The Rho/Rho-kinase signaling pathway plays an important role in mediating Ca2+ sensitization of vascular smooth muscle. The effect of anesthetics on Rho/Rho-kinase-mediated vasoconstriction has not been determined to date. This study is designed to examine the possible inhibitory effects of sevoflurane on the Rho/Rho-kinase pathway by measuring guanosine 5'-[gamma-thio]triphosphate (GTP gamma S)-stimulated contraction and translocation of RhoA (one of the three Rho subtypes) and Rock-2 (one of the two Rho-kinase subtypes) from the cytosol to the membrane in rat aortic smooth muscle.
GTP gamma S-induced contraction of rat aortic endothelium-denuded rings was measured using an isometric force transducer, and GTP gamma S-stimulated membrane translocation of RhoA and Rock-2 in smooth muscle cells was detected with Western blotting in the presence and absence of sevoflurane.
GTP gamma S (10(-4) m) induced a sustained contraction, which was significantly inhibited by the Rho-kinase inhibitor, Y27632 (3 x 10(-6) m). Before treatment with GTP gamma S, RhoA and Rock-2 were detected primarily in the cytosolic fraction. GTP gamma S (10(-4) m) stimulated the translocation of RhoA and Rock-2 from the cytosol to the membrane, which was sustained for more than 60 min. Sevoflurane (1.7, 3.4, and 5.1%) concentration dependently inhibited the GTP gamma S-induced constriction of rat aortic smooth muscle with a reduction of constriction of 52-75% (P < 0.01, n = 8), and attenuated the translocation of RhoA and Rock-2 by 31-66% and 34-78%, respectively (P < 0.05-0.01, respectively; n = 4).
The current findings show that sevoflurane depresses the GTP gamma S-stimulated contraction and translocation of both Rho and Rho-kinase from the cytosol in a concentration-dependent manner, indicating that sevoflurane is able to inhibit vasoconstriction mediated by the Rho/Rho-kinase pathway in rat aortic smooth muscle.
THE Ca2+-mediated phosphorylation of myosin light chain and the protein kinase C (PKC)–mediated Ca2+sensitization of contractile proteins are considered to be the major mechanisms for eliciting and regulating contraction of vascular smooth muscle (for review, see Horowitz et al. 1). Accumulating evidence suggests that volatile anesthetics alter the vasoconstriction evoked by various excitatory agonists through changes in the intracellular Ca2+concentration ([Ca2+]i) and/or by modulating the activation of PKC. For example, halothane increases tension by inducing the release of Ca2+from intracellular stores 2and by enhancing activation of PKC 3in endothelium-denuded rabbit pulmonary arterial strips. Isoflurane increases the Ca2+-stimulated contraction by activating the Ca2+-independent PKC in denuded rabbit femoral 4and pulmonary 5arterial strips. The halothane- and isoflurane-induced relaxation in pulmonary 3,5and femoral arteries 4involves the activation of Ca2+-dependent PKC and a Ca2+/calmodulin-dependent protein kinase II. Sevoflurane inhibits KCl- and norepinephrine-induced contraction likely by both reducing [Ca2+]iand decreasing Ca2+sensitivity of the myofilaments in the mesenteric resistance artery. 6Recent advances in molecular biology have found that an additional signaling pathway, including the downstream effectors of Rho, Rho-kinase, and myosin light chain phosphatase, also plays an important role in mediating vascular smooth muscle contraction. Rho, a small monomeric G-protein, modulates the Ca2+sensitization of vascular smooth muscle by activating Rho-kinase, resulting in inhibition of myosin light chain phosphatase activity (for reviews, see Kawano et al. , 7Somlyo et al. , 8and Fukata et al. 9). After activation, both Rho and Rho-kinase are translocated from the cytosol to the membrane in vascular smooth muscle cells; thus, the increased presence of Rho and Rho-kinase in the membrane fraction can be used to evaluate their role in vasoconstriction. 10–13Guanosine 5′-[(-thio]triphosphate (GTPγS), the nonhydrolysable guanosine 5′-triphosphate (GTP) analog, is able to directly bind to and activate monomeric G-proteins and has been used to activate the Rho/Rho-kinase pathway. 10,11,14,15GTPγS at a saturating concentration (10−4m) increases the Ca2+sensitivity of the contractile system by about threefold, regardless of muscle type. 16–18Whether anesthetics affect the Rho/Rho-kinase–mediated vascular contraction has not been determined to date. This study is designed to examine the possible inhibitory effects of sevoflurane, a new volatile anesthetic, on the Rho/Rho-kinase signaling pathway by measuring both GTPγS-stimulated contraction and membrane translocation of RhoA (one of the three Rho subtypes) and Rock-2 (one of the two Rho-kinase subtypes) from the cytosol in isolated rat aortic smooth muscle.
Materials and Methods
Isometric Force Measurement
The protocol was approved by the Wakayama Medical University Animal Care and Use Committee. Male Wistar rats (250–350 g) were anesthetized with halothane and euthanized by exsanguination from the common carotid artery. The descending thoracic aorta was carefully dissected, and adherent fat and connecting tissue were removed. The prepared aorta was cut into rings 3–4 mm in length. The endothelium was removed by gentle rubbing of the internal surface with a stainless steel needle. Rings were mounted vertically between two hooks, with the upper hook connected to the lever of an isometric force transducer. Rings were bathed in 10-ml organ chambers containing Krebs bicarbonate solution (KBS, pH 7.35–7.45) with the following composition (mm): NaCl, 118.2; KCl, 4.6; CaCl2, 2.5; KH2PO4, 1.2; MgSO4, 1.2; NaHCO3, 24.8; and dextrose 10. KBS in the chamber was gassed with a mixture of 95% O2and 5% CO2and was maintained at a temperature of 36.5°–37.5°C. The rings were maintained at a resting tension of 3.0 g and were equilibrated for 60 min in the control KBS, which was replaced every 20 min. Sevoflurane was introduced into the gas mixture using an agent-specific vaporizer (Penlon; Sigma, Abingdon, Oxon, United Kingdom). The concentration of the resulting gas mixture was monitored and adjusted using an Atom 303 anesthetic agent monitor (Atom, Tokyo, Japan). The concentrations of sevoflurane in KBS were measured by gas chromatography (Shimazu Seisakasho, Kyoto, Japan) and were determined to be 0.17 ± 0.03, 0.28 ± 0.02, and 0.41 ± 0.03 mm at sevoflurane concentrations of 1.7, 3.4, and 5.1%, respectively.After equilibration, all aortic rings were exposed to KBS containing 3 × 10−2m KCl to assess their overall contractile responsiveness. Removal of the endothelium was confirmed in 3 × 10−7m phenylephrine-precontracted vessels by the lack of relaxation to 10−5m acetylcholine. GTPγS at a concentration of 10−4m was selected to stimulate rat aortic rings in this study. In some preparations, Y27632 (3 × 10−6m), a highly specific Rho-kinase inhibitor, was used either 20 min before GTPγS application or after attainment of a sustained GTPγS-induced contraction. To measure the effects of sevoflurane on GTPγS-elicited vasoconstriction, some preparations were exposed to 1.7, 3.4, or 5.1% sevoflurane for 15 min before treatment with GTPγS or after a sustained GTPγS-stimulated contraction was achieved. The inhibitory effect of sevoflurane on contraction was expressed as the percentage of completely recovered response after cessation of sevoflurane administration.
Measurement of RhoA and Rock-2 Translocation
Rat thoracic aortas were dissected as described previously and opened longitudinally. The endothelium was removed gently with a needle. The prepared aortas were bathed with oxygenated KBS and were equilibrated for 60 min before exposure to the agents. To determine the time course of the GTPγS-induced membrane translocation of RhoA and Rock-2, some aortas were exposed to GTPγS (10−4m) for 0, 1, 5, 20, or 60 min and were then rapidly frozen with dry ice. To measure the dose effect of sevoflurane on GTPγS-stimulated translocation of RhoA and Rock-2, some aortas were treated with 1.7, 3.4, or 5.1% sevoflurane for 15 min before exposure to GTPγS and were quickly frozen 20 min after treatment with GTPγS. Frozen aortas were cut into small pieces and were homogenized in ice-cold lysis buffer (10−3m Tris · HCl, pH 7.5, 5 × 10−3m MgCl2, 2 × 10−3m EDTA, and 10−1m NaCl), with 10−3m 4-(2-aminoethyl)benzonesulfonyl fluoride, 20 μg/ml leupeptin, and 20 μg/ml aprotinin. 10Homogenates were centrifuged at 13,000 g for 3 min at 4°C, and the supernatant was collected and centrifuged at 100,000 g for 30 min at 4°C. The supernatant (cytosolic fraction) was removed, and the pellet (membrane fraction) was resuspended using the same buffer. The protein concentrations of each fraction were determined using the bicinchoninic acid method.
Equal amounts of total protein were used for every sample in each experiment. Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and were transferred to a nitrocellulose membrane. The membrane was treated with anti-RhoA and anti-Rock-2 antibodies (1:2,000 and 1:500, respectively) for 4 h, followed by incubation with horseradish peroxidase–conjugated antibody (1:2,000) for 1.5 h. Immunoreactive bands were detected using chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ) and were assessed with image analysis software (NIH Image 1.62; National Institutes of Health, Bethesda, MD). The amount of RhoA and Rock-2 on the membrane was expressed as a percentage of the total value (i.e. , membrane fraction plus cytosolic fraction).
GTPγS and sevoflurane were purchased from Sigma-Aldrich Fine Chemicals (St. Louis, MO) and Maruishi Pharmaceutical Company Limited (Osaka, Japan), respectively. Y27632 was provided by Calbiochem-Novabiochem Corporation (San Diego, CA). Polyclonal antibodies against RhoA and Rock-2 and the secondary antibody labeled with horseradish peroxidase were supplied by Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Other reagents for the isometric tension experiments and Western blotting were all of analytic grade. The agents were dissolved in distilled water, and the concentrations of the drugs were expressed as the final molar concentration.
The data were presented as mean ± SD. The sample size (n) refers to the number of rats from which the aortas were harvested. Two-factorial ANOVA was used to compare the effects of the different sevoflurane concentrations using the software program StatView (SAS Institute, Inc., Cary, NC). P values <0.05 were considered to be statistically significant.
GTPγS-induced Contraction and Membrane Translocation of RhoA and Rock-2 from the Cytosol
GTPγS (10−4m) induced a sustained contraction of endothelium-denuded rat aortic rings, reaching the maximal response about 20 min after application (fig. 1A), which was maintained for more than 2 h. This response was significantly inhibited by Y27632 (3 × 10−6m) given before (fig. 1B) or after (fig. 1A) application of GTPγS.
Two immunoreactive bands with molecular weights of 24 and 150 kDa, which corresponded to RhoA and Rock-2, respectively, were detected on the nitrocellulose membrane. Before treatment with GTPγS, almost all the RhoA and Rock-2 were detected only in the cytosolic fraction. Membrane translocation of both RhoA (fig. 2A) and Rock-2 (fig. 2B) began immediately at 1 min, continued to increase at 5 min, increased to the maximal level at 20 min, and was sustained up to 60 min after treatment with 10−4m GTPγS. Translocation of RhoA seemed to be more complete than that of Rock-2.
Inhibitory Effect of Sevoflurane on GTPγS-stimulated Contraction and Membrane Translocation of RhoA and Rock-2
Sevoflurane concentration dependently depressed the GTPγS (10−4m)-induced contraction of endothelium-denuded rat aortic rings when administered before (fig. 3A) or after (fig. 3B) application of GTPγS; with reductions of 51.9 ± 7.5, 66.7 ± 7.8 and 75.3 ± 4.6% in response to 1.7, 3.4, and 5.1% sevoflurane, respectively (P < 0.01, n = 8;fig. 3C). The contractile response of the rings completely recovered within a few minutes after cessation of sevoflurane exposure.
The GTPγS-stimulated membrane translocation of RhoA was attenuated by sevoflurane in a dose-dependent fashion, with decreases of 31.0 ± 7.3 (P < 0.05), 48.7 ± 12.3 (P < 0.01), and 65.6 ± 10.5% (P < 0.01) in response to 1.7, 3.4, and 5.1% sevoflurane, respectively (n = 4;fig. 4). Similar to the decrease of RhoA, membrane translocation of Rock-2 was also inhibited in a dose-dependent manner, with decreases of 33.7 ± 12.7 (P < 0.05), 48.9 ± 12.4 (P < 0.01), and 78.6 ± 11.2% (P < 0.01) in response to 1.7, 3.4, and 5.1% sevoflurane, respectively (n = 4;fig. 5).
GTPγS-stimulated, Rho/Rho-kinase Pathway–mediated Contraction
Rho and Rho-kinase are broadly distributed in the cardiovascular system, including the myocardium, 19conductance vessels, 12resistance vasculature, 20,21and capacitance vessels, 10and they play an important role in hemodynamic regulation. Rat aortic smooth muscle was used to examine the role of Rho and Rho-kinase in GTPγS-stimulated contraction in our study. Y27632, a highly specific Rho-kinase inhibitor with Kivalues of 0.14, 26, and greater than 250 μm for Rho-kinase, PKC, and myosin light chain kinase, respectively, 22strongly inhibited the sustained GTPγS-induced contraction in our study, indicating that Rho-kinase is involved in the GTPγS-generated contraction. The detection of membrane translocation of RhoA and Rock-2 observed in our experiment further verifies that GTPγS is able to activate the Rho/Rho-kinase signal pathway and induces contraction of vascular smooth muscle, a finding consistent with the results from previous investigations. 10,11
Rho belongs to the Rho subfamily of the Ras superfamily of monomeric GTP-binding proteins. It is involved in the regulation of various cellular functions such as stress fiber and focal adhesion formation, cell morphology, cell aggregation, cadherin-mediated cell–cell adhesion, cell motility, cytokinesis, membrane ruffling, neurite retraction, microvilli formation, and smooth muscle contraction (for reviews, see Fukata et al. , 9Takai et al. , 23and Bishop and Hall 24). The importance of Rho-mediated Ca2+sensitization of vascular smooth muscle has been recognized in recent years. Like other GTP-binding proteins, Rho exhibits both guanine 5′-diphosphate (GDP)/GTP binding activity and GTPase activity and functions as a molecular switch, cycling between a GDP-bound inactive state (Rho · GDP) and a GTP-bound active state (Rho · GTP). In the unstimulated situation, the inactive cytosolic form of Rho is combined with guanine nucleotide dissociation inhibitor. After stimulation with certain agonists, the activated Gα12/13subunit of the heterotrimeric G-protein binds to and activates p115Rho guanine nucleotide exchange factor, one of the specific guanine nucleotide exchange factors for Rho. Subsequently, activated p115Rho guanine nucleotide exchange factor catalyzes the inactive cytosolic Rho · GDP · guanine nucleotide dissociation inhibitor complex and stimulates nucleotide exchange on Rho (GTP replaces GDP), followed by dissociation of Rho · GTP from the guanine nucleotide dissociation inhibitor and translocation of Rho · GTP to the plasma membrane. In the presence of activated Rho · GTP, Rho-kinase also translocates to the membrane from the cytosol and becomes activated. Therefore, the proportion of Rho and Rho-kinase in the membrane fraction can be used to measure their activity. 10–13Activated Rho-kinase subsequently phosphorylates the myosin-binding subunit of myosin light chain phosphatase and depresses its activity, 25thus attenuating myosin light chain dephosphorylation, increasing the level of phosphorylated myosin light chain, and, finally, enhancing contraction without increasing [Ca2+]i(i.e. , Rho/Rho-kinase enhances Ca2+sensitivity of myosin;fig. 6).
Inhibitory Effect of Sevoflurane on GTPγS-induced Rho/Rho-kinase–mediated Contraction of Vascular Smooth Muscle
The important finding of this investigation was that sevoflurane significantly inhibited GTPγS-stimulated contraction and membrane translocation of both Rho and Rho-kinase in rat aortic smooth muscle in a concentration-dependent manner. This indicates that sevoflurane is able to attenuate Rho/Rho-kinase–mediated Ca2+sensitization of vascular smooth muscle even at clinically relevant concentrations (1.7–3.4%) of sevoflurane.
Vascular smooth muscle contraction is mediated by multiple signal pathways, and anesthetics are most likely to affect vascular contraction through multiple mechanisms. It has been established that sevoflurane inhibits agonist-induced vasoconstriction by altering [Ca2+]iand/or changing PKC activity. For instance, sevoflurane decreases the norepinephrine-stimulated increase in [Ca2+]ilargely by inhibiting transmembrane Ca2+influx through voltage-gated Ca2+channels but not through increasing inositol 1,4,5-triphosphate–induced Ca2+release from the intracellular stores in isolated rat 6and rabbit 26mesenteric resistance arteries. In a previous investigation that we conducted, sevoflurane suppressed the angiotensin II–stimulated, PKC-mediated, but not Ca2+-regulated contraction of rat aortic smooth muscle in a dose-dependent manner (unpublished data, Hatano H, Professor and Chairman, Department of Anesthesiology, Wakayama Medical University, Wakayama City, Japan). In the present investigation, sevoflurane has been shown for the first time to depress GTPγS-stimulated Rho/Rho-kinase–mediated contraction of rat aortic smooth muscle. These results provide further evidence to support the complexity of mechanisms through which anesthetics act on the vasculature.
Sevoflurane, at subanesthetic doses, inhibits the exchange of GTPγS for GDP bound to the Gα subunits of heterotrimeric G-proteins and markedly enhances the dissociation of GTPγS. 27Sanuki et al. 28suggest that sevoflurane depresses the β-adrenergic signal transduction system by reducing ligand receptor binding and disrupting the relation between the receptor and G-proteins in the rat myocardial membrane. Therefore, heterotrimeric G-proteins may be one of the targets on which sevoflurane acts. Based on the present finding that sevoflurane significantly inhibits GTPγS-induced activation of Rho and Rho-kinase and on the fact that GTPγS is able to directly bind to monomeric G-proteins, it is suggested that monomeric G-proteins, including Rho, are probably also targets of sevoflurane, which remains be determined in future studies.
In summary, our findings that sevoflurane concentration dependently inhibited GTPγS-stimulated contraction and membrane translocation of both Rho and Rho-kinase suggest for the first time that sevoflurane depresses the Rho/Rho-kinase–mediated contraction of rat aortic smooth muscle.
The authors thank Shunji Itoh, M.D., Research Assistant, and Prof. Akira Ooshima, M.D., Ph.D., First Department of Pathology, Wakayama Medical University, Japan, for their guidance and help in assaying Rho and Rho-kinase translocation.