Effect of Volatile Anesthetics with and without Verapamil on Intracellular Activity in Vascular Smooth Muscle

After obtaining approval of our institute's animal care committee, the descending thoracic aorta was isolated from male Sprague-Dawley rats (weighing 250–300 g) that were anesthetized with isoflurane. The aorta was cut into spiral strips approximately 7–10-mm long and 1–1.5-mm wide under a dissecting microscope and placed in a normal physiologic salt solution (PSS) containing (in mM): NACl, 136.9; KCl, 5.4; CaCl2, 1.5; MgCl2, 1.0; glucose, 5.5; NaHCO3, 23.8; and ethyl-enediaminetetraacetic acid, 0.01. The endothelium was removed by gently rubbing the intimal surface with a cotton swab moistened with PSS. The muscle strips were treated with 5 micro Meter acetoxymethyl ester of fura-2 solution (fura-2/AM) for 3–5 h at room temperature (23–25 degrees C). A noncytotoxic detergent, 0.05% Cremophor EL (Sigma Chemical, St. Louis, MO), was added to increase the solubility of fura-2/AM. A high KCl solution was made from PSS by substituting NaCl with equimolar KCl. The solutions were aerated with a 95% O²and 5% CO²mixture at 37 degrees C, pH 7.4.

Experiments were performed using a fluorimeter designed to measure the surface fluorescence of living tissues (CAF-100, Japan Spectroscopic, Tokyo, Japan) as described previously. [3,11]In short, the muscle strip was held horizontally in a temperature-controlled organ bath to measure muscle tension and [Ca²+]ⁱ. One end of the strip was connected to a strain-gauge transducer (TB-612T, Nihon Kohden, Tokyo, Japan) to measure muscle tension. A passive tension of 0.5–0.7 g was applied and allowed to equilibrate before the experiments were begun. In a preliminary study, we determined that this passive tension produced the maximum contractile response to variable concentrations of KCl. The intimal surface of the muscle strip was illuminated alternately at 50 Hz at excitation wavelengths of 340 nm+/-10 nm and 380 nm+/- 10 nm, and the amount of fluorescence at 500 nm+/-20 nm induced by 340 nm excitation (F340) and that induced by 380 nm excitation (F380) was measured. We did not calculate the absolute amount of [Ca²+]ⁱbecause the calculation could have contained an approximately 10% error due to endogenous fluorescent substances such as reduced form of nicotinamide adenine dinucleotide, [12]and also because we do not know the dissociation constant of fura-2 and Calcium²+ in the cell. Instead, the ratio of F340 to F380 (R340/380) was used to indicate the [Ca sup 2+]ⁱ.

Isometric contraction was induced either by 30 nM norepinephrine or 10 micro Meter PGF2 alpha, and the values for muscle tension and R340/380 5 min after the addition of each agonist were taken as the reference (100%). Then, 0%(control), 1%, 2%, and 3% halothane or 2% and 4% isoflurane, delivered by a vaporizer, was introduced into the O²/CO sub 2 mixture for 8 min (n = 8 for each condition, n means the number of the animal). The changes in muscle tension and R340/380 were compared with the reference values. The effects of 100 nM and 1 micro Meter verapamil on norepinephrine or PGF2 alpha-induced contraction also were examined during an identical time course. Only one concentration of either anesthetic or verapamil was examined in each muscle strip. In a preliminary experiment, we confirmed that the concentrations of the agonists used here were those that induced approximately 60–80% of the maximum contractile tension. In a second set of experiments, the same procedure was repeated for the anesthetics (not verapamil), but this time the muscle strips were pretreated with 1 micro Meter verapamil (n = 8 for each condition).

In the third set of experiments, the effects of the anesthetics were examined in nonreceptor-mediated contractions evoked with 1 micro Meter DPB. 12-Deoxyphorbol 13-isobutylate evoked contraction without an increase in R340/380 in the presence of verapamil. Therefore, reference values (100%) for muscle tension and R340/380 were obtained during an isometric contraction induced by 51 mM KCl in each muscle. The muscle strip was then washed with PSS several times so that the tension and R340/380 returned to the resting levels. Isometric contraction was again elicited by 1 micro Meter DPB. Five minutes after the start of the contraction, either halothane at 0%(control), 1%, 2%, and 3% or isoflurane at 2% and 4% was introduced into the O²/CO²mixture for 8 min (n = 8 for each condition). The changes in muscle tension and R340/380 5 min after the addition of DPB and 8 min after the introduction of halothane or isoflurane were expressed as a percentage of the reference value. In other muscle strips, DPB-induced contraction was obtained in the presence of 1 micro Meter verapamil, and the effects of halothane or isoflurane were examined in the identical time course (n = 8 each). In this experiment, the 51 mM KCl reference was measured before the administration of verapamil to compare the responses in the absence and presence of verapamil.

The concentration of halothane and isoflurane in the gas mixture was monitored continuously with a precalibrated anesthesia monitor (Model 303, Atom, Tokyo, Japan). The concentrations of halothane and isoflurane in the bath fluid were determined by testing 2-micro liter aliquots of the PSS using gas chromatography (GC-12A, Shimadzu, Tokyo, Japan) as reported previously. [11].

The following drugs and chemicals were used: fura-2/AM (Dojindo Laboratories, Kumamoto, Japan), Cremophor EL, norepinephrine, verapamil, 12-deoxyphorbol 13-isobutylate. Prostaglandin F2 alpha was donated by Ono Pharmaceuticals (Osaka, Japan).

The results of the experiments were expressed as the mean+/- standard error of the mean. Regression lines were obtained by the least-squares method. Statistical evaluation was performed by analysis of variance, and a P value of less than 0.05 was considered significant. To take into account the problem of multiple comparison if significant variance ratios were obtained using analysis of variance, the least significant differences were calculated with Scheffe's test.

Thirty nanomolar norepinephrine and 10 micro Meter PGF2 alpha showed a similar time course in inducing muscle contraction and [Ca²+]ⁱelevation. Both drugs gradually increased muscle tension to a final value (13 min after administration) approximately 15% higher than the reference (5 min) value (from 7%+/-3% to 21%+/-3%, P < 0.005). In contrast, the precipitous increment in R340/380 decreased gradually, and the final values were about 20% lower than the reference value (from 16%+/-2% to 22%+/-1%, P < 0.005). After washing the muscle strip with PSS several times, muscle tension and R340/380 returned to the resting levels (Figure 1(A)).

Halothane, isoflurane, and verapamil decreased the increases in muscle tension and R340/380 that had been induced by norepinephrine and PGF2 alpha (Figure 1(B) and Figure 2). The effects were concentration-dependent, except for the effect of isoflurane on R340/380 during PGF2 alpha-induced contraction. Halothane and isoflurane inhibited PGF2 alpha-induced increases in muscle tension and R340/380 more potently than those induced by norepinephrine (P < 0.05). Verapamil also inhibited PGF2 alpha-induced increases in R340/380 more than norepinephrine-induced increases (P < 0.01). However, no significant differences in muscle tension were observed between norepinephrine- and PGF2 alpha-induced contractions, probably because PGF2 alpha elicited significantly greater contractions than did norepinephrine during the verapamil experiment (P < 0.01).

Minute-by-minute changes in the [Ca²]ⁱ-tension relationship during norepinephrine- and PGF2 alpha-induced contractions are shown in Figure 3. Each point is the mean percentage of the corresponding R340/380 and tension after the administration of 3% halothane, 4% isoflurane, and 1 micro Meter verapamil. Although R340/380 started to decrease within 1 min of administration of the drugs, muscle tension continued to increase for another 1 or 2 min (Figure 1(B)). We therefore calculated the regression lines from the data obtained at 3 through 8 min after the administration of the respective drug. During PGF2 alpha-induced contractions, there was a significant difference in the slope and y intercept between the verapamil- and the anesthetic-induced regression lines (P < 0.005). During norepinephrine-induced contractions, however, no significant differences were observed between the regression lines produced by verapamil and by the volatile anesthetics (Table 1). Furthermore, we obtained significant differences in the slope and y intercept between norepinephrine- and PGF2 alpha-induced contractions after the administration of the anesthetic (P < 0.005). The result showed that the PGF2 alpha-induced contraction was more prone to relax than the norepinephrine-induced contraction at a given decrease in [Ca²+]ⁱelicited by the anesthetics.

Prior administration of 1 micro Meter verapamil did not influence resting muscle tension and R340/380. However, verapamil reduced the 30 nM norepinephrine- and the 10 micro Meter PGF2 alpha-induced increases in muscle tension and R340/380, showing that a part of norepinephrine- and PGF2 alpha-induced increase in [Ca²+]ⁱwas verapamil-sensitive. In the presence of verapamil, norepinephrine- and PGF2 alpha-induced increments in R340/380 decreased gradually to give final results 20%+/-2% and 26%+/-1% less than the reference values, respectively. Norepinephrine-induced muscle contractions also decreased gradually after 5 min, and the final value was 5%+/-4% less than the reference value in the presence of verapamil. However, with verapamil, PGF2 alpha-induced increases in muscle tension further increased the tension by 43%+/-1% over the reference value. These results suggest that PGF2 alpha increases Calcium²+ sensitivity of the contractile elements more potently than does norepinephrine.

Halothane enhanced the norepinephrine-induced increments in muscle tension and R340/380 in the presence of verapamil (Figure 1(C) and Figure 4(A)). At 3%, halothane augmented the increase in R340/380 by approximately 46%(from 81%+/-1% to 118%+/-8%), whereas it increased muscle tension by 25%(from 95%+/-4% to 119%+/-6%). Isoflurane also significantly enhanced the norepinephrine-induced increment in R340/380 in the presence of verapamil. Nevertheless, the norepinephrine-induced increase in muscle tension was enhanced only during 2% isoflurane administration (Figure 4(B)). In contrast to the norepinephrine-induced contraction, halothane and isoflurane did not increase R340/380 during PGF2 alpha-induced contractions under verapamil. However, both anesthetics significantly decreased increases in muscle tension in a dose-dependent manner (Figure 1(D), Figure 4(C and D)).

A 1-micro Meter dose of DPB increased muscle tension and R340/380; the increases were, respectively, 171%+/-7% and 72%+/-3% of those increases induced by the 51 mM KCl solution at 5 min after DPB administration. No significant differences were observed among the groups investigated. Therefore, we compared only the values obtained at 13 min after the administration of DPB (which corresponded to the values 8 min after the administration of either anesthetic).

The increase in R340/380 induced by DPB was temporary, the value 13 min after DPB administration was only 13%+/-8% of that induced by 51 mM KCl. In contrast to the R340/380, the increase in muscle tension was remarkable: 13 min after the addition of DPB the tension was 228%+/-20% of that induced by the 51 mM KCl solution, showing that DPB elevated Calcium²+ sensitivity of the contractile elements. Halothane at 2% and 3% and isoflurane at 4% significantly decreased the DPB-induced increase in muscle tension (Figure 5(A and B)). However, the DPB-induced increment in R340/380 was significantly enhanced by 2% and 3% halothane or 2% and 4% isoflurane, showing that the anesthetics modulate [Ca²+]ⁱ-tension relationship even during DPB-induced muscle contraction.

In the presence of 1 micro Meter verapamil, DPB induced little increase in R340/380; the value returned to the baseline 13 min after the administration of DPB. An increase in muscle tension, however, lasted even in the absence of an increase in R340/380. Muscle contraction in the presence of verapamil was significantly less than that in the absence of verapamil (P < 0.05). In the presence of verapamil, halothane at 2% and 3% and isoflurane at 2% and 4% significantly enhanced the DPB-induced increment in R340/380 (P < 0.01), but a significant increase in muscle tension was observed only during 2% isoflurane administration (P < 0.05;Figure 5(C and D)). This result, too, suggests that both anesthetics modulate [Ca²+]ⁱ-tension relationship in the presence of verapamil.

Our experiments indicated that the volatile anesthetics and the Calcium²+ channel blocker had a similar effect in inducing vascular smooth muscle relaxation. Both types of drugs decreased muscle contraction concomitantly with a decrease in [Ca²+]ⁱ. However, simultaneous measurement of [Ca²+]ⁱand muscle tension revealed differences between the effects of the anesthetics and those of verapamil during norepinephrine- and PGF2 alpha-induced contraction. Furthermore, the verapamil pretreatment allowed us to characterize the differences between contractions induced by norepinephrine from that induced by PGF2 alpha.

Stimulation of many types of receptors activates phospholipase C enzyme in vascular smooth muscle, resulting in hydrolysis of membrane-bound inositol lipids and formation of at least two second messengers, diacylglycerol and inositol-1,4,5-triphosphate. [13,14]Diacylglycerol acts by stimulating PKC, which enhances myofilament Calcium sup 2+ sensitivity. Inositol-1,4,5-triphosphate diffuses into the cytosol to release Calcium²+ from the intracellular stores. It may also be tetraphosphorylated to I(1,3,4,5)P4, which may have a role, in conjunction with inositol-1,4,5-triphosphate, in activating Calcium²+ entry from outside the cell. Extracellular Calcium²+ also may enter the cell through the Calcium²+ release-activated channels, which are probably termed as receptor-operated channels, activated by depletion of intracellular stores of Calcium²+. Norepinephrine and PGF2 alpha, therefore, increase [Ca²+]ⁱby opening the sarcolemmal Calcium sup 2+ channels as well as by releasing Calcium²+ from intracellular storage sites. [13–15].

This study revealed that both halothane and isoflurane could increase [Ca²+]ⁱduring agonist-induced contraction under specific circumstances. A similar contractile response has been observed with enflurane during muscle contraction elicited by a submaximum dose of norepinephrine in the rabbit aortic ring [5]; however, we did not observe the response in the absence of verapamil. In addition to inhibiting Calcium²+ entry into the cytoplasm, verapamil also suppresses inositol-1,4,5-triphosphate formation and the subsequent release of Calcium²+ from the sarcoplasmic reticulum during alpha adrenoceptor-induced contraction, probably at the level of G proteins and phospholipase C. [8]Therefore, in the presence of verapamil, a greater amount of Calcium²+ must remain in the sarcoplasmic reticulum during norepinephrine-induced contraction. This Calcium²+ should be released by the administration of the anesthetics. [3,11]This may explain why halothane and isoflurane increased [Ca²+]ⁱand muscle tension during norepinephrine-induced contraction in the presence of verapamil.

Nevertheless, our findings did not clarify why neither halothane nor isoflurane influenced [Ca²+]ⁱduring PGF2 alpha-induced contraction in the presence of verapamil. Norepinephrine and PGF2 alpha evoked similar contractile responses in PSS. [15]In a Calcium²+-free solution, although PGF2 alpha elicited a transient increase in [Ca²+]ⁱ, it did not induce transient muscle contraction as did norepinephrine [15]or phenylephrine (an alpha¹adrenoceptor agonist). [16]These reports suggest that PGF2 alpha and the alpha adrenoceptor agonists may release Calcium²+ from different intracellular Calcium²+ storage sites. [16,17]Therefore, the amount of Calcium²+ remaining, particularly in the anesthetic-releasable Calcium²+ stores, might differ during norepinephrine- and PGF2 alpha-induced contraction. Another possible explanation is that PGF2 alpha-induced Calcium²+ release may not be inhibited by verapamil. As a result, the intracellular Calcium²+ stores might be mobilized by the administration of PGF2 alpha, even in the presence of verapamil. Further work is required to solve this problem.

Halothane seemed to be more potent than isoflurane in increasing [Ca²+]ⁱduring norepinephrine-induced contraction. This finding is consistent with our previous study in which only halothane increased [Ca²+]ⁱduring KCl-induced contraction. [3]However, the DPB experiment indicates that a high concentration of isoflurane also releases much Calcium²+ from the intracellular storage site. This release became obvious, probably because DPB itself does not elicit apparent Calcium²+ release from the sarcoplasmic reticulum. [15]Therefore, the Calcium²+-releasing effect of isoflurane could be masked by the simultaneous inhibition of the sarcolemmal Calcium²+ channels during KCl-induced contraction. As a result, isoflurane could not increase [Ca²+]ⁱduring KCl-induced contraction.

During DPB-induced enhancement of PKC activity in the presence of verapamil, the volatile anesthetics induced a slightly greater increase in Calcium²+ release than that without verapamil, but they produced a far greater increase in muscle tension. Some of the increase in tension could be ascribed to the increased Calcium²+ sensitivity of the contractile elements produced by DPB. However, the increases in tension were too large compared to those in [Ca²+]ⁱespecially during 2% halothane and 2% isoflurane administration, indicating that some other mechanisms should be involved in the disparity between tension and [Ca²+]ⁱ. One of the possible mechanisms is that [Ca²+]ⁱ-tension relationship is not linear when [Ca²+]ⁱis low. We reported that muscle tension did not develop until [Ca²+]ⁱincreased to approximately 40–50% of KCl-induced reference response. [11]Another possible mechanism involves the different [Ca²+]ⁱwhen the volatile anesthetics are administered. In the absence of verapamil, DPB increased tension and [Ca²+]ⁱ; both of them were inhibited by the administration of the volatile anesthetics. In contrast, muscle contraction was not accompanied by an increase in [Ca²+]ⁱduring DPB-induced contraction with verapamil. Increase in [Ca²+] sub i activates not only myosin light chain (MLC) kinase but also Calcium sup 2+- and calmodulin-dependent protein kinase II. The latter kinase phosphorylates MLC kinase to decrease its activity. [18]Therefore, this Calcium²+-desensitizing mechanism should not be activated during DPB-induced contraction with verapamil.

It can be difficult to examine Calcium²+ sensitivity in these experiments because [Ca²+]ⁱwas dynamically changing with initial development of muscle tension, so that the [Ca²+]ⁱ-tension relationship is disproportionate. [19]During relaxation, a linear decline of tension and [Ca²+]ⁱcan be seen. Therefore, the [Ca²+]ⁱas determined by fura-2 was employed to generate [Ca sup 2+]ⁱ-tension regression lines from the more steady-state relationship observed during the relaxation phase of norepinephrine- and PGF2 alpha-induced contractions. These regression lines were then employed to compare effects on Calcium²+ sensitivity, which are enhanced by the receptor agonists. The volatile anesthetics apparently induce greater suppression in muscle tension for given changes in [Ca²+]ⁱas compared to verapamil during the PGF2 alpha-induced contractions. This accords with the subsequent experiment, in which volatile anesthetics attenuated muscle tension without a significant change in [Ca²+]ⁱ.

At least two mechanisms have been proposed for Calcium²+ sensitization during agonist-induced smooth muscle contraction: MLC phosphorylation-dependent and -independent mechanisms. [20,21]The MLC phosphorylation-dependent mechanism involves an altered balance between the activities of MLC kinase and MLC phosphatase. [22]Receptor agonists inhibit MLC phosphatase through a mechanism coupled to G protein, resulting in greater MLC phosphorylation at a given [Ca²+]ⁱ. [22,23]They also inhibit Calcium²+ desensitizing mechanisms by activating PKC. [14,24]Deoxyphorbol 13-isobutylate is a membrane-permeable activator of PKC. Therefore, the MLC phosphorylation-dependent mechanism is likely to be involved in Calcium sup 2+ sensitization induced by these drugs. In addition, norepinephrine, PGF2 alpha, and DPB can increase Calcium²+ sensitivity through the MLC phosphorylation-independent mechanism. [20,21].

Possible mechanisms that suppress Calcium²+ sensitivity of the contractile elements by volatile anesthetics involve an increase in cytosolic cyclic 3',5'-adenosine monophosphate [25,26]and cyclic 3',5'-guanosine monophosphate contents, [27]and a decrease in membrane-bound PKC. [26]Halothane and isoflurane modulated [Ca²+] sub i -tension relationship during PGF2 alpha- and DPB-induced contraction in the absence of verapamil, suggesting that the anesthetics influence MLC phosphorylation-dependent mechanism, or altered balance between the activities of MLC kinase and MLC phosphatase. We have not determined, however, why the volatile anesthetics did not modulate [Ca²+]ⁱ-tension relationship during norepinephrine-induced contraction. Norepinephrine increases Calcium²+ sensitivity less potently than does PGF2 alpha. [14]Also, the Calcium²+ sensitization caused by the MLC phosphorylation-independent mechanism is weak during norepinephrine-induced contraction compared to PGF2 alpha- and DPB-induced contraction. [20]These reports indicate that the mechanism to increase Calcium²+ sensitivity is different between norepinephrine and PGF2 alpha. It is possible, therefore, that halothane and isoflurane may proportionally influence both Calcium²+ sensitizing and Calcium²+ desensitizing processes during norepinephrine-induced contraction. Further study is necessary to clarify how the anesthetics affect Calcium sup 2+ sensitivity of the contractile elements during agonist-induced contraction.

Ozhan et al. [28]reported that halothane and isoflurane reduced contractions evoked by serotonin and Fluorine sup - but not those induced by the phorbol ester, phorbol dibutyrate (which is structurally analogous to diacylglycerol), in the porcine coronary artery. In our previous studies, we reported that halothane and isoflurane reduced the Calcium²+ sensitivity of the contractile elements during KCl-induced contraction. [3]Current experiments further revealed that halothane and isoflurane modulated the DPB-induced [Ca²+]ⁱ-tension relationship, indicating that the effects of volatile anesthetics cannot be confined to the proximal part of the signal transduction pathway. Therefore, simultaneous measurement of muscle tension and [Ca²+]ⁱseems important to ascertain the effects of volatile anesthetics on smooth muscle contraction because volatile anesthetics possibly increase [Ca²+]ⁱ.

In conclusion, halothane and isoflurane suppressed norepinephrine and PGF2 alpha-induced increases in muscle tension and [Ca sup 2+]ⁱ. The pretreatment with verapamil unmasked the intracellular action of the anesthetics: halothane and isoflurane released Calcium²+, possibly from the intracellular storage site during norepinephrine-induced contraction. Both anesthetics modulated [Ca²+] sub i -tension relationship during PGF2 alpha- and DPB-induced contraction. Further study is necessary to clarify the differential effects of the anesthetics on norepinephrine- and PGF2 alpha-induced contraction. Our findings strongly indicate that vasorelaxing effects of the anesthetics cannot be confined to the proximal part of the signal transduction pathway.

The authors thank Professor A. Namiki, MD, PhD, Chairman, Department of Anesthesiology, Sapporo Medical University School of Medicine, for the direction of this research and review of the manuscript.