Sevoflurane Inhibits Angiotensin II–induced, Protein Kinase C–mediated but Not Ca²⁺-elicited Contraction of Rat Aortic Smooth Muscle

With the approval of the Wakayama Medical University Animal Care and Use Committee (Wakayama City, Japan), male Wistar rats (weight, 250–350 g) were anesthetized with halothane and euthanized by exsanguination from the common carotid artery. The descending thoracic aorta was dissected carefully, and adherent fat and connective tissue was removed. The prepared aorta was cut into rings 3–4 mm in length, and the endothelium was denuded by gentle rubbing of the internal surface with a stainless steel needle to examine the contraction of rat aortic smooth muscle. The prepared rings were mounted vertically on force-displacement transducers (Nihondenki-sanei Co., Tokyo, Japan) and were bathed in 10-ml organ chambers containing Krebs bicarbonate solution (KBS) with 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. KBS in the chamber was gassed with a mixture of 95% O²–5% CO²to keep the pH value within the range 7.35–7.45 and was maintained at 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.

After equilibration, all the aortic rings were exposed to KBS containing KCl (3 × 10⁻²m) to assess the integrity of smooth muscle cells and their overall contractile responsiveness. Removal of the endothelium was confirmed in phenylephrine (3 × 10⁻⁷m)–precontracted rings by the lack of relaxation to 10⁻⁵m acetylcholine.

A submaximal concentration, 10⁻⁷m, of angiotensin II was used to induce contraction in the study. In a preliminary experiment, tachyphylaxis of the rings to angiotensin II was demonstrated (i.e ., the contractile response to the first application of angiotensin II was higher than that to the second and the third applications, and the responses to the last two applications were almost same [data not shown]). Therefore, the contractile response to the second application of angiotensin II was used as control. The response to the third application was used to test the effects of agents. The rings were incubated in normal KBS, Ca²⁺-free KBS, KBS with the selective Ca²⁺-dependent PKC (cPKC) inhibitor Gö 6976 (10⁻⁵m), the nonselective PKC-inhibitory Ro 31-8425 (10⁻⁵m), or sevoflurane (1.7, 3.4, and 5.1%) for 15 min. Angiotensin II was then applied, and resultant tension was measured.

Endothelium-denuded aortic strips approximately 5 mm long and 3.5 mm wide were prepared from the isolated rat descending thoracic aorta and were treated with 10⁻⁵m acetoxymethyl ester of fura-2 solution for 6–9 h at room temperature (19°-23°C). A noncytotoxic detergent, 0.1% cremophor, was added to the solution to increase the solubility of acetoxymethyl ester.

The determination of [Ca²⁺]ⁱwas performed using a fluorometer designed to measure the surface fluorescence of living tissues (CAF-110; Japan Spectroscopic, Tokyo, Japan). The muscle strip was held horizontally in a temperature-controlled organ bath. One end of the strip was connected to an isometric force transducer to measure muscle tension. A passive tension of 3.0 g was applied, and the muscle strip was allowed to equilibrate for 60 min before the start of the experiment. The intimal surface of the muscle strip was illuminated at 50 Hz, alternately at excitation wavelengths of 340 and 380 nm, and the amount of fluorescence at 510 nm induced by 340 nm excitation (F340) and that induced by 380 nm excitation (F380) was measured. The ratio of F340 to F380 (F340:F380) was used to indicate the [Ca²⁺]ⁱ.

Isometric contraction and F340:F380 induced by 3 × 10⁻²m KCl were measured, and the values were taken as the reference (100%). Sevoflurane (0 [control], 3.4, 5.1, or 8.5%) was introduced into the 95% O²–5% CO²mixture for 15 min. Thereafter, angiotensin II (10⁻⁷m) was administered when F340:F380 returned to the resting level. The changes in muscle tension and F340:F380 were recorded simultaneously and were expressed as a percentage of the reference values.

Isolated endothelium-denuded aortas were bathed in 95% O²–5% CO²KBS and equilibrated for 60 min. To examine the time course of the angiotensin II–induced phosphorylation of classic PKC-α (cPKC-α) and novel PKC-ε (nPKC-ε), aortas were exposed to angiotensin II (10⁻⁷m) for 0, 2, 4, 6, 8, or 10 min and then rapidly frozen with dry ice. The time course study showed that phosphorylation of cPKC-α reached a peak level 4 min after angiotensin II application, and a phosphorylated nPKC-ε band was not detected. Therefore, the dose effect of sevoflurane on angiotensin II–stimulated phosphorylation of only cPKC-α was examined 4 min after angiotensin II application in the following experiment. Aortas were incubated with 0 (control), 1.7, 3.4, or 5.1% sevoflurane for 15 min and were quickly frozen 4 min after exposure to angiotensin II.

The frozen tissues were homogenized in ice-cold lysis buffer with 0.2% Triton X-100. 4Homogenates were centrifuged at 1,000 g  for 30 min at 4°C, and the supernatant was collected and centrifuged at 10,000 g  for 30 min at 4°C. The supernatants, containing both the cytosolic and membrane fractions, were assayed for protein concentration using the bicinchoninic acid method and were used for subsequent detection of PKC phosphorylation. The lysis buffer contained 250 mm sucrose, 5 mm EDTA, 10 μg/ml aprotinin, 20 μg/ml soybean tyrosine inhibitor, 100 μg/ml phenylmethanesulfonyl fluoride, 100 μg/ml leupeptin, 10 mm HEPES (pH 7.4), and 5 μm each of phosphoserine, phosphothreonine, phosphotyrosine, β-glycerophosphate, p-nitrophenylphosphate, and sodium orthovanadate. 4 

In each experiment, samples were used at equal content of total protein. Proteins were separated by sodium-dodecyl-sulfate polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Membranes were treated with anti–cPKC-α, anti–nPKC-ε (1:10,000 and 1:2,000, respectively), anti–phosphorylated cPKC-α, or anti–phosphorylated nPKC-ε antibodies (1:1,000) for 4 h, followed by incubation with horseradish peroxidase–conjugated antibody (1:2,000) for 1.5 h. The densities of 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 levels of phosphorylated cPKC-α were expressed as a percentage of the cPKC-α density.

Sevoflurane was introduced into the gas mixture through an agent-specific vaporizer (Penlon Limited, Abingdon, Oxon, United Kingdom) as needed. The concentration in 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).

Because the same organ-bathing chambers were used to incubate the tissues for the tension measurement experiment and for PKC detection experiment, sevoflurane concentrations in KBS were considered to be the same if equal concentrations of sevoflurane were added to the gas mixture. The equilibrated concentrations of sevoflurane in the KBS were 0.17 ± 0.05, 0.35 ± 0.02, and 0.50 ± 0.03 mm at concentrations of 1.7, 3.4, and 5.1% in the gas mixture, respectively (n = 8–12). For the [Ca²⁺]ⁱmeasurement experiment, the surface area of the organ-bathing chamber in which the aortic strips were incubated was larger; therefore, the vaporizing amount of sevoflurane from the KBS was greater, and the actual concentration of sevoflurane in KBS was lower than that for the tension measurement and PKC detection experiments, although an equal concentration of sevoflurane was added to the gas mixture. The concentrations of sevoflurane in the KBS for the [Ca²⁺]ⁱmeasurement experiment were 0.14 ± 0.01, 0.27 ± 0.02, 0.38 ± 0.06, and 0.62 ± 0.08 mm at sevoflurane concentrations of 1.7, 3.4, 5.1, and 8.5% in the gas mixture, respectively (n = 3). Because it was difficult to adjust the concentration of sevoflurane in the KBS to equivalent levels for the experiments of tension measurement, PKC detection, and the experiment of [Ca²⁺]ⁱmeasurement and because sevoflurane did not significantly influence the angiotensin II–induced increase in [Ca²⁺]ⁱat concentrations of 3.4 and 5.1% in the current study, we attempted an even higher concentration (8.5%).

Angiotensin II, Gö 6976, and Ro 31-8425 were purchased from Sigma-Aldrich Fine Chemicals (St. Louis, MO). Sevoflurane was purchased from Dainabot Company Limited (Osaka, Japan). Polyclonal anti–cPKC-α, anti–nPKC-ε, anti–phosphorylated cPKC-α, and anti–phosphorylated nPKC-ε antibodies and the secondary antibody labeled with horseradish peroxidase were supplied by Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Other reagents for the mechanical experiment, Ca²⁺measurement, and Western blotting were all of analytical grade. Angiotensin II was dissolved using distilled water, and Gö 6976 and Ro 31-8425 were dissolved using dimethyl sulfoxide.

The data are presented as mean ± SD. The sample size (n values) represents the number of rats from which aortic rings (for tension measurement), aortic strips (for [Ca²⁺]ⁱmeasurement), or aortas (for Western blotting) were taken. Two-factorial analysis of variance was used to compare the effects of the different sevoflurane concentrations on the angiotensin II–induced contraction, [Ca²⁺]ⁱ, and phosphorylation of cPKC-α, using the software program StatView (SAS Institute Inc., Cary, NC). P  values less than 0.05 were considered statistically significant.

Angiotensin II (10⁻⁷m) induced a transient contraction of rat aortic endothelium–denuded rings, reaching the maximal level of 69.0 ± 3.1% (n = 10) of the 3 × 10⁻²m KCl–induced contraction 3.6 ± 0.2 min after application (fig. 1A). Angiotensin II (10⁻⁷m) also elicited a transient increase in [Ca²⁺]ⁱ, immediately reaching a peak level of 78.6 ± 15.5% (n = 7) of that induced by 3 × 10⁻²m KCl (fig. 1B). The absence of extracellular Ca²⁺almost completely abolished the angiotensin II (10⁻⁷m)–induced contraction (12.2 ± 5.3% of control, P < 0.001, n = 6;fig. 2). The selective cPKC inhibitor Gö 6976 (10⁻⁵m) significantly attenuated (63.3 ± 7.9% of control, P < 0.01, n = 6), but the nonselective PKC inhibitor Ro 31-8425 (10⁻⁵m) did not significantly inhibit the angiotensin II–induced contractile response (94.5 ± 7.6% of control, P > 0.05, n = 6;fig. 2).

The bands with molecular weights of approximately 80 and 90 kDa were identified as cPKC-α and nPKC-ε, respectively. In the same experiment, the densities of the cPKC-α band or the nPKC-ε band were almost identical for each sample. Similar to the contraction time course, angiotensin II (10⁻⁷m) transiently increased the phosphorylation of cPKC-α, with the maximal level achieved 4 min after application (fig. 3A). A phosphorylated nPKC-ε band was not detected in the presence of angiotensin II (fig. 3B).

Sevoflurane concentration-dependently inhibited the angiotensin II (10⁻⁷m)–induced contraction, with reductions of 14.2 ± 5.2% (P > 0.05), 26.7 ± 8.9% (P < 0.05), and 38.5 ± 12.8% (P < 0.01) in response to 1.7, 3.4, and 5.1% sevoflurane, respectively (n = 8, fig. 4).

The presence of sevoflurane did not significantly affect the angiotensin II (10⁻⁷m)–elicited increase in [Ca²⁺]ⁱ, with levels of 78.6 ± 15.5, 63.1 ± 12.8, 68.5 ± 15.0, and 64.1 ± 10.5% of that induced by 3 × 10⁻²m KCl in response to 0, 3.4, 5.1, and 8.5% sevoflurane (P > 0.05, n = 6–9;fig. 5), respectively, but were accompanied with a dose-dependent attenuation in contraction, which was measured spontaneously in the same experiments (data not shown).

Sevoflurane depressed the angiotensin II (10⁻⁷m)–stimulated phosphorylation of cPKC-α in a concentration-dependent manner, with decreases of 20.5 ± 14.2% (P > 0.05), 37.0 ± 17.8% (P < 0.05), and 62.5 ± 12.2% (P < 0.01) in response to 1.7, 3.4, and 5.1% sevoflurane, respectively (n = 4;fig. 6).

The main findings of this study are as follows: Angiotensin II (10⁻⁷m) induced a rapid increase in [Ca²⁺]ⁱ, a transient phosphorylation of cPKC-α, and a consequently transient contraction, which was almost completely abolished by removing extracellular Ca²⁺and was significantly inhibited by the selective cPKC inhibitor Gö 6976 (10⁻⁵m) but not by the nonselective PKC inhibitor Ro 31-8425 (10⁻⁵m). Phosphorylation of nPKC-ε in the presence of angiotensin II was not detected in the current study. Sevoflurane dose-dependently attenuated the angiotensin II (10⁻⁷m)–stimulated contraction and the phosphorylation of cPKC-α but not the angiotensin II–elicited increase in [Ca²⁺]ⁱ.

The potent peptide pressor hormone angiotensin II is important in the maintenance of vascular tone. The contractile action of angiotensin II is initiated on binding to AT¹receptors on the surface of vascular smooth muscle cells (VSMCs). 5After activation of phospholipase C, phosphatidylinositol-4,5-bisphosphate is hydrolyzed, and inositol 1,4,5-trisphosphate and diacylglycerol are produced. Inositol 1,4,5-trisphosphate stimulates the release of Ca²⁺from the sarcoplasmic reticulum. Angiotensin II also activates L-gated Ca²⁺channels and enhances Ca²⁺influx. Increased [Ca²⁺]ⁱelicits the rapid contraction of VSMCs. Diacylglycerol activates PKC, increasing the Ca²⁺sensitivity of contraction-associated proteins. 6 

The key role of Ca²⁺in the angiotensin II–induced vascular contraction was confirmed in our study, which showed that angiotensin II elicited a rapid increase in [Ca²⁺]ⁱ, faster than the resulting contraction, and that angiotensin II produced a lower contractile response in the absence of extracellular Ca²⁺. The current study also verified the importance of PKC in the angiotensin II–mediated vascular contraction. Significant inhibition was demonstrable with the selective cPKC inhibitor Gö 6976 but not with the nonselective PKC inhibitor Ro 31-8425 (which probably primarily inhibits nPKC activity), suggesting that Ca²⁺-dependent cPKC is involved in the angiotensin II–induced contraction. This correlates with the angiotensin II–stimulated transient increase in [Ca²⁺]ⁱand the consequently transient contraction. Ca²⁺-independent nPKC is considered to primarily mediate the sustained contraction of smooth muscle.

Protein kinase C  refers to a family of at least 11 different closely related serine–threonine kinases. Ca²⁺-dependent (or classic) PKC (cPKC-α, β^I, β^II, and γ) requires Ca²⁺, phospholipid, and diacylglycerol for activity. Ca²⁺-independent (or novel) PKC (nPKC-δ, -ε, -η, and -θ) exhibits kinase activity, requiring only diacylglycerol and phospholipid. Atypical PKC (aPKC-τ, -λ, and -ζ) can be activated by diacylglycerol or phorbol ester in the absence of Ca²⁺and phospholipid. 2 

The distribution of PKC isoenzymes varies and depends on the species, vascular domains, or both. cPKC-α, cPKC-β, nPKC-δ, nPKC-ε, and aPKC-ζ have been demonstrated in VSMCs, although not all of these seem to be expressed in all vascular smooth muscle tissues. 7–9The most predominant isoenzymes, cPKC-α and nPKC-ε, were selected to be detected using specific antibodies with Western blotting in this study.

After stimulation of angiotensin II, PKC translocates to the membrane and is autophosphorylated. Autophosphorylation increases the enzyme’s activity and affinity for substrates, effectors, or both. 3The extent of PKC phosphorylation has been proposed to represent a useful indicator of its activation state. 10–13Accordingly, the degree of phosphorylation of cPKC-α and nPKC-ε was used to represent the activation of both of them in the current study.

The detectable cPKC-α and nPKC-ε bands with Western blotting in this study indicate that cPKC-α and nPKC-ε exist in rat aortic smooth muscle. The failure to detect phosphorylated nPKC-ε in the presence of angiotensin II and a similar time course for both cPKC-α phosphorylation and the contraction induced by angiotensin II further verified that a Ca²⁺-dependent cPKC but not (or at least unpredominantly) Ca²⁺-independent nPKC is involved in the angiotensin II–induced contraction of rat aortic smooth muscle.

The effects of anesthetics on the contractile response of VSMCs vary, depending on anesthetics, agonists, species, and vascular domains. A large body of evidence has revealed that among the complex mechanisms of contraction, Ca²⁺and PKC are the major targets of anesthetics. Isoflurane has been demonstrated to inhibit serotonin- and endothelin-induced Ca²⁺mobilization, without inhibition of the phorbol ester–activated PKC in cultured rat aortic smooth muscle cells, 14but to enhance the phorbol ester-stimulated cytosolic PKC-mediated constriction of rat coronary artery smooth muscle. 15In skinned pulmonary arteries, nPKC primarily mediates the isoflurane-induced contraction, but both cPKC and nPKC take part in the regulation of isoflurane-induced relaxation. 16Halothane dose-dependently decreases Ca²⁺accumulation in the sarcoplasmic reticulum and increases Ca²⁺release from the sarcoplasmic reticulum in rabbit aortic smooth muscle cells 17and attenuates the phorbol ester–stimulated cytosolic and membrane PKC-mediated contraction of rat coronary smooth muscle. 15In skinned pulmonary arteries, the halothane-induced contraction is predominantly mediated via  nPKC, but the relaxation is predominantly regulated via  the Ca²⁺-calmodulin–dependent protein kinase II. 18 

The results of the current study have shown, for the first time, that sevoflurane dose-dependently inhibited the angiotensin II–induced phosphorylation of cPKC-α but not the increase in [Ca²⁺]ⁱ, suggesting that sevoflurane depresses the angiotensin II–induced, Ca²⁺-dependent cPKC-mediated but not Ca²⁺-elicited contraction of rat aortic smooth muscle, even at clinically relevant concentrations (1.7–3.4%). Also using Western blotting, Zhong et al . 4have shown that isoflurane increases the PKC activator phorbol-12-myristate-13-acetate–induced membrane translocation of nPKC-ε but not cPKC-α in cultured rat aortic smooth muscle cells, suggesting that isoflurane activates phorbol-12-myristate-13-acetate–stimulated nPKC but not cPKC. Differing from the mechanisms of the effects of sevoflurane on angiotensin II–induced contraction in this study, Akata et al . 19have demonstrated that sevoflurane inhibits norepinephrine-induced contraction by both reducing Ca²⁺influx and depressing the sensitivity of smooth muscle cells to Ca²⁺, without significant attenuation of Ca²⁺release from the sarcoplasmic reticulum in smooth muscle cells of mesenteric resistance arteries. Inconsistent with the current finding that sevoflurane did not affect the angiotensin II–stimulated increase in [Ca²⁺]ⁱ, isoflurane has been shown to dose-dependently decrease the angiotensin II–induced intracellular mobilization of Ca²⁺by inhibiting Ca²⁺release from internal stores and Ca²⁺influx through nifedipine-insensitive Ca²⁺channels in cultured rat aortic smooth muscle. 20Therefore, the complex mechanisms of VSMC contraction suggests that anesthetics probably affect vascular contraction via  the interruption of multiple signaling pathways. Whether sevoflurane affects other contraction-associated mechanisms remains to be clarified in future investigations.

In summary, the current study has demonstrated that Ca²⁺and cPKC, especially cPKC-α, are involved in the angiotensin II–induced transient contraction of rat aortic smooth muscle and that sevoflurane dose-dependently inhibits the angiotensin II–induced, cPKC-α–mediated but not the Ca²⁺-elicited contraction of rat aortic 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 experiment of Western blotting.