µM), 8-Br-cGMP (100 µM), SNP (10 µM), and Rp-8-Br-cGMPS (100 µM) on actin cytoskeleton organization, quantified by the F:G-actin ratio. Results were expressed as percentage of control values (designated 100%) determined in the absence of inhibitor (1st column at far left).

View larger version (69K): [in this window] [in a new window]   Fig. 4.   Loss of SNP-induced actin disorganization in passaged aortic smooth muscle cells correlates with loss of endogenous cGK I and is reversed by transfected cGK I. A, SNP (10 µM)-induced actin disorganization, expressed as percentage of the response observed in passage 1, gradually decreased in successive cell passages (P1-P5), remaining stable after P5 (black bars). cGK I transfection of passage 8 cells completely restored the inhibitory effect of SNP (open bar). B, Western blot analysis demonstrating successive loss of endogenous cGK I from higher passages of aortic smooth muscle cells. Shown are lysates from cells at passage 1 (P1), passage 4 (P4), and passage 6 (P6). C, F-actin (FITC-conjugated phalloidin) staining of P8 aortic cells (left panels) in the absence (control) and presence of SNP (10 µM) demonstrated that SNP induced disorganization of the actin cytoskeleton in cGK I-transfected aortic smooth muscle cells (identified by beads) but not in untransfected cells (cells without beads). Right panels, anti-CD8 antibody-labeled beads identify cells containing CD8 co-transfected with cGK I. (Magnification × 600.)

cGK Phosphorylates RhoA in Vitro-- Examination of the amino acid sequence of the RhoA revealed the presence of a consensus site for cGK phosphorylation that contains Ser-188 and is located at the C terminus of the protein, just upstream of Cys-190 to which the prenyl moiety is covalently linked to mediate membrane attachment of RhoA. To assess whether RhoA was a target for cGK-mediated phosphorylation, recombinant RhoA and GG-RhoA were incubated with recombinant cGK and subjected to SDS-PAGE. Autoradiography indicated that both RhoA and GG-RhoA were indeed a substrate for cGK (Fig. 5A, upper panel). To identify further the residue that was phosphorylated by cGK, we performed similar experiments using a RhoA mutant in which the Ser-188 was substituted by an Ala residue (RhoA^Ala-188). This substitution prevented phosphorylation of RhoA by cGK, indicating that cGK-mediated phosphorylation occurred on Ser-188 (Fig. 5A, lower panel).

View larger version (41K): [in this window] [in a new window]   Fig. 5.   In vitro phosphorylation of RhoA by cGK. A, autoradiography showing phosphorylation of RhoA or geranylgeranylated RhoA (GG-RhoA) (upper panel) and the absence of phosphorylation of RhoAAla-188 or geranylgeranylated RhoAAla-188 (GG-RhoAA188) (lower panel) by cGK in the presence of 0.1 µM cGMP. B, Western blot analysis of the distribution of RhoA in cytosolic and membrane fractions prepared from rat aorta in control conditions and in the presence of PE (10 µM) with or without SNP (10 µM). SNP inhibited PE-induced membrane association of RhoA.

SNP Inhibition of Membrane Anchoring of RhoA-- Phosphorylation of RhoA on Ser-188 by cyclic AMP-activated kinase has been shown to induce the extraction of the membrane-associated RhoA into the cytosol (26). We investigated the distribution of RhoA in cytosolic and cell membrane fractions prepared from endothelium-denuded aorta stimulated with PE in the absence and the presence of SNP by SDS-PAGE and Western blot with monoclonal anti-RhoA antibody (Fig. 5B). Stimulation of aorta with PE (10 µM) increased the amount of RhoA in the membrane fraction, whereas treatment with 10 µM SNP 10 min before PE caused RhoA depletion from the membrane fraction of PE-stimulated preparation. The amount of membrane-associated RhoA was even less than that observed in unstimulated control conditions suggesting that activation of cGMP/cGK pathway inhibits the membrane anchoring of RhoA. Activation of RhoA by G-protein-coupled receptor agonists requires translocation of inactive cytosolic RhoA to the membrane (31, 32), thus inhibition of membrane attachment of RhoA may be involved in the cGMP/cGK-mediated inhibition of RhoA-dependent processes.

cGMP/cGK-dependent RhoA Phosphorylation Prevents RhoA-induced Ca²⁺ Sensitization-- RhoA-dependent Ca²⁺ sensitization was induced by adding recombinant GG-RhoA or GG-RhoA^Ala-188 loaded with GTPS to permeabilized muscle strips (0.1 mg/ml, Fig. 6A). Non-geranylgeranylated RhoA loaded with GDP did not induce a significant rise in tension indicating that the observed effects of recombinant GG-RhoA and GG-RhoA^Ala-188 were not due to contaminants of protein preparations (Fig. 6B). The rise in tension induced by GG-RhoA was inhibited by the addition of 10 µM 8-Br-cGMP (Fig. 6A, upper trace, and B). On the contrary, 8-Br-cGMP (10 µM) had no effect on the GG-RhoA^Ala-188-induced rise in tension (Fig. 6A, lower trace, and B). In addition, preincubation of GG-RhoA with cGK for 30 min (activated by 0.1 µM cGMP) prevented the rise in tension induced by GG-RhoA (Fig. 6B). Subsequent stimulation with GTPS was still able to produce Ca²⁺ sensitization presumably by stimulating endogenous RhoA (Fig. 6A).

View larger version (20K): [in this window] [in a new window]   Fig. 6.   cGMP/cGK pathway inhibited RhoA (but not RhoAAla-188)-induced Ca2+ sensitization in -escin-permeabilized smooth muscle. A, typical trace showing that application of 10 µM 8-Br-cGMP to permeabilized muscle strips from guinea pig portal vein relaxed the tension rise induced by GG-RhoA (0.1 mg/ml) (upper trace) but had no effect on that produced by the GG-RhoAAla-188 phosphorylation site mutant (lower trace). Application of GTPS was still able to induce Ca2+ sensitization probably by activating endogenous RhoA. B, quantification of the effect on tension of GG-RhoA and GG-RhoAAla-188 in the absence and presence of 8-Br-cGMP and of RhoA pretreated with activated (0.1 µM cGMP) cGK. Negative control experiment using non-GG-RhoA (nGG, loaded with GDP) has also been performed. The rise in tension was expressed as percentage of the pCa 4.5-induced tension (*, p < 0.05).

Expression of RhoA Mutated at Ser-188 Prevents cGMP/cGK-dependent Disorganization of Actin Cytoskeleton-- To examine whether the phosphorylation of RhoA on Ser-188 is also responsible for the effect of cGMP on actin cytoskeleton, we have transfected aortic smooth muscle cells with RhoA and RhoA^Ala-188 mutants. RhoA has been co-expressed with CD8, and transfected cells were identified with anti-CD8 antibody-coated beads (Fig. 7). If phosphorylation of RhoA on Ser-188 induces inhibition of its activity, overexpression of RhoA^Ala-188 should prevent 8-Br-cGMP-induced actin disassembly. In cells expressing recombinant wild type RhoA, 8-Br-cGMP induced actin fiber disassembly as it did in non-transfected cells. On the contrary, 8-Br-cGMP did not disorganize the actin cytoskeleton of cells expressing RhoA^Ala-188 (Fig. 7A). In cells expressing constitutively active RhoA^Val-14, disassembly of actin induced by 8-Br-cGMP was also reduced in comparison to that obtained in non-transfected cells (Fig. 7C), indicating that expression of the active RhoA mutant partially antagonized 8-Br-cGMP effect. Such a protective effect of RhoA^Val-14 against the effect of phosphorylation by cAMP-dependent kinase has been reported and could be related to the observation that active GTP-RhoA was not as good a substrate as GDP-RhoA (33). However, a greater inhibition of 8-Br-cGMP-induced response was observed in cells transfected with the phosphorylation-resistant RhoA^{Val-14,Ala-188} mutant. In cells expressing RhoA^{Val-14,Ala-188}, actin filaments remained organized in parallel fibers in the presence of 8-Br-cGMP (Fig. 7, B and C). These results indicate that phosphorylation of RhoA on Ser-188 by cGK is involved in the disassembly of actin cytoskeleton induced by cGMP-dependent pathway.

View larger version (82K): [in this window] [in a new window]   Fig. 7.   Expression of RhoAAla-188 mutants prevents 8-Br-cGMP-induced actin cytoskeleton disorganization in aortic smooth muscle cells. F-actin staining of aortic smooth muscle cells showing that 8-Br-cGMP (100 µM) did not disorganize actin cytoskeleton organization in cells expressing RhoAAla-188 or RhoAVal-14,Ala-188 (A and B, cells labeled with beads), whereas an inhibitory effect was observed in untransfected cells (A and B, cells without beads). Magnification × 600. Right panels, anti-CD8 antibody-labeled beads identify cells containing CD8 co-transfected with cGK I. C, quantification of actin cytoskeleton disorganization measured in the presence of 8-Br-cGMP in cells expressing wild type RhoA, RhoAAla-188, RhoAVal-14, or RhoAVal-14,Ala-188. Measurements were made in 20-40 cells for each conditions, in three different batches of cells. Results are expressed as percentage of control, in the absence of 8-Br-cGMP.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The present data provide evidence that RhoA-mediated Ca²⁺ sensitization, contraction, and actin stress fiber organization are inhibited in smooth muscle cells by the cGMP/cGK pathway, via phosphorylation of RhoA. Since recent studies demonstrate a prominent role for RhoA in the vasoconstrictor action of agonists that stimulates G-protein-coupled receptors (3-6), RhoA-dependent mechanisms appear to be important targets for the NO/cGMP inhibitory signaling pathway turned on by vasodilators.

It was previously reported that 8-Br-cGMP could relax both Ca²⁺-dependent contraction induced by submaximal Ca²⁺ concentration and Ca²⁺ sensitization induced by GTPS in -toxin-permeabilized smooth muscle (7, 15). In the present study, we observed that 8-Br-cGMP-induced relaxation in -escin-permeabilized smooth muscle contracted with GTPS more effectively than that contracted by submaximal Ca²⁺ concentration (Fig. 2). This discrepancy between results obtained in -toxin versus -escin-permeabilized muscle could be related to the recent observation that submaximal Ca²⁺ concentration-induced contraction in -toxin-permeabilized smooth muscle was inhibited by the Rho kinase inhibitor Y-27632 (7). The sensitivity of Ca²⁺-induced contraction to Y-27632 or to 8-Br-cGMP was lost after more extensive permeabilization with Triton X-100 (7, 14, 15) or with -escin (Fig. 2). This indicates that a "basal" activation of RhoA and Rho kinase contributes to "the Ca²⁺-induced" contraction in -toxin-permeabilized muscle and that inhibition of the RhoA pathway by 8-Br-cGMP could contribute at least in part to 8-Br-cGMP relaxation of submaximal Ca²⁺-induced contraction in -toxin-permeabilized muscle. Preservation of strong coupling between receptors and intracellular signaling pathways in -toxin-permeabilized muscle, and the presence of millimolar ATP concentrations in experimental solutions that stimulate P2Y receptors coupled to the activation of RhoA² could contribute to the observed basal activation of RhoA.

Several observations suggest that 8-Br-cGMP-induced inhibition of RhoA-dependent Ca²⁺ sensitization and actin cytoskeleton organization in our studies was due to phosphorylation of RhoA by cGK. First, the cGMP-induced actin disassembly lost in cultured myocytes that no longer expressed cGK I is restored after transfection of these cells with cGK I cDNA. In addition, cGMP-induced actin disorganization was inhibited by the cGK-selective inhibitor R_p-8-Br-cGMPS. Second, RhoA is in vitro phosphorylated by cGK on Ser-188 in a consensus site for phosphorylation by cGK previously shown to be used by cAMP-dependent kinase (26, 33). Interestingly, very recently Surks et al. (16) reported that among the proteins that co-immunoprecipitated with the myosin-binding subunit of MLCP and were phosphorylated by cGK I was an unidentified 20-26-kDa protein. This size corresponds to that of RhoA, which was previously shown to interact with the myosin-binding subunit of MLCP (34). Therefore, according to our results, this unidentified phosphoprotein might be RhoA. Experiments performed to analyze in situ phosphorylation of RhoA in ³²P-loaded endothelium-denuded muscle strips of aorta stimulated with SNP or 8-Br-cGMP did not yield a clear and reproducible 21-23-kDa phosphorylated band in lysates immunoprecipitated with anti-RhoA antibody. Similar difficulties have been previously encountered with respect to detection of cAMP-dependent protein kinase-mediated phosphorylation of RhoA in intact cells (26). Third, the contracting effect of RhoA in -escin-permeabilized muscle was prevented by in vitro phosphorylation of RhoA with cGK prior to use. In addition, cGMP relaxed the RhoA-induced rise in tension but not tension produced by the phosphorylation-resistant RhoA^Ala-188 mutant. Fourth, actin fiber disassembly was not inhibited by the cGMP/cGK signaling pathway in vascular smooth muscle cells expressing the phosphorylation-resistant RhoA^Ala-188 mutant. Measurements of RhoA distribution in vascular smooth muscle indicated that cGK phosphorylation of RhoA removed activated RhoA from the membrane to the cytosol, as has been described for cAMP-dependent phosphorylation of RhoA (26). In the latter work, the phosphorylation of RhoA increased its interaction with guanine nucleotide dissociation inhibitor even in its GTP-bound state, leading to the termination of RhoA activation. However, phosphorylation of RhoA by cAMP-dependent kinase also reduced its interaction with Rho kinase (33). Therefore, cGK phosphorylation of RhoA may induce other changes in RhoA functions, in addition to cytosolic sequestration of RhoA, that contribute to the inhibition of RhoA-mediated cellular effects. Furthermore, numerous regulatory proteins could also be targets of cGK, and their possible involvement in the inhibitory effect of cGK on RhoA function has not been examined.

The inhibitory action of cGMP/cGK on RhoA provides a new mechanism through which endothelial NO could relax vascular smooth muscle. Fig. 8 depicts the signaling pathway, including RhoA inactivation, by which the cGMP/cGK signaling pathway induces vascular relaxation. In addition to cGK-mediated inactivation of RhoA, cGK-induced phosphorylation of telokin and the consequent increase of MLCP activity (18) can participate in cGK-induced Ca²⁺ desensitization. As in the case of Ca²⁺ signaling, RhoA-dependent Ca²⁺ sensitivity of the contractile apparatus is likewise determined by the sum of the effects of vasoconstrictors, and the opposing actions of cGMP/cGK signaling set in motion by NO released from endothelial cells. Endothelial impairment, defined as a decrease in NO production, is one of the earliest manifestations of hypertension, atherosclerosis, and pulmonary hypertension (35-37). This dysfunction could potentially decrease negative NO control of RhoA, thus shifting signal equilibrium in favor of RhoA stimulation, leading to Ca²⁺ sensitization of the contractile apparatus and vascular hypercontractility. In addition to modulating contraction, RhoA is also involved in the control of other cellular functions such as vascular smooth muscle proliferation and migration (38), two phenomena closely associated with the pathogenesis of hypertension, atherosclerosis, and restenosis of graft rejection (39-41). Thus cGMP/cGK phosphorylation and inhibition of RhoA may also mediate antiproliferative and antimigratory effects of NO (42-43). By mimicking NO effects, specific inhibitors of RhoA may be useful as therapeutic agents for compensating the endothelium impairment and smooth muscle alterations associated with myriad of vascular diseases.

View larger version (35K): [in this window] [in a new window]   Fig. 8.   Opposing actions of NO/cGK and vasoconstrictors on vascular smooth muscle cell contraction. Rho active, membrane-bound active RhoA; Rho-P inactive, cytosolic phosphorylated inactive RhoA; MLC-P, phosphorylated myosin light chain; MLCP-P, phosphorylated MLC phosphatase; CAM, calmodulin; MLCK, MLC kinase.

	ACKNOWLEDGEMENTS

We thank Dr. P. Boquet for the gift of C3 exoenzyme and Yoshitomi Pharmaceutical Industries, Ltd., for the gift of the Rho kinase inhibitor Y-27632.

	FOOTNOTES

^* This work was supported by grants from the Region Pays de Loire, the Institut de Recherches Internationales Servier, INSERM (to G. L., P. C., and J. B.), the Ministère de l'Education Nationale et de la Recherche (to P. P., H. L.  J., and C. C.-T.), and the Deutsche Forschungsgemeinschaft (to S. L.).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.

To whom correspondence may be addressed: Laboratoire de Physiologie Cellulaire et Moléculaire, Inserm U-533, Faculté des Sciences, 2 Rue de la Houssinière, BP 92208 44322 Nantes Cedex 3, France. Tel./Fax: 33 2 51 12 56 14; E-mail: Pierre.Pacaud@nat.svt. univ-nantes.fr or gervaix.loirand@nat.svt.univ-nantes.fr.

Published, JBC Papers in Press, April 26, 2000 DOI 10.1074/jbc.M000753200

² V. Sauzeau, H. Le Jeune, C. Cario-Toumaniantz, P. Chardin, P. Pacaud, and G. Loirand, unpublished results.

	ABBREVIATIONS

The abbreviations used are: MLC, 20-kDa myosin light chain; MLCK, myosin light chain kinase; MLCP, myosin light chain phosphatase; F-actin, filamentous actin; NO, nitric oxide; cGK, cGMP-dependent protein kinase; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; D600, methoxyverapamil; TSG, thapsigargin; PE, penylephrine; SNP, sodium nitroprusside; GG-RhoA, geranylgeranylated-RhoA; FITC, fluorescein isothiocyanate; 8-Br, 8-Bromo; GPS, guanosine 5'-3-O-(thio)triphosphate; GG, geranylgeranylated.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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