Comparison of Volatile Anesthetic Actions on Intracellular Calcium Stores of Vascular Smooth Muscle: Investigation in Isolated Systemic Resistance Arteries

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. 11,21Briefly, 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. 23Our method on the fura-2 fluorometry was also previously detailed. 24,25Briefly, 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. 24,25Changes 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. 25–27The ratio (R^340/380) 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, 28quenched both the F³⁴⁰and F³⁸⁰signals and thereby decreased the R^340/380 in fura-2 (2 μm)-containing Ca²⁺free solution or normal PSS (regardless of the presence of the strips). Therefore, the changes in R^340/380during application of the high concentrations of procaine were not evaluated. In all of the measurements except those with procaine, as we recently showed, 25changes 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 

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.

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 R^340/380caused 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, 4,28,29was 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 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. 11,30An 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. 11,30Therefore, 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. 33Therefore, 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.

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 , 23the 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. 35Therefore, the ratio of F³⁴⁰to F³⁸⁰(R^340/380), 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 R^340/380were fitted according to a logistic model described by De Lean et al. , 36and 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 

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.

Caffeine and norepinephrine both produced transient increases in R^340/380and force in the Ca²⁺-free solution in the fura-2–loaded strips. The maximal increases in R^340/380and 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 R^340/380and 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 R^340/380and 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 R^340/380and force caused by subsequently applied norepinephrine (n = 4).

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 R^340/380and force and inhibited increases in R^340/380and force caused by subsequently applied caffeine (fig. 2). In some strips, 5% isoflurane caused a very small increase in R^340/380in 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).

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 R^340/380induced by each anesthetic were inhibited strongly or eliminated, as were the anesthetic inhibitions of the increases in either force or R^340/380induced 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 R^340/380and 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 R^340/380and 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 R^340/380and 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.

Halothane (≥ 3%), applied during Ca²⁺loading, inhibited the caffeine-induced increases in R^340/380and 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 R^340/380in 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 R^340/380and force in either the fura-2–nonloaded or –loaded strips (fig. 4).

Both halothane (≥ 3%) and enflurane (5%), applied during Ca²⁺loading with procaine, enhanced the caffeine-induced increases in R^340/380and 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 R^340/380and 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).

Halothane (≥ 1%), enflurane (≥ 1%) and isoflurane (≥ 3%) all enhanced, although only sevoflurane (≥ 3%) attenuated, the caffeine-induced increase in R^340/380and force (fig. 5).

The four anesthetics influenced the norepinephrine response variously. Halothane (≥ 3%) inhibited the norepinephrine-induced increases in both R^340/380and force (fig. 6). However, enflurane did not influence the norepinephrine-induced increases in both R^340/380and force (fig. 6). Isoflurane (≥ 3%) enhanced only the norepinephrine-induced increase in force but not its increase in R^340/380(fig. 6). Sevoflurane (≥ 3%) enhanced the norepinephrine-induced increases in both R^340/380and force (fig. 6).

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 R^340/380([Ca²⁺]ⁱ) were parallel with the simultaneously observed anesthetic effects on force, as specifically discussed below.

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. 8–12,15,16,18This study confirms the procaine sensitivity of the Ca²⁺-releasing action of halothane that we previously proposed based on results from contraction experiments. 12In 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 R^340/380in 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 11,14showed 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. 17,19,37As the norepinephrine-induced increases in R^340/380in 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. 2,38Therefore, 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, 12procaine (in millimolar concentrations), proposed to inhibit the CICR, 4,28,29also 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.  7,8,14,16previously 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, 1these anesthetics may enhance opening of the RyR–CICR channels. 7,8,14,16Therefore, the ability of these anesthetics to release Ca²⁺from the ryanodine-sensitive SR, universally observed in both conduit and resistance arteries, 8–12,15,16,18may 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. 39,40The 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. 39,40Although halothane stimulated IP³formation in erythrocyte membranes, 41to our knowledge, no convincing evidence is presently available to indicate that volatile anesthetics stimulate IP³formation and thereby cause the Ca²⁺release in VSM cells.

Both halothane and enflurane, applied during Ca²⁺loading, inhibited the caffeine-induced increase in R^340/380as 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 R^340/380, 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 R^340/380, 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 7,15,16and also our previous findings in contraction experiments with the same artery. 11,12However, 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, 15but not with those on isoflurane obtained in contraction experiments at 23°C in isolated aorta. 14In the latter, isoflurane, like halothane and enflurane, also inhibited the Ca²⁺uptake. 14Because isoflurane caused Ca²⁺release in the aorta, 14the observed inhibitory effect of isoflurane on Ca²⁺uptake might also be caused by its Ca²⁺-releasing action.

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 R^340/380induced 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 R^340/380, 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. 7,8,14,15However, these anesthetics were shown to influence myofilament Ca²⁺sensitivity in VSM. 11,17,18,25,42In particular, both enflurane and isoflurane appear to increase the myofilament Ca²⁺sensitivity. 18,42Therefore, 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, 7–9,14,15the 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. 19In this study, halothane inhibited the norepinephrine-induced increase in R^340/380, 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 9suggested 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 9because 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. 20However, in this study, isoflurane did not influence the norepinephrine-induced increase in R^340/380, suggesting that isoflurane does not influence the IICR. This appears to be consistent with a previous finding in isolated aorta. 9The 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 R^340/380, 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 R^340/380(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 R^340/380suggests 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 R^340/380after treatment with verapamil, a Ca²⁺channel blocker. 25As verapamil was previously suggested to influence the Ca²⁺release from intracellular stores, 43the 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 R^340/380caused by lower concentrations of norepinephrine (≤ 1 μm). However, as the increase in R^340/380caused by the low concentrations of norepinephrine in the Ca²⁺solution were rather small and somewhat unstable, we did not investigate this issue further.

Not all of the observed anesthetic effects on R^340/380were parallel with the simultaneously observed anesthetic effects on force. Enflurane (5%), applied during Ca²⁺loading, inhibited the caffeine-induced increase in R^340/380but not the caffeine-induced increase in force. Because the [Ca²⁺]ⁱ–force relation is hyperbolic, the enflurane-induced slight (≈ 10%) decrease in R^340/380might not cause any significant decrease in force. Alternatively, because enflurane appeared to increase myofilament Ca²⁺sensitivity in VSM, 18the possible prolonged effect of enflurane on the Ca²⁺sensitivity might counteract its SR-depleting effect.

Isoflurane did not influence the norepinephrine-induced increase in R^340/380but 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.