Tyrosine kinase-catalyzed protein tyrosine phosphorylation plays an important role in initiating and modulating vascular smooth muscle contraction. The aim of the current study was to examine the effects of isoflurane on sodium orthovanadate (Na3VO4), a potent protein tyrosine phosphatase inhibitor-induced, tyrosine phosphorylation-mediated contraction of rat aortic smooth muscle.
The Na3VO4-induced contraction of rat aortic smooth muscle and tyrosine phosphorylation of proteins including phospholipase Cgamma-1 (PLCgamma-1) and p44/p42 mitogen-activated protein kinase (MAPK) were assessed in the presence of different concentrations of isoflurane, using isometric force measurement and Western blotting methods, respectively.
Na3VO4 (10(-4) m) induced a gradually sustained contraction and significant increase in protein tyrosine phosphorylation of a set of substrates including PLCgamma-1 and p42MAPK, all of which were markedly inhibited by genistein (5 x 10(-5) m), a tyrosine kinase inhibitor. Isoflurane (1.2-3.5%) dose-dependently depressed the Na3VO4-induced contraction (P < 0.05-0.005; n = 8). Isoflurane also attenuated the total density of the Na3VO4-induced, tyrosine-phosphorylated substrate bands and the density of tyrosine-phosphorylated PLCgamma-1 band and p42MAPK band (P < 0.05-0.005; n = 4) in a concentration-dependent manner.
The findings of the current study, that isoflurane dose-dependently inhibits both the Na3VO4-stimulated contraction and tyrosine phosphorylation of a set of proteins including PLCgamma-1 and p42MAPK in rat aortic smooth muscle, suggest that isoflurane depresses protein tyrosine phosphorylation-modulated contraction of vascular smooth muscle, especially that mediated by the tyrosine-phosphorylated PLCgamma-1 and MAPK signaling pathways.
REGULATION of smooth muscle contraction involves complex and overlapping regulatory mechanisms. Smooth muscle contraction is primarily regulated by alterations in intracellular Ca2+concentration ([Ca2+]i). Increases in [Ca2+]i, after the release of Ca2+from the sarcoplasmic reticulum, extracellular Ca2+influx, or both, triggers rapid contraction of vascular smooth muscle through the Ca2+/calmodulin/myosin light chain pathway,1whereas a decrease in [Ca2+]icauses relaxation of smooth muscle. Meanwhile, contraction is maintained and potentiated by Ca2+sensitization of the contractile proteins through several signaling transduction pathways. For example, protein kinase C (PKC) enhances contraction by inhibiting myosin light chain phosphatase, thus reducing dephosphorylation of the myosin light chain.2p44/p42 mitogen-activated protein kinase (p44/p42MAPK), also referred to as extracellular signal–regulated kinase (ERK1/2), potentiates contraction by phosphorylating caldesmon and increasing the binding of actin and myosin.3
In recent decades, a large body of evidence has demonstrated that protein tyrosine phosphorylation by protein tyrosine kinases (PTKs) activates and triggers many intracellular signaling pathways,4including phospholipase Cγ-1 (PLCγ-1)–mediated diacylglycerol/inositol trisphosphate/Ca2+pathway5–8and p44/p42MAPK pathways,9–14resulting in a series of cell events including vascular smooth muscle contraction. Various agonists such as growth factors, angiotensin II, and norepinephrine induce smooth muscle contraction primarily by eliciting PTK-mediated protein tyrosine phosphorylation.5–19Sodium orthovanadate (Na3VO4), a potent protein tyrosine phosphatase inhibitor,20also induces smooth muscle contraction by reducing dephosphorylation of tyrosine-phosphorylated proteins and enhancing the net level of protein tyrosine phosphorylation. Na3VO4has been effectively used to study the protein tyrosine phosphorylation signaling pathway.21,22
Volatile anesthetics have been shown to alter vascular smooth muscle contraction by changing [Ca2+]ivia affection of Ca2+mobilization and/or Ca2+influx23–26and/or by influencing Ca2+sensitization of contractile proteins (such as PKC,27–29Ca2+-calmodulin–dependent protein kinase II,30MAPK,31Rho/Rho-kinase,32and others). Responsiveness of these cellular targets varies depending on the anesthetic agents, stimuli, species, and vascular types. Little information is available regarding whether the alteration in vascular tension by anesthetics is attributable to the direct interruption of these targets, their upstream effectors, or both. Because protein tyrosine phosphorylation plays an important role in initiating and mediating the smooth muscle contraction–associated signaling pathways including PLC pathway and MAPK pathway, it is hypothesized that protein tyrosine phosphorylation by PTKs is a likely target that is interrupted by anesthetics, resulting in alterations of the activity of its downstream effectors, such as PLCγ and p44/p42MAPK. The current study is designed to examine the effects of isoflurane on the protein tyrosine phosphorylation–mediated vascular smooth muscle contraction elicited with Na3VO4, using isometric force measurement and detection of protein tyrosine phosphorylation, especially tyrosine phosphorylation of PLCγ-1 and p44/p42MAPK.
Materials and Methods
Isometric Force Measurement
With the approval of the Animal Care and Use Committee of Wakayama Medical University (Wakayama City, Japan), male Wistar rats (weight, 250–350 g) were anesthetized with halothane and were exsanguinated by bleeding from the common carotid artery. Descending thoracic aortas were carefully removed and rapidly immersed in ice-cold Krebs bicarbonate solution (pH 7.35–7.45) of the following composition: 118.2 mm NaCl, 4.6 mm KCl, 2.5 mm CaCl2, 1.2 mm KH2PO4, 1.2 mm MgSO4, 24.8 mm NaHCO3, and 10.0 mm dextrose. The adherent connective tissue was cleaned, and rings (3–4 mm in length) were obtained. After removal of the endothelium by gently rubbing the intimal surface with a stainless steel needle, the rings were mounted between wire hooks in a 10-ml organ bath containing Krebs bicarbonate solution warmed at 37°C and aerated continuously with 95% O2–5% CO2. The lower wire hook was fixed within the organ bath, and the upper hook was fixed to an isometric force transducer (Nihondenki-sanei Co., Tokyo, Japan). Isometric force development was recorded by using a force recorder.
Isoflurane was introduced into the gas mixture using an agent-specific vaporizer (Penlon Limited, 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 isoflurane in Krebs bicarbonate solution were measured by gas chromatography (Shimazu Seisakasho, Kyoto, Japan) and were determined to be 0.19 ± 0.01 mm (0.9% or 0.6 minimum alveolar concentration [MAC]), 0.39 ± 0.01 mm (1.8% or 1.3 MAC), and 0.56 ± 0.03 mm (2.6% or 1.9 MAC) at isoflurane concentrations of 1.2% (1 MAC), 2.3% (2 MAC), and 3.5% (3 MAC) in the gas mixture.
Each aortic ring was allowed to equilibrate for 60 min (with the organ bath solution changed every 20 min) at a resting tension of 3 g. Then rings were exposed to Krebs bicarbonate solution containing KCl (3 × 10−2m) to assess their overall contractile responsiveness. Rings that did not develop at least 2 g contractile active force were discarded. Removal of the endothelium was confirmed by the lack of relaxation to acetylcholine (10−5m) in rings precontracted with phenylephrine (3 × 10−7m).
In a first series of experiments, the time course of the Na3VO4(10−4m)–induced contraction was measured. Genistein (5 × 10−5m), a tyrosine kinase inhibitor, was applied either 15 min before Na3VO4treatment or after attainment of a sustained Na3VO4-induced contraction to assess the Na3VO4-induced, tyrosine phosphorylation–mediated contraction. To examine the effects of isoflurane on the Na3VO4-induced contraction, rings were exposed to 1.2, 2.3, or 3.5% isoflurane for 15 min before treatment with Na3VO4or after sustained Na3VO4-stimulated contraction had been achieved. The inhibitory effect of isoflurane on contraction was expressed as a percentage of the control.
Detection of Protein Kinase Phosphorylation
Rat descending thoracic aortas (approximately 3.5 cm in length) were excised. The vessels were carefully freed of all extraneous structures and opened longitudinally, and the endothelium was removed gently with a stainless needle. The prepared aortas were bathed in organ chambers with oxygenated Krebs bicarbonate solution and were equilibrated for 60 min before exposure to the agents. One aorta was used for each sample.
The time course of the Na3VO4-induced protein tyrosine phosphorylation was determined in the first experiment. Aortas were incubated in 10−4m Na3VO4for 0, 2, 5, 10, 20, or 30 min and were then rapidly frozen with dry ice. The detection results showed that tyrosine phosphorylation of the substrates (including PLCγ-1 and p42MAPK) reached a peak level 20 min after Na3VO4application. In the following testing experiments, aortas were pretreated with genistein (5 × 10−5m), 0, 1.2, 2.3, or 3.5% isoflurane for 15 min and were quickly frozen 20 min after Na3VO4(10−4m) application.
Frozen aortas were cut into small pieces and were homogenized in ice-cold lysis buffer (50 mm HEPES, pΗ 7.5, 1% Triton X-100, 50 mm NaCl, 50 mm sodium fluoride, 5 mm EDTA, 10 mm sodium pyrophosphate, 1 mm phenylmethanesulfonyl fluoride, 1 mm Na3VO4, 10 μg/ml leupeptin, and 20 μg/ml aprotinin).15Homogenates were centrifuged at 15,000g for 15 min at 4°C. The supernatant was collected, and protein concentration was determined using bicinchoninic acid method.33Solutions of the protein extracts were prepared by mixing them with an equal volume of 2× sample buffer (125 mm Tris, pH 6.8, 10% glycerol, 2% β-mercaptoethanol, and 0.08% bromophenol blue). The diluted samples then were heated in boiling water for 3 min and were stored at −80°C.
In each experiment, samples were used at equivalent total protein content. Proteins were separated by 7.5% sodium-dodecyl-sulfate polyacrylamide gel electrophoresis and electroblotted onto nitrocellulose membranes. The electroblotted membranes were incubated in blocking buffer (containing 20 mm Tris, pH 7.5, 150 mm NaCl, 3% bovine serum albumin, and 0.02% sodium azide) overnight at 4°C, and were then incubated with a phospho-tyrosine monoclonal antibody (p-Tyr-100, 1:2,000), phospho-PLCγ-1 (Tyr783, 1:1,000), or phospho-ERK (p44/p42MAPK) antibody (Tyr204, 1:1,000) for 4 h, followed by incubation with horseradish peroxidase–conjugated antibody (1:2,000) for 1.5 h. The densities of the immunoreactive bands were detected using chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ) and were assessed with image analysis software (NIH Image 1.62, Bethesda, MD). The band densities of tyrosine kinase–phosphorylated substrates, PLCγ-1, and MAPK were expressed relative to the basal control level (referred to as 1).
Na3VO4and genistein were purchased from Sigma-Aldrich Fine Chemicals (St. Louis, MO). Isoflurane was obtained from Dainabot Company Limited (Osaka, Japan). Phospho-tyrosine monoclonal antibody (p-Tyr-100) and phospho-PLCγ-1 antibody (Tyr783) were bought from Cell Signaling Technology Inc. (Beverly, MA). Phospho-ERK (Tyr204) antibody and the secondary antibody labeled with horseradish peroxidase were supplied by Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Other reagents for the tension measurement and Western blotting were all of analytical grade.
The results are presented as mean ± SD. The sample size (n values) represents the number of aortic rings (for tension measurement) or of aortas (for Western blotting), which equals to the number of rats used. Two-factorial analysis of variance was used to compare the effects of the different isoflurane concentrations on the Na3VO4-induced contraction, the band densities of tyrosine-phosphorylated substrates, PLCγ-1, and p44/p42MAPK, using the software program StatView (SAS Institute Inc., Cary, NC). P values less than 0.05 were considered statistically significant.
Na3VO4-induced Contraction and Protein Tyrosine Phosphorylation
Sodium orthovanadate (10−4m) induced a sustained contraction of rat aortic endothelium-denuded rings, reaching a maximal level comparable to that of the 3 × 10−2m KCl–induced contraction, 12.8 ± 3.2 min after its application, and maintained a stable contraction for at least 2 h (fig. 1A). Genistein (5 × 10−5m) reversibly and significantly depressed the Na3VO4-induced contraction when applied 15 min before (fig. 1B) or after (fig. 1C) treatment with Na3VO4.
A set of tyrosine-phosphorylated protein bands between 42 and 190 kd molecular weight, especially bands of 42, 45, 71, 88, 116, and 155 kd, were detected after stimulation with Na3VO4. The band density reached peak level 20 min after Na3VO4application and was significantly reduced by genistein (5 × 10−5m) (fig. 2A), which was consistent with the time course of the Na3VO4-induced vascular contraction.
The bands of molecular weight of 155 kd and 44/42 kd were identified as PLCγ-1 (fig. 2B) and p44/p42MAPK (fig. 2C) by phospho-PLCγ-1 antibody (Tyr783) and phospho-ERK (Tyr204) antibody, respectively. The densities of the phospho-PLCγ-1 and phospho-p42MAPK bands reached peak level 20 min after treatment with Na3VO4and were markedly attenuated by genistein (5 × 10−5m), with a similar time course to that of the tyrosine-phosphorylated substrates, but no significant change in the density of p44MAPK band was observed with Na3VO4treatment (figs. 2B–D).
Effects of Isoflurane on Na3VO4-induced Contraction and Protein Tyrosine Phosphorylation
Isoflurane concentration-dependently and reversibly depressed the Na3VO4(10−4m)–induced contraction of rat aortic smooth muscle rings, with reductions of 18.3 ± 4.8% (P < 0.05), 71.4 ± 5.5% (P < 0.005), and 84.8 ± 3.6% (P < 0.005) in response to 1.2, 2.3, and 3.5% isoflurane gas mixture, respectively. The tension recovered to control level after termination of isoflurane exposure (n = 8; figs. 3A–C).
Isoflurane decreased the band density of the Na3VO4(10−4m)–elicited tyrosine phosphorylation of total substrates in a concentration-related manner, with total decreases of 29.1 ± 5.4% (P < 0.05), 63.3 ± 4.9% (P < 0.005), and 73.1 ± 3.7% (P < 0.005) in response to 1.2, 2.3, and 3.5% isoflurane gas mixture, respectively (n = 4; fig. 4A).
The densities of tyrosine-phosphorylated PLCγ-1 band and p42MAPK band were also attenuated dose-dependently by isoflurane, with reductions of 31.3 ± 4.4% (P < 0.05), 60.4 ± 6.0% (P < 0.005), and 84.3 ± 4.9% (P < 0.005) for PLCγ-1 band density (n = 4; fig. 4B) and of 17.5 ± 2.9% (P > 0.05), 50.4 ± 7.3% (P < 0.005), and 72.9 ± 8.6% (P < 0.005) for p42MAPK band density in response to 1.2, 2.3, and 3.5% isoflurane gas mixture, respectively. However, isoflurane up to 3.5% did not affect the density of tyrosine-phosphorylated p44MAPK band (n = 4; figs. 4C and D).
Protein Tyrosine Kinase–catalyzed, Tyrosine Phosphorylation–mediated Contraction of Vascular Smooth Muscle
Protein tyrosine kinases are important elements of cellular signal transduction pathways, and three general subclasses have now been found: (1) the membrane receptor PTKs, which are regulated by extracellular ligands, such as epidermal growth factor and platelet-derived growth factor5,6,9,34; (2) cytosolic nonreceptor PTKs, such as the proto-oncogene products Abl and Fes, which seem to be located in the cytosol, and relatively little is known about their effects; and (3) the membrane-associated nonreceptor PTKs, such as p60c-src , which are lapidated and bound to the cytoplasmic face of the plasma membrane. They are activated by various agonists such as angiotensin II,7,8,10,14,15,17,35endothelin,35,36arginine-vasopressin,17,18and noradrenaline11,17through G protein–coupled receptors.
Protein tyrosine phosphorylation by PTKs modulates many signaling transduction pathways, and is thought to represent the beginning of a cascade of events. The amount or extent of protein tyrosine phosphorylation is regulated not only by the rate of phosphorylation catalyzed by PTKs, but also by the rate of protein tyrosine dephosphorylation catalyzed by protein tyrosine phosphatases. Therefore, both agonist-induced increases in protein tyrosine phosphorylation and protein tyrosine phosphatase inhibitor–produced decreases in protein tyrosine dephosphorylation are able to augment the net level of tyrosine kinase–phosphorylated proteins, resulting in contraction of vascular smooth muscle cells. The reduction of protein tyrosine phosphorylation by tyrosine kinase inhibitors, such as genistein, tyrphostins, and geldanomycin, depresses signal transduction and causes a relaxation of vascular smooth muscle cells.16,37,38In the current study, rat aortic smooth muscle was stimulated by the potent protein tyrosine phosphatase inhibitor Na3VO4to induce constant tyrosine phosphorylation of many proteins and sustained vascular contraction, which was significantly attenuated in the presence of genistein (figs. 1 and 2).
The activation of PTKs by agonists or depression of tyrosine phosphatase by tyrosine phosphatase inhibitors, such as Na3VO4, enhances tyrosine phosphorylation of a set of substrates ranging from 40 to 205 kd,4,15,17,11,21varied depending on the stimuli, species, and vascular types. These target substrates are involved in various responses, such as cell growth, division, proliferation, and smooth muscle cell contraction, most of which have not yet been identified. In the current study, Na3VO4increased protein tyrosine phosphorylation of a set of substrates, especially of those with molecular weights of 42, 71, 88, 116, 155, and 190 kd, which was significantly decreased in the presence of genistein (fig. 2A). In regard to the mechanisms of smooth muscle contraction, PLCγ-15–8and p44/p42MAPKs (or ERK1/2)9–14are phosphorylated by tyrosine kinases, consequently activating the diacylglycerol/inositol trisphosphate/Ca2+pathway and p44/p42MAPK pathway, finally increasing [Ca2+]iand potentiating myofilament Ca2+sensitivity. The increase in tyrosine phosphorylation of PLCγ-1 and p42MAPK by Na3VO4was confirmed with specific antibodies in our experiment (figs. 2B and C). However, the alteration of tyrosine-phosphorylated p44MAPK by Na3VO4was not detected in this study, which is consistent with the findings that p42MAPK but not p44MAPK was involved in the serotonin-induced tyrosine phosphorylation.12However, angiotensin II–induced and endothelin-induced tyrosine phosphorylation resulted in the expression of both p44MAPK and p42MAPK.35It may depend on the agonists used, the experimental condition, or vascular beds. The Na3VO4-induced vascular contraction in our study likely involves in the mechanisms of PTK-phosphorylated PLCγ-1–mediated and p42MAPK-mediated increase in [Ca2+]iand Ca2+sensitivity.
The tyrosine phosphorylation of PLCγ-1 activates inositol trisphosphate/Ca2+/myosin light chain kinase/myosin light chain 20 signaling pathway and diacylglycerol/PKC signaling pathway, resulting in vascular contraction. Both tyrosine and threonine phosphorylation of p44/p42MAPK by PTKs and MAPK kinase, respectively, phosphorylate caldesmon and separate its binding to actin, potentiating smooth muscle contraction (fig. 5).
Effects of Anesthetics on Protein Tyrosine Phosphorylation of Vascular Smooth Muscle
The important finding in the current study is that isoflurane inhibited both the Na3VO4-stimulated contraction and tyrosine phosphorylation of a set of proteins including PLCγ-1 and p42MAPK in a concentration-dependent manner, although the extent of the inhibitory effect on these parameters was not the same. This inhibition on tyrosine phosphorylation of a set of proteins and PLCγ-1 was significant even at a low concentration of isoflurane (1 MAC). It seems that the inhibitory potential on PLCγ-1 was greater than that on p42MAPK.
Many studies have been conducted on the effects and the mechanisms of anesthetics on vascular smooth muscle contraction, most of which have focused on Ca2+mobilization and Ca2+sensitivity. In cultured fetal rat aortic smooth muscle cells, 2% isoflurane and 2% halothane attenuated the serotonin-induced Ca2+mobilization (or [Ca2+]i) by approximately 26% and 43%, respectively.25Akata et al. 26reported that 3% isoflurane inhibited both the norepinephrine-induced contraction and increase in [Ca2+]iby approximately 30% but only attenuated the 40 mm KCl–induced contraction by approximately 30%, without significant effect on [Ca2+]iin isolated mesenteric resistance arteries. In another study, Akata et al. 23also demonstrated that the inhibition of sevoflurane on the norepinephrine- and KCl-induced contraction of isolated mesenteric resistance arteries occurred mainly by depression of the voltage-gated Ca2+influx and by inhibiting the myofilament Ca2+sensitivity. We demonstrated that sevoflurane concentration-dependently suppressed both the GTPγS-stimulated contraction and membrane translocation of Rho and Rho-kinase in aortic smooth muscle, thus suppressing the Rho/Rho-kinase signaling pathway–mediated Ca2+sensitization.31We also reported that sevoflurane depressed both the angiotensin II–induced contraction and the phosphorylation of Ca2+-dependent PKCα, without affecting [Ca2+]iin rat aortic smooth muscle, suggesting that sevoflurane inhibited the angiotensin II–induced, Ca2+-dependent PKCα-mediated vascular contraction.29However, other investigators showed that isoflurane decreased the angiotensin II–induced mobilization of intracellular Ca2+, Ca2+release from internal stores, and Ca2+influx through nifedipine-insensitive Ca2+channels.24Based on these findings, volatile anesthetics seem to have the ability to alter vascular smooth muscle contraction by interrupting different signaling pathways: Ca2+-mediated signaling pathway, Ca2+sensitization mechanisms, or other mechanisms, depending on the anesthetic agents, agonists, species, and vascular types.
Protein tyrosine kinases are the upstream effectors of some signaling pathways, and protein tyrosine phosphorylation is an important mechanism to activate and initiate a cascade of cellular responses. The current study showed that isoflurane inhibited the Na3VO4-induced contraction and protein tyrosine phosphorylation of many enzymes, including PLCγ-1 and p42MAPK, even at clinically relevant concentration. We speculate that the isoflurane-evoked decrease in [Ca2+]iand the depression on Ca2+sensitivity, as suggested by other studies23–29is modulated, at least in part, by inhibiting the protein tyrosine phosphorylation of PLCγ-1, p42MAPK.
In summary, the current study demonstrates for the first time that isoflurane concentration-dependently inhibits both the Na3VO4-stimulated contraction and tyrosine phosphorylation of a set of proteins including PLCγ-1 and p42MAPK in rat aortic smooth muscle. This suggests that isoflurane depresses protein tyrosine phosphorylation–modulated contraction, especially tyrosine-phosphorylated PLCγ-1 and p42MAPK signaling pathway–mediated contraction of vascular smooth muscle.
The authors thank Shunji Itoh, M.D. (Assistant Professor, First Department of Pathology, Wakayama Medical University, Wakayama City, Japan), and Akira Ooshima, M.D., Ph.D. (Professor and Chairman of the First Department of Pathology, Wakayama Medical University), for their guidance and help in the Western blotting experiment.