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From the Department of Anesthesiology, Wakayama Medical University, Wakayama, Japan.
Address correspondence and reprint requests to Koji Ogawa, MD, Department of Anesthesiology, Wakayama Medical University, 811-1 Kimiidera, Wakayama 641-0012, Japan. Address e-mail to ogawak{at}wakayama-med.ac.jp.
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Abstract |
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 Top Abstract IntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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METHODS: The effects of isoflurane on vasoconstriction, increase in [Ca2+]i, and phosphorylation of PKC in response to Ang II (10–7 M) were investigated, using an isometric force transducer, a fluorometer, and Western blotting, respectively.
RESULTS: Ang II elicited a transient contraction of rat aortic smooth muscle that was associated with an increase in [Ca2+]i and PKC phosphorylation. Isoflurane (1.2%–3.5%) inhibited Ang II-induced contraction of rat aortic smooth muscle in a concentration-dependent manner (P < 0.05 at 1.2%, P < 0.01 at 2.3% and 3.5% isoflurane, n = 6). Isoflurane also inhibited elevation of [Ca2+]i in response to Ang II (P < 0.01 at 2.3% and 3.5% isoflurane, n = 6), but failed to affect Ang II-induced phosphorylation of PKC at concentrations up to 3.5% (n = 7).
CONCLUSION: These results suggest that, unlike sevoflurane, the inhibitory effect of isoflurane on Ang II-induced contraction is mainly mediated by attenuation of the Ca2+-mediated signaling pathway.
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Introduction |
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TopAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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Volatile anesthetics decrease arterial blood pressure, in part, by direct relaxation of blood vessels. Although both sevoflurane and isoflurane can reduce Ang II-induced vascular contraction (2), the precise mechanisms are not fully understood. We have demonstrated that sevoflurane inhibits the contractile response to Ang II mainly by inhibiting PKC phosphorylation that regulates myofilament Ca2+ sensitivity without significant alteration of [Ca2+]i in rat aortic smooth muscle (3). However, it remains unclear whether the cellular mechanism underlying the inhibitory effect of isoflurane is similar to that of sevoflurane. The goal of the present study was to elucidate the mechanism by which isoflurane inhibits Ang II-induced vasoconstriction by measurements of isometric force and [Ca2+]i, and detection of PKC phosphorylation using Western blotting.
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METHODS |
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TopAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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Male Wistar rats weighing 250–400 g were anesthetized with halothane and exsanguinated by cutting through the common carotid artery. The chest of each rat was opened and the descending portion of the thoracic aorta was isolated. The aorta was cleared of excess fat and connective tissue and then cut into rings 3–4 mm in length. Four to six rings were typically harvested from one rat. The endothelium was removed by gently rubbing the luminal surface with a stainless steel needle. The aortic rings were fixed vertically between hooks in 10-mL organ baths containing a Krebs bicarbonate solution (KBS). The constituents of KBS included: NaCl 118.2 mM, KCl 4.8 mM, CaCl2 2.5 mM, KH2PO4 1.2 mM, MgSO4 1.2 mM, NaHCO3 24.8 mM, and dextrose 10 mM. The solution was continuously bubbled with a mixture of 95% O2 and 5% CO2 to maintain the pH within the range of 7.35–7.45, and the temperature of the solution was kept at 37°C. The hook anchoring the upper end of the rings was connected to the lever of a force-displacement transducer (NEC San-ei Instruments, Tokyo, Japan), and the lower hook was fixed to the bottom of the organ bath. Changes in isometric force development were amplified and displayed on chart recorders (Recti-Horiz-8K, NEC San-ei Instruments, Tokyo, Japan). The resting tension was adjusted to 3.0g, which was found in our previous studies to be optimal for inducing maximal contraction (2–5). Before the start of the experiments, the rings were allowed to equilibrate for 60 min, during which time the bathing solution was replaced every 15 min.
After equilibration, the aortic rings were incubated with KCl (30 mM) to assess their overall contractile responsiveness. Removal of the endothelium was verified by a lack of the relaxation response to acetylcholine (10–6 M) in rings precontracted with phenylephrine (3 x 10–7 M). Only aortic rings that developed at least 1.0g contractile force in response to KCl (30 mM) and exerted no relaxation response to acetylcholine were used for further experiments. Isoflurane was introduced into the gas mixture through agent-specific vaporizers (Penlon, Abingdon, Oxon, UK). The concentration in the resulting gas mixture was monitored and adjusted using a calibrated Atom 303 anesthetic agent monitor (Atom, Tokyo, Japan). The concentrations of the isoflurane in the bathing solution, as measured by gas chromatography (Shimadzu CO, Kyoto, Japan) were the same as the concentrations of isoflurane in our study (2), which used the same experimental system: 0.19 ± 0.01, 0.39 ± 0.01, and 0.56 ± 0.03 mM, corresponding to gas mixture concentrations of 1.2% (1 human MAC), 2.3% (2 MAC), and 3.5% (3 MAC), respectively (n = 8–12).
Ang II (10–7 M) was used to induce vascular contraction, based upon our previous investigations (2,3,5). To assess the effects of isoflurane on Ang II-induced contraction, four aortic rings from each of six different rats were randomly exposed to 0, 1.2, 2.3, or 3.5% isoflurane for 15 min before Ang II challenge (n = 6). Each ring was exposed to only one concentration of isoflurane. Isometric force development in response to Ang II was expressed as a percentage relative to that induced by KCl (30 mM).
For measurement of [Ca2+]i in aortic smooth muscle, endothelium-denuded aortic strips, approximately 5 mm long and 3.5 mm wide, were prepared from the isolated rat descending thoracic aorta. Two to three strips were typically harvested from one rat. The strips were incubated for 6 h in KBS containing acetoxymethyl ester of fura-2 (10–5 M) at room temperature (20°C to 22°C). A noncytotoxic detergent, cremophor (0.1%), was added to the solution to increase the solubility of acetoxymethyl ester. After the loading period, the preparations were washed three times with KBS. Each aortic strip was held horizontally in a temperature-controlled (37°C) organ bath that was continuously perfused with Krebs bicarbonate solution aerated with a mixture of 95% O2 and 5% CO2. Fluorescence measurements were performed using a dual-wavelength spectrofluorometer (CAF-110, Japan Spectroscopic, Tokyo, Japan) at excitation wavelengths of 340 and 380 nm and an emission wavelength of 510 nm. The 340 to 380 nm fluorescence ratio was used as an indicator of [Ca2+]i. Change in 340/380 ratio in response to KCl (30 mM) was observed first and the value was used as a standard of reference (100%). After washing with bathing solution, isoflurane at concentrations of 0, 1.2, 2.3, or 3.5% were introduced into the aerated gas mixture for 15 min, followed by the addition of Ang II (10–7 M) to the bathing solution. The change in 340/380 ratio was recorded and expressed as a percentage of the reference value.
For measurement of PKC phosphorylation, descending thoracic aortas (approximately 3.5 cm in length) were excised from the rats, carefully cleared of fat and connective tissue, and opened longitudinally. The endothelium was removed by gently rubbing with a stainless steel needle. One strip was obtained from each animal. The preparations were incubated in KBS bubbled with a mixture of 95% O2 and 5% CO2 and equilibrated for 60 min before the start of experiment.
Based on the findings from our previous study, in which phosphorylation of PKC reached a peak level at 4 min after application of Ang II (3), the effects of isoflurane on Ang II-induced stimulated PKC phosphorylation were examined 4 min after Ang II application. The effects of isoflurane on PKC phosphorylation in response to Ang II were examined by randomly pre-exposing 21 aortas from 21 different animals to isoflurane at concentrations of 0, 1.2, or 3.5% for 15 min, and then freezing the tissue with liquid nitrogen 4 min after application of Ang II (10–7 M). Another seven aortic strips from seven different animals were not exposed to isoflurane but incubated with a PKC inhibitor, bisindolylmaleimide 1 (BIS 1, 10–5 M) and then frozen with liquid nitrogen 4 min after application of Ang II (10–7 M). Seven additional strips without Ang II and isoflurane treatment, served as a baseline control.
Frozen aortas were cut into small pieces, and then homogenized in an ice-cold lysis buffer (50 mM HEPES, pH 7.5, 1% Triton X-100, 50 mM NaCI, 50 mM NaF, 5 mM EDTA, 10 mM sodium pyrophosphate, 1 mM phenylmethanesulfonyl fluoride, 1 mM Na3VO4, 10 µg/mL leupeptin, and 20 µg/mL aprotinin) (6). Tissue homogenates were centrifuged at 15,000g for 15 min at 4°C. The supernatant was collected, and the protein concentration was determined using the bicinchoninic acid method (7). Solutions of the protein extracts were combined with an equal volume of 2x sample buffer (125 mM Tris, pH 6.8, 10% glycerol, 2% ß-mercaptoethanol, and 0.08% bromophenol blue). The diluted samples were then heated with boiled water for 3 min and stored at –80°C for later measurement.
In each experiment, samples were used at an equivalent total protein content (25–30 µg). Proteins were separated by 10% sodium-dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) 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 PKC-antibody (1:1000) and phospho-PKC antibody (pan, ßIISer660, 1:1000) for 4 h, followed by incubation with horseradish peroxidase-conjugated antibody (1:2000) for 1.5 h. The densities of the immunoreactive bands were detected using the enhanced chemiluminescence system (Amersham Pharmacia Biotec, Piscataway, NJ) and were assessed with image analysis software (NIH Image 1.62, Bethesda, MD). Autophosphorylation of PKC was used as an indicator of PKC activation and expressed as the percentage relative to the baseline control level (referred to 100%).
All drugs were of the highest purity commercially available. Ang II (human) and BIS 1 were purchased from Sigma-Aldrich Fine Chemicals (St. Louis, MO). Isoflurane was obtained from Dinabot CO (Osaka, Japan). Polyclonal antibody against PKC (H-300) and the secondary antibody labeled with horseradish peroxidase were supplied by Santa Cruz Biotechnology, (Santa Cruz, CA), and polyclonal antibody against phospho-PKC (pan, ßIISer660) by Cell Signaling Technology, (Beverly, MA).
All data are presented as mean ± sd. The sample size (n) represents the number of aortic rings and strips for measurements of muscle tension and [Ca2+]i, respectively, and the number of aortas for Western blotting, which is equivalent to the number of rats used in each protocol. Two-way analysis of variance with Scheffé F-test for post hoc comparison was used to compare the effects of different concentrations of isoflurane on Ang II-induced contraction, [Ca2+]I, and PKC phosphorylation using the StatView software program (Version 5.0, SAS Institute, Cary, NC). P < 0.05 were considered to be statistically significant.
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RESULTS |
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TopAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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Isoflurane inhibited the contractile response to Ang II in a concentration-dependent manner, with levels of 92% ± 2% (P < 0.05), 74% ± 9% (P < 0.01), and 40% ± 5% (P < 0.01) of that induced by KCl (30 mM) at concentrations of 1.2, 2.3, and 3.5% isoflurane, respectively (n = 6, Fig. 2A). The presence of isoflurane also attenuated the increase of [Ca2+]i in response to Ang II, with [Ca2+]i levels of 65% ± 6% (P > 0.05), 49% ± 7% (P < 0.01), and 41% ± 8% (P < 0.01) of that induced by KCl (30 mM) at concentrations of 1.2, 2.3, and 3.5% isoflurane, respectively (n = 6, Fig. 2B).
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Ang II (10–7 M) activated PKC autophosphorylation of rat aortic smooth muscle with a maximum level of 169% ± 12% of the unstimulated baseline value in the absence of Ang II (P < 0.05). Treatment with BIS I (10–5 M), a PKC inhibitor, alone did not induce any change in the density of the phospho-PKC bands. However, PKC phosphorylation in response to Ang II was completely abolished by BIS I, with the level of 101% ± 16% of the baseline value (P < 0.05). Isoflurane up to a concentration of 3.5% did not affect Ang II-induced PKC phosphorylation, with levels of 157% ± 20% and 154% ± 23% of the baseline control value at isoflurane concentrations of 1.2% and 3.5%, respectively (n = 7, Fig. 3).
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DISCUSSION |
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TopAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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Ang II is one of the key components of renin– angiotensin–aldosterone system that regulates vascular tension and arterial blood pressure. The Ang II-mediated signaling pathway in vascular tissue has been well established (1,8). Briefly, Ang II binds to the angiotensin type 1 receptor on the surface of vascular smooth muscle cells and rapidly activates multiple phospholipases to hydrolyze membrane phospholipids. Activation of phospholipase C generates inositol 1,4,5-triphosphate and diacylglycerol, which stimulates Ca2+ release from intracellular calcium stores and PKC, respectively. Ang II also activates Ca2+ influx from extracellular space. PKC, in turn, increases the Ca2+ sensitivity of the contraction-associated proteins (9). Thus, Ang II-induced vasoconstriction is determined by both Ca2+-mediated and Ca2+ sensitization mechanisms.
PKC represents a family of at least 11 different closely related serine–threonine kinases (10). Several isoforms have been identified in systemic vascular tissue, and their expression can vary depending on species, the type or size or vessels, and the agonist used (11,12). In our previous study (3), we demonstrated that PKC
and PKC
are identified in the vascular smooth muscle in Wistar rats, and that Ang II stimulates the expression of PKC but not PKC. Based on our previous findings, it is likely that PKC expression in response to Ang II observed in the present study mainly consists of PKC, although measurement of the isozymes of PKC was not performed.
Volatile anesthetics have been shown to inhibit Ang II-induced vascular contraction in a concentration-dependent manner (2). We have shown that sevoflurane inhibits the contractile response to Ang II by attenuating PKC phosphorylation without significantly affecting [Ca2+]i (3). In contrast, the inhibitory effects of isoflurane on Ang II-induced contraction are mediated mainly by a decrease in [Ca2+]i but not by a decrease in PKC phosphorylation as seen in the present study. Consistent with our results, Samain et al. (13) have demonstrated that isoflurane inhibits an Ang II-stimulated increase in [Ca2+]i by inhibiting both Ca2+ release from internal stores and Ca2+ influx from the extracellular space in cultured rat aortic smooth muscle cells, although PKC activity was not measured. Although isoflurane and sevoflurane inhibited the contractile response to Ang II to the same extent, the cellular mechanism behind this effect appears to be completely different. The precise mechanism underlying the difference between the vascular effects of isoflurane versus sevoflurane remains to be clarified.
Volatile anesthetics can affect the expression of some protein kinases other than PKC. Sevoflurane has been reported to inhibit the contraction and membrane translocation of RhoA and Rock2 in response to GTP
S, a Rho/Rho-kinase pathway activator (4). Ang II-induced contraction of aortic smooth muscle is associated with stimulation of p44/42 mitogen-activated protein kinase as well as PKC (1,8,14). Notably, sevoflurane inhibits phosphorylation of p44/42 mitogen-activated protein kinase in response to Ang II at clinically relevant concentrations (5). Both isoflurane and sevoflurane possess the ability to affect protein kinases. We have previously demonstrated that in rat aortic smooth muscle isoflurane reversibly inhibits contraction and tyrosine phosphorylation in response to sodium orthovanadate, a potent protein tyrosine phosphatase inhibitor (15). Furthermore, sevoflurane has also been shown to inhibit sodium orthovanadate-induced protein tyrosine phosphorylation, however, to a more limited extent compared with isoflurane (16). Taking the available evidence as a whole, the ability of volatile anesthetics to alter the contraction of vascular smooth muscle appears to be modulated through different combinations of mechanisms, including Ca2+-mediated signaling pathways and Ca2+ sensitization, with the exact combination dependent on the specific anesthetics, the agonist used, the species being treated, and the type and size of vessels.
The major limitation of this in vitro study is that the present findings were obtained from endothelium-denuded vessels. Endothelium plays an important role in regulating systemic vascular tone in in vivo conditions. We have demonstrated that Ang II-induced vascular contraction is greater in endothelium-denuded aortic rings than in rings with intact endothelium (2), probably because Ang II also stimulates nitric oxide release from endothelium (17). We have also shown that the inhibitory effect of volatile anesthetics on Ang II-induced contraction is enhanced after removal of endothelium (2), suggesting the presence of endothelium may interfere with the present results. However, our primary goal of this study was to elucidate the cellular mechanism by which isoflurane inhibits Ang II-induced contraction of vascular smooth muscle. Hence, we used endothelium-denuded preparations to avoid the influence of endothelium. The findings from the current study cannot be directly extrapolated to in vivo conditions or humans. However, it has been reported in patients that induction of anesthesia sometimes causes profound and refractory hypotension in those treated with angiotensin-converting enzyme (18,19) or Ang II receptor antagonists (20,21). The inhibitory effects of anesthetics on Ang II-induced vascular contraction may contribute to severe hypotension after induction of anesthesia in patients treated with angiotensin system inhibitors.
In summary, the current study demonstrates that isoflurane, at clinically relevant concentrations, inhibits Ang II-induced contraction of rat aortic smooth muscle. This inhibition was associated with a decrease in [Ca2+]i but not with an alteration of PKC phosphorylation. Unlike sevoflurane, the inhibitory effect of isoflurane on vascular contraction is mainly mediated by an attenuation of the increase in [Ca2+]i in response to Ang II. Although isoflurane inhibits Ang II-induced vascular contraction to a similar extent compared with sevoflurane, the cellular mechanism of each of these anesthetics appears to differ.
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Footnotes |
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Supported in part by grant-in-aid No. 16591557 for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan, Tokyo, Japan.
Presented in part at the annual meeting of the American Society of Anesthesiologists, Chicago, Illinois, October 14, 2006.
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REFERENCES |
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TopAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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-thio] triphosphate-stimulated, Rho/Rho-kinase-mediated contraction of isolated rat aortic smooth muscle. Anesthesiology 2003;99:646–51.[ISI][Medline]
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