This Article Abstract Full Text (PDF) Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kandabashi, T. Articles by Takeshita, A. Search for Related Content PubMed PubMed Citation Articles by Kandabashi, T. Articles by Takeshita, A. Related Collections Cell signalling/signal transduction Chronic ischemic heart disease Mechanism of atherosclerosis/growth factors

(Arteriosclerosis, Thrombosis, and Vascular Biology. 2002;22:243.)
© 2002 American Heart Association, Inc.

Vascular Biology

Involvement of Rho-Kinase in Agonists-Induced Contractions of Arteriosclerotic Human Arteries

Tadashi Kandabashi; Hiroaki Shimokawa; Yasushi Mukai; Tetsuya Matoba; Ikuko Kunihiro; Keiko Morikawa; Masaaki Ito; Shosuke Takahashi; Kozo Kaibuchi; Akira Takeshita

From the Department of Cardiovascular Medicine (T.K., H.S., Y.M., T.M., I.K., K.M., AT) and Department of Anesthesiology (S.T.), Kyushu University Graduate School of Medical Sciences, Fukuoka; First Department of Internal Medicine, Mie Univsersity School of Medicine, Tsu, Japan (M.I.), and Department of Cell Pharmacology, Nagoya University Graduate School of Medicine, Nagoya, Japan (K.K.).

Address correspondence to Hiroaki Shimokawa, MD, PhD, Department of Cardiovascular Medicine, Kyushu University Graduate School of Medical Sciences, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. E-mail shimo{at}cardiol.med.kyushu-u.ac.jp

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Coronary artery spasm plays an important role in the pathogenesis of a wide variety of ischemic heart diseases. We have recently demonstrated that Rho-kinase plays a key role in the spasm in our porcine model. However, it remains to be elucidated whether Rho-kinase–mediated pathway also contributes to vasoconstriction of human arteries. From 15 patients who underwent coronary artery bypass operation, segments of isolated left internal thoracic arteries were obtained, and the endothelium was gently removed. Serotonin and histamine caused contractions, which were markedly inhibited by a specific Rho-kinase inhibitor, hydroxyfasudil. Western blot analysis showed that, during the serotonin-induced contractions, the extent of phosphorylation of myosin-binding subunit of myosin phosphatase (MBS, one of the major substrates of Rho-kinase) was significantly increased in the specimens. Hydroxyfasudil again significantly suppressed the serotonin-induced increase in MBS phosphorylation. There was a significant positive correlation between the extent of MBS phosphorylation and that of the serotonin-induced contractions and between hydroxyfasudil-sensitive components of the contractions and the extent of arteriosclerosis. These results indicate that Rho-kinase plays an important role in vascular smooth muscle contractions of arteriosclerotic human arteries, suggesting that Rho-kinase could be regarded as an important target for the treatment of arteriosclerotic vascular diseases in humans.

Key Words: signal transduction • Rho-kinase • human artery • vascular smooth muscle

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Coronary artery spasm plays an important role in the pathogenesis of a wide variety of ischemic heart diseases, not only in variant angina but also in effort and rest angina, acute myocardial infarction, ventricular arrhythmias, and sudden death.^1–4 The spasm is caused primarily by hypercontraction of vascular smooth muscle cells (VSMCs); however, the intracellular mechanism for the hypercontraction is still unknown, and the elucidation of this mechanism remains an important clinical issue.⁵

Phosphorylation of myosin light chain (MLC) is one of the essential steps for VSMC contraction,^6–8 which is initiated by Ca²⁺/calmodulin (CaM)-activated MLC kinase (MLCK) with subsequent phosphorylation of the 20-kDa regulatory MLC.^6–8 The level of MLC phosphorylation is determined by the balance between MLC phosphorylation by MLCK and dephosphorylation by MLC phosphatase (MLCPh). Several key molecules are involved in the regulation of MLCPh activity, including Rho-kinase/ROK/ROCKII^9–12 that is activated by the small GTPase Rho and CPI-17 (C-kinase–activated phosphatase inhibitor) that is specifically expressed in VSMC.^13–15

We have previously developed a porcine model of coronary spasm, in which long-term adventitial treatment with interleukin1-ß (IL-1ß), one of the major inflammatory cytokines, induces arteriosclerotic changes and vasospastic responses of the coronary artery.^16–22 In this model, Rho-kinase inhibits MLCPh activity by phosphorylating its myosin binding subunit (MBS)^21–23 resulting in the increased MLC mono- and diphosphorylation and coronary artery hyperconstriction.^20,21 We have recently demonstrated that Rho-kinase also plays an important role in the pathogenesis of coronary arteriosclerosis.^21–24 However, it remains to be examined whether Rho-kinase is involved in VSMC contraction of arteriosclerotic human arteries.

This study was thus designed to examine whether Rho-kinase and CPI-17 play an important role in VSMC contractions of human arteries.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
This study was reviewed and approved by the committee of the ethics on human research at the Cardiovascular Institute of the Kyushu University Graduate School of Medical Sciences. Because the left thoracic artery obtained during coronary artery bypass operation was classified as a surgical specimen, its use was exempted by the committee from required patient consent.

Sampling and Preparation of Human Arteries
Fifteen patients who underwent coronary artery bypass operation were included in this study. During the operation, distal segments of the left internal thoracic arteries (LITA) were obtained. These samples were carefully cleaned of any perivascular tissue in cold physiological salt solution of the following composition (mmol/L); NaCl 121, KCl 4.7, NaHCO₃ 24.7, MgSO₄ 12.2, CaCl₂ 2.5, KH₂PO₄ 1.2, and glucose 5.8. Physiological salt solution was aerated with 95% O₂ and 5% CO₂. For all experimental protocols, the endothelium was removed by gently rubbing of the luminal surface with a cotton swab in order to examine VSMC functions alone.²² All samples were cut into rings measuring approximately 4 mm in length.

Organ Chamber Experiments
Arterial rings were fixed vertically between two hooks in an organ bath of 20 mL capacity filled with Krebs solution. The hook anchoring the upper end of the strip was connected to the lever of a force transducer (Nihon-Kohden). Each preparation was stretched in a stepwise manner to an optimal length at which the force induced by 62 mmol/L KCl became maximum and constant.^20–22 The contractions to serotonin (1 nmol/L to 10 µmol/L) and to histamine (1 µmol/L) were then examined in the absence and presence of hydroxyfasudil (3 µmol/L), a specific inhibitor of Rho-kinase,²¹ which was added 15 minutes before the addition of the agonists. The inhibitory effect of hydroxyfasudil on the response to serotonin of each specimen was expressed as hydroxyfasudil-sensitive component calculated by the following equation; (F_c-F_h)/F_cx100(%), where F_c and F_h are the developed force to serotonin in the absence and presence of hydroxyfasudil, respectively. The developed tension was normalized and expressed as a percentage of that attained in the last contraction with 62 mmol/L KCl.^20–22

Histopathological Analysis of Human LITA
At the end of the organ chamber experiments, the arterial specimens were treated with sodium nitroprusside (5 µmol/L) and were relaxed beyond the basal level. Then the arterial rings were dismounted from the hook and fixed with 5% formaldehyde for 30 minutes. After fixation, the arterial specimens were dehydrated, embedded in paraffin, and cut into 5-µm-thick slices. These sections were stained with hematoxylin-eosin and van Gieson’s elastic staining for photomicroscopy. Three areas were measured by using a computer-assisted picture system. The intimal area (A_i) and medial area (A_m) were calculated by using the following formula: A_i=A_ie,-A_l, A_m=A_ee-A_ie,, where A_ee, A_ie, and A_l are the area within the external elastic lamina (EEL), internal elastic lamina (IEL), and the lumen area, respectively. The degree of neointimal formation was expressed by two parameters; one is percent intima (% intima) calculated by using the following equation: A_i/A_ie,x100(%), and another is intima/media ratio (I/M ratio) calculated by the following equation: A_i/A_mx100(%). In addition, for quantitative analysis of medial thickening, wall-to-lumen ration (W/L ratio) was also calculated by using the following equation: ([A_e/A_l]^1/2-1)x100(%).

Measurement of Rho-Kinase Activity and CPI-17 Phosphorylation
One of the major sites for phosphorylation of MBS by Rho-kinase both in vitro and in vivo has been identified as Ser-854,²⁵ and we have developed the antibody that specifically recognizes MBS phosphorylated at this site.²⁵ The extent of MBS phosphorylation in the strip was measured by SDS-PAGE, followed by electrophoretic transfer of the proteins to a nitrocellulose membrane. The amounts of phosphorylated MBS (MBS-P) in each sample were quantified by using immunoblot procedures. Briefly, the arterial specimens were removed from the hook at the maximal contractions and were immediately frozen by immersion in acetone containing 10% trichloracetic acid (TCA) cooled with dry ice, for Western blot analysis of MBS phosphorylations. The frozen arterial specimens obtained in the organ chamber experiments were washed three times with acetone containing dithiothreitol (10 mmol/L) to remove the TCA and dried. The dried ring was cut into small pieces, exposed to 200 µL of SDS-PAGE sample buffer for protein extraction. The extracted samples (20 µg of protein in each sample) were subjected to SDS-PAGE/immunoblot analysis by using the specific MBS-P antibody. The region containing MBS-P was visualized with an ECL Western Blotting Luminol Reagent (Santa Cruz Biotechnology). Similarly, the amounts of phosphorylated CPI-17 (CPI-17-P) in some samples were measured by immunoblot procedures by using anti-CPI-17-P antibody.¹⁴

Drugs
The following drugs were used: 5-hydroxytryptamine (serotonin) and histamine (Sigma Chemical), hydroxyfasudil (Asahi Chemical) and sarpogrelate (Mitsubishi-Tokyo Pharmaceutical).

Statistical Analysis
The results are expressed as mean±SEM. Throughout the text, n represents the number of arteries sampled. A repeated-measure ANOVA was performed to evaluate the global statistical significance, and if a significant F value was found, the Scheffe post hoc test was performed to identify the difference among the groups.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Rho-Kinase and Vascular Reactivity in Human LITA
Serotonin concentration dependently caused contractions of the human arterial segments without endothelium, which reached a maximum in 5 to 8 minutes (Figure 1). These contractile responses were almost abolished by the pretreatment with sarpogrelate, a selective 5-HT_2A receptor antagonist (10 µmol/L) (70±8% versus 6±1% in the absence and presence of sarpogrelate, respectively, n=3, P<0.01). Serotonin-induced VSMC contractions of the human arteries were markedly inhibited by hydroxyfasudil (3 µmol/L) (Figure 1). Similarly, histamine (1 µmol/L) caused contractions of the human arterial segments, which were also significantly inhibited by hydroxyfasudil (77±9% versus 48±8% in the absence and presence of hydroxyfasudil, respectively, n=3, P<0.01).

View larger version (14K): [in this window] [in a new window]   Figure 1. Serotonin-induced contractions of human internal thoracic arteries in the presence (open circles) and absence (filled circles) of a specific Rho-kinase inhibitor, hydroxyfasudil. Results are expressed as mean±SEM.

Rho-Kinase Activity in Human LITA
The extent of MBS phosphorylation was measured when the serotonin-induced contraction of each ring reached maximum.^21–22 Western blot analysis showed that MBS phosphorylation was significantly increased on stimulation by serotonin than under the control conditions (Figure 2). In addition, enhanced MBS phosphorylation was markedly inhibited by pretreatment with hydroxyfasudil (3 µmol/L) to the levels under control conditions (Figure 2). Importantly, there was a highly significant positive correlation between the extent of MBS phosphorylations and that of the serotonin-induced contractions (Figure 3).

View larger version (23K): [in this window] [in a new window]   Figure 2. Western blot analysis for MBS phosphorylations of human internal thoracic arteries with or without serotonin. MBS phosphorylation was significantly increased on stimulation by serotonin as compared with control conditions. Hydroxyfasudil (3 µmol/L) significantly suppressed MBS phosphorylation in response to serotonin. Results are expressed as mean±SEM.

View larger version (17K): [in this window] [in a new window]   Figure 3. Correlation between the extent of MBS phosphorylations and that of serotonin-induced contractions with or without hydroxyfasudil (3 µmol/L). There was a highly significant positive correlation between the two values.

CPI-17 Phosphorylation During Serotonin-Induced Contraction in Human LITA
The extent of CPI-17 phosphorylation was also measured in some specimens. Western blot analysis showed that CPI-17 phosphorylation was significantly increased during the serotonin-induced contraction (Figure 4). However, pretreatment with hydroxyfasudil failed to inhibit the increase in CPI-17 phosphorylation on stimulation by serotonin (Figure 4). In addition, there was no significant correlation between the extent of CPI-17 phosphorylations and that of the serotonin-induced contractions (r=0.35, not significant).

View larger version (23K): [in this window] [in a new window]   Figure 4. Western blot analysis for CPI-17 phosphorylations of human internal thoracic arteries with or without serotonin. CPI-17 phosphorylation was significantly increased on stimulation by serotonin as compared with control conditions. Hydroxyfasudil (3 µmol/L) did not inhibit the CPI-17 phosphorylation in response to serotonin. Results are expressed as mean±SEM.

A significant correlation was noted between the hydroxyfasudil-sensitive component of the serotonin-induced contraction and the extent of arteriosclerosis in human LITA (Figure 5).

View larger version (18K): [in this window] [in a new window]   Figure 5. Correlation between the hydroxyfasudil-sensitive component of serotonin-induced contractions and the indices of arteriosclerosis. There was a highly significant positive correlation between the hydroxyfasudil-sensitive component of serotonin-induced contractions and the indices of arteriosclerosis, such as intima:media (I/M) ratio (A), % intima (B), and wall:lumen (W/L) ratio (C).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The major findings of this study were that (1) VSMC contractions induced by serotonin and by histamine were significantly inhibited by the specific Rho-kinase inhibitor hydroxyfasudil in human LITA; (2) during the serotonin-induced contractions, the extents of phosphorylations of both MBS and CPI-17 were significantly increased; however, hydroxyfasudil inhibited only the MBS phosphorylations but not the CPI-17 ones, (3) there was a significant correlation between the extent of serotonin-induced contractions and the extent of MBS phosphorylations but not that of CPI-17; and (4) there was a significant correlation between the extent of Rho-kinase–mediated component of the serotonin-induced contractions and the extent of arteriosclerosis of human LITA. To the best of our knowledge, this is the first study that demonstrates the Rho-kinase–mediated inhibition of MLCPh activity in human arteries.

Involvement of Rho-Kinase and CPI-17 in Agonist-Induced VSMC Contractions of Human Arteries
We have previously demonstrated that, in our porcine model with IL-1ß, coronary artery spasm is associated with enhanced MLC mono- and diphosphorylations along with a significant increase in MBS phosphorylation.^20,21 Those changes were markedly suppressed by Rho-kinase inhibitors in our porcine model, where the expression and activity of the kinase were upregulated (Figure 6).^20–22 The inhibitory effect of hydroxyfasudil is not nonspecific because it does not inhibit contraction responses of normal porcine coronary arteries both in vivo and vitro^5,21 or those of human arteries when the extent of arteriosclerosis is minimal (in the present study). The level of MLC phosphorylations is determined by a balance between MLC phosphorylations by MLCK and its dephosphorylation by MLCPh^7,26 and the generation of diphosphorylated MLC is caused by inhibition of MLCPh in VSMC.²⁷ Thus, Rho-kinase plays a key role in the pathogenesis of coronary spasm in our porcine model by enhancing MLC phosphorylations through MLCPh inhibition (Figure 6). ^5,20–22

View larger version (32K): [in this window] [in a new window]   Figure 6. Working hypothesis on the intracellular mechanisms for hypercontraction of arteriosclerotic human artery. PLC indicates phospholipase C; DG, diacylglycerol; IP3, inositol 1,4,5-triphosphate; CaM, calmodulin; Cat, catalytic subunit; M20, 20-kDa regulatory subunit; +, stimulation; and -, inhibition. For the occurrence of hypercontraction, such as vasospasm, Rho-kinase–mediated pathway may play an important role, whereas the contribution of intracellular Ca2+ release may be minimal. Rho-kinase suppresses MLCPh through its MBS phosphorylations and subsequently causes hypercontractions or vasospasm of arteriosclerotic human artery. Rho-kinase and CPI-17 may exist in different intracellular signaling pathways, whereas the involvement of CPI-17-mediated pathway in agonists-induced hypercontraction of arteriosclerotic human artery may be minimal.

The present study demonstrated that Rho-kinase is also substantially involved in the contractions of human arteries induced by serotonin and histamine. Recently, it was reported that Rho-kinase may be involved in agonists-induced contractions of human arteries; however, no mechanistic consideration was made in this study.²⁸ In the present study, we were able to demonstrate that MBS phosphorylation was significantly increased during the serotonin-induced contractions of human VSMC, indicating that Rho-kinase is activated on stimulation by serotonin with resultant inhibition of MLCPh. In contrast, CPI-17 may not play a primary role in the contractions of human VSMC because the significant increase in CPI-17 phosphorylation during serotonin-induced contraction was not inhibited by hydroxyfasudil. These results suggest that, in the signal transduction for human VSMC contraction, Rho-kinase and CPI-17 are involved in different pathways and that Rho-kinase, but not CPI-17, plays an important role in the contractions of human VSMC (Figure 6). It was previously reported that 10 µmol/L of Y-27632, another Rho-kinase inhibitor, partially suppressed CPI-17 phosphorylation in rabbit femoral arteries²⁹ and in porcine aorta.³⁰ The discrepancy between our present finding and those previous reports is probably the result of the differences in the kind and concentrations of Rho-kinase inhibitors used and blood vessels tested, and of the presence or absence of atherosclerosis.

Role of Rho-Kinase in the Vasoconstriction of Human Arteries In Vivo
We have recently demonstrated that Rho-kinase is involved in the increased peripheral vascular resistance in patients with hypertension.³¹ We also have recently demonstrated that intracoronary fasudil markedly suppresses coronary artery spasm in patients with vasospastic angina.³² These results indicate that Rho-kinase is substantially involved in enhanced vasoconstriction in humans in vivo.

Effect of Arteriosclerosis on the Involvement of Rho-Kinase in the Contractions of Human VSMCs
Rho-kinase not only mediates VSMC contraction through MLCPh inhibition, but it also mediates various cellular functions that may stimulate the process of arteriosclerosis.⁵ Indeed, we have recently demonstrated that Rho-kinase is substantially involved in the pathogenesis of arteriosclerosis in various porcine models in vivo^33,34 and that the long-term inhibition of the molecule, with either gene transfer²³ or a Rho-kinase inhibitor,²⁴ induces a regression of arteriosclerosis in vivo. We have also demonstrated that Rho-kinase plays an important role in the pathogenesis of hypertensive vascular disease (eg, medial thickening and perivascular fibrosis).³⁵ In the present study, there was a highly significant positive correlation between the extent of involvement of Rho-kinase in serotonin-induced contractions of human arteries and the extent of arteriosclerosis. These results suggest that Rho-kinase may also play an important role in the pathogenesis of arteriosclerosis in humans.

Limitations of the Study
Several limitations can be mentioned for the present study. First, we used only human internal thoracic artery but not human coronary artery with coronary spasm for ethical reasons. However, the present study and our recent finding that intracoronary fasudil markedly suppresses coronary spasm in humans³² strongly suggest that Rho-kinase is also substantially involved in the pathogenesis of the spasm in humans. Second, we cannot totally rule out the contribution of CPI-17 to the contractions of human arteries, especially in vivo. For this purpose, a selective inhibitor of CPI-17 needs to be developed.

In summary, the present study demonstrated that Rho-kinase is substantially involved in the agonists-induced contractions of human arteries and that the contribution of Rho-kinase to the contractions increases as the extent of arteriosclerosis progresses. These results, together with our recent findings,^{20–24,31–35} suggest that Rho-kinase could be regarded as a novel therapeutic target for the treatment of arteriosclerotic cardiovascular diseases in humans.

	Acknowledgments

 
This work supported in part by grants-in-aid from the Japanese Ministry of Education, Science, Sports, Culture, and Technology, Tokyo, Japan (09470169, 10177223, 10357006, 12032215, and 12470158). The authors wish to thank Dr H. Yasui (The Department of Cardiovascular Surgery, Kyushu University Graduate School of Medical Sciences), and Dr Y. Kawachi (The Department of Cardiovascular Surgery, National Hospital Kyushu Medical Center) for cooperation in this study and Dr T. Kitazawa (The Department of Physiology and Biophysics, Georgetown University Medical Center) for valuable comments on our work. We also thank M. Sonoda, S. Tomita, and E. Gunshima for excellent technical assistance.

	Footnotes

 
Consulting Editor for this article was Alan M. Fogelman, MD, Professor of Medicine and Executive Chair, Departments of Medicine and Cardiology, UCLA School of Medicine, Los Angeles, Calif.

Received September 26, 2001; accepted November 20, 2001.

	References

Top Abstract Introduction Methods Results Discussion References

 
1. Maseri A, Severi S, Nes MD, L’Abbate A, Chierchia S, Marzilli M, Ballestra AM, Parodi O, Biagini A, Distante A. "Variant" angina: one aspect of a continuous spectrum of vasospastic myocardial ischemia: pathogenetic mechanisms, estimated incidence and clinical and coronary arteriographic findings in 138 patients. Am J Cardiol. 1978; 42: 1019–1035.[CrossRef][Medline] [Order article via Infotrieve]

2. Hillis LD, Braunwald E. Coronary-artery spasm. N Engl J Med. 1978; 299: 695–702.[Medline] [Order article via Infotrieve]

3. Shepherd JT, Vanhoutte PM. Spasm of the coronary arteries: causes and consequences (the scientist’s viewpoint). Mayo Clin Proc. 1985; 60: 33–46.[Medline] [Order article via Infotrieve]

4. Fuster V, Badimon L, Badimon JJ, Chesebro JH. The pathogenesis of coronary artery disease and the acute coronary syndromes. N Engl J Med. 1992; 326: 310–318.[Medline] [Order article via Infotrieve]

5. Shimokawa H. Cellular and molecular mechanisms of coronary artery spasm: lessons from animal models. Jpn Circ J. 2000; 64: 1–12.[CrossRef][Medline] [Order article via Infotrieve]

6. Kamm KE, Stull JT. Regulation of smooth muscle contractile elements by second messengers. Ann Rev Physiol. 1989; 51: 299–313.[CrossRef][Medline] [Order article via Infotrieve]

7. Somlyo AP, Somlyo AV. Signal transduction and regulation in smooth muscle. Nature. 1994; 372: 231–236.[CrossRef][Medline] [Order article via Infotrieve]

8. Murphy RA. What is special about smooth muscle? The significance of covalent crossbridge regulation. FASEB J. 1994; 8: 311–318.[Abstract]

9. Kimura K, Ito M, Amano M, Chihara K, Fukata Y, Nakafuku M, Yamamori B, Feng J, Nakano T, Okawa K, et al. Regulation of myosin phosphatase by Rho and Rho-associated kinase (Rho-kinase). Science. 1996; 273: 245–248.[Abstract]

10. Leung T, Manser E, Tan L, Lim L. A novel serine-threonine kinase binding the Ras-related RhoA GTPase which translocates the kinase to peripheral membranes. J Biol Chem. 1995; 270: 29051–29054.[Abstract/Free Full Text]

11. Ishizaki T, Maekawa M, Fujisawa K, Okawa K, Iwamatsu A, Fijita A, Watanabe N, Saito Y, Kakizuka A, Morii N, et al. The small GTP-binding protein Rho binds to activates a 160 kDa Ser/Thr protein kinase homologous to myotonic dystrophy kinase. EMBO J. 1996; 15: 1885–1893.[Medline] [Order article via Infotrieve]

12. Fukata Y, Amano M, Kaibuchi K. Rho-Rho-kinase pathway in smooth muscle contraction and cytoskeletal reorganization of non-muscle cells. Trends Pharmacol Sci. 2001; 22: 32–39.[CrossRef][Medline] [Order article via Infotrieve]

13. Eto M, Ohmori T, Suzuki M, Furuya K, Morita F. A novel protein phosphatase-1 inhibitory protein potentiated by protein kinase C: isolation from porcine aorta media and characterization. J Biochem. 1995; 118: 1104–1107.[Abstract/Free Full Text]

14. Koyama M, Ito M, Feng J, Seko T, Shiraki K, Takase K, Hartshorne DJ, Nakano T. Phosphorylation of CPI-17, an inhibitory phosphoprotein of smooth muscle myosin phosphatase, by Rho-kinase. FEBS Lett. 2000; 475: 197–200.[CrossRef][Medline] [Order article via Infotrieve]

15. Kitazawa T, Takizawa N, Ikebe M, Eto M. Reconstitution of protein kinase C-induced contractile Ca²⁺ sensitization in triton X-100-demembranated rabbit arterial smooth muscle. J Physiol. 1999; 520 (Pt 1): 139–152.[Medline] [Order article via Infotrieve]

16. Shimokawa H, Ito A, Fukumoto Y, Kadokami T, Nakaike R, Sakata M, Takayanagi T, Egashira K, Takeshita A. Chronic treatment with interleukin-1ß induces coronary intimal lesions and vasospastic responses in pigs in vivo: the role of platelet-derived growth factor. J Clin Invest. 1996; 97: 769–776.[Medline] [Order article via Infotrieve]

17. Ito A, Shimokawa H, Kadokami T, Fukumoto Y, Owada MK, Shiraishi T, Nakaike R, Takayanagi T, Egashira K, Takeshita A. Tyrosine kinase inhibitor suppresses coronary arteriosclerotic changes and vasospastic responses induced by chronic treatment with interleukin-1ß in pigs in vivo. J Clin Invest. 1995; 96: 1288–1294.[Medline] [Order article via Infotrieve]

18. Kadokami T, Shimokawa H, Fukumoto Y, Ito A, takayanagi T, Egashira K, Takeshita A. Coronary artery spasm does not depend on the intracellular calcium store but is substantially mediated by the protein kinase C-mediated pathway in a swine model with interleukin-1ß in vivo. Circulation. 1996; 94: 190–196.[Abstract/Free Full Text]

19. Fukumoto Y, Shimokawa H, Kozai T, Kadokami T, Kuwata K, Yonemitsu Y, Kuga T, Egashira K, Sueishi K, Takeshita A. Vasculoprotective role of inducible nitric oxide synthase at inflammatory coronary lesions induced by chronic treatment with interleukin-1ß in pigs in vivo. Circulation. 1997; 96: 3104–3111.[Abstract/Free Full Text]

20. Katsumata N, Shimokawa H, Seto M, Kozai T, Yamawaki T, Kuwata K, Egashira K, Ikegaki I, Asano T, Sasaki Y, Takeshita A. Enhanced myosin light chain phosphorylations as a central mechanism for coronary artery spasm in a swine model with interleukin-1ß. Circulation. 1997; 96: 4357–4363.[Abstract/Free Full Text]

21. Shimokawa H, Seto M, Katsumata N, Amano M, Kozai T, Yamawaki T, Kuwata K, Kandabashi T, Egashira K, Ikegaki I, et al. Rho-kinase-mediated pathway induces enhanced myosin light chain phosphorylations in a swine model of coronary artery spasm. Cardiovasc Res. 1999; 43: 1029–1039.[Abstract/Free Full Text]

22. Kandabashi T, Shimokawa H, Miyata K, Kunihiro I, Kawano Y, Fukata Y, Higo T, Egashira K, Takahashi S, Kaibuchi K, Takeshita A. Inhibition of myosin phosphatase by upregulated rho-kinase plays a key role for coronary artery spasm in a porcine model with interleukin- 1ß. Circulation. 2000; 101: 1319–1323.[Abstract/Free Full Text]

23. Morishige K, Shimokawa H, Eto Y, Kandabashi T, Miyata K, Matsumoto Y, Hoshijima M, Kaibuchi K, Takeshita A. Adenovirus-mediated transfer of dominant-negative Rho-kinase induces a regression of coronary arteriosclerosis in pigs in vivo. Arterioscler Thromb Vasc Biol. 2001; 21: 548–554.[Abstract/Free Full Text]

24. Shimokawa H, Morishige K, Miyata K, Kandabashi T, Eto Y, Ikegaki I, Asano T, Kaibuchi K, Takeshita A. Long-term inhibition of Rho-kinase induces a regression of arteriosclerotic coronary lesions in a porcine model in vivo. Cardiovasc Res. 2001; 51: 169–177.[Abstract/Free Full Text]

25. Kawano Y, Fukata Y, Oshiro N, Amano M, Nakamura T, Ito M, Matsumura F, Inagaki M, Kaibuchi K. Phosphorylation of myosin-binding subunit (MBS) of myosin phosphatase by Rho-kinase in vivo. J Cell Biol. 1999; 147: 1023–1038.[Abstract/Free Full Text]

26. Ikebe M, Hartshorne DJ, Elzinga M. Phosphorylation of the 20,000-dalton light chain of smooth muscle myosin by the calcium-activated, phospholipid-dependent protein kinase: phosphorylation sites and effects of phosphorylation. J Biol Chem. 1987; 262: 9569–9573.[Abstract/Free Full Text]

27. Seto M, Sakurada K, Kamm KE, Stull JT, Sasaki Y. Myosin light chain diphosphorylation is enhanced by growth promotion of cultured smooth muscle cells. Eur J Physiol. 1996; 432: 7–13.[CrossRef][Medline] [Order article via Infotrieve]

28. Batchelor TJ, Sadaba JR, Ishola A, Pacaud P, Munsch CM, Beech DJ. Rho-kinase inhibitors prevent agonist-induced vasospasm in human internal mammary artery. Br J Pharmacol. 2001; 132: 302–308.[CrossRef][Medline] [Order article via Infotrieve]

29. Kitazawa T, Eto M, Woodsome TP, Brautigen DL. Agonists trigger G protein-mediated activation of the CPI-17 inhibitor phosphoprotein of myosin light chain phosphatase to enhance vascular smooth muscle contractility. J Biol Chem. 2000; 275: 9897–9900.[Abstract/Free Full Text]

30. Eto M, Kitazawa T, Yazawa M, et al. Histamine-induced vasoconstriction involves phosphorylation of a specific inhibitor protein for myosin phosphatase, by protein kinase C alpha and delta isoforms. J Biol Chem. 2001; 276: 29072–29078.[Abstract/Free Full Text]

31. Masumoto A, Hirooka Y, Shimokawa H, Hironaga K, Setoguchi S, Takeshita A. Possible involvement of Rho-kinase in the pathogenesis of hypertension in humans. Hypertension. 2001; 38; 1307–1310.[Medline] [Order article via Infotrieve]

32. Shimokawa H, Mohri M, Masumoto A. Rho-kinase inhibitor suppresses coronary artery spasm in humans. Circulation. 2001; 104 (Suppl II): II-601.Abstract 2842.

33. Miyata K, Shimokawa H, Kandabashi T, Higo T, Morishige K, Eto Y, Egashira K, Kaibuchi K, Takeshita A. Rho-kinase is involved in macrophage-mediated formation of coronary vascular lesions in pigs in vivo. Arterioscler Thromb Vasc Biol. 2000; 20: 2351–2358.[Abstract/Free Full Text]

34. Eto Y, Shimokawa H, Hiroki J, Morishige K, Kandabashi T, Matsumoto Y, Amano M, Hoshijima M, Kaibuchi K, Takeshita A. Gene transfer of dominant negative Rho-kinase suppresses neointimal formation after balloon injury in pigs. Am J Physiol. 2000; 278: H1744–1750.

35. Mukai Y, Shimokawa H, Matoba T, Kandabashi T, Satoh S, Hiroki J, Kaibuchi K, Takeshita A. Involvement of Rho-kinase in hypertensive vascular disease: a novel therapeutic target in hypertension. FASEB J. 2001; 15: 1062–1064.[Free Full Text]

This article has been cited by other articles:

				R. Lu, A. Alioua, Y. Kumar, P. Kundu, M. Eghbali, N. V. Weisstaub, J. A. Gingrich, E. Stefani, and L. Toro c-Src tyrosine kinase, a critical component for 5-HT2A receptor-mediated contraction in rat aorta J. Physiol., August 15, 2008; 586(16): 3855 - 3869. [Abstract] [Full Text] [PDF]

				T. Hizume, K. Morikawa, A. Takaki, K. Abe, K. Sunagawa, M. Amano, K. Kaibuchi, C. Kubo, and H. Shimokawa Sustained Elevation of Serum Cortisol Level Causes Sensitization of Coronary Vasoconstricting Responses in Pigs In Vivo: A Possible Link Between Stress and Coronary Vasospasm Circ. Res., September 29, 2006; 99(7): 767 - 775. [Abstract] [Full Text] [PDF]

				T. P. Woodsome, A. Polzin, K. Kitazawa, M. Eto, and T. Kitazawa Agonist- and depolarization-induced signals for myosin light chain phosphorylation and force generation of cultured vascular smooth muscle cells J. Cell Sci., May 1, 2006; 119(9): 1769 - 1780. [Abstract] [Full Text] [PDF]

				G. Loirand, P. Guerin, and P. Pacaud Rho Kinases in Cardiovascular Physiology and Pathophysiology Circ. Res., February 17, 2006; 98(3): 322 - 334. [Abstract] [Full Text] [PDF]

				H. K. Surks, N. Riddick, and K.-i. Ohtani M-RIP Targets Myosin Phosphatase to Stress Fibers to Regulate Myosin Light Chain Phosphorylation in Vascular Smooth Muscle Cells J. Biol. Chem., December 30, 2005; 280(52): 42543 - 42551. [Abstract] [Full Text] [PDF]

				R. H. P. Hilgers and R. C. Webb Molecular Aspects of Arterial Smooth Muscle Contraction: Focus on Rho Experimental Biology and Medicine, December 1, 2005; 230(11): 829 - 835. [Abstract] [Full Text] [PDF]

				R. M. Vicari, B. Chaitman, D. Keefe, W. B. Smith, S. G. Chrysant, M. J. Tonkon, N. Bittar, R. J. Weiss, H. Morales-Ballejo, U. Thadani, et al. Efficacy and Safety of Fasudil in Patients With Stable Angina: A Double-Blind, Placebo-Controlled, Phase 2 Trial J. Am. Coll. Cardiol., November 15, 2005; 46(10): 1803 - 1811. [Abstract] [Full Text] [PDF]

				H. Shimokawa and A. Takeshita Rho-Kinase Is an Important Therapeutic Target in Cardiovascular Medicine Arterioscler Thromb Vasc Biol, September 1, 2005; 25(9): 1767 - 1775. [Abstract] [Full Text] [PDF]

				A. Endo, H. K. Surks, S. Mochizuki, N. Mochizuki, and M. E. Mendelsohn Identification and Characterization of Zipper-interacting Protein Kinase as the Unique Vascular Smooth Muscle Myosin Phosphatase-associated Kinase J. Biol. Chem., October 1, 2004; 279(40): 42055 - 42061. [Abstract] [Full Text] [PDF]

				T. Nagaoka, Y. Morio, N. Casanova, N. Bauer, S. Gebb, I. McMurtry, and M. Oka Rho/Rho kinase signaling mediates increased basal pulmonary vascular tone in chronically hypoxic rats Am J Physiol Lung Cell Mol Physiol, October 1, 2004; 287(4): L665 - L672. [Abstract] [Full Text] [PDF]

				E. A. Wehrwein, C. A. Northcott, R. D. Loberg, and S. W. Watts Rho/Rho Kinase and Phosphoinositide 3-Kinase Are Parallel Pathways in the Development of Spontaneous Arterial Tone in Deoxycorticosterone Acetate-Salt Hypertension J. Pharmacol. Exp. Ther., June 1, 2004; 309(3): 1011 - 1019. [Abstract] [Full Text] [PDF]

				H. Kawamura, K. Yokote, S. Asaumi, K. Kobayashi, M. Fujimoto, Y. Maezawa, Y. Saito, and S. Mori High Glucose-Induced Upregulation of Osteopontin Is Mediated via Rho/Rho Kinase Pathway in Cultured Rat Aortic Smooth Muscle Cells Arterioscler Thromb Vasc Biol, February 1, 2004; 24(2): 276 - 281. [Abstract] [Full Text]

				T. Kandabashi, H. Shimokawa, K. Miyata, I. Kunihiro, Y. Eto, K. Morishige, Y. Matsumoto, K. Obara, K. Nakayama, S. Takahashi, et al. Evidence for Protein Kinase C-Mediated Activation of Rho- Kinase in a Porcine Model of Coronary Artery Spasm Arterioscler Thromb Vasc Biol, December 1, 2003; 23(12): 2209 - 2214. [Abstract] [Full Text] [PDF]

				H. Ogita, S. Kunimoto, Y. Kamioka, H. Sawa, M. Masuda, and N. Mochizuki EphA4-Mediated Rho Activation via Vsm-RhoGEF Expressed Specifically in Vascular Smooth Muscle Cells Circ. Res., July 11, 2003; 93(1): 23 - 31. [Abstract] [Full Text] [PDF]

				J. Galle, A. Mameghani, S.-S. Bolz, S. Gambaryan, M. Gorg, T. Quaschning, U. Raff, H. Barth, S. Seibold, C. Wanner, et al. Oxidized LDL and its Compound Lysophosphatidylcholine Potentiate AngII-Induced Vasoconstriction by Stimulation of RhoA J. Am. Soc. Nephrol., June 1, 2003; 14(6): 1471 - 1479. [Abstract] [Full Text] [PDF]

				T. Seko, M. Ito, Y. Kureishi, R. Okamoto, N. Moriki, K. Onishi, N. Isaka, D. J. Hartshorne, and T. Nakano Activation of RhoA and Inhibition of Myosin Phosphatase as Important Components in Hypertension in Vascular Smooth Muscle Circ. Res., March 7, 2003; 92(4): 411 - 418. [Abstract] [Full Text] [PDF]

				A. Masumoto, M. Mohri, H. Shimokawa, L. Urakami, M. Usui, and A. Takeshita Suppression of Coronary Artery Spasm by the Rho-Kinase Inhibitor Fasudil in Patients With Vasospastic Angina Circulation, April 2, 2002; 105(13): 1545 - 1547. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kandabashi, T. Articles by Takeshita, A. Search for Related Content PubMed PubMed Citation Articles by Kandabashi, T. Articles by Takeshita, A. Related Collections Cell signalling/signal transduction Chronic ischemic heart disease Mechanism of atherosclerosis/growth factors