Mechanism of RhoA/Rho kinase activation in endothelin-1- induced contraction in rabbit basilar artery

Departments of Neurosurgery, Physiology, and Biophysics, University of Mississippi Medical Center, Jackson, Mississippi 39216

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

This study was undertaken to demonstrate the role of the RhoA/Rho kinase pathway in endothelin-1 (ET-1)-induced contraction of the rabbit basilar artery. Isometric tension and Western blot were used to examine ET-1-induced contraction and RhoA activation. The upstream effect on ET-1-induced RhoA activity was determined by using ET_A and ET_B receptor antagonists, protein kinase C (PKC), tyrosine kinase, and phosphatidylinositol-3 kinase inhibitors. The downstream effect of ET-1-induced contraction and RhoA activity was studied in the presence of the Rho kinase inhibitor Y-27632. The effect of Rho kinase inhibitor on ET-1-induced myosin light chain (MLC) phosphorylation was investigated by using urea-glycerol-PAGE immunoblotting. We found 1) ET-1 increased RhoA activity (membrane binding RhoA) in a concentration-dependent manner; 2) ET_A, but not ET_B, receptor antagonist abolished the effect of ET-1 on RhoA activation; 3) phosphodylinositol-3 kinase inhibitor, but not PKC and tyrosine kinase inhibitors, reduced ET-1-induced RhoA activation; 4) Rho kinase inhibitor Y-27632 (10 µM) inhibited ET-1-induced contraction; and 5) ET-1 increased the level of MLC phosphorylation. Rho kinase inhibitor Y-27632 reduced the effect of ET-1 on MLC phosphorylation. This study demonstrated that RhoA/Rho kinase activation is involved in ET-1-induced contraction in the rabbit basilar artery. Phosphodylinositol-3 kinase and MLC might be the upstream and downstream factors of RhoA activation.

cerebral vasospasm; rabbit basilar artery

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

CEREBRAL VASOSPASM is a leading cause and a frequent complication of the morbidity and mortality in patients with subarachnoid hemorrhage (SAH). Cerebral vasospasm is characterized by a delayed and prolonged contraction of the major cerebral arteries (13, 39). The cause of vasospasm might be vasoactive substances released into the subarachnoid space by the dissolution of the resultant blood clot (14, 32) or by vasoactive agents released from the vessel walls. There is evidence that endothelin-1 (ET-1) is a major cause of cerebral vasospasm after SAH (40). The levels of ET-1 in bloody cerebrospinal fluid are elevated in patients with SAH. ET-1 causes long-lasting vasoconstriction. ET-1 receptor antagonists, endothelin-converting enzyme inhibitors, and ET-1 antisense oligonucleotide prevent cerebral vasospasm in animal models (40).

It is commonly accepted that increased intracellular calcium is a determinant for contraction in vascular smooth muscle in response to agonists (14). Recent studies have shown that most of these agonists are able to modulate contraction by altering myofilament calcium sensitivity or through calcium independent pathways. The involvement of protein kinase C (PKC) (2, 6, 11), tyrosine kinase (18), phosphatidylinositol (PI)-3 kinase (41), and also RhoA/Rho kinase in calcium sensitization has been reported in vascular smooth muscle (5, 20). In addition, cross talk between these different kinase pathways may be the key signaling event of calcium sensitization of the contractile activation of vascular smooth muscle (25). RhoA/Rho kinase is an important pathway to mediate myofilament Ca²⁺ sensitization of smooth muscle cells (16), and RhoA/Rho kinase has been suggested to be involved in cerebral vasospasm (15, 26, 30, 35). However, the possible role of the RhoA pathway in ET-1-induced contraction is not clear. In this study, we investigated the effect of ET-1 on RhoA activation and the types of ET receptors involved, and the implication of PKC, tyrosine kinase, and PI-3 kinase in the mechanisms controlling ET-1-induced contraction was also studied.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Materials. ET-1 was purchased from Alexis. Staurosporine (PKC inhibitor), genistein (tyrosine kinase inhibitor), and LY-294002 (PI-3 kinase inhibitor) were purchased from BioMol. Y-27632 (Rho kinase inhibitor) was purchased from TOCRIS. Anti-RhoA (26C4), ROKI, and ROKII antibodies were purchased from Santa Cruz Biotechnology. BQ-610, BQ-788, monoclonal anti-myosin (light chain 20K) antibody, and other chemicals were purchased from Sigma Chemical.

Western blot. Thirty male New Zealand White rabbits were anesthetized by an injection of thiopental (20 mg/kg iv) and euthanatized by exsanguination. The basilar arteries were removed from the base of the brain and incubated with ET-1 with different concentrations and for different time periods in the Krebs-Henseleit buffer. Some arteries were treated with specific antagonists for 30 min before being treated with ET-1. After treatment, the arteries were immediately frozen in liquid nitrogen. Western blot analysis was performed as previously described (28). Strips of basilar arteries were homogenized in lysis buffer. After the sample was centrifuged at 15,000 g for 1 h to generate membrane and cytosolic fractions, equal amounts of protein from the membrane fractions were loaded into each lane of SDS-12% polyacrylamide gels. The gels were then electrophoresed, transferred to a nitrocellulose membrane, and finally analyzed by Western blotting by using mouse monoclone anti- RhoA antibody (Santa Cruz, CA). As an internal control, -actin was blotted in the same membrane.

The Animal Care and Use Committee approved all the procedures using rabbit tissues.

Isometric tension. The basilar arteries were removed and cut into 2-mm rings in a dissecting chamber filled with modified Krebs-Henseleit bicarbonate solution containing (in mM) 120 NaCl, 4.5 KCl, 1 MgSO₄, 27 NaHCO₃, 1.2 KH₂PO₄, 2.5 CaCl₂, and 10 dextrose and bubbled with 95% O₂-5% CO₂. We removed endothelial cells by gently rubbing the rings with a steel hook.

Rings were suspended at the resting tension of 400 mg (Radnoti transducer, Radnoti Glass) between stainless steel hooks in 10 water-jacketted tissue baths (Radnoti Glass) in modified Krebs-Henseleit biocarbonate buffer with 95% O₂-5% CO₂ at 37°C. Rings were incubated for 90 min until a stable rest tension was achieved, and the solution was changed every 20 min to remove metabolites. The tissues were challenged with 60 mM KCl twice at 30-min intervals before the experiment. Isometric force transducers were connected to arterial rings, and contraction was recorded with an eight-channel MacLab 8E and stored on a Power Macintosh computer.

Measurements of myosin light chain phosphorylation. The extent of myosin light chain (MLC) phosphorylation in the rabbit basilar artery strips was measured by glycerol-PAGE followed by electrophoretic transfer of the proteins to a nitrocellulose membrane. The amounts of the phosphorylation form were quantified by immunoblot procedures, as described by Persechini et al. (19).

ET-1- and antagonist-treated rings were frozen by immersion in acetone containing 10% trichloroacetic acid (TCA) and 10 mM dithiothreitol (DTT) cooled with dry ice. Frozen tissues were washed twice with acetone containing 10 mM DTT to remove the TCA and then dried. The dried ring was cut into small pieces and exposed to the urea sample buffer (20 mM Tris, 22 mM glycine, 10 mM DTT, 8.3 M urea, and 0.1% bromophenol blue) for purposes of extraction, and then the proteins were processed for urea-glycerol-PAGE and immumoblotting with anti-MLC monoclonal antibody and horseradish peroxidase-conjugated second antibody. Quantitative evaluation of phosphorylated MLC was performed by densitometric analysis using Quantity One software (Bio-Rad). The percentage of the phosphorylated form in total MLC was calculated to indicate the extent of MLC phosphorylation.

Statistical analysis. The data were expressed as means ± SE. Statistical analysis was performed using one-way ANOVA. Differences were considered to be significant when the P value was <0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

ET-1 increased RhoA activity in the rabbit basilar artery. Membrane binding RhoA protein levels were measured in the basilar arteries treated with different concentrations of ET-1 and at different time periods (Fig. 1). The amount of the membrane binding RhoA fraction was markedly enhanced in the concentration-dependent manner. ET-1 at 10-100 nM significantly increased RhoA activity (P < 0.01). When the rings were treated with 10 nM ET-1 for different time periods, RhoA activity was increased but not in a time-dependent fashion.

View larger version (39K): [in this window] [in a new window]   Fig. 1.   Time- and concentration-dependent effect of endothelin (ET)-1 on the membrane RhoA activity. Left, representative immunoblot band is shown as well as a corresponding -actin band. Right, graph displaying protein band density (% of control). A: concentration-dependent effect of ET-1 at 1, 10, and 100 nM on the RhoA band density for 10 min. B: time-dependent effect of ET-1 (10 nM) at 5, 10, and 30 min on RhoA band density. Values are means ± SE. * and ** P < 0.05 and 0.01, respectively (vs. control, n = 4).

ET_A and ET_B receptor antagonist on ET-1-induced RhoA activation. Figure 2 shows that ET_A receptor antagonist BQ-610 (1 µM), but not ET_B antagonist BQ-788 (1 µM), completely abolished ET-1-induced RhoA activation (P < 0.01).

View larger version (36K): [in this window] [in a new window]   Fig. 2.   Effects of ET receptor antagonists on the RhoA activity induced by ET-1 (10 nM). A: representative immunoblot band is shown as well as a corresponding -actin band. B: graph displaying density of the protein (% of control). Arteries were preincubated with ETA receptor antagonist BQ-610 (1 µM) and ETB receptor antagonist BQ-788 (1 µM) for 30 min before they were treated with ET-1 (10 nM) for 10 min. Values are means ± SE. ** P < 0.01 vs. control, n = 6.

PKC, tyrosine kinase, and PI-3 kinase inhibitors on ET-1-induced RhoA activation. Figure 3 shows that the PI-3 kinase inhibitor LY-294002 (50 µM), but not the PKC and tyrosine kinase inhibitors staurosporine (30 nM) and genistein (100 µM), significantly inhibited the ET-1-induced RhoA activation (P < 0.05).

View larger version (26K): [in this window] [in a new window]   Fig. 3.   Effects of protein kinase C (PKC), tyrosine kinase, and phosphatidylinositol (PI)-3 kinase inhibitors on RhoA activity induced by ET-1 (10 nM). A: representative immunoblot band is shown as well as a corresponding -actin band. B: graph displaying density of the protein (% of control). Arteries were preincubated with PKC inhibitor staurosporine (Staus; 30 nM), tyrosine kinase inhibitor genistein (Gen; 100 µM), or PI-3 kinase inhibitor LY-294002 (LY; 50 µM) for 30 min and then were treated with ET-1 (10 nM) for 10 min. Values are means ± SE. ** P < 0.01 vs. control, n = 4.

Rho kinase inhibitor on ET-1-induced contraction and RhoA/Rho kinase activities. The effect of Y-27632 on RhoA, ROKI, and ROKII activities (on the membrane) was measured. Y-27632 (10 µM) significantly inhibited ROKI and ROKII (P < 0.05) but not RhoA activities (P > 0.05) (Fig. 4).

View larger version (23K): [in this window] [in a new window]   Fig. 4.   Effect of Rho kinase inhibitor on RhoA/Rho kinase activity. Left, representative immunoblot band for RhoA (A), ROKI (B), and ROKII (C) are shown as well as a corresponding -actin band. They are all stripped from the same membrane. Right, graph displaying corresponding density of the protein (% of control). Arteries were preincubated with Rho kinase inhibitor Y-27362 (10 µM) for 30 min and then were treated with ET-1 (10 nM) for 10 min. Values are means ± SE. * and ** P < 0.05 and 0.01, respectively (vs. control, n = 3).

ET-1 produced a concentration-dependent contraction in the rabbit basilar arteries. Preincubation of the rings with Rho kinase inhibitor Y-27632 (10-50 µM) significantly inhibited ET-1-induced contraction (P < 0.01), even though the effect of Y-27632 was more pronounced at lower ET-1 concentrations (1-10 nM) (Fig. 5).

View larger version (20K): [in this window] [in a new window]   Fig. 5.   Effect of Rho kinase inhibitor on ET-1-induced contraction. A: original trace of Rho kinase inhibitor Y-27632 on ET-1-induced concentration-dependent contraction. Solid curve is ET-1-induced contraction in the absence of Y-27632. Dotted curve is in the presence of 10 µM Y-27632 and medium dash curve is in the presence of 50 µM Y-27632. B: summary of the effect of Y-27632 on ET-1-induced contraction. Rings were incubated in Y-27632 at 10 µM or 50 µM for 30 min before application of ET-1. Values are means ± SE. * and ** P < 0.05 and 0.01, respectively (vs. control, n = 5).

Rho kinase inhibitor on ET-1-induced MLC phosphorylation. The extent of MLC phosphorylation at the resting level (without stimulation) was 25.3 ± 3.1% (n = 3). ET-1 (10 nM, for 10 min) increased the extent of MLC phosphorylation to 43.6 ± 1.2% (n = 3, P < 0.05). Y-27632 (10 µM) abolished the effect of ET-1 on MLC phosphorylation and reduced the level to 15.9 ± 3.6%. Similarly, Y-27632 (10 µM) reduced ET-1-induced contraction from 122.3 ± 7.82% to 78.1 ± 11.2% (Fig. 6).

View larger version (28K): [in this window] [in a new window]   Fig. 6.   Effect of Rho kinase inhibitor on myosin light chain (MLC) phosphorylation and force in ET-1-induced contraction. A: representative photograph of MLC immunoblot. B: summary of the MLC phosphorylation and force generation either at the resting level, induced by ET-1 (10 nM) alone for 10 min, or induced by ET-1 (10 nM) in the presence of Y-27632 (10 µM, 30 min preincubation) for 10 min. Values are means ± SE. * and ** P < 0.05 and 0.01, respectively (vs. control, n = 4).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The following observations were made: first, ET-1 produced RhoA activation in the rabbit basilar artery by activation of ET_A, not by ET_B receptors; second, the upstream effect on ET-1-induced RhoA activation might involve PI-3 kinase but not PKC or tyrosine kinase; and finally, the downstream effect of ET-1-induced RhoA activation involves Rho kinase and MLC phosphorylation.

ET-1 and RhoA/Rho kinase pathway in cerebral vasospasm. Current clinical and experimental investigations support the hypothesis that ET-1 is a major cause of cerebral vasospasm after SAH. ET-1 level was found to increase significantly in the cerebrospinal fluid and plasma of patients after SAH (33). ET-1 causes long-lasting vasoconstriction, similar to cerebral vasospasm following SAH, and the constrictor effect of ET-1 was enhanced after SAH (40). Endothelin receptor antagonists, endothelin-converting enzyme inhibitors, and ET-1 antisense oligonucleotide can prevent cerebral vasospasm after SAH in animal models (40).

ET-1-induced contraction might be mediated by an elevation of intracellular Ca²⁺ and by an increased Ca²⁺ sensitivity. RhoA/Rho kinase pathway plays a very important role in the Ca²⁺ sensitivity in cerebral arteries. Ca²⁺ activates MLC kinase to increase MLC phosphorylation. Activated RhoA appears to inhibit MLC phosphatase activity via Rho kinase and to increase the level of MLC phosphorylation (16, 17). There are some evidences that RhoA/Rho kinases are involved in cerebral vasospasm. In a rat double hemorrhage model, RhoA and Rho kinase mRNA levels were upregulated in the rat spastic basilar artery on day 7 (15). Fasudil hydrochlorides and hydroxyfasudil, nonspecific inhibitors for Rho kinase, reduced cerebral vasospasm after SAH in experimental animals and in humans (30, 35). Furthermore, the specific Rho kinase inhibitor Y-27632 reversed cerebral vasospasm in a dog double hemorrhage model (26). In the present study, we found that ET-1 activates RhoA, which leads to the activation of Rho kinase and MLC phosphorylation in the rabbit basilar artery.

RhoA/Rho kinase pathway in ET-1-induced contraction. The vasoactive function of ET-1 is mediated by three different receptor subtypes: ET_A, ET_B1, and ET_B2. The ET_A receptor subtype is located in vascular smooth muscle cells and mediates the vasocontrictive effect of endothelins (1, 24). The ET_B1 receptor subtype is present in vascular endothelial cells and mediates endothelium-derived relaxation (21). The ET_B2 receptor subtype is located in smooth muscle cells and causes vasoconstriction (3, 4, 29, 36). The relative subtype distribution varies depending on the vessel type. In cerebral arteries, the ET_A receptor subtype predominates, whereas ET_B is responsible for the remaining contraction (10, 41). Our results showed that ET-1-induced RhoA activation was blocked by a ET_A antagonist but not by a ET_B antagonist, indicating ET-1-induced RhoA activation was mediated by ET_A receptors, even though the possible contribution from a ET_B1 receptor cannot be excluded.

Translocation of RhoA from the cytosol to the membrane of the cells represents activation of RhoA. In the present study, we used Western blots to measure RhoA concentration on the membrane to represent RhoA activity. Our results (Fig. 1) showed that 1 nM concentration of ET-1 had a tendency to increase RhoA activity, which could not induce contraction (Fig. 5), whereas 10 and 100 nM ET-1 could significantly increase RhoA activity, which also could induce vascular contraction. Time response (5, 10, and 30 min) for ET-1-induced RhoA activation also was shown from our results. These suggested that ET-1 increased RhoA activity of the rabbit basilar artery.

PI-3 kinase has been reported to be involved in the ET-1-induced contraction because ET-1 increased PI-3 kinase activity (38) and the PI-3 kinase inhibitor inhibited ET-1-induced contraction in the rabbit basilar artery in one of our previous publications (41). Our results showed that PI-3 kinase inhibitor LY-294002 significantly inhibited ET-1-induced RhoA activation, suggesting that PI-3 kinase is upstream of RhoA in ET-1-induced basilar artery contraction, which is consistent with other reports (22). Tyrosine kinase inhibitor genistein failed to inhibit RhoA activation in the present study, which suggests that RhoA activation by ET-1 is independent of tyrosine kinase. This result is consistent with the observations of Wang and Bitar (38) and Sakurada et al. (23) that tyrosine kinase is not involved in ET-1-induced RhoA activation in the rabbit aorta and rectosigmoid smooth muscle. There are evidences that RhoA regulates Ca²⁺ sensitivity in smooth muscle via PKC-mitogen-activated protein kinase pathway or through a PKC-mediated effect on MLC phosphatase (8, 9). We tested this hypothesis in an ET-1-induced contraction and found that strauosporine, a PKC inhibitor, failed to inhibit RhoA activation, suggesting that RhoA might be upstream of PKC. This view was supported by an observation that Rho inhibitors block PKC translocation/activation in endothelial or epithelial cells, suggesting a RhoA requirement for PKC activation and translocation (7).

RhoA-induced vascular contraction is mediated by its downstream Rho kinase. We found that Y-27632 markedly inhibited ET-1-induced contraction in the rabbit basilar artery. Because the inhibitory effect of Y-27632 was mainly on the contraction induced by lower concentrations of ET-1, other mechanisms could be involved in ET-1-induced contraction. Vascular contraction results from MLC phosphorylation, which is the last step of the intracellular signal transduction pathway for smooth muscle contraction. Activation of Rho kinase inhibits MLC phosphatase, thus increasing the level of phosphorylated MLC. We measured both the MLC phophorylation and the force generated by ET-1 in the rabbit basilar artery. ET-1 increased MLC phosphorylation and force at the same time. Rho kinase inhibitor Y-27632 inhibited ET-1-induced MLC phosphoralation and correspondingly decreased the force.

The apparent involvement of the RhoA/Rho kinase pathway in mediating ET-1-induced contraction suggests this pathway could be an important target for further drug development. There are suggestions that the Rho kinase inhibitor might be useful in the treatment of hypertension (37), coronary artery spasm (12, 31), and cerebral vasospasm following SAH (26, 34), as well as neointimal proliferation in arteries subjected to injury by balloon angioplasty (27). The combination of the relaxant and antiproliferative effect of RhoA/Rho kinase inhibitors should confer significant therapeutic benefit.

	FOOTNOTES

Address for reprint requests and other correspondence: J. H. Zhang, Dept. of Neurosurgery, Louisiana State Univ. Health Science Center, 1501 Kings Highway, Shreveport, LA 71130-3932 (E-mail: johnzhang3910{at}yahoo.com).

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

May 9, 2002;10.1152/ajpheart.00141.2002

Received 21 February 2002; accepted in final form 6 May 2002.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

1.   Arai, H, Hori S, Aramori I, Ohkubo H, and Nakanishi S. Cloning and expression of a cDNA encoding an endothelin receptor. Nature 348: 730-732, 1990[Medline].

2.   Buus, CL, Aalkjaer C, Nilsson H, Juul B, Moller JV, and Mulvany MJ. Mechanisms of Ca²⁺ sensitization of force production by noradrenaline in rat mesenteric small arteries. J Physiol 510: 577-590, 1998[Abstract/Free Full Text].

3.   Clozel, M, Gray GA, Breu V, Loffler BM, and Osterwalder R. The endothelin ET_B receptor mediates both vasodilation and vasoconstriction in vivo. Biochem Biophys Res Commun 186: 867-873, 1992[Web of Science][Medline].

4.   Davenport, AP, O'Reilly G, Molenaar P, Maguire JJ, Kuc RE, Sharkey A, Bacon CR, and Ferro A. Human endothelin receptors characterized using reverse transcriptase-polymerase chain reaction, in situ hybridization, and subtype-selective ligands BQ123 and BQ3020: evidence for expression of ET_B receptors in human vascular smooth muscle. J Cardiovasc Pharmacol 22, Suppl8: S22-S25, 1993.

5.   Fu, X, Gong MC, Jia T, Somlyo AV, and Somlyo AP. The effects of the Rho-kinase inhibitor Y-27632 on arachidonic acid-, GTPgammaS-, and phorbol ester-induced Ca²⁺-sensitization of smooth muscle. FEBS Lett 440: 183-187, 1998[Web of Science][Medline].

6.   Gailly, P, Gong MC, Somlyo AV, and Somlyo AP. Possible role of atypical protein kinase C activated by arachidonic acid in Ca²⁺ sensitization of rabbit smooth muscle. J Physiol 500: 95-109, 1997[Abstract/Free Full Text].

7.   Hippenstiel, S, Kratz T, Krull M, Seybold J, Eichel-Streiber C, and Suttorp N. Rho protein inhibition blocks protein kinase C translocation and activation. Biochem Biophys Res Commun 245: 830-834, 1998[Web of Science][Medline].

8.   Hirata, K, Kikuchi A, Sasaki T, Kuroda S, Kaibuchi K, Matsuura Y, Seki H, Saida K, and Takai Y. Involvement of rho p21 in the GTP-enhanced calcium ion sensitivity of smooth muscle contraction. J Biol Chem 267: 8719-8722, 1992[Abstract/Free Full Text].

9.   Horowitz, A, Menice CB, Laporte R, and Morgan KG. Mechanisms of smooth muscle contraction. Physiol Rev 76: 967-1003, 1996[Abstract/Free Full Text].

10.   Iwasaki, T, Hayasaki-Kajiwara Y, Shimamura T, Naya N, and Nakajima M. Endothelin receptor subtype antagonist activity of S-0139 in various isolated rabbit and canine arteries. Eur J Pharmacol 400: 255-262, 2000[Web of Science][Medline].

11.   Jensen, PE. Calphostin C-sensitive enhancements of force by lysophosphatidylinositol and diacylglycerols in mesenteric arteries from the rat. Br J Pharmacol 119: 15-22, 1996[Web of Science][Medline].

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

13.   Kassell, NF, Sasaki T, Colohan AR, and Nazar G. Cerebral vasospasm following aneurysmal subarachnoid hemorrhage. Stroke 16: 562-572, 1985[Abstract/Free Full Text].

14.   Macdonald, RL, and Weir BK. A review of hemoglobin and the pathogenesis of cerebral vasospasm. Stroke 22: 971-982, 1991[Abstract/Free Full Text].

15.   Miyagi, Y, Carpenter RC, Meguro T, Parent AD, and Zhang JH. Upregulation of rho A and rho kinase messenger RNAs in the basilar artery of a rat model of subarachnoid hemorrhage. J Neurosurg 93: 471-476, 2000[Web of Science][Medline].

16.   Narumiya, S, Ishizaki T, and Watanabe N. Rho effectors and reorganization of actin cytoskeleton. FEBS Lett 410: 68-72, 1997[Web of Science][Medline].

17.   Noda, M, Yasuda-Fukazawa C, Moriishi K, Kato T, Okuda T, Kurokawa K, and Takuwa Y. Involvement of rho in GTP gamma S-induced enhancement of phosphorylation of 20 kDa myosin light chain in vascular smooth muscle cells: inhibition of phosphatase activity. FEBS Lett 367: 246-250, 1995[Web of Science][Medline].

18.   Ohanian, J, Ohanian V, Shaw L, Bruce C, and Heagerty AM. Involvement of tyrosine phosphorylation in endothelin-1-induced calcium- sensitization in rat small mesenteric arteries. Br J Pharmacol 120: 653-661, 1997[Web of Science][Medline].

19.   Persechini, A, Kamm KE, and Stull JT. Different phosphorylated forms of myosin in contracting tracheal smooth muscle. J Biol Chem 261: 6293-6299, 1986[Abstract/Free Full Text].

20.   Pfitzer, G, and Arner A. Involvement of small GTPases in the regulation of smooth muscle contraction. Acta Physiol Scand 164: 449-456, 1998[Web of Science][Medline].

21.   Randall, MD, Douglas SA, and Hiley CR. Vascular activities of endothelin-1 and some alanyl substituted analogues in resistance beds of the rat. Br J Pharmacol 98: 685-699, 1989[Web of Science][Medline].

22.   Ren, XD, and Schwartz MA. Regulation of inositol lipid kinases by Rho and Rac. Curr Opin Genet Dev 8: 63-67, 1998[Web of Science][Medline].

23.   Sakurada, S, Okamoto H, Takuwa N, Sugimoto N, and Takuwa Y. Rho activation in excitatory agonist-stimulated vascular smooth muscle. Am J Physiol Cell Physiol 281: C571-C578, 2001[Abstract/Free Full Text].

24.   Sakurai, T, Yanagisawa M, Takuwa Y, Miyazaki H, Kimura S, Goto K, and Masaki T. Cloning of a cDNA encoding a non-isopeptide-selective subtype of the endothelin receptor. Nature 348: 732-735, 1990[Medline].

25.   Sasaki, M, Hattori Y, Tomita F, Moriishi K, Kanno M, Kohya T, Oguma K, and Kitabatake A. Tyrosine phosphorylation as a convergent pathway of heterotrimeric G protein- and Rho protein-mediated Ca²⁺ sensitization of smooth muscle of rabbit mesenteric artery. Br J Pharmacol 125: 1651-1660, 1998[Web of Science][Medline].

26.   Sato, M, Tani E, Fujikawa H, and Kaibuchi K. Involvement of Rho-kinase-mediated phosphorylation of myosin light chain in enhancement of cerebral vasospasm. Circ Res 87: 195-200, 2000[Abstract/Free Full Text].

27.   Sawada, N, Itoh H, Ueyama K, Yamashita J, Doi K, Chun TH, Inoue M, Masatsugu K, Saito T, Fukunaga Y, Sakaguchi S, Arai H, Ohno N, Komeda M, and Nakao K. Inhibition of rho-associated kinase results in suppression of neointimal formation of balloon-injured arteries. Circulation 101: 2030-2033, 2000[Abstract/Free Full Text].

28.   Seasholtz, TM, Majumdar M, Kaplan DD, and Brown JH. Rho and Rho kinase mediate thrombin-stimulated vascular smooth muscle cell DNA synthesis and migration. Circ Res 84: 1186-1193, 1999[Abstract/Free Full Text].

29.   Shetty, SS, Okada T, Webb RL, DelGrande D, and Lappe RW. Functionally distinct endothelin B receptors in vascular endothelium and smooth muscle. Biochem Biophys Res Commun 191: 459-464, 1993[Web of Science][Medline].

30.   Shibuya, M, Suzuki Y, Sugita K, Saito I, Sasaki T, Takakura K, Nagata I, Kikuchi H, Takemae T, and Hidaka H. Effect of AT877 on cerebral vasospasm after aneurysmal subarachnoid hemorrhage. Results of a prospective placebo-controlled double-blind trial. J Neurosurg 76: 571-577, 1992[Web of Science][Medline].

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

32.   Sima, B, Weir BK, Macdonald RL, and Zhang H. Extracellular nucleotide-induced [Ca²⁺]_i elevation in rat basilar smooth muscle cells. Stroke 28: 2053-2058, 1997[Abstract/Free Full Text].

33.   Suzuki, H, Sato S, Suzuki Y, Takekoshi K, Ishihara N, and Shimoda S. Increased endothelin concentration in CSF from patients with subarachnoid hemorrhage. Acta Neurol Scand 81: 553-554, 1990[Web of Science][Medline].

34.   Tachibana, E, Harada T, Shibuya M, Saito K, Takayasu M, Suzuki Y, and Yoshida J. Intra-arterial infusion of fasudil hydrochloride for treating vasospasm following subarachnoid haemorrhage. Acta Neurochir (Wien) 141: 13-19, 1999[Medline].

35.   Takayasu, M, Suzuki Y, Shibuya M, Asano T, Kanamori M, Okada T, Kageyama N, and Hidaka H. The effects of HA compound calcium antagonists on delayed cerebral vasospasm in dogs. J Neurosurg 65: 80-85, 1986[Web of Science][Medline].

36.   Teerlink, JR, Breu V, Sprecher U, Clozel M, and Clozel JP. Potent vasoconstriction mediated by endothelin ET_B receptors in canine coronary arteries. Circ Res 74: 105-114, 1994[Abstract/Free Full Text].

37.   Uehata, M, Ishizaki T, Satoh H, Ono T, Kawahara T, Morishita T, Tamakawa H, Yamagami K, Inui J, Maekawa M, and Narumiya S. Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension. Nature 389: 990-994, 1997[Medline].

38.   Wang, P, and Bitar KN. Rho A regulates sustained smooth muscle contraction through cytoskeletal reorganization of HSP27. Am J Physiol Gastrointest Liver Physiol 275: G1454-G1462, 1998[Abstract/Free Full Text].

39.   Weir, B, Macdonald RL, and Stoodley M. Etiology of cerebral vasospasm. Acta Neurochir Suppl (Wien) 72: 27-46, 1999[Medline].

40.   Zimmermann, M, and Seifert V. Endothelin and subarachnoid hemorrhage: an overview. Neurosurgery 43: 863-875, 1998[Web of Science][Medline].

41.   Zubkov, AY, Rollins KS, Parent AD, Zhang J, and Bryan Jr RM Mechanism of endothelin-1-induced contraction in rabbit basilar artery. Stroke 31: 526-533, 2000[Abstract/Free Full Text].