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Pain Medicine  |   May 2001
Comparison of Volatile Anesthetic Actions on Intracellular Calcium Stores of Vascular Smooth Muscle: Investigation in Isolated Systemic Resistance Arteries
Author Affiliations & Notes
  • Takashi Akata, M.D., Ph.D.
    *
  • Mikio Nakashima, M.D., Ph.D.
  • Kaoru Izumi, M.D.
  • * Lecturer, ‡ Postgraduate Student, Department of Anesthesiology and Critical Care Medicine, Faculty of Medicine, Kyushu University. † Associate Professor, Surgical Operating Center, Saga Medical School.
  • Received from the Department of Anesthesiology and Critical Care Medicine, Faculty of Medicine, Kyushu University, Fukuoka, Japan.
Article Information
Pain Medicine
Pain Medicine   |   May 2001
Comparison of Volatile Anesthetic Actions on Intracellular Calcium Stores of Vascular Smooth Muscle: Investigation in Isolated Systemic Resistance Arteries
Anesthesiology 5 2001, Vol.94, 840-850. doi:
Anesthesiology 5 2001, Vol.94, 840-850. doi:
IN vascular smooth muscle (VSM), Ca2+is stored intracellularly in sarcoplasmic reticulum (SR), which contains at least two types of Ca2+-release channels: inositol 1,4,5-triphosphate (IP3) receptor–Ca2+-release channel and ryanodine receptor (RyR)–Ca2+-release channel. 1,2 The IP3-receptor channel is believed to play a primary physiologic role in Ca2+mobilization. 3 The Ca2+-induced Ca2+release (CICR) may occur through the RyR channel, although its physiologic role is not fully understood. 1,2,4,5 However, it is generally agreed that the SR plays a pivotal role in the regulation of intracellular Ca2+concentration [Ca2+]i, a major determinant of vascular tone, not only as a supply of the activator Ca2+but also as a buffer against VSM activation by Ca2+. 3,6 
Evidence is accumulating that volatile anesthetics at clinical concentrations impair functional integrity of the SR and thereby alter vascular homeostasis. 7–16 Halothane, enflurane, or isoflurane appeared to stimulate Ca2+release or leakage from either ryanodine-sensitive 9–18 or IP3-sensitive SR, 17,19,20 causing transient or sustained increases in vascular tone. The anesthetic-induced Ca2+release from ryanodine-sensitive SR may be caused by activation of the RyR–Ca2+-release channels. 7,8,14,16 In addition, these anesthetics may inhibit Ca2+uptake by the SR, decreasing the amount of Ca2+in SR (i.e.  , Ca2+availability for VSM contraction). 7,8,12,14–16 In cultured VSM cells, halothane and isoflurane appeared to attenuate inositol phosphate formation and thereby inhibit the IP3-induced Ca2+release. 19,20 Furthermore, halothane was recently shown to have a stimulating action on the Ca2+uptake in addition to its Ca2+-releasing action. 12 
Previous studies have investigated volatile anesthetic actions on the SR of VSM by measuring changes in either force or [Ca2+]i. 7–9,12–17 However, it is not always straightforward to interpret results from the force experiments, in which changes in force do not necessarily indicate changes in the [Ca2+]i, because of the possible anesthetic effects on myofilament Ca2+sensitivity and the nonlinear relation between force and [Ca2+]i. Some investigators have already examined the anesthetic actions on the SR of conduit arteries by measuring the [Ca2+]i. 9,13,16,17 However, the anesthetic actions on SR in small arteries, the major sites of regulation of vascular resistance and thus of tissue perfusion and of arterial blood pressure, have not yet been studied in the [Ca2+]imeasurement experiments. In addition, less is known about the action of sevoflurane on the SR. 15 We thus investigated the actions of volatile anesthetics, including sevoflurane, on the SR of VSM in isolated systemic resistance arteries, using fura-2 fluorometry.
Materials and Methods
Force and Ca2+Measurements
With approval from the Kyushu University Animal Care and Use Committee (Fukuoka, Japan), an endothelium-denuded strip was prepared from the third-order branch of male Sprague-Dawley rat (250–350 g) mesenteric artery (≈ 150–200 μm in diameter), and isometric force was measured by attaching the strip to a strain gauge transducer as previously detailed. 11,21 Briefly, the strip was horizontally mounted in a chamber attached to the stage of a microscope, and the resting tension was adjusted to obtain a maximal response to KCl. The solution was changed by infusing it into one end while aspirating simultaneously from the other end. Functional removal of endothelium was verified by lack of acetycholine (10 μm)-induced vasorelaxation, as previously described. 22 
In most of the experiments, changes in [Ca2+]iwere measured simultaneously with those in force, using fura-2, a fluorescent Ca2+-indicator dye. 23 Our method on the fura-2 fluorometry was also previously detailed. 24,25 Briefly, to enable loading of the fura-2 into the VSM cells, the strips were incubated in normal physiologic salt solution (PSS) containing 10 μm acetoxymethyl ester of fura-2 (fura-2–AM) and 2% albumin for approximately 2 h at approximately 35°C. After this period, the solution containing fura-2–AM was washed out with normal PSS for approximately 1 h to ensure sufficient esterification of fura-2–AM in the cells and to equilibrate the strips before the measurements. 24,25 Changes in the fluorescence intensity of the fura-2–Ca2+complex were measured by a fluorimeter equipped with a dual wavelength excitation device (CAM-230; Japan Spectroscopic, Tokyo, Japan) connected to the microscope with optical fibers. The microscope was focused on the VSM layers, and the tissue was illuminated with ultraviolet lights of the wavelengths of 340 and 380 nm alternatively limited to a frequency of 1,000 Hz. The fura-2 fluorescence signals induced by excitation at 340 and 380 nm were collected through the 20-times objective lens (Plan Fluor; Nikon, Tokyo, Japan) and measured through a 500-nm filter with a photomultiplier. The background fluorescence (including autofluorescence of the strip) as excited by 340- and 380-nm ultraviolet light, was obtained after completion of each experiment by breaking the cell membranes with Triton-X-100 (1%) and subsequently quenching the fura-2 fluorescence signals with MnCl2(20 mm) as reported previously. 25–27 The ratio (R340/380) of fura-2 fluorescence intensities excited by 340 nm (F340) to those excited by 380 nm (F380) was calculated after subtracting the background fluorescence.
None of the agents used during Ca2+measurements, except procaine, influenced the fluorescence signals. High concentrations (≥ 3 mm) of procaine, as previously reported in fluo-3-fluorometry, 28 quenched both the F340and F380signals and thereby decreased the R340/380 in fura-2 (2 μm)-containing Ca2+free solution or normal PSS (regardless of the presence of the strips). Therefore, the changes in R340/380during application of the high concentrations of procaine were not evaluated. In all of the measurements except those with procaine, as we recently showed, 25 changes in F340and F380were constantly in opposite directions. Therefore, the observed changes in F340and F380would reflect changes in the [Ca2+]ibut not motion artifacts. All experiments with the fura-2–loaded strips were conducted during the period in which constant vascular responses were obtained, i.e.  , for approximately 3 h. 25 
Solutions and Drugs
The ionic concentrations of HEPES-buffered PSS were as follows: 138 mm NaCl, 5.0 mm KCl, 1.2 mm MgCl2, 1.5 mm CaCl2, 10 mm HEPES, and 10 mm glucose. The pH was adjusted with NaOH to 7.35 at 35°C. The Ca2+-free solution was prepared by removing CaCl2with or without adding 2 mm EGTA.
HEPES, norepinephrine, acetylcholine, and albumin (bovine) were obtained from Sigma Chemical Co. (St. Louis, MO). Caffeine and EGTA were obtained from Nacalai Tesque (Kyoto, Japan). Fura-2–AM was purchased from Dojindo Laboratories (Kumamoto, Japan). Ryanodine was purchased from Agri Systems International (Wind Gap, PA). Halothane and sevoflurane were obtained from Takeda Pharmaceutical Co. (Osaka, Japan) and Kodama Pharmaceutical Co. (Osaka, Japan), respectively. Enflurane and isoflurane were obtained from Dainihon Pharmaceutical Co. (Osaka, Japan). All other reagents were of the highest grade commercially available.
Experimental Design
The effects of halothane, enflurane, isoflurane, and sevoflurane at 1–5% on the SR were examined in both fura-2–nonloaded and –loaded strips, in the latter of which isometric force and [Ca2+]iwere simultaneously measured. All experiments were conducted at 35°C to prevent early deterioration of the strips, as previously described. 11,21 
We first characterized the SR of this artery by examining the effects of ryanodine on increases in R340/380caused by either caffeine or norepinephrine in the Ca2+-free solution. We also investigated this issue by consecutively applying caffeine and norepinephrine in the Ca2+-free solution in the fura-2–loaded strips.
We next examined the ability of those anesthetics to cause Ca2+release from the SR in both the fura-2–nonloaded and –loaded strips. In this series of experiments, after loading the SR with Ca2+by incubating the strips in PSS (1.5 mm Ca2+) for 8 min, each anesthetic and caffeine were consecutively applied in the Ca2+-free solution at a 5-min interval. In some experiments, procaine, previously suggested to inhibit CICR, 4,28,29 was applied before and during application of either halothane or enflurane.
We then examined the effects of those anesthetics on Ca2+uptake by the SR. For these experiments, after depletion of the SR by caffeine, each anesthetic was applied during Ca2+loading (1.5 mm Ca2+, 8 min), and the amount of Ca2+in SR was estimated from an increase in either force or [Ca2+]icaused by caffeine 2 min after removal of extracellular Ca2+. In some experiments, halothane or enflurane was applied during Ca2+loading with or without procaine, which inhibited the Ca2+releasing action of these anesthetics in the aforementioned experiments.
We finally examined the actions of those anesthetics on either CICR or IP3-induced Ca2+release (IICR) mechanism by examining the effects of each anesthetic on an increase in [Ca2+]ievoked by either caffeine or norepinephrine; after the Ca2+loading, each anesthetic was applied for 5 min before and during the application of either caffeine or norepinephrine in the Ca2+-free solution.
Volatile Anesthetic Delivery and Analysis
Volatile anesthetics were delivered via  calibrated agent-specific vaporizers in line with the air gas aerating the solutions. Each solution was equilibrated with each anesthetic for at least 15 min before introduction to the chamber, which was covered with a thin glass plate to prevent the equilibration gas from escaping into the atmosphere. Using gas chromatography, we previously reported concentrations of each anesthetic in the PSS produced by multiple concentrations of each anesthetic during the same experimental condition; the obtained values were within 90% of theoretical values predicted by the partition coefficient of each anesthetic in Krebs solution or water. 11,30 An excellent linear relation was obtained between the aqueous concentrations of each anesthetic (y) and its concentrations (volume percent) in the gas mixture (x): halothane, y = 0.0033 + 0.30x, r = 0.999; enflurane, y = 0.0019 + 0.29x, r = 0.997; isoflurane, y =−0.0068 + 0.21x, r = 0.998; sevoflurane, y = 0.0028 + 0.13x, r = 0.999. 11,30 Therefore, the concentrations produced by 1–5% halothane, enflurane, isoflurane, and sevoflurane in the PSS can be predicted as 0.3–1.5, 0.29–1.45, 0.21–1.05, and 0.13–0.67 mm, respectively. Recently reported concentrations of halothane, isoflurane, and sevoflurane in blood sampled from this rat during steady state anesthesia with 1 minimum alveolar concentration of each anesthetic (1.0, 1.5, and 2.8% for halothane, isoflurane, and sevoflurane, respectively 31,32) were 0.70, 0.65, and 0.66 mm, respectively. 33 Therefore, the aqueous concentrations of these three anesthetics examined in our study can be considered as clinical concentrations. To our knowledge, no information is available regarding the concentration of enflurane in blood sampled from this rat during steady state anesthesia with enflurane. However, the theoretical blood concentration of enflurane in this rat during anesthesia with 1 minimum alveolar concentration (2.45%34) enflurane calculated using the blood–gas partition coefficient (1.91) is approximately 1.84 mm, which is larger than the concentration produced by 5% enflurane in the PSS in this study, i.e.  , 1.45 mm. Therefore, the concentrations produced by 3–5% enflurane in the PSS in this study also can be considered as clinical concentrations.
Calculation and Data Analysis
Although absolute values of [Ca2+]ihave been calculated based on the fura-2 fluorescence ratio and the dissociation constant of fura-2 for Ca2+binding obtained in vitro  , 23 the dissociation constant of fura-2 for Ca2+binding in cytoplasm appears to be significantly different from that measured in the absence of protein because more than half of the fura-2 molecules in cytoplasm are protein-bound. 35 Therefore, the ratio of F340to F380(R340/380), calculated after subtracting the background fluorescence, was used as an indicator of [Ca2+]i, as previously described. 24–27 
The concentration–response data for the anesthetic-induced increase in either force or R340/380were fitted according to a logistic model described by De Lean et al.  , 36 and the EC50values were derived from the least-squares fit using the aforementioned model. Because the relation between actual concentrations of volatile anesthetics in the solutions and anesthetic concentrations (volume percent) in the gas mixture is theoretically linear, the anesthetic concentrations on the x-axis are displayed as volume percent for the anesthetic concentration–response relations as previously described. 11,12,25 
Statistics
All results are expressed as the mean ± SD. The n denotes the number of preparations (= the number of animals). Data were analyzed using analysis of variance, the Scheffé F test, and the Student t  test. Comparisons among groups were performed by two-factor analysis of variance for repeated measures. When overall differences were detected, individual comparisons among groups at each concentration were performed by the Scheffé F test. Comparisons within each group were made by one-factor analysis of variance for repeated measures, and post hoc  comparisons were made using the Scheffé F test for multiple comparisons. All other necessary comparisons between two groups were made by the Student t  test. P  < 0.05 was considered significant.
Results
Characterization of the Sarcoplasmic Reticulum Ca2+Stores
Caffeine and norepinephrine both produced transient increases in R340/380and force in the Ca2+-free solution in the fura-2–loaded strips. The maximal increases in R340/380and force caused by caffeine (20 mm, maximum) were 158.3 ± 13.2% and 65.5 ± 7.2%, respectively, of that caused by KCl (40 mm) in normal PSS (n = 4). The maximal increases in R340/380and force caused by norepinephrine (10 μm, maximum) were 136.9 ± 19.5% and 109.1 ± 18.1%, respectively, of that caused by KCl (40 mm) in normal PSS (n = 4). Treatment with ryanodine (10 μm, 20 min) consistently eliminated these increases in R340/380and force caused by either caffeine or norepinephrine in the Ca2+-free solution (n = 4). In addition, when caffeine (20 mm) and norepinephrine (10 μm) were consecutively applied in the Ca2+-free solution, caffeine consistently eliminated the increases in R340/380and force caused by subsequently applied norepinephrine (n = 4).
Ability of Volatile Anesthetics to Stimulate Ca2+Release from the Sarcoplasmic Reticulum
When each anesthetic and caffeine were consecutively applied in the Ca2+-free solution in the fura-2–nonloaded strips, both halothane and enflurane, but not isoflurane and sevoflurane, caused transient contractions and inhibited the contractions caused by subsequently applied caffeine (fig. 1). Similarly, when each anesthetic and caffeine were consecutively applied in the Ca2+-free solution in the fura-2–loaded strips, both halothane and enflurane, but not isoflurane and sevoflurane, caused transient increases in R340/380and force and inhibited increases in R340/380and force caused by subsequently applied caffeine (fig. 2). In some strips, 5% isoflurane caused a very small increase in R340/380in the Ca2+-free solution (fig. 2); however, the effect was not statistically significant. No significant differences were observed in the vasoconstricting actions of both halothane and enflurane at 3 and 5% between the fura-2–nonloaded and –loaded strips. Significant differences were observed in the vasoconstricting effects in both the fura-2–nonloaded and –loaded strips and also in the Ca2+-releasing effects in the fura-2–loaded strips between halothane and enflurane, and the order of potency on all of these actions was halothane > enflurane (> isoflurane, sevoflurane) (figs. 1 and 2).
Fig. 1. Contractions in the fura-2–nonloaded strips produced by consecutive applications of either halothane (HAL), enflurane (ENF), isoflurane (ISO), or sevoflurane (SEV) and caffeine in the Ca2+-free solution (n = 4). (A  ) Examples. (Left  ) Control caffeine contractions (CONT). (Right  ) Anesthetic effects. Arrows indicate the time points when the extracellular Ca2+was removed after the Ca2+load. (B  and C  ) Concentration–response data for the consecutive applications of each anesthetic (B  ) and caffeine (C  ). The contractions induced by each anesthetic or caffeine were normalized to the control caffeine contraction (100%). The EC50values for the halothane and enflurane contractions were 1.20 and 3.88%, respectively. * P  < 0.05 versus  control within each group. #P  < 0.05 between halothane and enflurane groups at each concentration.
Fig. 1. Contractions in the fura-2–nonloaded strips produced by consecutive applications of either halothane (HAL), enflurane (ENF), isoflurane (ISO), or sevoflurane (SEV) and caffeine in the Ca2+-free solution (n = 4). (A 
	) Examples. (Left 
	) Control caffeine contractions (CONT). (Right 
	) Anesthetic effects. Arrows indicate the time points when the extracellular Ca2+was removed after the Ca2+load. (B 
	and C 
	) Concentration–response data for the consecutive applications of each anesthetic (B 
	) and caffeine (C 
	). The contractions induced by each anesthetic or caffeine were normalized to the control caffeine contraction (100%). The EC50values for the halothane and enflurane contractions were 1.20 and 3.88%, respectively. * P 
	< 0.05 versus 
	control within each group. #P 
	< 0.05 between halothane and enflurane groups at each concentration.
Fig. 1. Contractions in the fura-2–nonloaded strips produced by consecutive applications of either halothane (HAL), enflurane (ENF), isoflurane (ISO), or sevoflurane (SEV) and caffeine in the Ca2+-free solution (n = 4). (A  ) Examples. (Left  ) Control caffeine contractions (CONT). (Right  ) Anesthetic effects. Arrows indicate the time points when the extracellular Ca2+was removed after the Ca2+load. (B  and C  ) Concentration–response data for the consecutive applications of each anesthetic (B  ) and caffeine (C  ). The contractions induced by each anesthetic or caffeine were normalized to the control caffeine contraction (100%). The EC50values for the halothane and enflurane contractions were 1.20 and 3.88%, respectively. * P  < 0.05 versus  control within each group. #P  < 0.05 between halothane and enflurane groups at each concentration.
×
Fig. 2. Changes in R340/380and force in the fura-2–loaded strips produced by consecutive applications of either halothane (HAL), enflurane (ENF), isoflurane (ISO), or sevoflurane (SEV) and caffeine in the Ca2+-free solution (n = 5 or 6). (A  ) Examples: the responses with (+) or without (−) (control) the previous treatment with each anesthetic (5%) are depicted with gray and black lines, respectively. Because the changes in force observed in this experiment were identical to those shown in the figure 1, only the changes in R340/380are shown. 0Ca-2G = Ca2+-free, 2-mm EGTA solution. Arrows indicate the peaks of responses. (B  and C  ) Concentration–response data for the consecutive applications of each anesthetic (B  ) and caffeine (C  ). The increases in R340/380and force induced by each anesthetic or caffeine were normalized to control (no anesthetic treatment) caffeine-induced increases in R340/380and force, respectively. The EC50values for the halothane-induced increases in R340/380and force were 1.82 and 2.06%, respectively. * P  < 0.05 versus  control within each group. #P  < 0.05 between halothane and enflurane groups at each concentration.
Fig. 2. Changes in R340/380and force in the fura-2–loaded strips produced by consecutive applications of either halothane (HAL), enflurane (ENF), isoflurane (ISO), or sevoflurane (SEV) and caffeine in the Ca2+-free solution (n = 5 or 6). (A 
	) Examples: the responses with (+) or without (−) (control) the previous treatment with each anesthetic (5%) are depicted with gray and black lines, respectively. Because the changes in force observed in this experiment were identical to those shown in the figure 1, only the changes in R340/380are shown. 0Ca-2G = Ca2+-free, 2-mm EGTA solution. Arrows indicate the peaks of responses. (B 
	and C 
	) Concentration–response data for the consecutive applications of each anesthetic (B 
	) and caffeine (C 
	). The increases in R340/380and force induced by each anesthetic or caffeine were normalized to control (no anesthetic treatment) caffeine-induced increases in R340/380and force, respectively. The EC50values for the halothane-induced increases in R340/380and force were 1.82 and 2.06%, respectively. * P 
	< 0.05 versus 
	control within each group. #P 
	< 0.05 between halothane and enflurane groups at each concentration.
Fig. 2. Changes in R340/380and force in the fura-2–loaded strips produced by consecutive applications of either halothane (HAL), enflurane (ENF), isoflurane (ISO), or sevoflurane (SEV) and caffeine in the Ca2+-free solution (n = 5 or 6). (A  ) Examples: the responses with (+) or without (−) (control) the previous treatment with each anesthetic (5%) are depicted with gray and black lines, respectively. Because the changes in force observed in this experiment were identical to those shown in the figure 1, only the changes in R340/380are shown. 0Ca-2G = Ca2+-free, 2-mm EGTA solution. Arrows indicate the peaks of responses. (B  and C  ) Concentration–response data for the consecutive applications of each anesthetic (B  ) and caffeine (C  ). The increases in R340/380and force induced by each anesthetic or caffeine were normalized to control (no anesthetic treatment) caffeine-induced increases in R340/380and force, respectively. The EC50values for the halothane-induced increases in R340/380and force were 1.82 and 2.06%, respectively. * P  < 0.05 versus  control within each group. #P  < 0.05 between halothane and enflurane groups at each concentration.
×
When procaine was applied before and during application of halothane (5%) or enflurane (5%) in the experiments in which each anesthetic and caffeine were consecutively applied in the Ca2+-free solution in either the fura-2–nonloaded or –loaded strips, the increases in either force or R340/380induced by each anesthetic were inhibited strongly or eliminated, as were the anesthetic inhibitions of the increases in either force or R340/380induced by subsequently applied caffeine (fig. 3). The halothane contractions were consistently eliminated by procaine (10 mm) in both the fura-2–nonloaded (n = 4) and –loaded (n = 5) strips (fig. 3). The amplitude of caffeine contraction after previous treatment with halothane + procaine in the fura-2–nonloaded strips was 101.9 ± 7.46% (P  > 0.05, n = 4) of control; the caffeine-induced increases in R340/380and force after previous treatment with halothane + procaine in the fura-2–loaded strips were 92.7 ± 5.3% (P  < 0.05, n = 5) and 98.3 ± 13.7% (P  > 0.05, n = 5), respectively, of control. The enflurane-induced increases in R340/380and force in the presence of procaine (1 mm) in the fura-2–loaded strips were 19.5 ± 14.4% (P  < 0.05, n = 5) and 5.7 ± 12.8% (P  < 0.05, n = 5), respectively, of control, while the caffeine-induced increases in R340/380and force after previous treatment with enflurane + procaine (1 mm) were 94.3 ± 9.3% (P  > 0.05, n = 5) and 103.1 ± 11.4% (P  > 0.05, n = 5), respectively, of control.
Fig. 3. Effects of procaine applied with either halothane (HAL, A  ) or enflurane (ENF, B  ) on responses to consecutively applied each anesthetic and caffeine (20 mm) in the Ca2+-free solution. (A-a  ) An example of the effect of procaine on contractions induced by consecutive application of halothane and caffeine in the fura-2–nonloaded strips. Identical results were obtained in the fura-2–loaded strips (not shown). (A-b  ) A typical example of the effect of procaine on changes in R340/380induced by consecutive application of halothane and caffeine in the fura-2–loaded strips. The changes in R340/380during application of procaine do not reflect those in the [Ca2+]ibecause of its quenching effect on the fluorescence (see Methods). (B-a, b  ) Examples (a  ) and the analyzed data (b  ; n = 3) on the effects of procaine (applied with enflurane) on contractions induced by the consecutive application of enflurane and caffeine in the fura-2–nonloaded strips. The contractions induced by enflurane (open circles) or caffeine (closed circles) were normalized to a control caffeine-induced contraction (100%; see the uppermost trace of B-a  ). CONT = control (no procaine) responses to the consecutive application of enflurane and caffeine. The IC50value for the procaine-induced inhibition of the enflurane contraction was 82.1 μm. * P  < 0.05 versus  control within each group. (B-c  ) An example of the inhibition by procaine of the enflurane-induced increase in R340/380in the fura-2–loaded strips. +/− procaine = in the presence (+, gray) or absence (−, black) of procaine. All arrows indicate the time points when the extracellular Ca2+was removed after the Ca2+load. 0Ca-2G = Ca2+-free, 2-mm EGTA solution.
Fig. 3. Effects of procaine applied with either halothane (HAL, A 
	) or enflurane (ENF, B 
	) on responses to consecutively applied each anesthetic and caffeine (20 mm) in the Ca2+-free solution. (A-a 
	) An example of the effect of procaine on contractions induced by consecutive application of halothane and caffeine in the fura-2–nonloaded strips. Identical results were obtained in the fura-2–loaded strips (not shown). (A-b 
	) A typical example of the effect of procaine on changes in R340/380induced by consecutive application of halothane and caffeine in the fura-2–loaded strips. The changes in R340/380during application of procaine do not reflect those in the [Ca2+]ibecause of its quenching effect on the fluorescence (see Methods). (B-a, b 
	) Examples (a 
	) and the analyzed data (b 
	; n = 3) on the effects of procaine (applied with enflurane) on contractions induced by the consecutive application of enflurane and caffeine in the fura-2–nonloaded strips. The contractions induced by enflurane (open circles) or caffeine (closed circles) were normalized to a control caffeine-induced contraction (100%; see the uppermost trace of B-a 
	). CONT = control (no procaine) responses to the consecutive application of enflurane and caffeine. The IC50value for the procaine-induced inhibition of the enflurane contraction was 82.1 μm. * P 
	< 0.05 versus 
	control within each group. (B-c 
	) An example of the inhibition by procaine of the enflurane-induced increase in R340/380in the fura-2–loaded strips. +/− procaine = in the presence (+, gray) or absence (−, black) of procaine. All arrows indicate the time points when the extracellular Ca2+was removed after the Ca2+load. 0Ca-2G = Ca2+-free, 2-mm EGTA solution.
Fig. 3. Effects of procaine applied with either halothane (HAL, A  ) or enflurane (ENF, B  ) on responses to consecutively applied each anesthetic and caffeine (20 mm) in the Ca2+-free solution. (A-a  ) An example of the effect of procaine on contractions induced by consecutive application of halothane and caffeine in the fura-2–nonloaded strips. Identical results were obtained in the fura-2–loaded strips (not shown). (A-b  ) A typical example of the effect of procaine on changes in R340/380induced by consecutive application of halothane and caffeine in the fura-2–loaded strips. The changes in R340/380during application of procaine do not reflect those in the [Ca2+]ibecause of its quenching effect on the fluorescence (see Methods). (B-a, b  ) Examples (a  ) and the analyzed data (b  ; n = 3) on the effects of procaine (applied with enflurane) on contractions induced by the consecutive application of enflurane and caffeine in the fura-2–nonloaded strips. The contractions induced by enflurane (open circles) or caffeine (closed circles) were normalized to a control caffeine-induced contraction (100%; see the uppermost trace of B-a  ). CONT = control (no procaine) responses to the consecutive application of enflurane and caffeine. The IC50value for the procaine-induced inhibition of the enflurane contraction was 82.1 μm. * P  < 0.05 versus  control within each group. (B-c  ) An example of the inhibition by procaine of the enflurane-induced increase in R340/380in the fura-2–loaded strips. +/− procaine = in the presence (+, gray) or absence (−, black) of procaine. All arrows indicate the time points when the extracellular Ca2+was removed after the Ca2+load. 0Ca-2G = Ca2+-free, 2-mm EGTA solution.
×
Effects of Volatile Anesthetics on Ca2+Uptake by the Sarcoplasmic Reticulum
Halothane (≥ 3%), applied during Ca2+loading, inhibited the caffeine-induced increases in R340/380and force in either the fura-2–nonloaded or –loaded strips, indicating a decrease in amount of Ca2+in the SR (fig. 4). Enflurane (5%), applied during Ca2+loading, also inhibited the caffeine-induced increases in R340/380in the fura-2–loaded strips but did not inhibit the caffeine-induced increases in force in either the fura-2–nonloaded or –loaded strips (fig. 4). Neither isoflurane (5%) nor sevoflurane (5%), applied during Ca2+loading, influenced the caffeine-induced increases in R340/380and force in either the fura-2–nonloaded or –loaded strips (fig. 4).
Fig. 4. Effects of volatile anesthetics on Ca2+uptake by SR. (A  ) Effects of halothane (HAL), enflurane (ENF), isoflurane (ISO), and sevoflurane (SEV), applied during the Ca2+loading, on the caffeine (20 mm)-induced contraction, an estimate for the amount of stored Ca2+, in either absence (left  ) or presence (right  ) of procaine (P; 10 mm for halothane, 1 mm for enflurane) in the fura-2–nonloaded strips (n = 4). * P  < 0.05 versus  control (100%) within each group. #P  < 0.05 versus  the enflurane group at each concentration. ‡P  < 0.05 versus  the procaine-treated group at each concentration in each anesthetic group. (B  ) Effects of halothane (HAL), enflurane (ENF), isoflurane (ISO), and sevoflurane (SEV), applied during Ca2+loading, on the caffeine (20 mm)-induced increases in R340/380(open column) and force (gray column), estimates for the amount of stored Ca2+, in either absence or presence of procaine (PROC; 10 mm for halothane, 1 mm for enflurane) in the fura-2–loaded strips. Effects of procaine alone, applied during Ca2+loading phase, on the caffeine response are also shown (n = 4 or 5). * P  < 0.05 versus  control (100%).
Fig. 4. Effects of volatile anesthetics on Ca2+uptake by SR. (A 
	) Effects of halothane (HAL), enflurane (ENF), isoflurane (ISO), and sevoflurane (SEV), applied during the Ca2+loading, on the caffeine (20 mm)-induced contraction, an estimate for the amount of stored Ca2+, in either absence (left 
	) or presence (right 
	) of procaine (P; 10 mm for halothane, 1 mm for enflurane) in the fura-2–nonloaded strips (n = 4). * P 
	< 0.05 versus 
	control (100%) within each group. #P 
	< 0.05 versus 
	the enflurane group at each concentration. ‡P 
	< 0.05 versus 
	the procaine-treated group at each concentration in each anesthetic group. (B 
	) Effects of halothane (HAL), enflurane (ENF), isoflurane (ISO), and sevoflurane (SEV), applied during Ca2+loading, on the caffeine (20 mm)-induced increases in R340/380(open column) and force (gray column), estimates for the amount of stored Ca2+, in either absence or presence of procaine (PROC; 10 mm for halothane, 1 mm for enflurane) in the fura-2–loaded strips. Effects of procaine alone, applied during Ca2+loading phase, on the caffeine response are also shown (n = 4 or 5). * P 
	< 0.05 versus 
	control (100%).
Fig. 4. Effects of volatile anesthetics on Ca2+uptake by SR. (A  ) Effects of halothane (HAL), enflurane (ENF), isoflurane (ISO), and sevoflurane (SEV), applied during the Ca2+loading, on the caffeine (20 mm)-induced contraction, an estimate for the amount of stored Ca2+, in either absence (left  ) or presence (right  ) of procaine (P; 10 mm for halothane, 1 mm for enflurane) in the fura-2–nonloaded strips (n = 4). * P  < 0.05 versus  control (100%) within each group. #P  < 0.05 versus  the enflurane group at each concentration. ‡P  < 0.05 versus  the procaine-treated group at each concentration in each anesthetic group. (B  ) Effects of halothane (HAL), enflurane (ENF), isoflurane (ISO), and sevoflurane (SEV), applied during Ca2+loading, on the caffeine (20 mm)-induced increases in R340/380(open column) and force (gray column), estimates for the amount of stored Ca2+, in either absence or presence of procaine (PROC; 10 mm for halothane, 1 mm for enflurane) in the fura-2–loaded strips. Effects of procaine alone, applied during Ca2+loading phase, on the caffeine response are also shown (n = 4 or 5). * P  < 0.05 versus  control (100%).
×
Both halothane (≥ 3%) and enflurane (5%), applied during Ca2+loading with procaine, enhanced the caffeine-induced increases in R340/380and force in either the fura-2–loaded or –nonloaded strips (fig. 4). In control experiments, procaine (1 and 10 mm), applied during the Ca2+loading phase, did not influence the caffeine-induced increases in R340/380and force in either the fura-2–nonloaded or –loaded strips (fig. 4); the caffeine-induced increases in force after treatment with 1 and 10 mm procaine in the fura-2–nonloaded strips were 104.1 ± 3.7% and 99.5 ± 5.2%, respectively, of control (P  > 0.05, n = 4).
Effects of Volatile Anesthetics on Caffeine- or Norepinephrine-induced Ca2+Release Mechanisms
Halothane (≥ 1%), enflurane (≥ 1%) and isoflurane (≥ 3%) all enhanced, although only sevoflurane (≥ 3%) attenuated, the caffeine-induced increase in R340/380and force (fig. 5).
Fig. 5. Effects of halothane (HAL), enflurane (ENF), isoflurane (ISO), and sevoflurane (SEV) on the caffeine-induced increases in R340/380and force in the Ca2+-free solution (n = 5). (A  ) Examples: the responses in either presence (+) or absence (−) of each anesthetic are depicted with gray and black lines, respectively. 0Ca-2G = Ca2+-free, 2-mm EGTA solution. Arrows indicate the peaks of responses. (B  and C  ) Concentration–response data for the anesthetic-induced increases in R340/380and force (B  ) and the caffeine-induced increases in R340/380and force in the presence of each anesthetic (C  ). The increases in R340/380and force induced by each anesthetic or caffeine were normalized to control caffeine-induced increases in R340/380and force, respectively. The EC50values for the halothane-induced increases in R340/380and force were 1.16 and 1.20%, respectively. * P  < 0.05 versus  control within each group. #P  < 0.05 between halothane and enflurane groups at each concentration.
Fig. 5. Effects of halothane (HAL), enflurane (ENF), isoflurane (ISO), and sevoflurane (SEV) on the caffeine-induced increases in R340/380and force in the Ca2+-free solution (n = 5). (A 
	) Examples: the responses in either presence (+) or absence (−) of each anesthetic are depicted with gray and black lines, respectively. 0Ca-2G = Ca2+-free, 2-mm EGTA solution. Arrows indicate the peaks of responses. (B 
	and C 
	) Concentration–response data for the anesthetic-induced increases in R340/380and force (B 
	) and the caffeine-induced increases in R340/380and force in the presence of each anesthetic (C 
	). The increases in R340/380and force induced by each anesthetic or caffeine were normalized to control caffeine-induced increases in R340/380and force, respectively. The EC50values for the halothane-induced increases in R340/380and force were 1.16 and 1.20%, respectively. * P 
	< 0.05 versus 
	control within each group. #P 
	< 0.05 between halothane and enflurane groups at each concentration.
Fig. 5. Effects of halothane (HAL), enflurane (ENF), isoflurane (ISO), and sevoflurane (SEV) on the caffeine-induced increases in R340/380and force in the Ca2+-free solution (n = 5). (A  ) Examples: the responses in either presence (+) or absence (−) of each anesthetic are depicted with gray and black lines, respectively. 0Ca-2G = Ca2+-free, 2-mm EGTA solution. Arrows indicate the peaks of responses. (B  and C  ) Concentration–response data for the anesthetic-induced increases in R340/380and force (B  ) and the caffeine-induced increases in R340/380and force in the presence of each anesthetic (C  ). The increases in R340/380and force induced by each anesthetic or caffeine were normalized to control caffeine-induced increases in R340/380and force, respectively. The EC50values for the halothane-induced increases in R340/380and force were 1.16 and 1.20%, respectively. * P  < 0.05 versus  control within each group. #P  < 0.05 between halothane and enflurane groups at each concentration.
×
The four anesthetics influenced the norepinephrine response variously. Halothane (≥ 3%) inhibited the norepinephrine-induced increases in both R340/380and force (fig. 6). However, enflurane did not influence the norepinephrine-induced increases in both R340/380and force (fig. 6). Isoflurane (≥ 3%) enhanced only the norepinephrine-induced increase in force but not its increase in R340/380(fig. 6). Sevoflurane (≥ 3%) enhanced the norepinephrine-induced increases in both R340/380and force (fig. 6).
Fig. 6. Effects of halothane (HAL), enflurane (ENF), isoflurane (ISO), and sevoflurane (SEV) on the norepinephrine-induced increases in R340/380and force in the Ca2+-free solution (n = 4 or 5). (A  ) Examples: the responses in either presence (+) or absence (−) of each anesthetic were depicted with gray and black lines, respectively. 0Ca-2G = Ca2+-free, 2-mm EGTA solution. Arrows indicate the peaks of responses. (B  and C  ) Concentration–response data for the anesthetic-induced increases in R340/380and force (B  ) and the norepinephrine-induced increases in R340/380and force in the presence of each anesthetic (C  ). The increases in R340/380and force induced by each anesthetic or caffeine were normalized to the control norepinephrine-induced increases in R340/380and force, respectively. * P  < 0.05 versus  control within each group. #P  < 0.05 between halothane and enflurane groups at each concentration.
Fig. 6. Effects of halothane (HAL), enflurane (ENF), isoflurane (ISO), and sevoflurane (SEV) on the norepinephrine-induced increases in R340/380and force in the Ca2+-free solution (n = 4 or 5). (A 
	) Examples: the responses in either presence (+) or absence (−) of each anesthetic were depicted with gray and black lines, respectively. 0Ca-2G = Ca2+-free, 2-mm EGTA solution. Arrows indicate the peaks of responses. (B 
	and C 
	) Concentration–response data for the anesthetic-induced increases in R340/380and force (B 
	) and the norepinephrine-induced increases in R340/380and force in the presence of each anesthetic (C 
	). The increases in R340/380and force induced by each anesthetic or caffeine were normalized to the control norepinephrine-induced increases in R340/380and force, respectively. * P 
	< 0.05 versus 
	control within each group. #P 
	< 0.05 between halothane and enflurane groups at each concentration.
Fig. 6. Effects of halothane (HAL), enflurane (ENF), isoflurane (ISO), and sevoflurane (SEV) on the norepinephrine-induced increases in R340/380and force in the Ca2+-free solution (n = 4 or 5). (A  ) Examples: the responses in either presence (+) or absence (−) of each anesthetic were depicted with gray and black lines, respectively. 0Ca-2G = Ca2+-free, 2-mm EGTA solution. Arrows indicate the peaks of responses. (B  and C  ) Concentration–response data for the anesthetic-induced increases in R340/380and force (B  ) and the norepinephrine-induced increases in R340/380and force in the presence of each anesthetic (C  ). The increases in R340/380and force induced by each anesthetic or caffeine were normalized to the control norepinephrine-induced increases in R340/380and force, respectively. * P  < 0.05 versus  control within each group. #P  < 0.05 between halothane and enflurane groups at each concentration.
×
Discussion
Using fura-2 fluorometry, this study provides direct evidence that halothane, enflurane, isoflurane, and sevoflurane at clinical concentrations differentially influence the SR functions in isolated systemic resistance arteries. Presumably because of the volatile anesthetic effects on myofilament Ca2+sensitivity and the nonlinear relation between [Ca2+]iand force, not all of the observed anesthetic effects on R340/380([Ca2+]i) were parallel with the simultaneously observed anesthetic effects on force, as specifically discussed below.
The Ca2+-releasing Action of Volatile Anesthetics
Results from the experiments in which each anesthetic and caffeine were consecutively applied in the Ca2+-free solution indicate that both halothane and enflurane stimulate Ca2+release from the ryanodine–caffeine-sensitive SR, as previously suggested in both conduit and resistance arteries. 8–12,15,16,18 This study confirms the procaine sensitivity of the Ca2+-releasing action of halothane that we previously proposed based on results from contraction experiments. 12 In addition, this study, for the first time, demonstrates that the Ca2+-releasing action of enflurane is also sensitive to procaine. Neither isoflurane nor sevoflurane caused any significant increases in R340/380in the Ca2+-free solution, indicating that both anesthetics (5%, 35°C) do not stimulate Ca2+release from the SR. However, previous experiments performed at 22–23°C 11,14 showed that high concentrations (≥ 2%) of isoflurane cause ryanodine-sensitive, endothelium-independent contractions. Isoflurane may stimulate Ca2+release from the ryanodine-sensitive SR if its aqueous concentration is high or the tissue temperature is low.
Previous studies suggested that halothane and isoflurane may stimulate Ca2+leakage from the IP3-sensitive SR. 17,19,37 As the norepinephrine-induced increases in R340/380in the Ca2+-free solution were eliminated by previous treatment with either ryanodine or caffeine, the IP3-sensitive SR appears to be overlapped with the ryanodine-sensitive SR in this artery, consistent with previous studies suggesting a structural overlap between the both types of SR in smooth muscle. 2,38 Therefore, the anesthetic effects on the ryanodine–caffeine-sensitive SR are undistinguishable from those on the norepinephrine–IP3-sensitive SR in this study.
In our previous experiments, 12 procaine (in millimolar concentrations), proposed to inhibit the CICR, 4,28,29 also inhibited the Ca2+-releasing actions of phenylephrine and IP3, suggesting that procaine is a nonspecific inhibitor of Ca2+release. Therefore, we cannot discuss the mechanisms of the Ca2+-releasing action of halothane or enflurane from their procaine sensitivity. Su et al.  7,8,14,16 previously showed that, in isolated conduit arteries, halothane, enflurane, and isoflurane all facilitated the ryanodine depletion of the SR. As ryanodine depletes the SR by binding to the RyR–CICR channels in an open state and then locking them open, 1 these anesthetics may enhance opening of the RyR–CICR channels. 7,8,14,16 Therefore, the ability of these anesthetics to release Ca2+from the ryanodine-sensitive SR, universally observed in both conduit and resistance arteries, 8–12,15,16,18 may be a result of activation of the RyR–CICR channels. There is direct evidence to indicate that both halothane and enflurane activate the RyR–CICR channels of cardiac SR: in isolated SR vesicles from cardiac muscle, both anesthetics stimulated the [3H]ryanodine binding, an index for the open state probability of the RyR–CICR channels. 39,40 The order of potency for the Ca2+-releasing effects of volatile anesthetics from the ryanodine-sensitive SR observed in this artery (i.e.  , halothane > enflurane [>isoflurane]) is consistent with previous studies in which halothane and enflurane, but not isoflurane, stimulated the [3H]ryanodine binding to cardiac SR. 39,40 Although halothane stimulated IP3formation in erythrocyte membranes, 41 to our knowledge, no convincing evidence is presently available to indicate that volatile anesthetics stimulate IP3formation and thereby cause the Ca2+release in VSM cells.
The Actions of Volatile Anesthetics on Ca2+Uptake
Both halothane and enflurane, applied during Ca2+loading, inhibited the caffeine-induced increase in R340/380as an estimate for the amount of Ca2+in SR, indicating that both anesthetics inhibit Ca2+uptake by the SR. In contrast, neither isoflurane nor sevoflurane, applied during Ca2+loading, influenced the caffeine-induced increase in R340/380, indicating that these anesthetics do not influence the Ca2+uptake. Interestingly, halothane and enflurane, applied during the Ca2+loading with procaine at concentrations sufficient to block their Ca2+-releasing action, conversely enhanced the caffeine- induced increase in R340/380, indicating that these anesthetics enhance the Ca2+uptake after blockade of their Ca2+-releasing action. This suggests that both anesthetics have a stimulating action on Ca2+uptake in addition to the Ca2+-releasing action, and that their inhibitory effects on Ca2+uptake in the absence of procaine were caused by their Ca2+-releasing action but not by inhibition of SR Ca2+–adenosine triphosphatase activity. These suggestions on halothane support previous findings in conduit arteries 7,15,16 and also our previous findings in contraction experiments with the same artery. 11,12 However, the action of enflurane on the Ca2+uptake in systemic resistance artery was, for the first time, described in this study. Our data on the effects of isoflurane or sevoflurane on Ca2+uptake are consistent with the previous findings in contraction experiments at 37°C in isolated mesenteric arteries, 15 but not with those on isoflurane obtained in contraction experiments at 23°C in isolated aorta. 14 In the latter, isoflurane, like halothane and enflurane, also inhibited the Ca2+uptake. 14 Because isoflurane caused Ca2+release in the aorta, 14 the observed inhibitory effect of isoflurane on Ca2+uptake might also be caused by its Ca2+-releasing action.
Effects of Volatile Anesthetics on Ca2+-releasing Mechanisms
Although no direct evidence is presently available, as previously discussed, halothane, enflurane, and isoflurane all appear to activate the RyR–CICR channels in VSM. In support of this idea, in the current study, the increase in R340/380induced by caffeine, believed to activate the CICR, was enhanced in the presence of these anesthetics (despite the reduced amount of Ca2+in the SR on application of caffeine because of their Ca2+-releasing actions). This suggests that these anesthetics activate the CICR. However, sevoflurane inhibited the caffeine-induced increase in R340/380, indicating that sevoflurane attenuates the CICR (a novel finding of this study).
Previous studies have demonstrated that contractile response to caffeine was enhanced in the presence of halothane, enflurane, and isoflurane, suggesting that these anesthetics may enhance the CICR. 7,8,14,15 However, these anesthetics were shown to influence myofilament Ca2+sensitivity in VSM. 11,17,18,25,42 In particular, both enflurane and isoflurane appear to increase the myofilament Ca2+sensitivity. 18,42 Therefore, the enhanced contractile responses to caffeine in the presence of these anesthetics could reflect the anesthetic effects on both the Ca2+sensitivity and [Ca2+]i. In addition, because volatile anesthetics were simultaneously applied with caffeine in the previous studies, 7–9,14,15 the response to caffeine also could reflect the Ca2+-releasing action of the anesthetics. This study, in which changes in [Ca2+]iwere measured and each anesthetic was applied before application of caffeine, for the first time, definitely proves that the three anesthetics enhance the caffeine–CICR mechanism in VSM.
Halothane appeared to attenuate inositol phosphate formation and thereby inhibit the Ca2+release in the cultured VSM cells. 19 In this study, halothane inhibited the norepinephrine-induced increase in R340/380, apparently suggesting that halothane attenuates the IP3-induced Ca2+release. However, because the Ca2+amount in SR had been reduced on application of norepinephrine because of the previous treatment with halothane (which caused the Ca2+release), the effect of halothane on norepinephrine-induced Ca2+release mechanism was unclear from our data. Although a previous study 9 suggested that halothane may not influence the norepinephrine-induced increase in [Ca2+]iin isolated aorta, the effect of halothane on the norepinephrine-induced Ca2+release was also unclear in the previous study 9 because of the protocol used, i.e.  , as halothane was applied simultaneously with norepinephrine in the study, the observed norepinephrine-induced increase in [Ca2+]icould reflect not only the effect of halothane on norepinephrine-induced Ca2+release but also the Ca2+-releasing effect of halothane. Further investigation is needed to clarify this issue.
Isoflurane also appeared to attenuate inositol phosphate formation and thereby inhibit the Ca2+release in the cultured aortic VSM cells. 20 However, in this study, isoflurane did not influence the norepinephrine-induced increase in R340/380, suggesting that isoflurane does not influence the IICR. This appears to be consistent with a previous finding in isolated aorta. 9 The responsiveness of the cultured cells to isoflurane might be altered in the culture process.
Little is known about the effects of enflurane on the IICR. In this study, enflurane did not influence the norepinephrine-induced increase in R340/380, suggesting that enflurane may not influence the IICR. However, as enflurane also caused Ca2+release, the amount of Ca2+in the SR had been reduced on application of norepinephrine because of the previous treatment with enflurane. Therefore, there is a possibility that enflurane stimulates the IICR and thereby counteracted the attenuated norepinephrine response caused by the reduced amount of Ca2+in the SR. However, because the enflurane-induced increase in R340/380(i.e.  , the amount of Ca2+released from SR by enflurane) was rather small, this possibility appears to be minor.
The observed effect of sevoflurane on the norepinephrine-induced increase in R340/380suggests that sevoflurane stimulates the IICR. This is contradictory to our recent suggestion that sevoflurane does not influence the IICR, which was based on its inability to influence the norepinephrine (0.5 μm)-induced increase in R340/380after treatment with verapamil, a Ca2+channel blocker. 25 As verapamil was previously suggested to influence the Ca2+release from intracellular stores, 43 the sensitivity of Ca2+release mechanism to sevoflurane might be altered by the verapamil treatment in our previous study. Alternatively, sevoflurane may enhance only the Ca2+release mechanism induced by higher concentrations of norepinephrine. We attempted to examine the effects of sevoflurane on the increase in R340/380caused by lower concentrations of norepinephrine (≤ 1 μm). However, as the increase in R340/380caused by the low concentrations of norepinephrine in the Ca2+solution were rather small and somewhat unstable, we did not investigate this issue further.
Comparison of the Data between the Ca2+Measurement and Force Recording Experiments
Not all of the observed anesthetic effects on R340/380were parallel with the simultaneously observed anesthetic effects on force. Enflurane (5%), applied during Ca2+loading, inhibited the caffeine-induced increase in R340/380but not the caffeine-induced increase in force. Because the [Ca2+]i–force relation is hyperbolic, the enflurane-induced slight (≈ 10%) decrease in R340/380might not cause any significant decrease in force. Alternatively, because enflurane appeared to increase myofilament Ca2+sensitivity in VSM, 18 the possible prolonged effect of enflurane on the Ca2+sensitivity might counteract its SR-depleting effect.
Isoflurane did not influence the norepinephrine-induced increase in R340/380but enhanced the norepinephrine-induced increase in force, suggesting that isoflurane enhances the myofilament Ca2+sensitivity. This is consistent with previous observations, where isoflurane enhanced Ca2+-activated contractions in skinned VSM. 42 
The changes in force in the presence of volatile anesthetics observed in the fura-2–nonloaded strips were identical to those observed in the fura-2–loaded strips, suggesting that fura-2 loading, a nonphysiologic intervention, does not significantly influence the volatile anesthetic actions on VSM cells.
In conclusion, in VSM cells of systemic resistance arteries, halothane, enflurane, isoflurane, and sevoflurane at clinical concentrations appear to influence either Ca2+mobilization or Ca2+removal from the cytoplasm through direct effects on the SR Ca2+stores, i.e.  , the effects on amount of Ca2+in the SR, Ca2+uptake process, or Ca2+-releasing mechanisms. Although both halothane and enflurane have opposing actions on the amount of Ca2+in the SR (i.e.  , Ca2+-releasing action and a stimulating action on Ca2+uptake), their overall effects appear to reduce the amount of Ca2+in the SR, which could influence vascular tone or reactivity both by decreasing Ca2+availability for VSM contraction and by enhancing the Ca2+-buffering capacity of SR. In addition, all four of these anesthetics may significantly influence vascular reactivity through their effects on the CICR or IICR mechanism. Finally, the observed differences in the direct effects on the SR among the four anesthetics may underlie the differences in their circulatory effects.
The authors thank Masae Yamakawa B.S., (Kyushu University Hospital, Fukuoka, Japan) for her kind assistance in this work.
References
Iino M: Calcium release mechanisms in smooth muscle. Jpn J Pharmacol 1990; 54: 345–54Iino, M
Lesh RE, Nixon GF, Fleischer S, Airey JA, Somlyo AP, Somlyo AV: Localization of ryanodine receptors in smooth muscle. Circ Res 1997; 82: 175–85Lesh, RE Nixon, GF Fleischer, S Airey, JA Somlyo, AP Somlyo, AV
Somlyo AP, Somlyo AV: Signal trasduction and regulation in smooth muscle. Nature 1994; 372: 231–6Somlyo, AP Somlyo, AV
Iino M: Calcium-induced calcium release mechanism in guinea pig taenia caeci. J Gen Physiol 1989; 37: 363–83Iino, M
Jagger JH, Wellman GC, Heppner TJ, Porter VA, Perez GJ, Gollasch M, Kleppisch T, Rubart M, Stevenson AS, Lederer WJ, Knot HJ, Bonev AD, Nelson MT: Ca2+channels, ryanodine receptors and Ca2+-activated K+channels: A functional unit for regulating arterial tone. Acta Physiol Scand 1998; 164: 577–87Jagger, JH Wellman, GC Heppner, TJ Porter, VA Perez, GJ Gollasch, M Kleppisch, T Rubart, M Stevenson, AS Lederer, WJ Knot, HJ Bonev, AD Nelson, MT
Chen Q, van Breemen C: Receptor mediated disruption of the smooth muscle SR buffer barrier function. Jpn J Pharmacol 1992; 58 (suppl II): 60–66Chen, Q van Breemen, C
Su JY, Zhang CC: Intracellular mechanisms of halothane’s effect on isolated aortic strips of the rabbit. A nesthesiology 1989; 71: 409–17Su, JY Zhang, CC
Su JY, Chang YI, Tang LJ: Mechanisms of action of enflurane on vascular smooth muscle: Comparison of rabbit aorta and femoral artery. A nesthesiology 1994; 81: 700–709Su, JY Chang, YI Tang, LJ
Tsuchida H, Namba H, Seki S, Fujita S, Tanaka S, Namiki A: Role of intracellular Ca2+pools in the effects of halothane and isoflurane on vascular smooth muscle contraction. Anesth Analg 1994; 78: 1067–76Tsuchida, H Namba, H Seki, S Fujita, S Tanaka, S Namiki, A
Boyle WA III, Maher GM: Endothelium-independent vasoconstricting and vasodilating actions of halothane on rat mesenteric resistance blood vessels. A nesthesiology 1995; 82: 221–35Boyle, WA Maher, GM
Akata T, Boyle WA III: Volatile anesthetic actions on contractile proteins in membrane-permeabilized small mesenteric arteries. A nesthesiology 1995; 82: 700–12Akata, T Boyle, WA
Akata T, Boyle WA III: Dual actions of halothane on intracellular calcium stores of vascular smooth muscle. A nesthesiology 1996; 84: 580–95Akata, T Boyle, WA
Namba H, Tsuchida H: Effect of volatile anesthetics with or without verapamil on intracellular activity in vascular smooth muscle. A nesthesiology 1996; 84: 1465–74Namba, H Tsuchida, H
Su JY: Mechanisms of action of isoflurane on contraction of rabbit conduit artery. A nesthesiology 1996; 82: 837–42Su, JY
Yamamoto M, Hatano Y, Kakuyama M, Tachibana T, Maeda H, Mori K: Different effects of halothane, isoflurane and sevoflurane on sarcoplasmic reticulum of vascular smooth muscle in dog mesenteric artery. Acta Anaesthesiol Scand 1997; 41: 376–80Yamamoto, M Hatano, Y Kakuyama, M Tachibana, T Maeda, H Mori, K
Su JY, Tang L-J: Effects of halothane on the sarcoplasmic reticulum Ca2+stores and contractile proteins in rabbit pulmonary arteries. A nesthesiology 1998; 88: 1096–106Su, JY Tang, L-J
Tsuchida H, Namba H, Yamakage M, Fujita S, Notsuki E, Namiki A: Effects of halothane and isoflurane on cytosolic calcium ion concentrations and contraction in vascular smooth muscle of the rat aorta. A nesthesiology 1993; 78: 531–40Tsuchida, H Namba, H Yamakage, M Fujita, S Notsuki, E Namiki, A
Kakuyama M, Hatano Y, Nakamura K, Toda H, Terasato K, Nishiwada M, Mori K: Halothane and enflurane constrict canine mesenteric arteries by releasing Ca2+from intracellular Ca2+stores. A nesthesiology 1994; 80: 1120–7Kakuyama, M Hatano, Y Nakamura, K Toda, H Terasato, K Nishiwada, M Mori, K
Sill JC, Uhl C, Eskuri S, Van Dyke RA, Tarara J: Halothane inhibits agonist-induced inositol phosphate and Ca2+signaling in A7r5 cultured vascular smooth muscle cells. Mol Pharmacol 1991; 40: 1006–13Sill, JC Uhl, C Eskuri, S Van Dyke, RA Tarara, J
Sill JC, Eskuri S, Nelson R, Van Dyke RA, Tarara J: The volatile anesthetic isoflurane attenuates Ca2+mobilization in cultured vascular smooth muscle cells. J Pharmacol Exp Ther 1993; 265: 74–80Sill, JC Eskuri, S Nelson, R Van Dyke, RA Tarara, J
Akata T, Yoshitake J, Nakashima M, Itoh T: Effects of protamine on vascular smooth muscle of rabbit mesenteric artery. A nesthesiology 1991; 75: 833–46Akata, T Yoshitake, J Nakashima, M Itoh, T
Akata T, Nakashima M, Kodama K, Boyle WA III, Takahashi S: Effects of volatile anesthetics on ACh-induced relaxation in the rabbit mesenteric resistance artery. A nesthesiology 1995; 82: 188–204Akata, T Nakashima, M Kodama, K Boyle, WA Takahashi, S
Grynkiewicz G, Poenie M, Tsien RY: A new generation of Ca2+indicators with greatly improved fluorescence properties. J Biol Chem 1985; 260: 3440–50Grynkiewicz, G Poenie, M Tsien, RY
Akata T, Kodama K, Evers A, Takahashi S: Protamine relaxes vascular smooth muscle by directly reducing cytosolic calcium concentrations in small resistance arteries. J Anesth 1996; 10: 252–9Akata, T Kodama, K Evers, A Takahashi, S
Akata T, Izumi K, Nakashima M: The action of sevoflurane on vascular smooth muscle of isolated mesenteric resistance arteries. Part II: Mechanisms of endothelium-independent vasorelaxation. A nesthesiology 2000; 92: 1441–53Akata, T Izumi, K Nakashima, M
Kageyama M, Mori T, Yanagisawa T, Taira N: Is staurosporine a specific inhibitor of protein kinase C in intact porcine coronary arteries? J Pharmacol Exp Ther 1991; 259: 1019–26Kageyama, M Mori, T Yanagisawa, T Taira, N
Yanagisawa T, Okada Y: KCl depolarization increases Ca2+sensitivity of contractile elements in coronary arterial smooth muscle. Am J Physiol 1994; 267: H614–21Yanagisawa, T Okada, Y
Somlyo AV, Horiuti K, Trentham DR, Kitazawa T, Somlyo AP: Kinetics of Ca2+release and contraction induced by photolysis of caged D-myo-inositol 1,4,5-triphosphate in smooth muscle. J Biol Chem 1992; 267: 22316–22Somlyo, AV Horiuti, K Trentham, DR Kitazawa, T Somlyo, AP
Endo M: Calcium release from the sarcoplasmic reticulum. Physiol Rev 1977; 57: 71–108Endo, M
Akata T, Kodama K, Takahashi S: Volatile anesthetic actions on norepinephrine-induced contraction of small splanchnic resistance arteries. Can J Anaesth 1995; 42: 1040–50Akata, T Kodama, K Takahashi, S
Drummond JC: MAC for halothane, enflurane, and isoflurane in the New Zealand white rabbit: And a test for the validity of MAC determinations. A nesthesiology 1985; 62: 336–8Drummond, JC
Taheri S, Halsey MJ, Liu J, Eger II EI, Koblin DD, Laster MJ: What solvent best represents the site of action of inhaled anesthetics in humans, rats, and dogs? Anesth Analg 1991; 72:627–34
Yamazaki M, Stekiel TA, Bosnjak ZJ, Kampine JP, Stekiel WJ: Effects of volatile anesthetic agents on in situ vascular smooth muscle transmembrane potential in resistance- and capacitance-regulating blood vessels. A nesthesiology 1998; 88: 1085–95Yamazaki, M Stekiel, TA Bosnjak, ZJ Kampine, JP Stekiel, WJ
Cole DJ, Kalichman MW, Shapiro HM, Drummond JC: The nonlinear potency of sub-MAC concentrations of nitrous oxide in decreasing the anesthetic requirement of enflurane, halothane, and isoflurane in rats. A nesthesiology 1990; 73: 93–9Cole, DJ Kalichman, MW Shapiro, HM Drummond, JC
Konishi M, Olson A, Hollingworth S, Baylor SM: Myoplasmic binding of fura-2 investigated by steady-state fluorescence and absorbance measurements. Biophys J 1988; 54: 1089–1104Konishi, M Olson, A Hollingworth, S Baylor, SM
De Lean AP, Munson PJ, Rodbard D: Simultaneous analysis of families of sigmoidal curves: Application to bioassay, radioligand assay, and physiological dose-response curves. Am J Physiol 1978; 235: E97–102De Lean, AP Munson, PJ Rodbard, D
Missiaen L, Declerck I, Droogmans G, Plessers L, De Smedt H, Raeymaekers L, Casteels R: Agonist-dependent Ca2+and Mn2+entry dependent on state of filling of Ca2+stores in aortic smooth muscle cells of rat. J Physiol (Lond) 1990; 427: 171–86Missiaen, L Declerck, I Droogmans, G Plessers, L De Smedt, H Raeymaekers, L Casteels, R
Wibo M, Godfraind T: Comparative localization of inositol 1,4,5-triphosphate and ryanodine receptors in intestinal smooth muscle: An analytical subfractionation study. Biochem J 1994; 297: 415–23Wibo, M Godfraind, T
Connelly TJ, Hayek R-E, Rusy BF, Coronado R: Volatile anesthetics selectively alter [3H]ryanodine binding to skeletal and cardiac ryanodine receptors. Biochem Biophys Res Com 1992; 186: 595–600Connelly, TJ Hayek, R-E Rusy, BF Coronado, R
Lynch C III, Frazer MJ: Anesthetic alteration of ryanodine binding by cardiac calcium release channels. Biochem Biophys Acta 1994; 1194: 109–117Lynch, C Frazer, MJ
Rooney TA, Hager R, Stubbs CD, Thomas AP: Halothane regulates G-protein-dependent phopholipase C activity in turkey erythrocyte membranes. J Biol Chem 1993; 268: 15550–6Rooney, TA Hager, R Stubbs, CD Thomas, AP
Toda H, Su JY: Mechanisms of isoflurane-increased submaximum Ca2+-activated force in rabbit skinned femoral arterial strips. A nesthesiology 1998; 89: 731–40Toda, H Su, JY
Kobayashi S, Gong MC, Somlyo AV, Somlyo AP: Ca2+channel blockers distinguish between G-protein-coupled pharmacomechanical Ca2+release and Ca2+sensitization. Am J Physiol 1991; 260: C364–70Kobayashi, S Gong, MC Somlyo, AV Somlyo, AP
Fig. 1. Contractions in the fura-2–nonloaded strips produced by consecutive applications of either halothane (HAL), enflurane (ENF), isoflurane (ISO), or sevoflurane (SEV) and caffeine in the Ca2+-free solution (n = 4). (A  ) Examples. (Left  ) Control caffeine contractions (CONT). (Right  ) Anesthetic effects. Arrows indicate the time points when the extracellular Ca2+was removed after the Ca2+load. (B  and C  ) Concentration–response data for the consecutive applications of each anesthetic (B  ) and caffeine (C  ). The contractions induced by each anesthetic or caffeine were normalized to the control caffeine contraction (100%). The EC50values for the halothane and enflurane contractions were 1.20 and 3.88%, respectively. * P  < 0.05 versus  control within each group. #P  < 0.05 between halothane and enflurane groups at each concentration.
Fig. 1. Contractions in the fura-2–nonloaded strips produced by consecutive applications of either halothane (HAL), enflurane (ENF), isoflurane (ISO), or sevoflurane (SEV) and caffeine in the Ca2+-free solution (n = 4). (A 
	) Examples. (Left 
	) Control caffeine contractions (CONT). (Right 
	) Anesthetic effects. Arrows indicate the time points when the extracellular Ca2+was removed after the Ca2+load. (B 
	and C 
	) Concentration–response data for the consecutive applications of each anesthetic (B 
	) and caffeine (C 
	). The contractions induced by each anesthetic or caffeine were normalized to the control caffeine contraction (100%). The EC50values for the halothane and enflurane contractions were 1.20 and 3.88%, respectively. * P 
	< 0.05 versus 
	control within each group. #P 
	< 0.05 between halothane and enflurane groups at each concentration.
Fig. 1. Contractions in the fura-2–nonloaded strips produced by consecutive applications of either halothane (HAL), enflurane (ENF), isoflurane (ISO), or sevoflurane (SEV) and caffeine in the Ca2+-free solution (n = 4). (A  ) Examples. (Left  ) Control caffeine contractions (CONT). (Right  ) Anesthetic effects. Arrows indicate the time points when the extracellular Ca2+was removed after the Ca2+load. (B  and C  ) Concentration–response data for the consecutive applications of each anesthetic (B  ) and caffeine (C  ). The contractions induced by each anesthetic or caffeine were normalized to the control caffeine contraction (100%). The EC50values for the halothane and enflurane contractions were 1.20 and 3.88%, respectively. * P  < 0.05 versus  control within each group. #P  < 0.05 between halothane and enflurane groups at each concentration.
×
Fig. 2. Changes in R340/380and force in the fura-2–loaded strips produced by consecutive applications of either halothane (HAL), enflurane (ENF), isoflurane (ISO), or sevoflurane (SEV) and caffeine in the Ca2+-free solution (n = 5 or 6). (A  ) Examples: the responses with (+) or without (−) (control) the previous treatment with each anesthetic (5%) are depicted with gray and black lines, respectively. Because the changes in force observed in this experiment were identical to those shown in the figure 1, only the changes in R340/380are shown. 0Ca-2G = Ca2+-free, 2-mm EGTA solution. Arrows indicate the peaks of responses. (B  and C  ) Concentration–response data for the consecutive applications of each anesthetic (B  ) and caffeine (C  ). The increases in R340/380and force induced by each anesthetic or caffeine were normalized to control (no anesthetic treatment) caffeine-induced increases in R340/380and force, respectively. The EC50values for the halothane-induced increases in R340/380and force were 1.82 and 2.06%, respectively. * P  < 0.05 versus  control within each group. #P  < 0.05 between halothane and enflurane groups at each concentration.
Fig. 2. Changes in R340/380and force in the fura-2–loaded strips produced by consecutive applications of either halothane (HAL), enflurane (ENF), isoflurane (ISO), or sevoflurane (SEV) and caffeine in the Ca2+-free solution (n = 5 or 6). (A 
	) Examples: the responses with (+) or without (−) (control) the previous treatment with each anesthetic (5%) are depicted with gray and black lines, respectively. Because the changes in force observed in this experiment were identical to those shown in the figure 1, only the changes in R340/380are shown. 0Ca-2G = Ca2+-free, 2-mm EGTA solution. Arrows indicate the peaks of responses. (B 
	and C 
	) Concentration–response data for the consecutive applications of each anesthetic (B 
	) and caffeine (C 
	). The increases in R340/380and force induced by each anesthetic or caffeine were normalized to control (no anesthetic treatment) caffeine-induced increases in R340/380and force, respectively. The EC50values for the halothane-induced increases in R340/380and force were 1.82 and 2.06%, respectively. * P 
	< 0.05 versus 
	control within each group. #P 
	< 0.05 between halothane and enflurane groups at each concentration.
Fig. 2. Changes in R340/380and force in the fura-2–loaded strips produced by consecutive applications of either halothane (HAL), enflurane (ENF), isoflurane (ISO), or sevoflurane (SEV) and caffeine in the Ca2+-free solution (n = 5 or 6). (A  ) Examples: the responses with (+) or without (−) (control) the previous treatment with each anesthetic (5%) are depicted with gray and black lines, respectively. Because the changes in force observed in this experiment were identical to those shown in the figure 1, only the changes in R340/380are shown. 0Ca-2G = Ca2+-free, 2-mm EGTA solution. Arrows indicate the peaks of responses. (B  and C  ) Concentration–response data for the consecutive applications of each anesthetic (B  ) and caffeine (C  ). The increases in R340/380and force induced by each anesthetic or caffeine were normalized to control (no anesthetic treatment) caffeine-induced increases in R340/380and force, respectively. The EC50values for the halothane-induced increases in R340/380and force were 1.82 and 2.06%, respectively. * P  < 0.05 versus  control within each group. #P  < 0.05 between halothane and enflurane groups at each concentration.
×
Fig. 3. Effects of procaine applied with either halothane (HAL, A  ) or enflurane (ENF, B  ) on responses to consecutively applied each anesthetic and caffeine (20 mm) in the Ca2+-free solution. (A-a  ) An example of the effect of procaine on contractions induced by consecutive application of halothane and caffeine in the fura-2–nonloaded strips. Identical results were obtained in the fura-2–loaded strips (not shown). (A-b  ) A typical example of the effect of procaine on changes in R340/380induced by consecutive application of halothane and caffeine in the fura-2–loaded strips. The changes in R340/380during application of procaine do not reflect those in the [Ca2+]ibecause of its quenching effect on the fluorescence (see Methods). (B-a, b  ) Examples (a  ) and the analyzed data (b  ; n = 3) on the effects of procaine (applied with enflurane) on contractions induced by the consecutive application of enflurane and caffeine in the fura-2–nonloaded strips. The contractions induced by enflurane (open circles) or caffeine (closed circles) were normalized to a control caffeine-induced contraction (100%; see the uppermost trace of B-a  ). CONT = control (no procaine) responses to the consecutive application of enflurane and caffeine. The IC50value for the procaine-induced inhibition of the enflurane contraction was 82.1 μm. * P  < 0.05 versus  control within each group. (B-c  ) An example of the inhibition by procaine of the enflurane-induced increase in R340/380in the fura-2–loaded strips. +/− procaine = in the presence (+, gray) or absence (−, black) of procaine. All arrows indicate the time points when the extracellular Ca2+was removed after the Ca2+load. 0Ca-2G = Ca2+-free, 2-mm EGTA solution.
Fig. 3. Effects of procaine applied with either halothane (HAL, A 
	) or enflurane (ENF, B 
	) on responses to consecutively applied each anesthetic and caffeine (20 mm) in the Ca2+-free solution. (A-a 
	) An example of the effect of procaine on contractions induced by consecutive application of halothane and caffeine in the fura-2–nonloaded strips. Identical results were obtained in the fura-2–loaded strips (not shown). (A-b 
	) A typical example of the effect of procaine on changes in R340/380induced by consecutive application of halothane and caffeine in the fura-2–loaded strips. The changes in R340/380during application of procaine do not reflect those in the [Ca2+]ibecause of its quenching effect on the fluorescence (see Methods). (B-a, b 
	) Examples (a 
	) and the analyzed data (b 
	; n = 3) on the effects of procaine (applied with enflurane) on contractions induced by the consecutive application of enflurane and caffeine in the fura-2–nonloaded strips. The contractions induced by enflurane (open circles) or caffeine (closed circles) were normalized to a control caffeine-induced contraction (100%; see the uppermost trace of B-a 
	). CONT = control (no procaine) responses to the consecutive application of enflurane and caffeine. The IC50value for the procaine-induced inhibition of the enflurane contraction was 82.1 μm. * P 
	< 0.05 versus 
	control within each group. (B-c 
	) An example of the inhibition by procaine of the enflurane-induced increase in R340/380in the fura-2–loaded strips. +/− procaine = in the presence (+, gray) or absence (−, black) of procaine. All arrows indicate the time points when the extracellular Ca2+was removed after the Ca2+load. 0Ca-2G = Ca2+-free, 2-mm EGTA solution.
Fig. 3. Effects of procaine applied with either halothane (HAL, A  ) or enflurane (ENF, B  ) on responses to consecutively applied each anesthetic and caffeine (20 mm) in the Ca2+-free solution. (A-a  ) An example of the effect of procaine on contractions induced by consecutive application of halothane and caffeine in the fura-2–nonloaded strips. Identical results were obtained in the fura-2–loaded strips (not shown). (A-b  ) A typical example of the effect of procaine on changes in R340/380induced by consecutive application of halothane and caffeine in the fura-2–loaded strips. The changes in R340/380during application of procaine do not reflect those in the [Ca2+]ibecause of its quenching effect on the fluorescence (see Methods). (B-a, b  ) Examples (a  ) and the analyzed data (b  ; n = 3) on the effects of procaine (applied with enflurane) on contractions induced by the consecutive application of enflurane and caffeine in the fura-2–nonloaded strips. The contractions induced by enflurane (open circles) or caffeine (closed circles) were normalized to a control caffeine-induced contraction (100%; see the uppermost trace of B-a  ). CONT = control (no procaine) responses to the consecutive application of enflurane and caffeine. The IC50value for the procaine-induced inhibition of the enflurane contraction was 82.1 μm. * P  < 0.05 versus  control within each group. (B-c  ) An example of the inhibition by procaine of the enflurane-induced increase in R340/380in the fura-2–loaded strips. +/− procaine = in the presence (+, gray) or absence (−, black) of procaine. All arrows indicate the time points when the extracellular Ca2+was removed after the Ca2+load. 0Ca-2G = Ca2+-free, 2-mm EGTA solution.
×
Fig. 4. Effects of volatile anesthetics on Ca2+uptake by SR. (A  ) Effects of halothane (HAL), enflurane (ENF), isoflurane (ISO), and sevoflurane (SEV), applied during the Ca2+loading, on the caffeine (20 mm)-induced contraction, an estimate for the amount of stored Ca2+, in either absence (left  ) or presence (right  ) of procaine (P; 10 mm for halothane, 1 mm for enflurane) in the fura-2–nonloaded strips (n = 4). * P  < 0.05 versus  control (100%) within each group. #P  < 0.05 versus  the enflurane group at each concentration. ‡P  < 0.05 versus  the procaine-treated group at each concentration in each anesthetic group. (B  ) Effects of halothane (HAL), enflurane (ENF), isoflurane (ISO), and sevoflurane (SEV), applied during Ca2+loading, on the caffeine (20 mm)-induced increases in R340/380(open column) and force (gray column), estimates for the amount of stored Ca2+, in either absence or presence of procaine (PROC; 10 mm for halothane, 1 mm for enflurane) in the fura-2–loaded strips. Effects of procaine alone, applied during Ca2+loading phase, on the caffeine response are also shown (n = 4 or 5). * P  < 0.05 versus  control (100%).
Fig. 4. Effects of volatile anesthetics on Ca2+uptake by SR. (A 
	) Effects of halothane (HAL), enflurane (ENF), isoflurane (ISO), and sevoflurane (SEV), applied during the Ca2+loading, on the caffeine (20 mm)-induced contraction, an estimate for the amount of stored Ca2+, in either absence (left 
	) or presence (right 
	) of procaine (P; 10 mm for halothane, 1 mm for enflurane) in the fura-2–nonloaded strips (n = 4). * P 
	< 0.05 versus 
	control (100%) within each group. #P 
	< 0.05 versus 
	the enflurane group at each concentration. ‡P 
	< 0.05 versus 
	the procaine-treated group at each concentration in each anesthetic group. (B 
	) Effects of halothane (HAL), enflurane (ENF), isoflurane (ISO), and sevoflurane (SEV), applied during Ca2+loading, on the caffeine (20 mm)-induced increases in R340/380(open column) and force (gray column), estimates for the amount of stored Ca2+, in either absence or presence of procaine (PROC; 10 mm for halothane, 1 mm for enflurane) in the fura-2–loaded strips. Effects of procaine alone, applied during Ca2+loading phase, on the caffeine response are also shown (n = 4 or 5). * P 
	< 0.05 versus 
	control (100%).
Fig. 4. Effects of volatile anesthetics on Ca2+uptake by SR. (A  ) Effects of halothane (HAL), enflurane (ENF), isoflurane (ISO), and sevoflurane (SEV), applied during the Ca2+loading, on the caffeine (20 mm)-induced contraction, an estimate for the amount of stored Ca2+, in either absence (left  ) or presence (right  ) of procaine (P; 10 mm for halothane, 1 mm for enflurane) in the fura-2–nonloaded strips (n = 4). * P  < 0.05 versus  control (100%) within each group. #P  < 0.05 versus  the enflurane group at each concentration. ‡P  < 0.05 versus  the procaine-treated group at each concentration in each anesthetic group. (B  ) Effects of halothane (HAL), enflurane (ENF), isoflurane (ISO), and sevoflurane (SEV), applied during Ca2+loading, on the caffeine (20 mm)-induced increases in R340/380(open column) and force (gray column), estimates for the amount of stored Ca2+, in either absence or presence of procaine (PROC; 10 mm for halothane, 1 mm for enflurane) in the fura-2–loaded strips. Effects of procaine alone, applied during Ca2+loading phase, on the caffeine response are also shown (n = 4 or 5). * P  < 0.05 versus  control (100%).
×
Fig. 5. Effects of halothane (HAL), enflurane (ENF), isoflurane (ISO), and sevoflurane (SEV) on the caffeine-induced increases in R340/380and force in the Ca2+-free solution (n = 5). (A  ) Examples: the responses in either presence (+) or absence (−) of each anesthetic are depicted with gray and black lines, respectively. 0Ca-2G = Ca2+-free, 2-mm EGTA solution. Arrows indicate the peaks of responses. (B  and C  ) Concentration–response data for the anesthetic-induced increases in R340/380and force (B  ) and the caffeine-induced increases in R340/380and force in the presence of each anesthetic (C  ). The increases in R340/380and force induced by each anesthetic or caffeine were normalized to control caffeine-induced increases in R340/380and force, respectively. The EC50values for the halothane-induced increases in R340/380and force were 1.16 and 1.20%, respectively. * P  < 0.05 versus  control within each group. #P  < 0.05 between halothane and enflurane groups at each concentration.
Fig. 5. Effects of halothane (HAL), enflurane (ENF), isoflurane (ISO), and sevoflurane (SEV) on the caffeine-induced increases in R340/380and force in the Ca2+-free solution (n = 5). (A 
	) Examples: the responses in either presence (+) or absence (−) of each anesthetic are depicted with gray and black lines, respectively. 0Ca-2G = Ca2+-free, 2-mm EGTA solution. Arrows indicate the peaks of responses. (B 
	and C 
	) Concentration–response data for the anesthetic-induced increases in R340/380and force (B 
	) and the caffeine-induced increases in R340/380and force in the presence of each anesthetic (C 
	). The increases in R340/380and force induced by each anesthetic or caffeine were normalized to control caffeine-induced increases in R340/380and force, respectively. The EC50values for the halothane-induced increases in R340/380and force were 1.16 and 1.20%, respectively. * P 
	< 0.05 versus 
	control within each group. #P 
	< 0.05 between halothane and enflurane groups at each concentration.
Fig. 5. Effects of halothane (HAL), enflurane (ENF), isoflurane (ISO), and sevoflurane (SEV) on the caffeine-induced increases in R340/380and force in the Ca2+-free solution (n = 5). (A  ) Examples: the responses in either presence (+) or absence (−) of each anesthetic are depicted with gray and black lines, respectively. 0Ca-2G = Ca2+-free, 2-mm EGTA solution. Arrows indicate the peaks of responses. (B  and C  ) Concentration–response data for the anesthetic-induced increases in R340/380and force (B  ) and the caffeine-induced increases in R340/380and force in the presence of each anesthetic (C  ). The increases in R340/380and force induced by each anesthetic or caffeine were normalized to control caffeine-induced increases in R340/380and force, respectively. The EC50values for the halothane-induced increases in R340/380and force were 1.16 and 1.20%, respectively. * P  < 0.05 versus  control within each group. #P  < 0.05 between halothane and enflurane groups at each concentration.
×
Fig. 6. Effects of halothane (HAL), enflurane (ENF), isoflurane (ISO), and sevoflurane (SEV) on the norepinephrine-induced increases in R340/380and force in the Ca2+-free solution (n = 4 or 5). (A  ) Examples: the responses in either presence (+) or absence (−) of each anesthetic were depicted with gray and black lines, respectively. 0Ca-2G = Ca2+-free, 2-mm EGTA solution. Arrows indicate the peaks of responses. (B  and C  ) Concentration–response data for the anesthetic-induced increases in R340/380and force (B  ) and the norepinephrine-induced increases in R340/380and force in the presence of each anesthetic (C  ). The increases in R340/380and force induced by each anesthetic or caffeine were normalized to the control norepinephrine-induced increases in R340/380and force, respectively. * P  < 0.05 versus  control within each group. #P  < 0.05 between halothane and enflurane groups at each concentration.
Fig. 6. Effects of halothane (HAL), enflurane (ENF), isoflurane (ISO), and sevoflurane (SEV) on the norepinephrine-induced increases in R340/380and force in the Ca2+-free solution (n = 4 or 5). (A 
	) Examples: the responses in either presence (+) or absence (−) of each anesthetic were depicted with gray and black lines, respectively. 0Ca-2G = Ca2+-free, 2-mm EGTA solution. Arrows indicate the peaks of responses. (B 
	and C 
	) Concentration–response data for the anesthetic-induced increases in R340/380and force (B 
	) and the norepinephrine-induced increases in R340/380and force in the presence of each anesthetic (C 
	). The increases in R340/380and force induced by each anesthetic or caffeine were normalized to the control norepinephrine-induced increases in R340/380and force, respectively. * P 
	< 0.05 versus 
	control within each group. #P 
	< 0.05 between halothane and enflurane groups at each concentration.
Fig. 6. Effects of halothane (HAL), enflurane (ENF), isoflurane (ISO), and sevoflurane (SEV) on the norepinephrine-induced increases in R340/380and force in the Ca2+-free solution (n = 4 or 5). (A  ) Examples: the responses in either presence (+) or absence (−) of each anesthetic were depicted with gray and black lines, respectively. 0Ca-2G = Ca2+-free, 2-mm EGTA solution. Arrows indicate the peaks of responses. (B  and C  ) Concentration–response data for the anesthetic-induced increases in R340/380and force (B  ) and the norepinephrine-induced increases in R340/380and force in the presence of each anesthetic (C  ). The increases in R340/380and force induced by each anesthetic or caffeine were normalized to the control norepinephrine-induced increases in R340/380and force, respectively. * P  < 0.05 versus  control within each group. #P  < 0.05 between halothane and enflurane groups at each concentration.
×