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Anesth Analg 2008; 106:1765-1771 © 2008 International Anesthesia Research Society doi: 10.1213/ane.0b013e31816f1fba
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Abstract |
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 Top Abstract IntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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BACKGROUND: Airway smooth muscle contraction is not caused by the increase in intracellular Ca2+ ([Ca2+]i) alone because agonist stimulation increases tension at the same [Ca2+]i (increase in Ca2+ sensitivity). The small G protein RhoA and Rho-kinase (ROCK) play important roles in the regulation of Ca2+ sensitivity. In this study, we investigated the effects of three ROCK inhibitors (fasudil, Y-27632, and H-1152) on rat airway smooth muscle contraction and the effects of ROCK inhibitors on propofol-induced bronchodilatory effects.
METHODS: Ring strips from intrapulmonary bronchus of male Wistar rats were placed in 400-µL organ baths containing Krebs–Henseleit solution. After obtaining stable contraction with 30 µM acetylcholine, (1) propofol (1 µM–1 mM) was cumulatively applied; (2) cumulative doses of Y-27632 (0.01–300 µM), fasudil (0.01–100 µM), or H-1152 (0.01–100 µM) were applied; (3) propofol (1 µM–1 mM), with Y-27632, fasudil or H-1152 (0.03 µM or 0.1 µM), was cumulatively applied.
RESULTS: (1) Propofol produced concentration-dependent relaxation of rat bronchial smooth muscle. (2) All ROCK inhibitors produced concentration-dependent relaxation. (3) 0.03 µM Y-27632 and fasudil had no significant effect on the concentration–response curve for propofol, while 0.1 µM of both agents significantly shifted concentration–response curves to the left and decreased EC50. H-1152 (both 0.03 µM and 0.1 µM) significantly sifted the concentration–response curve for propofol to the left and decreased EC50.
CONCLUSIONS: ROCK inhibitors, especially H-1152, can attenuate the contraction of rat airway smooth muscle. The combined use of ROCK inhibitors and propofol causes greater relaxation.
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Introduction |
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TopAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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Contraction of smooth muscle in response to physiologic agonists has previously been thought to be induced solely by an increase in cytosolic Ca2+ ([Ca2+]i) and the strength of contraction is regulated by [Ca2+]i. However, simultaneous measurement of [Ca2+]i and tension revealed that the relationship between [Ca2+]i and tension is not one-on-one, and that agonist stimulation increases tension at the same [Ca2+]i.1 Agonists-induced increases in tension at a given [Ca2+]i were confirmed in permeabilized smooth muscle fiber samples,2,3 and has been recognized as an increase in Ca2+ sensitivity.4
It is generally accepted that smooth muscle contraction is regulated by phosphorylation of myosin light chain (MLC), which is determined by a balance between the activities of MLC kinase and MLC phosphatase5 (Fig. 1). An agonist-induced increase in Ca2+ sensitivity has been reported to be caused by a decrease in MLC phosphatase activity, without affecting MLC kinase activity.6,7
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The small G protein, RhoA, plays an important role in the signal transduction pathway from the cell membrane. RhoA is accompanied by several downstream target molecules, and is involved in the expression of various cellular functions, including smooth muscle contraction. Rho-kinase (Rho-associate coiled-coil forming kinase; ROCK) is a target molecule of RhoA, and the RhoA-ROCK pathway has been suggested to play important roles in the signal transduction pathway and in various cellular functions, including the regulation of Ca2+ sensitivity in smooth muscle contraction.8
ROCK phosphorylates MLC9 and myosin-binding subunit of MLC phosphatase, resulting in the inhibition of MLC phosphatase activity.10 And, ROCK also phosphorylates and activates CPI-17 (protein kinase C-activated 17-kDa inhibitor protein of type 1 phosphatase), which mediates the inhibition of MLC phosphatase activity,11 and all of them are involved in an increase in MLC phosphorylation.
Many volatile and IV anesthetics that are used clinically are able to relax airway smooth muscle.1,2,7,12–18 However, volatile anesthetics are still considered to be more beneficial for anesthetic management of patients with airway hypersensitivity, such as asthma and chronic obstructive pulmonary disease, because they exhibit stronger bronchodilatory effects at clinical doses compared with IV anesthetics.
Volatile anesthetics have been confirmed to not only decrease [Ca2+]i but also inhibit the agonist-induced increase in Ca2+ sensitivity in permeabilized canine tracheal smooth muscle.2,14,16 Halothane interrupts the acetylcholine (ACh)-induced inhibition of MLC phosphatase activity, whereas the MLC kinase activity is not affected.7 This action causes the increase in MLC phosphatase activity and results in relaxation.
In contrast, although in vitro and in vivo bronchodilation have been reported with IV anesthetics, this relaxation is caused solely by decreasing [Ca2+]i with the inhibition of Ca2+ influx through L-type voltage-operated Ca2+ channels.18 The IV anesthetics, propofol, ketamine, and midazolam, have been confirmed to have no effect on the ACh-induced increases in Ca2+ sensitivity in permeabilized canine tracheal smooth muscle.16 These findings suggest that strong bronchodilatory effects caused by volatile anesthetics are partly related to the inhibition of G-protein-mediated increases in Ca2+ sensitivity, and the inhibition of Ca2+ sensitization is integral in the potent bronchodilatory effect.
Y-27632 was developed as a selective ROCK inhibitor, and has been widely used to elucidate various cellular mechanisms.19–21 Y-27632 inhibits the RhoA-mediated increase in Ca2+ sensitivity of the airway smooth muscle contraction,3 and it suggests that Y-27632 may act as a potent bronchodilator. However, Y-27632 is not particularly selective, meaning a relatively high concentration is necessary to inhibit ROCK. Thus, a more selective and potent drug will be necessary for the clinical application of ROCK inhibitors.
Recently H-1152 ((S)-(+)-2-methyl-1-[(4-methyl-5-isoquinoline) sulfonyl]-homopiperazine) was newly developed. This new ROCK inhibitor has been reported to be more selective (with a Ki value of 1.6 nM for ROCK)22 and potent than previous ROCK inhibitors, such as Y-27632 and fasudil.22,23
However, there has been no report of the influence of this novel ROCK inhibitor on the contraction of airway smooth muscle.
And, interestingly, one report showed that Y-27632 in relatively low concentrations strongly augmented the relaxant effect of β2-adrenoceptor agonists salbutamol and terbutaline in bovine airway smooth muscle.24
Therefore, in this study, we investigated the effects of ROCK inhibitors, including H-1152, on rat airway smooth muscle contraction by muscarinic receptor stimulation by ACh.
We also explored the influence of these ROCK inhibitors on the inhibitory effect of propofol on airway contraction to elucidate whether the addition of a ROCK inhibitor affects anesthetic-induced bronchodilatory effects.
We performed this study to test the following hypothesis: Although both propofol and ROCK inhibitors induce relaxation of airway smooth muscle independently, propofol-induced relaxation would be enhanced by concomitant administration of a low concentration ROCK inhibitor.
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METHODS |
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TopAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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Tissue Preparation
Male Wistar rats (6 wk of age, 180–220 g, specific pathogen-free) were used. All experiments were approved by the Institutional Animal Care and Use Committee at Okayama University (Okayama, Japan). Animals were killed by exsanguination from the abdominal aorta under sodium pentobarbital anesthesia (50 mg/kg, i.p.). The third branch of the intrapulmonary bronchus was isolated by a method previously described.25 In brief, the tissue was carefully cleaned of lung parenchyma and adhering connective tissue, and then cut into ring strips (about 200 µm width, 500 µm diameter). The epithelium was removed by gently rubbing with keen-edged tweezers under a stereomicroscope. The resultant tissue ring preparation was suspended in a 400-µL organ bath at a resting tension of 50 mg. The isometric contraction of the circular smooth muscle was measured with a force-displacement transducer (T7–8-240, Orientec, Japan) and recorder (DC-3100, NEC-sanei, Japan). The organ bath contained modified Krebs–Henseleit solution with the following composition: NaCl 118.0 mM, KCl 4.8 mM, CaCl2 2.5 mM, MgSO4 1.2 mM, NaHCO3 25.0 mM, KH2PO4 1.2 mM, and glucose 11.0 (pH 7.4). The buffer solution was oxygenated with 95% O2–5% CO2 at room temperature. During an equilibration period in the organ bath, the tissues were washed four times at 15- to 20-min intervals and were equilibrated slowly to a baseline tension of 50 mg.
Experimental Protocols
Effects of Propofol on ACh-Induced Contraction in Rat Bronchial Smooth Muscle
This protocol was designed to confirm the inhibitory effect of propofol on airway smooth muscle contraction, previously reported in other species,15,16 in our experimental environment. After obtaining a stable contraction with 30 µM ACh, which produced approximately 50% of the maximal force induced by 1 mM ACh in preliminary studies (data not shown), propofol (1 µM–1 mM) was cumulatively applied, and concentration–response curves were generated from each experiment. The effect of 10% intralipid, similar to the vehicle for propofol, on the contraction with ACh also was evaluated using the equivalent volume.
Effects of ROCK Inhibitors on Ach-Induced Contraction in Rat Bronchial Smooth Muscle
A second protocol was designed to determine the effect of ROCK inhibitors on rat bronchial smooth muscle contraction and what concentrations of drugs should be used in subsequent studies. After obtaining a stable contraction with 30 µM ACh, cumulative doses of Y-27632 (0.01–300 µM), fasudil (0.01–100 µM), or H-1152 (0.01–100 µM), were applied, and concentration–response curves for these agents were generated from each experiment.
Effects of ROCK Inhibitors on Propofol-Induced Relaxation in Rat Bronchial Smooth Muscle
The last protocol was designed to determine whether ROCK inhibitors in low concentrations affect propofol-induced inhibitory effects on rat bronchial smooth muscle contraction.
After obtaining a stable contraction with 30 µM ACh, propofol (1 µM–1 mM), with Y-27632 (0.03 µM or 0.1 µM), fasudil (0.03 µM or 0.1 µM), or H-1152 (0.03 µM or 0.1 µM) was cumulatively applied. Concentration–response curves for these agents were generated from each experiment.
Materials
Propofol was purchased from AstraZeneca Japan (Osaka, Japan). Propofol was diluted from an aqueous emulsion (5.6 x 10–2 M) in 10% (wt/vol) soybean oil, 2.25% glycerol, and 1.2% purified egg lecithin. All other drugs and chemicals were prepared in distilled filtered water. ACh and 10% intralipid were purchased from Sigma (St. Louis, MO). Fasudil, Y-27632, and H-1152 were purchased from Calbiochem (LA Jolla, CA).
Statistical Analysis
Forces were expressed as mean ± sd; n represents the number of rats. Relaxation produced by ROCK inhibitors or propofol was expressed as a percent change from the initial force produced by 30 µM ACh. Concentration–response curves obtained from each experiment were fitted by nonlinear regression with SigmaPlot software (for Windows Version 8.0, SPSS Inc., Chicago, IL, United States). The 50% effective concentrations (EC50) were calculated with Sigmaplot software for each experiment.
Statistical significance of difference between groups was determined by two-way analysis of variance (ANOVA), followed by Bonferroni multiple comparison test with SigmaStat software (for Windows Version 3.0, SPSS Inc.) and a value of P < 0.05 was considered significant.
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RESULTS |
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TopAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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Effects of Propofol on ACh-Induced Contraction in Rat Bronchial Smooth Muscle
Propofol produced concentration-dependent relaxation of rat bronchial smooth muscle precontracted with 30 µM ACh, whereas its vehicle intralipid did not affect contraction (Fig. 2). EC50 value for propofol was 158.2 ± 39.2 µM (n = 5).
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Effects of ROCK Inhibitors on ACh-Induced Contraction in Rat Bronchial Smooth Muscle
All ROCK inhibitors Y-27632, fasudil, and H-1152 produced concentration-dependent relaxation of rat bronchial smooth muscle precontracted with 30 µM ACh (Fig. 3). Significant differences in concentration–response curves, determined by two-way ANOVA, followed by Bonferroni multiple comparison test, were found (P < 0.001, <0.001, and <0.001 for Y-27632 vs fasudil, Y-27632 vs H-1152, and fasudil vs H-1152, respectively).
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EC50 values calculated from each experiment were 27.8 ± 23.1 µM, 7.4 ± 2.1 µM, and 0.9 ± 0.9 µM for Y-27632, fasudil, and H-1152, respectively (n = 5).
Effects of ROCK Inhibitors on Propofol-Induced Relaxation in Rat Bronchial Smooth Muscle
Both 0.03 µM Y-27632 and 0.03 µM fasudil had no significant effect on the concentration–response curve for propofol (P = 1.000 and 0.367 for Y-27632 vs propofol alone and fasudil vs propofol alone, respectively) (Figs. 4A and B). EC50 was 143.0 ± 50.2 µM and 115.7 ± 39.0 µM for Y-27632 (0.03 µM) and fasudil (0.03 µM), respectively (n = 5).
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In contrast, 0.1 µM of both agents significantly shifted the concentration–response curves to the left (P = 0.040 and 0.023 for Y-27632 vs propofol alone and fasudil vs propofol alone, respectively). The EC50 was 107.9 ± 42.3 µM and 101.3 ± 34.9 µM for Y-27632 and fasudil, respectively (n = 5).
H-1152 in both concentrations (0.03 µM and 0.1 µM) significantly shifted the concentration–response curve for propofol to the left (P < 0.001 and <0.001 for 0.03 µM and 0.1 µM, respectively). The EC50 was 92.0 ± 21.7 µM and 77.6 ± 10.2 µM for 0.03 µM H-1152 and 0.1 µM H-1152, respectively (n = 5) (Fig. 4C).
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DISCUSSION |
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TopAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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The major findings of this study were that (1) propofol has an inhibitory effect on rat bronchial smooth muscle contraction in a concentration-dependent manner, (2) ROCK inhibitors Y-27632, fasudil, and H-1152 caused concentration-dependent relaxation of ACh-precontracted rat bronchial smooth muscle, (3) consistent with previous reports,18,19 H-1152 was more potent than the other ROCK inhibitors, and (4) ROCK inhibitors in relatively low concentrations augmented the propofol-induced relaxation of rat bronchial smooth muscle contracted by ACh.
Effects of Propofol on ACh-Induced Contraction in Rat Bronchial Smooth Muscle
First, confirming previous reports in many species,15,16 propofol showed an inhibitory effect on rat bronchial smooth muscle contraction in a concentration-dependent manner (Fig. 2).
Since we did not perform simultaneous measurements of [Ca2+]i and tension, the results of the current study are not enough to elucidate the point where propofol acts as an inhibitor in the smooth muscle contraction mechanism (shown in Fig. 1).
However, although it was performed in different animal species, the first author previously showed that propofol did not affect Ca2+ sensitivity in a skinned canine tracheal smooth muscle preparation, which maintains [Ca2+]i at a constant level.16 And, another report has also demonstrated that propofol relaxes porcine airway smooth muscle by reducing [Ca2+]i with the inhibition of Ca2+ influx through L-type voltage-operated Ca2+ channels.15
We presume that propofol reduces [Ca2+]i, but does not affect Ca2+ sensitivity, in rat bronchial smooth muscle, similar to that in other species.
Effects of ROCK Inhibitors on ACh-Induced Contraction in Rat Bronchial Smooth Muscle
Second, we investigated the effects of three ROCK inhibitors on the bronchial smooth muscle contraction of rats. Confirming the result of a previous report,3 Y-27632 showed an inhibitory effect on rat bronchial smooth muscle contraction. And, we demonstrate for the first time that the novel ROCK inhibitor H-1152 has inhibitory effects on airway smooth muscle contraction, and also showed that, as previously reported in other tissue,22,23 H-1152 is also more potent than Y-27632 and fasudil in the inhibitory effects on airway smooth muscle contraction (Fig. 3).
Without the simultaneous measuring of [Ca2+]i with the force, these results are not enough to reveal the complete mechanism of these ROCK inhibitors-induced relaxation. However, a report by Chiba et al., showing that Y-27632 inhibits Ca2+ sensitivity in skinned rat bronchial smooth muscle with an experimental system entirely same as this study,3 strongly supports our presumption that these ROCK inhibitors commonly inhibit the Ca2+ sensitizing mechanism (Fig. 1) by inhibiting RhoA. Further studies will be needed to completely elucidate the mechanism of this result.
Effects of ROCK Inhibitors on Propofol-Induced Relaxation in Rat Bronchial Smooth Muscle
In the third protocol, a ROCK inhibitor at a very low concentration augmented the propofol-induced relaxation of airway smooth muscle (Figs. 4A–C).
Since ROCK inhibitors per se exhibit a relaxant effect on the contraction of airway smooth muscle (Fig. 3), the attenuation in the force elicited by these ROCK inhibitors themselves should be considered carefully in accounting for this "augmentation." Therefore, we used two relatively low concentrations (0.03 and 0.1 µM), approximately EC5-EC10 in all ROCK inhibitors investigated (Fig. 3), in this protocol. In the current results, for example, despite 0.03 µM H-1152 alone causing only 5% relaxation of ACh-induced contraction (Fig. 3), the combination of 0.03 µM H-1152 and 100 µM (10–4 M) propofol produced more than 50% relaxation (Fig. 4C), which exceeded the simple sum of relaxation by both 0.03 µM H-1152 (approximately 5%) (Fig. 3) and 100 µM propofol (approximately 30%) (Fig. 2).
Therefore, we think that this result was not just the additive effects of ROCK inhibitors and propofol, but a certain type of synergistic effect was caused in this experimental condition.
Further studies also need to elucidate the mechanism of this augmentation.
H-1152 at both 0.03 µM and 0.1 µM significantly shifted the concentration-response curve for propofol (i.e., the augmentation of propofol-induced relaxation), whereas 0.03 µM Y-27632 and fasudil had no significant effects (Figs. 4A–C). This result also suggests the high potency of H-1152 compared with Y-27632 and fasudil, and shows the same tendency as the result of the relaxant effects of a ROCK inhibitor alone (Fig. 3).
ROCK inhibitors are expected to be a new strategy for the treatment of various diseases associated with abnormal contraction of smooth muscle, such as hypertension, stroke, and angina pectoris.
In Japan and Korea, fasudil has been sued clinically for cerebral vasospasm after surgery for subarachnoid hemorrhage and associated cerebral ischemic symptoms for several years, and is a popular drug. Its use for various symptoms associated with abnormal contraction of smooth muscle, such as angina pectoris, is currently being investigated. This means that, in the near future, we have the possibility of managing the anesthesia of the patients treated with a ROCK inhibitor preoperatively, and the results of the current study may suggest that the interaction between ROCK inhibitors and anesthetics should be considered in the anesthetic management of these patients.
In conclusion, selective ROCK inhibitors, especially H-1152, can attenuate the ACh-induced contractile responses of rat bronchial smooth muscle.
The combined use of ROCK inhibitors and propofol causes greater relaxation in rat bronchial smooth muscle contracted by ACh.
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Footnotes |
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Accepted for publication January 31, 2008.
Presented, in part, at the International Anesthesia Research Society 80th Clinical and Scientific Congress held on March 24–28, 2006 in San Francisco, CA, United States, and 81st Clinical and Scientific Congress held on March 23–27, 2007 in Orlando, FL, United States.
The contents in this manuscript have not been published elsewhere and the paper is not being submitted elsewhere. And, this manuscript has been read and approved by all co-authors.
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REFERENCES |
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TopAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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- Kai T, Takahashi S, Kanaide H. Halothane counteracts acetylcholine-induced increase in Ca2+ sensitivity of the contractile apparatus in airway smooth muscle. Eur J Pharmacol 1996;315:313–18[Web of Science][Medline]
- Akao M, Hirasaki A, Jones KA, Wong GY, Bremerich DH, Warner DO. Halothane reduces myofilament Ca2+ sensitivity during muscarinic receptor stimulation of airway smooth muscle. Am J Physiol 1996;271:L719–L725[Web of Science][Medline]
- Chiba Y, Takeyama H, Sakai H, Misawa M. Effects of Y-27632 on acetylcholine-induced contraction of intact and permeabilized intrapulmonary bronchial smooth muscles in rats. Eur J Pharmacol 2001;427:77–82[Web of Science][Medline]
- Somlyo AP, Somlyo AV. Signal transduction and regulation in smooth muscle. Nature 1994;372:231–6[Medline]
- Hartshorne DJ. Biochemistry of the contractile process in smooth muscle. In: Johnson LR, ed. Physiology of the gastrointestinal tract. 2nd ed. New York: Raven press, 1987:423–82
- Kitazawa T, Masuo M, Somlyo AP, Gerthoffer M. G protein-mediated inhibition of myosin light-chain phosphatase in vascular smooth muscle. Proc Natl Acad Sci USA 1991;88:9307–10
[Abstract/Free Full Text] - Hanazaki M, Jones KA, Perkins WJ, Warner DO. Halothane increases smooth muscle protein phosphatase in airway smooth muscle. Anesthesiology 2001;94:129–36[Web of Science][Medline]
- 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;22:32–9[Medline]
- Amano M, Ito M, Kimura K, Fukata Y, Chihara K, Nakano T, Matsuura Y, Kaibuchi K. Phosphorylation and activation of myosin by Rho-associated kinase (Rho-kinase). J Biol Chem 1996;271:20246–9
[Abstract/Free Full Text] - Kimura K, Ito M, Amano M, Chihara K, Fukata Y, Nakafuku M, Yamamori B, Feng J, Nakano T, Okawa K, Iwamatsu A, Kaibuchi K. Regulation of myosin phosphatase by Rho and Rho-associated kinase (Rho-kinase). Science 1996;273:245–8[Abstract]
- 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[Web of Science][Medline]
- Jones KA, Housmans PR, Warner DO, Lorenz RR, Rehder K. Halothane alters cytosolic calcium transient in tracheal smooth muscle. Am J Physiol 1993;265:L80–L86[Web of Science][Medline]
- Yamakage M, Kohro S, Kawamata T, Namiki A. Inhibitory effects of four inhaled anesthetics on canine tracheal smooth muscle contraction and intracellular Ca2+ concentration. Anesth Analg 1993;77:67–72
[Abstract/Free Full Text] - Kai T, Bremerich DH, Jones KA, Warner DO. Drug-specific effects of volatile anesthetics on Ca2+ sensitization in airway smooth muscle. Anesth Analg 1998;87:425–9
[Abstract/Free Full Text] - Yamakage M, Hirshman CA, Croxton TL. Inhibitory effects of thiopental, ketamine, and propofol on voltage-dependent Ca2+ channels in porcine tracheal smooth muscle cells. Anesthesiology 1995;83:1274–82[Web of Science][Medline]
- Hanazaki M, Jones KA, Warner DO. Effects of intravenous anesthetics on Ca2+ sensitivity in canine tracheal smooth muscle. Anesthesiology 2000;92:133–9[Web of Science][Medline]
- Pabelick CM, Rehder K, Jones KA, Shumway R, Lindahl SG, Warner DO. Stereospecific effects of ketamine enantiomers on canine tracheal smooth muscle. Br J Pharmacol 1997;121:1378–82[Web of Science][Medline]
- Yamakage M, Matsuzaki T, Tsujiguchi N, Honma Y, Namiki A. Inhibitory effects of diazepam and midazolam on Ca2+ and K+ channels in canine tracheal smooth muscle cells. Anesthesiology 1999;90:197–207[Web of Science][Medline]
- Uehata M, Ishizaki T, Satoh H, Ono T, Kawahara T, Morishita T, Tamakawa H, Yamagami K, Inui J, Maekawa M, Narumiya S. Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension. Nature 1997;389:990–4[Medline]
- Hirose M, Ishizaki T, Watanabe N, Uehata M, Kranenburg O, Moolenaar WH, Matsumura F, Maekawa M, Bito H, Narumiya S. Molecular dissection of the Rho-associated protein kinase (p160ROCK)-regulated neurite remodeling in neuroblastoma N1E-115 cells. J Cell Biol 1998;141:1625–36
[Abstract/Free Full Text] - Uchida S, Watanabe G, Shimada Y, Maeda M, Kawabe A, Mori A, Arii S, Uehata M, Kishimoto T, Oikawa T, Imamura M. The suppression of small GTPase rho signal transduction pathway inhibits angiogenesis in vitro and in vivo. Biochem Biophys Res Commun 2000;269:633–40[Web of Science][Medline]
- Sasaki Y, Suzuki M, Hidaka H. The novel and specific Rho-kinase inhibitor (S)-(+)-2-methyl-1-[(4-methyl-5-isoquinoline)sulfonyl]-homopiperazine as a probing molecule for Rho-kinase-involved pathway. Pharmacol Ther 2002;93:225–32[Web of Science][Medline]
- Ikenoya M, Hidaka H, Hosoya T, Suzuki M, Yamamoto N, Sasaki Y. Inhibition of rho-kinase-induced myristoylated alanine-rich C kinase substrate (MARCKS) phosphorylation in human neuronal cells by H-1152, a novel and specific Rho-kinase inhibitor. J Neurochem 2002;81:9–16[Web of Science][Medline]
- Nakahara T, Moriuchi H, Yunoki M, Sakamato K, Ishii K. Y-27632 potentiates relaxant effects of beta 2-adrenoceptor agonists in bovine tracheal smooth muscle. Eur J Pharmacol 2000;389:103–6[Web of Science][Medline]
- Chiba Y, Takada Y, Miyamoto S, MitsuiSaito M, Karaki H, Misawa M. Augmented acetylcholine-induced, Rho-mediated Ca2+ sensitization of bronchial smooth muscle contraction in antigen-induced airway hyperresponsive rats. Br J Pharmacol 1999;127:597–600[Web of Science][Medline]
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