Mechanisms of Direct Inhibitory Action of Isoflurane on Vascular Smooth Muscle of Mesenteric Resistance Arteries

With approval from the Kyushu University Animal Care and Use Committee (Fukuoka, Japan), by use of the method previously detailed, 18endothelium-denuded strips were prepared from the third- or fourth-order branches of male Sprague-Dawley rat (250–350 g, 7–10 W) mesenteric arteries. These branches are known to contribute to the systemic vascular resistance. 19,20One strip was prepared from one animal.

Isometric force was measured by attaching the strip to a strain-gauge transducer as previously detailed. 8,18Briefly, the strip was mounted horizontally 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. Removal of endothelium was verified by the inability of acetylcholine (10 μm) to cause significant (10% or more) relaxation during contractions induced by norepinephrine (10 μm).

In the first series of experiments, changes in the [Ca²⁺]ⁱwere measured simultaneously with those in force by use of fura-2, a fluorescent Ca²⁺-indicator dye. 21Our method on the fura-2 fluorometry was also detailed previously. 22,23Briefly, to allow loading of the fura-2 into the VSMCs, 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. 22,23Changes in the fluorescence intensity of the fura-2-Ca²⁺complex were measured by a fluorometer equipped with a dual-wavelength excitation device (CAM-230; Japan Spectroscopic, Tokyo, Japan) connected to the microscope with optical fibers. The VSM tissue was illuminated with ultraviolet lights at wavelengths of 340 and 380 nm alternatively limited to a frequency of 1000 Hz. The fura-2 fluorescence signals induced by excitation at 340 and 380 nm were collected through the 10× objective lens (Plan Fluor; Nikon, Tokyo, Japan) and measured through a 500-nm filter with a photomultiplier. The background fluorescence 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). The ratio (R^340/380) of fura-2 fluorescence intensities excited by 340 nm (F³⁴⁰) to those excited by 380 nm (F³⁸⁰) was calculated after the background fluorescence had been subtracted. None of the agents used during Ca²⁺measurements influenced the fluorescence signals. In all the Ca²⁺measurements, as we showed previously, 23changes in F³⁴⁰and F³⁸⁰were constantly in opposite directions. All experiments with the fura-2-loaded strips were performed during the period in which constant vascular responses were obtained, i.e. , for approximately 3 h. 23 

In the next series of experiments, only isometric force was measured in the non-fura-2-loaded endothelium-denuded strips, the smooth muscle membrane of which was permeabilized with β-escin. To achieve the membrane permeabilization, the strips were incubated with β-escin (50 μm for 25 min) at room temperature (approximately 22°C) in relaxing solution after steady contractions induced by 40 mm K⁺had been measured. 8,18Ionomycin (0.3 μm) was present throughout the β-escin-permeabilized muscle experiments to eliminate the influence of intracellular Ca²⁺stores.

To prevent early deterioration of the thin vascular strips, the aforementioned experiments with membrane-intact and β-escin membrane-permeabilized strips were performed at 35°C and room temperature (approximately 22°C), respectively, as done previously. 8,18 

The ionic concentrations of the normal PSS were as follows (mm): NaCl 138, KCl 5.0, MgCl²1.2, CaCl²1.5, HEPES 10, and glucose 10. The pH was adjusted with NaOH to 7.35 at 22°C. The high-K⁺solutions were prepared by replacing NaCl with KCl isoosmotically and adding guanethidine (3 μm) 24to prevent norepinephrine outflow from the sympathetic nerve terminals. The Ca²⁺-free solution was prepared by removing CaCl²with or without adding EGTA.

The compositions of relaxing or activating solutions used in the β-escin-permeabilized muscle experiments were determined by solving multiequilibrium equations using a hydrogen ion activity coefficient of 0.75 and association constants for the various ions as detailed previously.⁸The composition of the relaxing solution was 80 mm potassium methanesulfonate (KMS), 20 mm PIPES, 7 mm Mg(MS)², 5 mm adenosine 5′-triphosphate, 10 mm creatinine phosphate, and 4 mm EGTA. The 4 mm EGTA-containing activating solution was prepared by adding a specific amount of Ca(MS)²to obtain the desired concentration of free Ca²⁺ions based on the calculations previously reported.⁸The pH was adjusted with KOH to 7.00 at 22°C, and the ionic strength was kept constant at 0.2 mm by adjusting the concentration of KMS. Guanosine 5′-triphosphate (50 μm) was present throughout the experimental periods to minimize rundown of contractile responses in the β-escin-permeabilized strips. 25 

Nifedipine stock solution (1 mm) was prepared in 70% ethanol under conditions of reduced illumination, whereas niflumic acid was prepared as a stock solution (10 mm) in dimethyl sulfoxide. 26 

Adenosine 5′-triphosphate, creatinine phosphate, guanosine 5′-triphosphate, HEPES, ionomycin, β-escin, norepinephrine, acetylcholine, nifedipine, and niflumic acid were obtained from Sigma Chemical Co. EGTA, PIPES-K², and methanesulfonic acid were obtained from Fluka Chemie AG. SKF-96365 was purchased from Calbiochem. Ryanodine was purchased from Agri Systems International. Fura-2/AM was purchased from Dojindo Laboratories. Isoflurane was obtained from Dainabot Co. All other reagents were of the highest grade commercially available.

In experiments with the fura-2-loaded strips, we first examined the effects of isoflurane on increases in [Ca²⁺]ⁱand force caused by norepinephrine or KCl, using protocols identical to those we used previously to examine the direct action of isoflurane on this artery. 12Because the sympathetic nervous system plays a central role in the maintenance of resting vascular tone in vivo , norepinephrine (0.5 μm [EC⁴⁰]) was chosen as a test stimulant as in our previous study. 12Conversely, KCl (40 mm) was used as a tool to activate VGCCs. Each stimulant was applied for 3 or 5 min (3 min for KCl; 5 min for norepinephrine) at 7- or 17-min intervals (7 min for KCl; 17 min for norepinephrine) so as to obtain reproducible responses, and then isoflurane was applied for 5 or 15 min (5 min for KCl; 15 min for norepinephrine) before and during the subsequent applications of either stimulant until the steady-state effects were observed (for 15 min for KCl; for 59 min for norepinephrine). Our rationale to use these protocols was detailed previously. 12,27 

The underlying mechanisms of contractile response to either KCl or norepinephrine could be different between its initial phase (during development of force) and its sustained phase (during maintenance of force). Therefore, in some experiments, isoflurane was applied to the strip precontracted with KCl (40 mm) after the vascular response ([Ca²⁺]ⁱand force) to KCl had reached a plateau. However, isoflurane was not applied to the strips precontracted with 0.5 μm norepinephrine, because vascular response ([Ca²⁺]ⁱand force) to 0.5 μm norepinephrine continued, gradually diminishing progressively after the maximum was reached, and a plateau was not shaped for some time.

In the above-described experiments, we investigated the vascular effects of 3 and 5% isoflurane, because these concentrations of isoflurane distinctly inhibited both norepinephrine- and KCl-induced contractions in our previous experiments with the non-fura-2-loaded, endothelium-denuded strips prepared from this artery. 12As detailed below, the aqueous concentrations produced by 3–5% isoflurane in our experiments would be considered as anesthetic (i.e. , clinically relevant) concentrations.

To investigate mechanisms of the observed [Ca²⁺]ⁱ-reducing effects of isoflurane, we next attempted to characterize the vascular response to either norepinephrine or KCl by evaluating the effects of ryanodine (10 μm), nifedipine (0.01–10 μm), SKF-96365 (0.3–10 μm), and niflumic acid (3–100 μm) on the norepinephrine- or KCl-induced increases in [Ca²⁺]ⁱand force. Ryanodine (10 μm, 20 min) was previously shown to deplete the intracellular Ca²⁺stores (presumably sarcoplasmic reticulum, SR) in this mesenteric artery 17and thus was used to eliminate the influence of SR on the vascular responses in this study. Nifedipine is a selective blocker of the L-type VGCCs, 28and SKF-96365 was reported previously to block receptor-operated Ca²⁺channels (ROCCs). 29,30Niflumic acid has been reported to selectively inhibit Ca²⁺-activated Cl⁻(Cl^−Ca) currents without influencing the VGCC activity in VSMCs. 26,31Preliminary experiments indicated that 5 min is sufficient for all of these channel blockers to exert their maximal effects on the response to norepinephrine or KCl. Thus, in the experiments with nifedipine, SKF-96365, or niflumic acid, the strips were incubated with each blocker for 5 min before and during subsequent application of either norepinephrine or KCl.

To investigate the effects of isoflurane on norepinephrine-induced plasmalemmal Ca²⁺influx, we examined the effects of isoflurane (3–5%) on the norepinephrine (0.5 μm)-induced increases in [Ca²⁺]ⁱand force after treatment with ryanodine (10 μm, 20 min). 17 

To investigate the effects of isoflurane on myofilament Ca²⁺sensitivity, we examined its effects on increases in [Ca²⁺]ⁱand force evoked by stepwise incremental increases in the extracellular Ca²⁺concentrations ([Ca²⁺]^e) from 0 to 5 mm during 40 mm K⁺depolarization or those evoked by stepwise incremental increases in the [Ca²⁺]^efrom 0 to 1.5 mm during stimulation with norepinephrine (0.5 μm) in the fura-2-loaded strips. Our rationale to use these protocols has been detailed previously. 23,32In our previous experiments 12as well as in the above-described experiments, 5 and 37 min were considered sufficient for isoflurane to exert its maximal (i.e. , steady-state) effects on the responses to KCl (40 mm) and norepinephrine (0.5 μm), respectively. Therefore, the strips were incubated for 10 and 40 min before and during subsequent applications of Ca²⁺in these experiments with 40 mm K⁺depolarization and norepinephrine stimulation, respectively. In the above-described experiments, both 3 and 5% isoflurane distinctly inhibited the KCl response, whereas only 5% isoflurane consistently produced distinct inhibition of the norepinephrine response. Thus, the effects of both 3 and 5% isoflurane were examined in the experiments with 40 mm K⁺depolarization, whereas only the effects of 5% isoflurane were examined in the experiments with norepinephrine.

Finally, to further investigate the effects of isoflurane on myofilament Ca²⁺sensitivity, we also examined the effects of isoflurane on the Ca²⁺-force relation in the β-escin-permeabilized, non-fura-2-loaded strips in the absence of norepinephrine; α-adrenergic receptor coupling was not retained in the β-escin-permeabilized strips prepared from this artery, as reported previously. 25,33 

Isoflurane was delivered via  a calibrated isoflurane vaporizer (Forawick; Muraco Medical Co., Tokyo, Japan) in line with the air gas aerating the HEPES-buffered solutions. Each solution was equilibrated with isoflurane for at least 15 min before introduction to the chamber, which was covered with thin glass plates to prevent the equilibration gas from escaping into the atmosphere. Using gas chromatography, we previously reported concentrations of isoflurane in the PSS produced by 0.5, 1.0, 1.5, and 3.0% isoflurane under exactly the same experimental conditions, 8,34and the values obtained were within 92% (91.8–98.5%) of theoretical values predicted by the partition coefficient of isoflurane in Krebs solution at 37°C (0.55). Excellent linear relationship was obtained between the aqueous concentrations of isoflurane (y) and its concentrations (vol%) in the gas mixture (x); y =−0.0068 + 0.21x, r = 0.998). 34Therefore, the concentrations produced by 1, 2, 3, and 5% isoflurane in the PSS can be predicted as 0.21, 0.42, 0.63, and 1.05 mm, respectively. The aqueous concentration produced by 3% isoflurane (i.e. , 0.63 mm) is almost equal to a previously reported concentration of isoflurane (0.65 mm) 13in blood sampled from this rat under steady-state anesthesia with 1.5% (1 minimum alveolar concentration [MAC] in this rat 35) isoflurane. Accordingly, the aqueous concentration produced by 5% isoflurane would correspond to its blood concentration in this rat under steady-state anesthesia with 2.5% (1.67 MAC in this rat 35) isoflurane. Because its partition coefficients in blood and Krebs solution at 37°C are 1.43 and 0.55, respectively, isoflurane is much more (approximately 2.6 times more) soluble in blood than in the buffer solution. On the basis of calculation using the blood/gas partition coefficient of isoflurane (i.e. , 1.43), its blood concentration would reach to 1.0 mm during steady-state anesthesia with 1.8% (approximately 1.5 MAC in human) isoflurane. Usually, 1.5 to 2.0 times MAC is required to maintain anesthesia with only a single inhalational agent and to suppress cardiovascular response to incision. 36We thus believe that the aqueous concentrations produced by 3–5% isoflurane in our experiments can be considered anesthetic (i.e. , clinically relevant) concentrations.

Although absolute values of [Ca²⁺]ⁱcould be calculated on the basis of the fura-2 fluorescence ratio and the dissociation constant of fura-2 for Ca²⁺binding obtained in vitro , 21the dissociation constant of fura-2 for Ca²⁺binding in cytoplasm is significantly different (threefold to fourfold increase) from that measured in the absence of protein, because more than half of the fura-2 molecules in cytoplasm are protein-bound. 37Therefore, we used the ratio of F³⁴⁰to F³⁸⁰(R³⁴⁰/³⁸⁰), which was calculated after the background fluorescence had been subtracted, as an indicator of [Ca²⁺]ⁱ.

Changes in the R^340/380and force were expressed as the percentage value of the reference (i.e. , values before application of isoflurane). The basal values in normal PSS were assumed to be 0% in all experiments. In experiments in which the strips were pretreated with isoflurane, its effects on the response to norepinephrine or KCl were evaluated 3 or 5 min (3 min for KCl; 5 min for norepinephrine) after application of each stimulant. In experiments in which isoflurane was applied to the strips precontracted with KCl, the effects of isoflurane were evaluated 20, 60, 120, and 180 s after its application.

The concentration-response data for the effects of nifedipine, SKF-96365, and niflumic acid and the Ca²⁺-force relation in the β-escin-permeabilized strips were fitted according to a four-parameter logistic model described by De Lean et al . 38The 50% inhibitory concentration (IC⁵⁰) and the 50% effective concentration (EC⁵⁰) were derived from the least-squares fit by use of the above-described model. Because the relationship between the R^340/380value (nonphysiologic value) and the [Ca²⁺]ⁱis not theoretically linear, the R^340/380values were not transformed to a logarithmic scale on the x axis in the representation of the R^340/380-force relationship, and attempts were not made to fit the data for the R^340/380-force relation according to the above-described logistic model. Because the relationship between actual concentrations of isoflurane in the solutions and anesthetic concentrations (vol%) in the gas mixture is theoretically linear, the anesthetic concentrations on the x axis are displayed as vol% for the isoflurane concentration-response relationships, as done previously. 8,27 

All results are expressed as mean ± SD. The term n denotes the number of strips. Data were analyzed by one- or two-factor ANOVA, Scheffé F test, contrast, Student t  test, and Welch's t  test. Comparisons among groups were performed by two-factor ANOVA for repeated measures. When overall differences were detected, individual comparisons among groups at each time or concentration were performed by either Scheffé F test (for multiple comparisons), two-tailed, unpaired Student t  test (for comparison between two groups with homogeneous population variances), or Welch's t  test (for comparison between two groups with heterogeneous population variances). Comparisons within each group were made by one-factor ANOVA for repeated measures, and post hoc  comparisons were made by use of the contrast for multiple comparisons. All other necessary comparisons between two groups, including comparisons between the present and our previous data 23in the Discussion, were made by either the Student t  test or Welch's t  test. As previously detailed, 23statistical analysis was not made in the overall comparison of the R^340/380-force relations. Alternatively, we attempted to find data points in the isoflurane-treated group where the increases in R^340/380are not significantly different from those of certain data points in the control group and then compare the force levels at the certain R^340/380levels between the control and isoflurane-treated groups. A value of P < 0.05 was considered significant.

Norepinephrine (0.5 μm) produced increases in both R^340/380and force in the normal PSS (fig. 1). The ryanodine treatment slightly but significantly inhibited the norepinephrine-induced increases in both R^340/380and force (P < 0.05; n = 10; R^340/380, 79.3 ± 6.9% of control; force, 96.7 ± 4.4% of control). The norepinephrine-induced increases in both R^340/380and force were inhibited during application of isoflurane (3–5%) in either the presence or absence of ryanodine (fig. 1). These inhibitions were still observed 15 min after washout of isoflurane from the chamber (table 1), and it took more than 15 min for VSMCs to recover from the inhibitions (fig. 1).

KCl (40 mm) also produced increases in both R^340/380and force in the normal PSS (fig. 2). The KCl-induced increase in force was inhibited by both 3 and 5% isoflurane; however, the KCl-induced increase in R^340/380was inhibited only by 5% isoflurane but not by 3% isoflurane (figs. 2 and 3). The ryanodine treatment did not significantly influence either the KCl-induced increases in both R^340/380and force or the isoflurane-induced inhibitions of the KCl-induced increases in both R^340/380and force (n = 5) (fig. 2).

Isoflurane (3–5%) did not influence the basal levels of either R^340/380or force; however, in the strips precontracted with KCl, it produced small transient increases in R^340/380and force, which were followed by sustained decreases in R^340/380and force, respectively (fig. 3). Such transient increases in R^340/380and force caused by 5% isoflurane were totally eliminated by the ryanodine treatment (n = 5).

In the ryanodine-treated strips, both nifedipine and SKF-96365 significantly inhibited both the norepinephrine- and KCl-induced increases in R^340/380(figs. 4 and 5), whereas niflumic acid (30–100 μm) inhibited only the norepinephrine-induced, not KCl-induced, increase in R^340/380(fig. 6). There was no concentration range in which SKF-96365 selectively inhibited the norepinephrine-induced increase in R^340/380(fig. 5).

In the fura-2-loaded strips, the stepwise increment of [Ca²⁺]^eduring KCl depolarization produced concentration-dependent increases in both R^340/380and force (fig. 7). These increases in R^340/380were inhibited only by 5% isoflurane, not by 3% isoflurane; however, the increases in force were inhibited by both 3 and 5% isoflurane (fig. 7). As a result, the R^340/380-force relation was shifted to the right after exposure to isoflurane (fig. 7). Despite the identical (P > 0.05) increases in R^340/380, the increases in force caused by 3 and 5 mm extracellular Ca²⁺during exposure to 3% isoflurane were smaller (P < 0.05) than those caused by 3 and 5 mm extracellular Ca²⁺before exposure to isoflurane, respectively (fig. 7). Similarly, regardless of the similar (P > 0.05) increases in R^340/380, the increase in force caused by 3 mm extracellular Ca²⁺during exposure to 5% isoflurane was smaller (P < 0.05) than that produced by 1.5 mm extracellular Ca²⁺before exposure to isoflurane (fig. 7).

In the fura-2-loaded strips, the stepwise increment of [Ca²⁺]^eduring stimulation with norepinephrine also produced concentration-dependent increases in R^340/380and force (fig. 8). These increases in both R^340/380and force were inhibited by 5% isoflurane, and the R^340/380-force relation was shifted modestly to the right during exposure to isoflurane (fig. 8). Despite the identical (P > 0.05) increase in R^340/380, the increases in force caused by 1.5 mm extracellular Ca²⁺after exposure to isoflurane (5%) were smaller (P < 0.05) than that caused by 0.5 mm extracellular Ca²⁺before exposure to isoflurane (fig. 8).

In the β-escin-treated strips, the stepwise increment of [Ca²⁺] in the bath solution produced concentration-dependent increases in force (fig. 9). Isoflurane (5%) did not significantly influence the Ca²⁺-force relation (fig. 9).

Using fura-2 fluorometry, this study, for the first time, provides direct evidence to indicate that isoflurane inhibits myofilament Ca²⁺sensitivity and plasmalemmal Ca²⁺influx during stimulation with either norepinephrine or KCl in VSMCs of systemic resistance arteries. In addition, this study demonstrates for the first time the novel possibility that isoflurane prevents activation of the Cl^−Cachannels and thereby causes a prolonged inhibition of norepinephrine-activated signaling pathway in VSMCs. Because this study was designed to gain access to the mechanisms behind the direct (i.e. , endothelium-independent) inhibitory action of isoflurane on mesenteric arterial VSMCs, the following discussion focuses on its underlying mechanisms but not its relevance to the overall circulatory effects of isoflurane in vivo , which was already discussed in our previous article. 12 

The results obtained indicate that in rat mesenteric resistance arteries, isoflurane at anesthetic concentrations directly inhibits VSM reactivity by inhibiting Ca²⁺mobilization and/or myofilament Ca²⁺sensitivity. Specifically, the low concentration (3%) of isoflurane presumably depresses contractile response to KCl exclusively by inhibiting the myofilament Ca²⁺sensitivity, whereas it appears to depress contractile response to norepinephrine at least in part by reducing the [Ca²⁺]ⁱ. The observed effects of the high concentration (5%) of isoflurane on the R^340/380-force relation suggest that the depressed contractile response to either KCl or norepinephrine during exposure to 5% isoflurane is a result of both reduction of the [Ca²⁺]ⁱand inhibition of the myofilament Ca²⁺sensitivity.

In this artery, treatment with ryanodine (10 μm) depletes not only the caffeine/ryanodine-sensitive SR but also the norepinephrine/inositol 1,4,5-triphosphate-sensitive SR. 17,32Thus, the norepinephrine-induced increase in R^340/380observed after ryanodine treatment is presumably because of plasmalemmal Ca²⁺influx. The effects of nifedipine on either norepinephrine- or KCl-induced increases in R^340/380observed after ryanodine treatment suggest that not only the KCl-induced but also the norepinephrine (0.5 μm)-induced Ca²⁺influxes are attributable exclusively to activation of the VGCCs. This is consistent with the previously reported threshold concentrations of norepinephrine for membrane depolarization in small mesenteric arteries (i.e. , 0.3–1 μm) 39–41and the recent recognition that VGCCs play a major role, whereas ROCCs play only a minor role, in the norepinephrine-induced Ca²⁺influx in rat mesenteric arteries. 42 

The ability of niflumic acid, a Cl^−Cachannel blocker, 26,31to selectively inhibit the norepinephrine-induced increase in R^340/380in the presence of ryanodine indicates that activation of the Cl^−Cachannels is also involved in the norepinephrine-induced Ca²⁺influx. In VSM, activation of the Cl⁻channels would result in membrane depolarization and hence activation of VGCCs, because the Cl⁻equilibrium potential (i.e. , approximately −20 to −30 mV) is more positive than both the resting membrane potential (i.e. , approximately −50 to −70 mV) and the threshold potential for opening VGCCs (i.e. , approximately −60 to −45 mV). 43Thus, as has been proposed in some vascular preparations, 26,44,45norepinephrine presumably activates the Cl^−Cachannels and thereby depolarizes the cell membrane, leading to the opening of VGCCs and hence contraction. In contrast, KCl directly depolarizes the cell membrane and thereby activates VGCCs. Previous studies have suggested that during stimulation with norepinephrine, Ca²⁺released from SR activates the Cl^−Cachannels. 44However, we speculate that the Cl^−Cachannels were presumably activated by Ca²⁺entering the cells through the nonselective cation channels during stimulation with norepinephrine in our experiments with the ryanodine-treated strips.

The ability of isoflurane to inhibit both the norepinephrine- and KCl-induced increases in R^340/380in the presence of ryanodine indicates that isoflurane inhibits both the norepinephrine- and KCl-induced plasmalemmal Ca²⁺influxes, which are presumably through VGCCs, as discussed above. Isoflurane was previously shown to inhibit the whole cell L-type Ca²⁺currents in cerebral or coronary arterial cells. 46,47Thus, in the mesenteric arterial cells, isoflurane also may inhibit the activation of VGCCs and thereby depress both the norepinephrine- and KCl-induced Ca²⁺influx. However, the observed difference in recovery time course after washout of isoflurane between the norepinephrine- and KCl-induced increases in R^340/380(after ryanodine treatment) indicates that the mechanisms behind the isoflurane-induced inhibition of the norepinephrine-induced Ca²⁺influx are at least in part different from those behind its inhibition of the KCl-induced Ca²⁺influx. Because activation of the Cl^−Cachannels appears to be involved in the norepinephrine-induced but not the KCl-induced Ca²⁺influx, isoflurane may prevent the activation of Cl^−Cachannels and hence the norepinephrine-induced Ca²⁺influx through VGCCs in a prolonged manner. In other words, isoflurane may act at some steps between receptor binding and activation of the Cl^−Cachannels to cause a prolonged inhibition of the norepinephrine-activated signaling pathway. Because of its high lipophilicity, isoflurane may remain present in the VSMCs for some time after its removal from the extracellular space to exert such a prolonged effect.

Ryanodine strongly inhibited the initial phase of the norepinephrine-induced increase in R^340/380but only modestly inhibited its tonic phase, as shown previously in this artery. 23,32This suggests that the norepinephrine-induced initial increase in [Ca²⁺]ⁱis attributable primarily to Ca²⁺release from SR, whereas its tonic increase in [Ca²⁺]ⁱis primarily a result of the plasmalemmal Ca²⁺influx. Because isoflurane does not seem to influence the norepinephrine-induced Ca²⁺release from SR in this artery, 17the isoflurane-induced inhibition of the norepinephrine-induced increase in [Ca²⁺]ⁱobserved in the absence of ryanodine was probably exclusively a result of its inhibition of the norepinephrine-induced Ca²⁺influx.

The inability of ryanodine to influence the KCl-induced increase in R^340/380suggests that the KCl-induced increase in [Ca²⁺]ⁱis exclusively a result of activation of the plasmalemmal Ca²⁺influx, consistent with our previous studies in this artery. 8,23,32Because isoflurane seems to enhance the caffeine-induced Ca²⁺release (presumably Ca²⁺-induced Ca²⁺release) from SR in this artery, 17if the Ca²⁺-induced Ca²⁺release mechanism operates significantly during the KCl response, the ryanodine treatment would enhance the isoflurane-induced inhibition of KCl-induced increase in R^340/380. However, it was not influenced by the ryanodine treatment, suggesting that the isoflurane-induced inhibition of the KCl-induced increase in [Ca²⁺]ⁱis exclusively a result of its effect on the plasmalemmal Ca²⁺influx. The Ca²⁺-induced Ca²⁺release mechanism may play only a minor, if any, role in the KCl response in this artery.

Isoflurane has been suggested to increase vascular tone through its direct effects on SR, 8,9plasmalemmal Ca²⁺influx, 48or the protein kinase C system. 11,49In this study, isoflurane caused small transient increases in R^340/380and force during stimulation with KCl (but not in the resting state). The observed increases in R^340/380and force were eliminated by the ryanodine treatment, indicating that the isoflurane-induced contraction results from the Ca²⁺release from SR, consistent with the previous proposal in rat aorta. 9Because the amount of Ca²⁺stored in SR would be increased during stimulation with KCl, which activates voltage-gated Ca²⁺influx and thereby causes a continuous increase in the [Ca²⁺]ⁱ, the inability of isoflurane to cause significant increases in R^340/380in the resting state might be explained by possibly decreased amounts of Ca²⁺in SR. In a recent study using membrane-permeabilized rabbit pulmonary arteries, 49isoflurane caused transient contraction similar to that observed in this artery. However, on the basis of sensitivity to protein kinase C inhibitors, the contraction in the pulmonary artery appeared to be caused by protein kinase C activation. 49The difference might be explained by the species or regional differences.

On the basis of sensitivity to SKF-96365, a putative inhibitor of ROCCs, 29,30isoflurane was previously proposed to enhance contractile response to phenylephrine by stimulating Ca²⁺influx through ROCCs in rat aorta. 48However, our results on SKF-96365 indicate that SKF-96365 does not serve as a selective inhibitor of ROCCs, consistent with the recent recognition that selective inhibitors of ROCCs or nonselective cation channels are not currently available. 50 

The observed difference in the effect of isoflurane on the Ca²⁺-force relation between the fura-2-loaded membrane-intact and the β-escin-permeabilized conditions suggests that its inhibition of the Ca²⁺activation of contractile proteins is presumably mediated by the intact plasma membrane or because of an effect on some intracellular regulatory mechanisms of contractile proteins that are impaired in the β-escin-permeabilized strips. It is currently unclear whether isoflurane inhibits only the Ca²⁺activation of contractile proteins (i.e. , basal Ca²⁺sensitivity of contractile proteins) or inhibits both the basal Ca²⁺sensitivity and the norepinephrine-induced Ca²⁺sensitizing mechanism(s). Further investigations are necessary to clarify this issue.

More than half of the fura-2 molecules in myoplasma are in a protein-bound form, 37possibly influencing responsiveness or sensitivity of the VSMCs to isoflurane. However, no significant differences were found in its steady-state effects on contractile responses to either norepinephrine or KCl between the fura-2-loaded and nonloaded strips (according to comparison between our previous data 12and the present data). Thus, the fura-2 loading, a nonphysiologic intervention as well, does not seem to significantly influence the action of isoflurane on VSMCs.

The direct vascular action of isoflurane observed in this study was substantially identical to that of sevoflurane previously observed in this artery. 23In our experiments, the aqueous concentrations produced by 3% isoflurane (i.e. , 0.63 mm) and 5% sevoflurane (i.e. , 0.67 mm) are almost equal to the respective anesthetic concentrations in blood sampled from this rat under steady-state anesthesia at 1 MAC (1.5% for isoflurane, 352.8% for sevoflurane 51), i.e. , 0.65 and 0.66 mm, respectively. 13We thus compared the effect of 3% isoflurane on contractile response to either norepinephrine or KCl with that of 5% sevoflurane observed in our previous study. 23No significant difference (P > 0.05) was observed in the inhibition of the norepinephrine (0.5 μm)-induced increase in either force or R^340/380between 3% isoflurane and 5% sevoflurane, suggesting that the inhibitory action of isoflurane on the norepinephrine response is identical to that of sevoflurane compared at the equivalent MAC. However, a significant difference was found in the inhibition of the KCl response between 3% isoflurane and 5% sevoflurane: namely, although no significant difference was observed in the inhibition of the KCl-induced increase in force between 3% isoflurane and 5% sevoflurane (P > 0.05), only 5% sevoflurane, not 3% isoflurane, significantly inhibited the KCl-induced increase in R^340/380. Thus, the mechanisms behind the depressed contractile response to KCl appear different between 3% isoflurane and 5% sevoflurane.

In conclusion, isoflurane directly inhibits VSM reactivity by inhibiting both Ca²⁺mobilization and myofilament Ca²⁺sensitivity in systemic resistance arteries. Isoflurane inhibits plasmalemmal Ca²⁺influx through VGCCs during stimulation with either KCl or norepinephrine. However, the mechanisms behind its inhibition of the voltage-gated Ca²⁺influx during stimulation with KCl are presumably at least in part different from those during stimulation with norepinephrine. During stimulation with norepinephrine, isoflurane may prevent activation of the Cl^−Cachannels and thereby inhibit the Ca²⁺influx through VGCCs in a prolonged manner. The presence of the plasma membrane appears essential for isoflurane to exert its inhibitory action on the Ca²⁺-induced activation of contractile proteins.

The authors thank Masae Yamakawa, Research Assistant, Kyushu University, Fukuoka, Japan, for her kind assistance in this work.