Yasuko Kureishi

Endothelial progenitor cells (EPCs) have been isolated from circulating mononuclear cells in peripheral blood and shown to incorporate into foci of neovascularization, consistent with postnatal vasculogenesis. These circulating EPCs are derived from bone marrow and are mobilized endogenously in response to tissue ischemia or exogenously by cytokine stimulation. We show here, using a chemotaxis assay of bone marrow mononuclear cells in vitro and EPC culture assay of peripheral blood from simvastatin-treated animals in vivo, that the HMG-CoA reductase inhibitor, simvastatin, augments the circulating population of EPCs. Direct evidence that this increased pool of circulating EPCs originates from bone marrow and may enhance neovascularization was demonstrated in simvastatin-treated mice transplanted with bone marrow from transgenic donors expressing beta-galactosidase transcriptionally regulated by the endothelial cell-specific Tie-2 promoter. The role of Akt signaling in mediating effects of statin on EPCs is suggested by the observation that simvastatin rapidly activates Akt protein kinase in EPCs, enhancing proliferative and migratory activities and cell survival. Furthermore, dominant negative Akt overexpression leads to functional blocking of EPC bioactivity. These findings establish that augmented mobilization of bone marrow-derived EPCs through stimulation of the Akt signaling pathway constitutes a novel function for HMG-CoA reductase inhibitors.

The finding that circulating endothelial progenitor cells (EPCs) may home to sites of neovascularization and differentiate into mature endothelial cells (ECs) in situ is consistent with "vasculogenesis" (1), a critical paradigm for establishment of the primordial vascular network in the embryo.Our findings (2-7), together with the recent reports from other investigators (8-14), suggest that growth and development of new blood vessels in the adult is not restricted to angiogenesis but encompasses both vasculogenesis and angiogenesis.Although several studies have established angiogenic properties of EPCs (3-5, 15), including physiological mobilization (4) in response to the angiogenic growth VEGF (5), the involved signaling pathways have remained enigmatic.Recently, Akt protein kinase has been shown to act downstream of the angiogenic growth factors VEGF and angiopoietin (16-18) to confer EC survival (18), migration (19), and production of endothelial cell NO (20,21) in response to VEGF.These suggest a potentially important role for Akt signaling in mediating the response of ECs to angiogenic stimuli.Indeed Kureishi et al. have recently shown that the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, simvastatin, rapidly activates Akt signaling in ECs, and that this stimulates EC bioactivity in vitro and enhances angiogenesis in vivo ( 22).Statins inhibit the activity of HMG-CoA reductase, which catalyzes the synthesis of mevalonate, a rate-limiting step in cholesterol biosynthesis.The clinical application of statins has already led to important improvements in primary and secondary prevention of coronary artery disease (CAD) in subjects with and without elevated cholesterol levels.Preclinical studies suggest that statins may promote angiogenesis in ischemic limbs ( 22) and protect against ischemia-reperfusion injury of the heart (23), through mechanisms that may be mediated by activation of Akt signaling and endothelium-derived nitric oxide (NO) production in normocholesterolemic animals.Accordingly, we investigated the hypothesis that Akt may constitute a key signaling pathway in the angio-

Small GTPase Rho plays pivotal roles in the Ca2+ sensitization of smooth muscle. However, the GTP-bound active form of Rho failed to exert Ca2+-sensitizing effects in extensively Triton X-100-permeabilized smooth muscle preparations, due to the loss of the important diffusible cofactor (Gong, M. C., Iizuka, K., Nixon, G., Browne, J. P., Hall, A., Eccleston, J. F., Sugai, M., Kobayashi, S., Somlyo, A. V., and Somlyo, A. P. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 1340–1345). Here we demonstrate the contractile effects of Rho-associated kinase (Rho-kinase), recently identified as a putative target of Rho, on the Triton X-100-permeabilized smooth muscle of rabbit portal vein. Introduction of the constitutively active form of Rho-kinase into the cytosol of Triton X-100-permeabilized smooth muscle provoked a contraction and a proportional increase in levels of monophosphorylation of myosin light chain in both the presence and the absence of cytosolic Ca2+. These effects of constitutively active Rho-kinase were wortmannin (a potent myosin light chain kinase inhibitor)-insensitive. Immunoblot analysis revealed that the amount of native Rho-kinase was markedly lower in Triton X-100-permeabilized tissue than in intact tissue. Our results demonstrate that Rho-kinase directly modulates smooth muscle contraction through myosin light chain phosphorylation, independently of the Ca2+-calmodulin-dependent myosin light chain kinase pathway. Small GTPase Rho plays pivotal roles in the Ca2+ sensitization of smooth muscle. However, the GTP-bound active form of Rho failed to exert Ca2+-sensitizing effects in extensively Triton X-100-permeabilized smooth muscle preparations, due to the loss of the important diffusible cofactor (Gong, M. C., Iizuka, K., Nixon, G., Browne, J. P., Hall, A., Eccleston, J. F., Sugai, M., Kobayashi, S., Somlyo, A. V., and Somlyo, A. P. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 1340–1345). Here we demonstrate the contractile effects of Rho-associated kinase (Rho-kinase), recently identified as a putative target of Rho, on the Triton X-100-permeabilized smooth muscle of rabbit portal vein. Introduction of the constitutively active form of Rho-kinase into the cytosol of Triton X-100-permeabilized smooth muscle provoked a contraction and a proportional increase in levels of monophosphorylation of myosin light chain in both the presence and the absence of cytosolic Ca2+. These effects of constitutively active Rho-kinase were wortmannin (a potent myosin light chain kinase inhibitor)-insensitive. Immunoblot analysis revealed that the amount of native Rho-kinase was markedly lower in Triton X-100-permeabilized tissue than in intact tissue. Our results demonstrate that Rho-kinase directly modulates smooth muscle contraction through myosin light chain phosphorylation, independently of the Ca2+-calmodulin-dependent myosin light chain kinase pathway. Smooth muscle contraction is primarily regulated by the levels of phosphorylation of myosin light chain (MLC), 1The abbreviations used are: MLC, myosin light chain; GTPγS, guanosine 5′-[γ-thio]triphosphate; Rho-kinase, Rho-associated serine/threonine kinase; CaM, calmodulin; CAT, the catalytic subunit of recombinant Rho-kinase; WM, wortmannin; DTT, dithiothreitol. which has heretofore been considered to be governed by a Ca2+-calmodulin (CaM)-dependent MLC kinase pathway (1Somlyo A.P. Somlyo A.V. Nature. 1994; 372: 231-236Crossref PubMed Scopus (1731) Google Scholar, 2Kamm K.E. Stull J.T. Annu. Rev. Pharmacol. Toxicol. 1985; 25: 593-603Crossref PubMed Google Scholar, 3Hartshorne D.J. Johnson D.R. Physiology of the Gastrointestinal Tract. 1. Raven Press, New York1987: 423-482Google Scholar, 4Sellers J.R. Adelstein R.S. Boyer P. Krebs E.G. The Enzyme. 18. Academic Press, San Diego, CA1987: 381-418Google Scholar). However, as the use of Ca2+ indicator revealed that the force/Ca2+ratio is variable, the Ca2+-CaM-dependent MLC kinase pathway cannot solely account for the mechanisms of agonist- or GTPγS-induced increases in the force/Ca2+ ratio, so-called Ca2+ sensitization (1Somlyo A.P. Somlyo A.V. Nature. 1994; 372: 231-236Crossref PubMed Scopus (1731) Google Scholar, 5Bradley A.B. Morgan K.G. J. Physiol. ( London ). 1987; 385: 437-448Crossref PubMed Scopus (133) Google Scholar, 6Kobayashi S. Kitazawa T. Somlyo A.V. Somlyo A.P. J. Biol. Chem. 1989; 264: 17997-18004Abstract Full Text PDF PubMed Google Scholar, 7Himpens B. Kitazawa T. Somlyo A.P. Pflügers Arch. Gen. Physiol. 1990; 417: 21-28Crossref Scopus (153) Google Scholar, 8Kitazawa T. Kobayashi S. Horiuchi K. Somlyo A.V. Somlyo A.P. J. Biol. Chem. 1989; 264: 5339-5342Abstract Full Text PDF PubMed Google Scholar, 9Kubota Y. Nomura M. Kamm K.E. Mumby M.C. Stull J.T. Am. J. Physiol. 1992; 262: C405-C410Crossref PubMed Google Scholar). Thus, an additional mechanism that can regulate Ca2+ sensitization of smooth muscle has been proposed. Using membrane permeabilization of smooth muscle, the possibility that monomeric Ras family G-proteins, such as Rho, contribute to Ca2+ sensitization of smooth muscle was demonstrated (10Hirata K. Kikuchi A. Sasaki T. Kuroda S. Kaibuchi K. Matsuura Y. Seki H. Saida K. Takai Y. J. Biol. Chem. 1991; 267: 8719-8722Google Scholar, 11Gong 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 (266) Google Scholar, 12Otto B. Steusloff A. Just I. Aktories K. Pfitzer G. J. Physiol. ( London ). 1996; 496: 317-329Crossref PubMed Scopus (106) Google Scholar). Direct activation of G-proteins by the application of GTPγS (8Kitazawa T. Kobayashi S. Horiuchi K. Somlyo A.V. Somlyo A.P. J. Biol. Chem. 1989; 264: 5339-5342Abstract Full Text PDF PubMed Google Scholar, 9Kubota Y. Nomura M. Kamm K.E. Mumby M.C. Stull J.T. Am. J. Physiol. 1992; 262: C405-C410Crossref PubMed Google Scholar), agonists (1Somlyo A.P. Somlyo A.V. Nature. 1994; 372: 231-236Crossref PubMed Scopus (1731) Google Scholar, 5Bradley A.B. Morgan K.G. J. Physiol. ( London ). 1987; 385: 437-448Crossref PubMed Scopus (133) Google Scholar, 6Kobayashi S. Kitazawa T. Somlyo A.V. Somlyo A.P. J. Biol. Chem. 1989; 264: 17997-18004Abstract Full Text PDF PubMed Google Scholar, 7Himpens B. Kitazawa T. Somlyo A.P. Pflügers Arch. Gen. Physiol. 1990; 417: 21-28Crossref Scopus (153) Google Scholar, 8Kitazawa T. Kobayashi S. Horiuchi K. Somlyo A.V. Somlyo A.P. J. Biol. Chem. 1989; 264: 5339-5342Abstract Full Text PDF PubMed Google Scholar), and GTP-activated Rho (10Hirata K. Kikuchi A. Sasaki T. Kuroda S. Kaibuchi K. Matsuura Y. Seki H. Saida K. Takai Y. J. Biol. Chem. 1991; 267: 8719-8722Google Scholar, 11Gong 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 (266) Google Scholar, 12Otto B. Steusloff A. Just I. Aktories K. Pfitzer G. J. Physiol. ( London ). 1996; 496: 317-329Crossref PubMed Scopus (106) Google Scholar) could exert Ca2+-sensitizing effects on saponin- or β-escin-permeabilized smooth muscle. However, the activated Rho failed to induce Ca2+ sensitization of extensively Triton X-100-permeabilized smooth muscle (11Gong 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 (266) Google Scholar). Considering that extensive Triton X-100-permeabilization allows higher molecular weight compounds to diffuse from the cytosol of smooth muscle of the rabbit portal vein (13Iizuka K. Ikebe M. Somlyo A.V. Somlyo A.P. Cell Calcium. 1994; 16: 431-445Crossref PubMed Scopus (49) Google Scholar), important diffusible factor(s) for the Ca2+ sensitization of smooth muscle might be lost during extensive permeabilization by Triton X-100, an event that would result in no response to activated Rho. We have recently reported that Rho-kinase, which is activated by GTP-bound active form of Rho (14Leung T. Manser E. Tan E. Lim L. J. Biol. Chem. 1995; 270: 29051-29054Abstract Full Text Full Text PDF PubMed Scopus (636) Google Scholar, 15Matsui T. Amano M. Yamamoto T. Chihara K. Nakafuku M. Ito M. Nakano T. Okawa K. Iwamatsu A. Kaibuchi K. EMBO J. 1996; 15: 2208-2216Crossref PubMed Scopus (938) Google Scholar, 16Ishizaki T. Maekawa M. Fujisawa K. Okawa K. Iwamatsu A. Fujita A. Watanabe N. Saito Y. Kakizuka A. Morii N. Narumiya S. EMBO J. 1996; 15: 1885-1893Crossref PubMed Scopus (792) Google Scholar), phosphorylates not only MLC, thereby activating myosin ATPase (17Amano M. Ito M. Kimura K. Fukata Y. Chihara K. Nakano T. Matsuura Y. Kaibuchi K. J. Biol. Chem. 1996; 271: 20246-20249Abstract Full Text Full Text PDF PubMed Scopus (1677) Google Scholar), but also myosin phosphatase, thus inactivating it in vitro (18Kimura K. Ito M. Amano M. Chihara K. Fukata Y. Nakafuku M. Yamamori B. Feng J.H. Nakano T. Okawa K. Iwamatsu A. Kaibuchi K. Science. 1996; 273: 245-248Crossref PubMed Scopus (2436) Google Scholar). These findings in a cell-free system, plus the previous reports of G-protein-mediating Ca2+ sensitization as described above, suggest that Rho-kinase may induce contraction and concomitant MLC phosphorylation of the smooth muscle. We examined the effects of the constitutively active form of Rho-kinase on smooth muscle extensively permeabilized by Triton X-100 and attempted to determine if Rho-kinase would be the factor lost during extensive Triton X-100 permeabilization. The catalytic subunit of recombinant Rho-kinase (CAT; molecular mass is about 80 kDa) was expressed as a glutathione S-transferase fusion protein and purified using a baculovirus system and a glutathione-Sepharose column (17Amano M. Ito M. Kimura K. Fukata Y. Chihara K. Nakano T. Matsuura Y. Kaibuchi K. J. Biol. Chem. 1996; 271: 20246-20249Abstract Full Text Full Text PDF PubMed Scopus (1677) Google Scholar). The kinase activity of the elute was determined by phosphorylation assay using S6 peptide as a substrate (15Matsui T. Amano M. Yamamoto T. Chihara K. Nakafuku M. Ito M. Nakano T. Okawa K. Iwamatsu A. Kaibuchi K. EMBO J. 1996; 15: 2208-2216Crossref PubMed Scopus (938) Google Scholar) in buffer containing 50 mm Tris-HCl, pH 7.5, 1 mm EDTA, 1 mm EGTA, 1 mm DTT, and 100 μm ATP (0.5–20 Gbq/mmol). CaM was purified from bovine brain by previously described method (19Walsh M.P. Valentine K.A. Ngai P.K. Carruthers C.A. Hollenberg M.D. Biochem. J. 1984; 224: 117-127Crossref PubMed Scopus (107) Google Scholar). Other materials and chemicals were obtained from commercial sources. Small strips of male rabbit (2–2.5 kg) portal veins were manually dissected (50–100 μm wide and 0.5–1 mm long), connected to an isometric force transducer (UL-2GR, Minebea, Japan), and mounted in a well (200 μl) on a bubble plate (6Kobayashi S. Kitazawa T. Somlyo A.V. Somlyo A.P. J. Biol. Chem. 1989; 264: 17997-18004Abstract Full Text PDF PubMed Google Scholar). After recording contractions evoked by 118 mm K+, the strips were incubated in relaxing solution, followed by 0.5% Triton X-100 for 20 min at 25 °C. The solutions have been described in detail elsewhere (6Kobayashi S. Kitazawa T. Somlyo A.V. Somlyo A.P. J. Biol. Chem. 1989; 264: 17997-18004Abstract Full Text PDF PubMed Google Scholar). CaM (0.5 μm) was added to all reactive solutions for experiments with chemical permeabilization. 0.5% Triton X-100-permeabilized and intact rabbit portal veins were homogenized in extraction buffer containing 50 mm Tris-HCl, pH 7.2, 400 mm NaCl, 2 mm EGTA, 1 mm EDTA, 1 mm DTT, 0.1 μm p-amidinophenylmethanesulfonyl fluoride hydrochloride, 10 μg/ml leupeptin, and 1 mm benzamidine. Each extract was centrifuged at 100,000 × g for 30 min at 4 °C, and the supernatant was subjected to SDS-polyacrylamide gel electrophoresis (20Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207240) Google Scholar) followed by immunoblotting (21Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988: 471-510Google Scholar). Anti-Rho-kinase rabbit polyclonal antibodies were generated against the glutathioneS-transferase fusion-catalytic domain of Rho-kinase (15Matsui T. Amano M. Yamamoto T. Chihara K. Nakafuku M. Ito M. Nakano T. Okawa K. Iwamatsu A. Kaibuchi K. EMBO J. 1996; 15: 2208-2216Crossref PubMed Scopus (938) Google Scholar), and anti-MLC rabbit polyclonal antibodies were provided by Dr. J. T. Stull (University of Texas Southwestern Medical Center, Dallas, TX). Immunostained proteins were visualized by Supersignal (Pierce). Densities of bands of immunostained proteins were quantitated using a scanning densitometer, Densitograph (Atto, Tokyo, Japan). After treatment with 5.1 μg/ml (0.06 μm) CAT and/or 10 μm wortmannin, the fringe-like strips of the rabbit portal veins permeabilized by Triton X-100 were quickly placed in a frozen slurry of acetone containing 10% trichloroacetic acid and 10 mm DTT to terminate the contractile responses. After depletion of trichloroacetic acid, the strips were homogenized in urea sample buffer containing 20 mm Tris base, 22 mmglycine, pH 8.6, 8 m urea, 10 mm DTT, 10% sucrose, and 0.1% bromphenol blue. The extracts were subjected to glycerol-urea polyacrylamide gel electrophoresis following immunoblotting using anti-MLC antibodies as documented (22Persechini A.K. Kamm K.E. Stull J.T. J. Biol. Chem. 1986; 261: 6293-6299Abstract Full Text PDF PubMed Google Scholar). Immunostained proteins were visualized colorimetrically with 4-chloro-1-naphthol and subjected to densitometrical quantitation. Introduction of CAT, which is not only the constitutively active form of Rho-kinase but also the highly homologous domain among rat (14Leung T. Manser E. Tan E. Lim L. J. Biol. Chem. 1995; 270: 29051-29054Abstract Full Text Full Text PDF PubMed Scopus (636) Google Scholar), bovine (15Matsui T. Amano M. Yamamoto T. Chihara K. Nakafuku M. Ito M. Nakano T. Okawa K. Iwamatsu A. Kaibuchi K. EMBO J. 1996; 15: 2208-2216Crossref PubMed Scopus (938) Google Scholar), human (16Ishizaki T. Maekawa M. Fujisawa K. Okawa K. Iwamatsu A. Fujita A. Watanabe N. Saito Y. Kakizuka A. Morii N. Narumiya S. EMBO J. 1996; 15: 1885-1893Crossref PubMed Scopus (792) Google Scholar), and mouse Rho-associated kinases (23Nakagawa O. Fujisawa K. Ishizaki T. Saito Y. Nakao K. Narumiya S. FEBS Lett. 1996; 392: 189-193Crossref PubMed Scopus (651) Google Scholar), into the cytosol of the extensively Triton X-100-permeabilized rabbit portal vein smooth muscle provoked a contraction both at a constant cytosolic Ca2+ (pCa 6.5; Fig.1 a) and at a nominally zero cytosolic Ca2+ buffered with 10 mm EGTA (pCa ≪ 8.0; Fig. 1 c). CAT exerted contraction, whereas the vehicle had no effect on the force (Fig. 1, a andc). These contractions were completely reversed by wash out of CAT, contrary to those induced by 10 μm microcystin-LR (24Somlyo A.P. Kitazawa T. Himpens B. Matthijs G. Horiuti K. Kobayashi S. Goldman Y.E. Somlyo A.V. Adv. Protein Phosphatases. 1989; 5: 181-195Google Scholar, 25Gong M.C. Cohen P. Kitazawa T. Ikebe M. Masuo M. Somlyo A.P. Somlyo A.V. J. Biol. Chem. 1992; 267: 14662-14668Abstract Full Text PDF PubMed Google Scholar) (Fig. 1 b). In the absence of cytosolic Ca2+ at pCa ≪ 8.0, the CAT-induced contraction was also reversible (data not shown). In neither intact nor α-toxin-permeabilized strips of the portal vein did CAT exert the contractile effects (data not shown). These observations are interpreted to mean that constitutively active CAT could be introduced into the cytosol of the smooth muscle only by extensive membrane permeabilization to induce a reversible contraction. MLC phosphorylation mediated by Ca2+-CaM-dependent MLC kinase pathway plays a primary role in smooth muscle contraction through myosin-actin-interaction and the consequent activation of myosin ATPase (2Kamm K.E. Stull J.T. Annu. Rev. Pharmacol. Toxicol. 1985; 25: 593-603Crossref PubMed Google Scholar, 3Hartshorne D.J. Johnson D.R. Physiology of the Gastrointestinal Tract. 1. Raven Press, New York1987: 423-482Google Scholar, 4Sellers J.R. Adelstein R.S. Boyer P. Krebs E.G. The Enzyme. 18. Academic Press, San Diego, CA1987: 381-418Google Scholar). To investigate involvement of Ca2+-CaM-dependent MLC kinase pathway in the CAT-induced contraction, we examined the effects of wortmannin (WM), a potent MLC kinase inhibitor (26Nakanishi S. Kakita S. Takahashi I. Kawahara K. Tsukuda E. Sano T. Yamada K. Yoshida M. Kase H. Matsuda Y. Hashimoto Y. Nonomura Y. J. Biol. Chem. 1992; 267: 2157-2163Abstract Full Text PDF PubMed Google Scholar) on force development induced by cumulative application of CAT (Fig. 2). In the presence of cytosolic Ca2+ at pCa 6.5, in which Ca2+-CaM-dependent MLC kinase should be active, 10 μm WM shifted the dose-response curve down and to the right. In the absence of cytosolic Ca2+ at pCa ≪ 8.0, in which MLC kinase would be hardly activated, 10 μm WM did not affect CAT-induced force development. In the absence of CAT, treatment of 10 μm WM completely inhibited the cytosolic Ca2+-provoked contraction atpCa 6.5, in the Triton X-100-permeabilized fibers. Considering our finding that WM did not affect the activity of CAT up to 100 μm in vitro (data not shown), this WM-sensitive component of CAT-induced contraction at pCa 6.5 seemed to be due to inhibition of the Ca2+-provoked contraction through the Ca2+-CaM-dependent MLC kinase pathway but not related to the CAT-mediated pathway. All these observations suggest that the CAT-induced contraction of smooth muscle of rabbit portal vein permeabilized by Triton X-100 is modulated independently by the Ca2+-CaM-dependent MLC kinase pathway. To clarify whether CAT induces contraction with a concomitant increase in levels of MLC phosphorylation, we examined the effects of CAT on MLC phosphorylation, using immunoblotting with anti-MLC polyclonal antibody (Fig. 3). As shown in lanes 1–3 of Fig.3 a, at pCa ≪ 8.0, monophosphorylation of MLC was detected only in the presence of CAT and was insensitive to 10 μm WM (42.77 ± 9.22% of the total amount of immunostained MLC (n = 4) in the absence of WM, 35.95 ± 3.39% (n = 4; p > 0.05) in the presence of WM, respectively). At pCa 6.5, shown inlanes 4–6 of Fig. 3 a, CAT potentiated the level of monophosphorylation of MLC (60.33 ± 1.42%, n= 4), which was partially inhibited by 10 μm WM (26.05 ± 5.18%, n = 4, p < 0.01). Based on the statistical analysis and the results in Fig. 2, this WM-sensitive component of CAT-induced MLC phosphorylation atpCa 6.5 also seemed to be due to inhibition of Ca2+-CaM-dependent MLC kinase activity. These results are consistent with that of counterparts of the effects of CAT on the contractile responses (Fig. 3 b). It was concluded that CAT potentiates the contractile response by increasing the extent of monophosphorylation of MLC. To determine if native Rho-kinase is one of the cofactors diffusible during permeabilization by Triton X-100, we examined the amounts of native Rho-kinase in intact and permeabilized fibers by immunoblot analysis using rabbit polyclonal antibodies against Rho-kinase. To standardize the densitometrical value, the ratio of densitometrical quantification of immunostaining of Rho-kinase to that of MLC was calculated in both intact and permeabilized fibers. As shown in Fig.4, the amounts of native Rho-kinase in the Triton X-100-permeabilized rabbit portal vein were markedly lower than those in intact tissue (0.06 ± 0.01 (n = 4) for permeabilized sample, 0.95 ± 0.02 (n = 4) for intact sample, respectively), whereas the amounts of the possible cytoskeletal proteins, such as MLC and myosin heavy chain in permeabilized fibers were similar to the counterparts of intact fibers. These results confirm that extensive permeabilization by Triton X-100 allows for the loss of cytosolic proteins, including Rho-kinase, whereas cytoskeletal proteins such as myosin are stable. Based on all of these findings taken together plus evidence that the direct activation of G-proteins did not exert contractile effects on the extensively Triton X-100-permeabilized smooth muscle (11Gong 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 (266) Google Scholar), we consider that Rho-kinase may be a valid candidate for the key molecule in G-protein-mediating smooth muscle contraction and may be the molecule lost during extensive permeabilization by Triton X-100. We demonstrate here what seems to be the evidence that Rho-kinase is a direct on the contractile of smooth muscle, independently of the Ca2+-CaM-dependent MLC kinase pathway. for MLC kinase Y. Nomura M. Kamm K.E. Mumby M.C. Stull J.T. Am. J. Physiol. 1992; 262: C405-C410Crossref PubMed Google Scholar, K. Ikebe M. Somlyo A.V. Somlyo A.P. Cell Calcium. 1994; 16: 431-445Crossref PubMed Scopus (49) Google Scholar), we no that the of kinases to the cytosol of permeabilized smooth muscle directly contractile responses with findings with the inhibition of myosin may be the mechanism of the G-protein-mediating Ca2+ sensitization of smooth muscle contraction (1Somlyo A.P. Somlyo A.V. Nature. 1994; 372: 231-236Crossref PubMed Scopus (1731) Google Scholar, 9Kubota Y. Nomura M. Kamm K.E. Mumby M.C. Stull J.T. Am. J. Physiol. 1992; 262: C405-C410Crossref PubMed Google Scholar, A.P. Kitazawa T. Himpens B. Matthijs G. Horiuti K. Kobayashi S. Goldman Y.E. Somlyo A.V. Adv. Protein Phosphatases. 1989; 5: 181-195Google Scholar, 25Gong M.C. Cohen P. Kitazawa T. Ikebe M. Masuo M. Somlyo A.P. Somlyo A.V. J. Biol. Chem. 1992; 267: 14662-14668Abstract Full Text PDF PubMed Google Scholar, T. Masuo M. Somlyo A.P. Proc. Natl. Acad. Sci. U. S. A. 1991; PubMed Scopus Google Scholar), the CAT-induced contraction of smooth muscle permeabilized by Triton X-100 may be also mediated by the inhibition of myosin is by our previous finding that Rho-kinase inhibited the activity of myosin through of subunit in vitro (18Kimura K. Ito M. Amano M. Chihara K. Fukata Y. Nakafuku M. Yamamori B. Feng J.H. Nakano T. Okawa K. Iwamatsu A. Kaibuchi K. Science. 1996; 273: 245-248Crossref PubMed Scopus (2436) Google Scholar). However, at cytosolic zero contraction of the permeabilized smooth muscle was by an MLC kinase inhibitor M.C. Cohen P. Kitazawa T. Ikebe M. Masuo M. Somlyo A.P. Somlyo A.V. J. Biol. Chem. 1992; 267: 14662-14668Abstract Full Text PDF PubMed Google Scholar), whereas the CAT-induced contraction was insensitive to it of the MLC kinase inhibitor to and myosin contractions the that myosin inhibition cannot account for the CAT-induced contraction at the cytosolic zero Ca2+. this together with our that Rho-kinase directly the phosphorylation of MLC and myosin in vitro (17Amano M. Ito M. Kimura K. Fukata Y. Chihara K. Nakano T. Matsuura Y. Kaibuchi K. J. Biol. Chem. 1996; 271: 20246-20249Abstract Full Text Full Text PDF PubMed Scopus (1677) Google Scholar), we suggest that the of CAT-induced contraction of Triton X-100-permeabilized rabbit portal vein might be a concomitant monophosphorylation of MLC directly induced by CAT independently of a Ca2+-CaM-dependent MLC kinase pathway. We that Rho-kinase is considered a valid key molecule in G-protein-mediating Ca2+ sensitization of smooth muscle contraction. We Dr. J. T. Stull for the D. J. and M. P. for and M. for on the

Two mechanisms are proposed to account for the inhibition of myosin phosphatase (MP) involved in Ca2+ sensitization of vascular muscle, ie, phosphorylation of either MYPT1, a target subunit of MP or CPI-17, an inhibitory phosphoprotein. In cultured vascular aorta smooth muscle cells (VSMCs), stimulation with angiotensin II activated RhoA, and this was blocked by pretreatment with 8-bromo-cGMP. VSMCs stimulated by angiotensin II, endothelin-1, or U-46619 significantly increased the phosphorylation levels of both MYPT1 (at Thr696) and CPI-17 (at Thr38). The angiotensin II-induced phosphorylation of MYPT1 was completely blocked by 8-bromo-cGMP or Y-27632 (a Rho-kinase inhibitor), but not by GF109203X (a PKC inhibitor). In contrast, phosphorylation of CPI-17 was inhibited only by GF109203X. Y-27632 dramatically corrected the hypertension in N(omega)-nitro-L-arginine methyl ester (L-NAME)-treated rats, and this hypertension also was sensitive to isosorbide mononitrate. The level of the active form of RhoA was significantly higher in aortas from L-NAME-treated rats. Expression of RhoA, Rho-kinase, MYPT1, CPI-17, and myosin light chain kinase were not significantly different in aortas from L-NAME-treated and control rats. Activation of RhoA without changes in levels of other signaling molecules were observed in three other rat models of hypertension, ie, stroke-prone spontaneously hypertensive rats, renal hypertensive rats, and DOCA-salt rats. These results suggest that independent of the cause of hypertension, a common point in downstream signaling and a critical component of hypertension is activation of RhoA and subsequent activation of Rho-kinase.