Title: Cyclic GMP-dependent Protein Kinase Signaling Pathway Inhibits RhoA-induced Ca2+ Sensitization of Contraction in Vascular Smooth Muscle
Abstract: 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 RhoAAla-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. 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 RhoAAla-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. 20-kDa myosin light chain myosin light chain kinase myosin light chain phosphatase filamentous actin nitric oxide cGMP-dependent protein kinase Dulbecco's modified Eagle's medium fetal calf serum methoxyverapamil thapsigargin penylephrine sodium nitroprusside geranylgeranylated-RhoA fluorescein isothiocyanate 8-Bromo guanosine 5′-3-O-(thio)triphosphate geranylgeranylated 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.Somlyo A.P. Nature. 1997; 389: 908-911Crossref PubMed Scopus (34) Google Scholar). The major regulatory mechanism of smooth muscle contraction is phosphorylation/dephosphorylation of the 20-kDa myosin light chain (MLC)1 (2.Somlyo A.P. Somlyo A.V. Nature. 1994; 372: 231-236Crossref PubMed Scopus (1751) Google Scholar). 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.Somlyo A.P. Nature. 1997; 389: 908-911Crossref PubMed Scopus (34) Google Scholar, 2.Somlyo A.P. Somlyo A.V. Nature. 1994; 372: 231-236Crossref PubMed Scopus (1751) Google Scholar). 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.Gong M.C. Iizuka K. Nixon G. Browne J.P. Hall A. Eccleston J.F. Sugai M. Kobayashi S. Somlyo A.V. Somlyo A.P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1340-1345Crossref PubMed Scopus (268) Google Scholar, 4.Otto B. Steusloff A. Just I. Aktories K. Pfitzer G. J. Physiol. (Lond.). 1996; 496: 317-329Crossref Scopus (106) Google Scholar, 5.Fujihara H. Walker L.A. Gong M.C. Lemichez E. Boquet P. Somlyo A.V. Somlyo A.P. Mol. Biol. Cell. 1997; 8: 2437-2447Crossref PubMed Scopus (144) Google Scholar, 6.Uehata M. Ishizaki T. Satoh H. Ono T. Kawahara T. Morishita T. Tamakawa H. Yamagami K. Inui J. Maekawa M. Narumiya S. Nature. 1997; 389: 990-994Crossref PubMed Scopus (2574) Google Scholar). 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.Gong M.C. Iizuka K. Nixon G. Browne J.P. Hall A. Eccleston J.F. Sugai M. Kobayashi S. Somlyo A.V. Somlyo A.P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1340-1345Crossref PubMed Scopus (268) Google Scholar, 6.Uehata M. Ishizaki T. Satoh H. Ono T. Kawahara T. Morishita T. Tamakawa H. Yamagami K. Inui J. Maekawa M. Narumiya S. Nature. 1997; 389: 990-994Crossref PubMed Scopus (2574) Google Scholar, 7.Fu X. Gong M.C. Jia T. Somlyo A.V. Somlyo A.P. FEBS Lett. 1998; 440: 183-187Crossref PubMed Scopus (196) Google Scholar). 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.Togashi H. Emala C.W. Hall I.P. Hirshman C.A. Am. J. Physiol. 1998; 274: L803-L809PubMed Google Scholar, 9.Hirshman C.A. Emala C.W. Am. J. Physiol. 1999; 21: L653-L661Google Scholar). 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.Furchgott R.F. Annu. Rev. Pharmacol. Toxicol. 1995; 35: 1-27Crossref PubMed Google Scholar). cGMP-induced relaxation involves activation of the cGMP-dependent protein kinase (cGK) (11.Lohmann S.M. Vaandrager A.B. Smolenski A. Walter U. De Jonge H.R. Trends Biochem. Sci. 1997; 22: 307-312Abstract Full Text PDF PubMed Scopus (355) Google Scholar, 12.Pfeifer A. Klatt P. Massberg S. Ny L. Sausbier M. Hirneiss C. Wang G.X. Korth M. Aszodi A. Andersson K.E. Krombach F. Mayerhofer A. Ruth P. Fassler R. Hofmann F. EMBO J. 1998; 17: 3045-3051Crossref PubMed Scopus (459) Google Scholar). 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.Lincoln T.M. Cornwell T.L. FASEB J. 1993; 7 (238): 328Crossref PubMed Scopus (546) Google Scholar), and “Ca2+desensitization” by stimulation of MLCP activity through unknown mechanisms (14.Wu X. Somlyo A.V. Somlyo A.P. Biochem. Biophys. Res. Commun. 1996; 220: 658-663Crossref PubMed Scopus (140) Google Scholar, 15.Lee M.R. Li L. Kitazawa T. J. Biol. Chem. 1997; 272: 5063-5068Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar). 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.Surks H.K. Mochizuki N. Kasai Y. Georgescu S.P Tang K.M. Ito M. Lincoln T.M. Mendelsohn M.E. Science. 1999; 286: 1583-1587Crossref PubMed Scopus (448) Google Scholar). 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.Nakamura M. Ichikawa K. Ito M. Yamamori B. Okinaka T. Isaka N. Yoshida Y. Fujita S. Nakano T. Cell. Signal. 1999; 11: 671-676Crossref PubMed Scopus (52) Google Scholar). Such an indirect mechanism involving telokin, a low molecular weight protein expressed in phasic smooth muscle, has been proposed (18.Wu X. Haystead T.A. Nakamoto R.K. Somlyo A.V. Somlyo A.P. J. Biol. Chem. 1998; 273: 11362-11369Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). 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.Wu X. Haystead T.A. Nakamoto R.K. Somlyo A.V. Somlyo A.P. J. Biol. Chem. 1998; 273: 11362-11369Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). 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.Narumiya S. J. Biochem. (Tokyo). 1996; 120: 215-228Crossref PubMed Scopus (362) Google Scholar). 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.Loirand G. Le Jeune H. Pernaton S. Pacaud P. Circulation. 1999; 100: I-623Google Scholar). 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. 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.Horiuti K. J. Physiol. (Lond.). 1988; 398: 131-148Crossref Scopus (81) Google Scholar) 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 β-escin (50–70 μm) in the relaxing solution for 35 min at 25 °C as described previously (22.Loirand G. Cario-Toumaniantz C. Chardin P. Pacaud P. J. Physiol. (Lond.). 1999; 516: 825-834Crossref Scopus (64) Google Scholar). 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.Loirand G. Cario-Toumaniantz C. Chardin P. Pacaud P. J. Physiol. (Lond.). 1999; 516: 825-834Crossref Scopus (64) Google Scholar). Smooth muscle cells from young rat (45 g) aorta were isolated by enzymatic dissociation as described previously (23.Guibert C. Pacaud P. Loirand G. Marthan R. Savineau J.P. Am. J. Physiol. 1996; 271: L450-L458PubMed Google Scholar). 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. 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 × gfor 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 mmTris-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). 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-α-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.Knowles G.C. McCulloch C.A.G. J. Histochem. Cytochem. 1992; 40: 1605-1612Crossref PubMed Scopus (83) Google Scholar) 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. RhoA and RhoAAla-188 were expressed in Escherichia coli, purified, then geranylgeranylated in vitro by type 1 geranylgeranyltransferase and loaded with GTPγS as described previously (22.Loirand G. Cario-Toumaniantz C. Chardin P. Pacaud P. J. Physiol. (Lond.). 1999; 516: 825-834Crossref Scopus (64) Google Scholar, 25.Nobes K. Lauritzen I. Mattei M.G. Paris S. Hall A. Chardin P. J. Cell Biol. 1998; 141: 187-197Crossref PubMed Scopus (311) Google Scholar, 26.Lang P. Gesbert F. Delespine-Carmagnat M. Stancou R. Pouchelet M. Bertoglio J. EMBO J. 1996; 15: 510-519Crossref PubMed Scopus (484) Google Scholar). Phosphorylation of recombinant RhoA and geranylgeranylated (GG) RhoA was determined in a kinase assay system using cGK Iα (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 [γ-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. Full-length RhoAWT, RhoAAla-188, RhoAVal-14, and RhoAVal-14,Ala-188 were cloned in pSG5 vector (Stratagene, La Jolla, CA), and full-length cGK Iα was cloned in pcDNA3 (Invitrogen, Groninger, The Netherlands). RhoA or cGKIα 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.Jurman M.E. Boland L.M. Yellen G. BioTechniques. 1994; 17: 876-881PubMed Google Scholar). Cells were then fixed and stained as described above. 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. 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. 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.Uehata M. Ishizaki T. Satoh H. Ono T. Kawahara T. Morishita T. Tamakawa H. Yamagami K. Inui J. Maekawa M. Narumiya S. Nature. 1997; 389: 990-994Crossref PubMed Scopus (2574) Google Scholar) 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.1 A 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.1 B 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.Pacaud P. Bolton T.B. J. Physiol. (Lond.). 1991; 441: 477-499Crossref Scopus (179) Google Scholar, 29.Pacaud P. Malam-Souley R. Loirand G. Desgranges C. Am. J. Physiol. 1995; 269: H30-H36PubMed Google Scholar). 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. 1 A), 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.1 B and Table I). Similar results were obtained with SNP (Fig. 1 C 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 β-escin-permeabilized smooth muscle strips.Table IInhibitory action of 8-Br-cGMP and SNP on the sustained rise in tension induced by PE (1 μm)IC50AortaPulmonary arteryPortal veinIC508-Br-cGMP80 μm113 μm141 μmIC50 SNP6.2 nm16.2 nm7.4 nmIC50 8-Br-cGMP after D600-TSG treatment18 μm36 μm44 μmIC50 SNP after D600-TSG treatment3.1 nm3.8 nm3.5 nmConcentrations of 8-Br-cGMP or SNP leading to half-maximal inhibition (IC50) of PE-induced tension were determined from cumulative concentration-response curve obtained from 5 to 6 experiments. Open table in a new tab Concentrations of 8-Br-cGMP or SNP leading to half-maximal inhibition (IC50) of PE-induced tension were determined from cumulative concentration-response curve obtained from 5 to 6 experiments. Ca2+-dependent contractions and Ca2+ sensitization of contractile proteins could be independently evoked in β-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 GTPγS atpCa 6.3 in guinea pig portal vein (Fig.2 A). 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.2 B). This indicates that Rho/Rho kinase pathway does not participate to the Ca2+-induced contraction in β-escin-permeabilized muscle, in contrast to previous observations in α-toxin-permeabilized muscle (7.Fu X. Gong M.C. Jia T. Somlyo A.V. Somlyo A.P. FEBS Lett. 1998; 440: 183-187Crossref PubMed Scopus (196) Google Scholar, 14.Wu X. Somlyo A.V. Somlyo A.P. Biochem. Biophys. Res. Commun. 1996; 220: 658-663Crossref PubMed Scopus (140) Google Scholar). The relaxing effect of 8-Br-cGMP on the contraction induced by GTPγS 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 GTPγS-induced Ca2+ sensitization was inhibited by C3 and Y-27632 (Fig.2 B), indicating that it depends on RhoA/Rho kinase activation. Similar results were obtained in β-escin-permeabilized strips from rabbit pulmonary artery in which 8-Br-cGMP relaxed GTPγS-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. Staining of aortic smooth muscle cell actin cytoskeleton with FITC-phalloidin revealed a dense and organized network of actin stress fibers (Fig.3 A) that is inhibited by the RhoA-inactivating exoenzyme C3 or the Rho kinase inhibitor Y-27632 (Fig. 3 C). 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 andC). This effect was inhibited by the cGK inhibitorR p-8-Br-cGMPS (100 μm). Similar results were obtained using a monoclonal anti-α-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.4 A). 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. 4 B). Such down-regulation of cGK I has previously been reported to occur upon smooth muscle cell passaging (30.Cornwell T.L. Lincoln T.M. J. Biol. Chem. 1989; 264: 1146-1155Abstract Full Text PDF PubMed Google Scholar). 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 Iα was expressed in aortic smooth muscle cells that have lost endogenous cGK. cGK Iα 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 andC) on actin cytoskeleton organization. cGK Iα 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. 4 C). Similar results were obtained with transfection of cGK Iβ (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