Volatile anesthetic agents have been shown to have contractile effects in vascular tissues during specific conditions. This study compared contractile effects of halothane and sevoflurane in rat aorta treated with verapamil. This study also tried to elucidate the mechanism of the contraction.
Endothelium-denuded rat thoracic aorta was used for recording of isometric tension and measurement of influx of 45Ca2+. All experiments were performed in the presence of verapamil. In recording of tension, rings were precontracted with a submaximum dose of phenylephrine, followed by exposure to halothane or sevoflurane. For measurement of influx of 45Ca2+, rat aortic strips were exposed to phenylephrine and then to additional halothane or sevoflurane. Influx of Ca2+ was estimated by incubating the strips in 45Ca2+-labeled solution for 2 min.
Halothane (0.5-4.0%) induced contraction in a dose-dependent manner, whereas sevoflurane (1-4%) had no effect on tension. Influx of 45Ca2+ was strongly enhanced by halothane at 1% and 2%, but only slightly at 4%, and was not affected by 1-4% sevoflurane. SK&F 96365, a blocker of voltage-independent Ca2+ channels, abolished contraction and influx of 45Ca2+ by 1% halothane. Depletion of Ca2+ from the sarcoplasmic reticulum with ryanodine or thapsigargin reduced the contraction induced by halothane at 4% but not that at 1% and 2%.
Halothane is suggested to cause contraction by enhancing influx of Ca2+ via voltage-independent Ca2+ channels at concentrations up to 2% and by inducing release of Ca2+ at 4%. Sevoflurane (1-4%) is devoid of these contractile effects.
THE halogenated volatile anesthetic agents halothane, enflurane, isoflurane, and sevoflurane modify vascular contractility by acting on various mechanisms of contraction. Although they are thought to inhibit vascular contraction, they also can contract vessels depending on the vessel type and on experimental conditions. In resistance arteries, such as mesenteric artery, anesthetic agents cause a transient contraction, which has been clearly explained by release of Ca2+from the sarcoplasmic reticulum (SR). 1–4In conductance arteries such as aorta, sustained contraction is induced by anesthetic agents when they are precontracted with α-agonists in the presence of blockers of voltage-operated Ca2+channels (VOCCs). 5–7In these studies in aortae, the preliminary dose of VOCC blockers was considered to mask the inhibitory effect of anesthetic agents on VOCCs, 8,9revealing a contractile effect of anesthetic agents, which is not fully elucidated however. Namba and Tsuchida 5suggested a role of the release of Ca2+for the contraction of rat aorta by halothane and isoflurane because of a simultaneous increase in the intracellular free Ca2+concentration ([Ca2+]i). In contrast, by using 45Ca2+as a tracer ion, we recently suggested that contraction of rat aorta by isoflurane was attributable to influx of Ca2+through Ca2+channels other than VOCCs and that the role of the release of Ca2+might be negligible. 7
Vascular smooth muscle cells have heterogenous Ca2+channels, VOCCs, and channels activated by receptor stimulation independent of depolarization. Although the latter channels were first named “receptor-operated Ca2+channels,”10we use the term “voltage-independent Ca2+channels” (VICCs) in this article in accordance with recent reviews. 11,12VICCs can be classified into several groups according to their mode of activation 11,12: (1) channels tightly coupled to the receptor; (2) depletion-operated Ca2+channels activated when the intracellular Ca2+store is empty; and (3) channels regulated via soluble messengers. These different types of channels can coexist in the same smooth muscle cell. 13–15Although VICCs have important roles in contraction, 10little is known about the effect of anesthetic agents on their activity.
The purpose of this study was to investigate the contractile effect of the other prevailing volatile anesthetic agents halothane and sevoflurane in rat aorta, especially in relation to the activity of VICCs. We conducted tension experiments and measurement of influx of 45Ca2+in endothelium-denuded rat aorta in the presence of verapamil.
The study was approved by the Kyoto University Animal Use Committee. Male Wistar rats (weight, 250–350 g) were anesthetized with pentobarbital, 50 mg/kg given intraperitoneally, and killed by exsanguination. The descending part of the thoracic aorta was isolated and cut into rings 5–6 mm wide. The endothelium was removed by rotating the rings around a rough-surfaced needle. The absence of functional endothelium was confirmed by the inability of 10 μM acetylcholine to inhibit phenylephrine contraction in each ring in tension experiments. In the subsequent experiments throughout, a supramaximal dose (10 μM) of verapamil was included in the bathing solution. This dose of verapamil completely blocked KCl-induced contraction and [Ca2+]iresponse in rat aorta, 7,16,17proving that VOCCs were functionally inhibited. As the effect of the anesthetic agents was observed in the continuous presence of verapamil, it should have been free from the influence of the minor nonspecific effects of verapamil, 17such as suppression of Na+or K+channels.
Recording of Isometric Tension
The aortic rings were mounted vertically in organ baths filled with 10 ml Krebs–Ringer’s solution aerated with a 95% O2/5% CO2gas mixture. Isometric tension was recorded with a force displacement transducer (San-ei, Tokyo, Japan). The rings were placed in the bath with an optimal resting tension of 3 g for 60 min to equilibrate. They were then incubated for 10 min with 10 μM verapamil and contracted with 300 nM phenylephrine. This dose of phenylephrine was shown in our preliminary study to elicit ≈60% of the maximal contraction and was considered to allow enhancement and suppression of the contraction induced by the anesthetic agents. Phenylephrine induced a sustained contraction, which reached a stable level within 15 min (fig. 1A). Twenty minutes after addition of phenylephrine, halothane or sevoflurane (1–4%) was introduced into the gas aerating the baths. Halothane and sevoflurane were vaporized using Fluotec 3 (Ohmeda, West Yorkshire, United Kingdom) and MK III-ST (Acoma, Tokyo, Japan), respectively, and their concentrations in the gas were adjusted while monitoring with an Atom 303 anesthetic agent monitor (Atom, Tokyo, Japan). In a previous study from our laboratory in which the concentration of halothane and sevoflurane in the bath solution was assayed by gas chromatography, the anesthetic agents were proven to reach stable levels within 10 min after introduction into the gas. 1,18Anesthetic agents were maintained for 15 min and then discontinued, after which recovery from their effects were observed for 20 min. The maximum tension increment after introduction of the anesthetic agents was expressed as a percent of the maximum contraction induced by phenylephrine before addition of anesthetic agents in each ring.
In some experiments with halothane, the composition of Krebs–Ringer’s solution was modified to control the movement of Ca2+. Reference contraction was obtained in each ring with 300 nM phenylephrine in the presence of 10 μM verapamil. After phenylephrine had been washed out and the tension had returned to its basal level, modified experiments were conducted. Modifications to the Krebs–Ringer’s solution were made, by replacing 2 mM Ca2+with 1 mM EGTA or by adding either 30 μM SK&F 96365 (blocker of VICCs), 20 μM ryanodine (blocker of Ca2+release channel of the SR), or 0.1 μM thapsigargin (inhibitor of the SR Ca2+-adenosine triphosphatase). All solutions contained 10 μM verapamil. In either experiment, the increase of maximum tension after addition of halothane was expressed as a percent of the reference contraction induced by phenylephrine.
Experiment on Influx of 45Ca2+
To estimate the amount of unidirectional influx of Ca2+into the smooth muscle, we used 45Ca2+as a tracer ion. Experiments were performed using a method described previously 7,19with some modifications. Aortic rings were prepared and deprived of endothelium as described earlier. They were then cut open into strips and bathed in HEPES-buffered saline solution (HBSS) containing 10 μM verapamil. Verapamil was included in the solution throughout the experiment. The strips were allowed to equilibrate for 60 min in the solution with continuous aeration with air at 37°C. The strips were then transferred to HBSS containing 300 nM phenylephrine. After 10 min, 0–4% halothane was introduced to the solution through the aeration line. Halothane was vaporized and its concentration monitored as described earlier. The strips were exposed to halothane for up to 20 min. Every 5 min during the course, the rings were transferred to another vial filled with HBSS containing 1 μCi/ml 45Ca2+in addition to phenylephrine and halothane. After exposure of the strips to 45Ca2+for 2 min, they were bathed in 2 mM EGTA-containing HBSS at 0°C for 50 min to remove extracellular 45Ca2+and then blotted and weighed. Intracellular 45Ca2+was extracted by incubating the strips in 1.5 ml of 5 mM EDTA at room temperature overnight. 45Ca2+in the solution was analyzed with a liquid scintillation counter after adding 3 ml of scintillation cocktail (Scintisol EX-H; Dojindo, Kumamoto, Japan). Data are expressed as the estimated total amount of influx of Ca2+(μmol) per kilogram aorta (wet weight). The number of minutes for each data point represents the time from the beginning of stimulation with phenylephrine until removal from the 45Ca2+solution. Therefore, strips were exposed to 45Ca2+for the last 2 min of the time indicated. Zero minutes means that strips were exposed to 45Ca2+without stimulation by phenylephrine. The same experiment was conducted with sevoflurane (0–4%), but its effect was analyzed only at the time point of 20 min, i.e. , after 10 min of exposure to the anesthetic agent.
Solutions and Drugs
The Krebs–Ringer’s solution was composed of (in mM) NaCl 120, KCl 5.0, CaCl22.0, MgCl21.0, NaHCO325.0, and dextrose 5.5; the p H of the solution was 7.3–7.4 when the solution was aerated with a 95% O2/5% CO2gas mixture. The HEPES-buffered saline solution was composed of (in mM) NaCl 140, KCl 5.0, CaCl22.0, MgCl21.0, dextrose 5.5, HEPES 5.0 and p H-adjusted to 7.30–7.35 with NaOH. The drugs used were halothane (Takeda Pharmaceutical Co., Osaka, Japan); sevoflurane (Maruishi Pharmaceutical Co., Osaka, Japan); phenylephrine, ryanodine, thapsigargin, and phentolamine (Sigma Chemical Co., St. Louis, MO); acetylcholine (Daiichi Pharmaceutical Co., Tokyo, Japan); verapamil, EGTA, and EDTA (Nacalai Tesque, Kyoto, Japan), and SK&F 96365 (BIOMOL; Plymouth Meeting, PA).
Data for anesthetic-induced contraction were expressed as median values with 25th and 75th percentiles because the maximum tension increment after anesthetic agents that we measured could not be lower than zero and did not follow a normal distribution. The other data were distributed normally and are expressed as mean ± SD. For comparison between two groups, t test was used. For comparison among more than two groups, one-way analysis of variance was used. When data were not distributed normally or when variances were not equal among groups, the Kruskall-Wallis test was used. In either test, if significance (P < 0.05) was detected among groups, each group was compared with the control using Dunnett’s test for post hoc multiple comparison. Differences at P < 0.05 were considered statistically significant.
Effect of Anesthetic Agents on Phenylephrine-evoked Contraction and Influx of Ca2+
For a control tension experiment, rat aortic rings pretreated with verapamil were exposed to halothane or sevoflurane (1%, 2%, or 4%; n = 4 each) at their basal tension. No contraction was observed in any of the rings examined (data not shown). We then tried to increase the concentration of anesthetic agent immediately by replacing the bath solution with already saturated solution, a method described by Kakuyama et al. 1to obtain a more definite contraction by halothane. Again, halothane or sevoflurane at 1%, 2%, or 4% failed to evoke any contraction (n = 4 each; data not shown).
Phenylephrine at 300 nM evoked a sustained contraction (fig. 1A), which averaged 0.844 g (SD = 0.209 g, n = 144) and did not differ significantly among groups. In this setting when halothane was introduced into the solution, the precontraction by phenylephrine was enhanced continuously (≤ 2%) or transiently (4%;fig. 1B). Sevoflurane 1–4%, failed to cause any contraction or relaxation (fig. 1C). When the maximum tension increment after use of anesthetic agents was plotted, halothane caused contraction in a linear, dose-dependent manner, whereas sevoflurane had no significant effect (fig. 1D).
The experiment on influx of 45Ca2+revealed a stable increase in influx of Ca2+by phenylephrine, which developed within 10 min (fig. 2A). As this influx occurred while VOCCs were blocked, it should be through VICCs activated by phenylephrine. Halothane 1% induced a marked and sustained increase in influx of Ca2+, showing a time course consistent with halothane-induced contraction (fig. 2A). Halothane 4%, however, which induced the largest contraction, caused only a slight and transient increase in influx of Ca2+(fig. 2A). Ten minutes after introduction, the effects of varied concentrations (1–4%) of halothane were compared (fig. 2B). Influx of Ca2+was significantly larger in the 1% and 2% halothane groups than in the control (0%). In contrast, 10 min of exposure to sevoflurane 1–4% did not change influx of Ca2+(fig. 2B). In the subsequent experiments, the mechanism of halothane-induced contraction and influx of Ca2+was investigated.
Effect of Removal of Ca2+
To verify the role of influx of Ca2+in halothane-induced contraction, tension experiments were conducted in the absence of extracellular Ca2+. Five minutes after the bath solution was replaced by Ca2+-free Krebs–Ringer’s solution, 300 nM phenylephrine was added to the bath, which induced a transient contraction (fig. 3), possibly attributable to release of Ca2+from the SR. As the contraction was not sustained, we had to start halothane when the tension was near the basal level. Halothane 1% or 4% induced virtually no change in the tension in any ring (n = 4 each;fig. 3).
Effect of SK&F 96365
To confirm that halothane-induced contraction and influx of Ca2+were mediated by VICCs, the effect of SK&F 96365, a blocker of VICCs, 11was examined. In vascular smooth muscle, its blocking effect on VICCs has been shown in patch–clamp and contraction studies. 15,20,21Although it also may have a weak VOCC-blocking action, 21it should not matter in the presence of the maximal dose of verapamil. Contraction was induced by 300 nM phenylephrine 10 min after adding SK&F 96365 to the bath, which was only transient as in Ca2+-free solution (fig. 4A), suggesting that SK&F 96365 effectively blocked influx of Ca2+via VICCs without interrupting release of Ca2+. Again, halothane 1% or 4% induced no change in tension (fig. 4A). The effect of SK&F 96365 was also examined in experiments on the influx of 45Ca2+. SK&F 96365 30 μM was included in the HBSS from the beginning of the equilibrium period throughout the experiment. Influx of Ca2+was estimated at 0, 10, or 20 min after addition of 300 nM phenylephrine, either with or without exposure to 1% halothane for the last 10 min. Phenylephrine did not significantly increase influx of Ca2+over the time course (fig. 4B). Moreover, halothane did not increase influx of Ca2+at 20 min compared with the control (fig. 4B).
Effect of Ryanodine and Thapsigargin
In another series of tension experiments, we used ryanodine and thapsigargin to block release of Ca2+by halothane. Ryanodine is an open-channel blocker of Ca2+-induced Ca2+release channel, which could abolish halothane-induced release of Ca2+in vascular smooth muscle cells, 1,2,4,22whereas thapsigargin inhibits Ca2+-adenosine triphosphatase of the SR, 23both resulting in depletion of stored Ca2+. When ryanodine or thapsigargin was added to the solution, a slowly developing contraction was induced, which has been explained by increased leakage of Ca2+from the SR or by influx of Ca2+attributable to SR depletion. 24,25After 40 min, when the contraction was stable, 300 nM phenylephrine and then halothane were applied to the ring (fig. 5A). Treatment with ryanodine, however, enhanced contraction by halothane up to 2%, but reduced it at 4% (figs. 5A and 5B). Thus, the maximal contraction was obtained at 2% (fig. 5B). Treatment with thapsigargin gave essentially the same result with a similar concentration–response curve (fig. 5B).
Influx of Ca2+without Phenylephrine
To examine the possibility that halothane specifically acts on α-receptors, we conducted an experiment on the influx of 45Ca2+without phenylephrine. Phentolamine, an α-receptor antagonist, was included at 10 μM in the HBSS from the beginning of the equilibrium period. Influx of Ca2+was estimated either without any treatment or after 10 min of exposure to 1% halothane. In either group, the rings were incubated in solutions containing 45Ca2+for the last 2 min. Estimated influx of Ca2+was significantly higher in halothane-exposed strips (44.6 ± 17.5 μmol/kg, n = 16, P < 0.05) than in control strips (33.6 ± 7.0 μmol/kg, n = 16). The 1% halothane-evoked fraction of influx of Ca2+was 11.0 μmol/kg, much less than that in the presence of phenylephrine (27.1 μmol/kg). This appeared to be consistent with the result of the tension experiment, in which halothane alone could not induce contraction without precontraction.
In rat aortic rings pretreated with verapamil, halothane at 0.5–4.0% enhanced phenylephrine-evoked contraction in a linear, dose-dependent manner, whereas 1–4% sevoflurane had no effect. The result with halothane is compatible with a previous study in which halothane enhanced norepinephrine-induced contraction in verapamil-treated rat aorta. 5Although the study showed a simultaneous increase in [Ca2+]iby using a Ca2+indicator dye, this technique does not define the mechanism of increased [Ca2+]i, which might be induced by anesthetic agents either through influx of Ca2+, 7release of Ca2+, 4,6,26inhibited extrusion of cytosolic Ca2+, 27,28or decreased uptake of cytosolic Ca2+into the SR. 4We performed an experiment on the influx of 45Ca2+because it was a useful method for evaluating influx of Ca2+alone, unaffected by movement of Ca2+inside the cell. 19The results of our experiment on the influx of 45Ca2+demonstrated that halothane 1% and 2% increased the influx of Ca2+, which had been submaximally preactivated by phenylephrine. The pathway of influx of Ca2+should be VICCs because VOCCs had been functionally inhibited by verapamil. Halothane 1% induced an even larger influx of Ca2+(27.1 μmol/kg) than phenylephrine (15.5 μmol/kg) despite that it caused only ≈10% of phenylephrine-induced contraction. This puzzling disproportion may be attributable to their opposite effects on Ca2+sensitivity of the contractile protein, i.e. , sensitization with α-receptor stimulation 16,17and desensitization with halothane. 3,29In contrast, influx of Ca2+induced by 4% halothane was unexpectedly low, yielding a curious parabolic-shaped dose–response curve different from that of contraction. In addition, 1–4% sevoflurane had no effect on influx of Ca2+, which was consistent with its tension result.
We postulated that the transient contraction induced by 4% halothane was attributable to release of Ca2+and tried to confirm this hypothesis. In previous studies, contraction by halothane has been ascribed to release of Ca2+from the SR, because the contraction was resistant to removal of extracellular Ca2+4,29or was abolished by ryanodine. 1–4,22In this study, 1% or 4% halothane did not evoke contraction in Ca2+-free solution; however, this did not necessarily prove that halothane did not release Ca2+from the SR, because soaking the ring in EGTA-containing solution should have reduced the basal [Ca2+]iand depleted intracellular Ca2+stores, 30which is an untoward condition to observe release of Ca2+by halothane. We therefore performed an experiment in normal solution using drugs that deplete the store of Ca2+. Ryanodine and thapsigargin reduced the contraction induced by 4% halothane but not that by 1% and 2% halothane. Although it has been suggested that smooth muscle cells may have a store of Ca2+that is resistant to ryanodine but sensitive to inositol 1,4,5-trisphosphate, 31thapsigargin is considered to deplete both types of store. 23Therefore our results suggest that release of Ca2+from the SR plays an important role in contraction by halothane at 4% but not at concentrations of ≤ 2%. After depletion of the store of Ca2+, the dose–response curve of contraction became more similar to that of halothane-induced influx of 45Ca2+and could be considered to reflect the activity of influx of Ca2+more directly than that without depletion of the store. A minor discrepancy between the experiments on tension and influx of 45Ca2+may be pointed out; 4% halothane still could cause contraction after depletion of the store, whereas it could not cause a large influx of Ca2+. This finding can be explained, however, by the different way of measurement; contraction was measured at its maximal point and influx of Ca2+at the fixed time, 10 min after use of halothane.
To confirm that halothane-induced influx of Ca2+was mediated by VICCs, we examined the effect of SK&F 96365, a blocker of VICCs, in the experiments on tension and influx of 45Ca2+. SK&F 96365 blocked the sustained phase of contraction induced by phenylephrine to evoke a phasic contraction similar to that in a Ca2+-free solution. Contraction induced by 1% or 4% halothane was also abolished. It was considered, however, that this condition may not be favorable for contraction by halothane, because it should deplete intracellular Ca2+by blocking Ca2+replenishment as in Ca2+-free solution. We then tested SK&F 96365 in experiments on the influx of 45Ca2+. This completely abolished influx of Ca2+by phenylephrine and 1% halothane. This is an additional finding that supports that halothane activates VICCs. SK&F 96365 is a nonspecific blocker of VICCs. 11As subtype-specific blocker is not yet established, we could not determine the subtype of VICCs sensitive to halothane.
The mechanism of VICC activation by halothane was not fully examined in this study. Halothane should not act on the α-receptor as an agonist because 1% halothane was able to activate influx of Ca2+even in the presence of phentolamine. In addition, it is unlikely that halothane augments the binding of phenylephrine to the α-receptor, because it has been shown that halothane did not increase, but rather inhibited, the binding of radioligands to α-receptors obtained from rat brain 32or rabbit myometrium. 33It seems, however, that activation of VICCs by halothane is not independent of the signal transduction system from the α-receptor to VICCs, because the amount of influx of Ca2+activated by halothane was higher in the presence of phenylephrine. Halothane might enhance activation of the signaling pathway distal to the level of the receptor, i.e. , G protein, intracellular second messengers, or the channel protein itself. Another possibility is that halothane evokes release of Ca2+, thus depleting stored Ca2+and stimulating influx of Ca2+via a depletion-operated mechanism. This has been suggested to account for the activated pathway of influx of Ca2+induced by halothane in canine airway smooth muscle. 34This may not be a primary mechanism of VICC activation in rat aorta, however, considering that abolishment of release of Ca2+by ryanodine or thapsigargin did not inhibit contraction by 1–2% halothane. It is necessary to elucidate the signal transduction pathway from the α-receptor to the channel before further conclusions can be made.
In previous studies using mesenteric arteries, low concentrations (< 0.75%) of halothane were shown to induce contraction by stimulating release of Ca2+even in a resting state. 1,2,4In contrast, in the current study using rat aorta, release of Ca2+was detected only when rings were precontracted in the presence of verapamil and the concentration of halothane was increased to 4%. The different responses might be ascribable to the different Ca2+regulatory machinery in these two tissues. The vascular smooth muscle from different size of vessels and tissue bed can display different forms of pharmacomechanical coupling mechanisms, such as relative roles of VICCs, VOCCs, and the SR as the source of Ca2+, sensitivity of the SR to halothane, the content of Ca2+in the SR, and the Ca2+-buffering capacity of the cytosol. Although influx of Ca2+has not been examined directly in studies using mesenteric arteries, some of the results have raised the possibility that halothane stimulates influx of Ca2+through the cell membrane. 1,4Therefore, it is possible that influx of Ca2+induced by halothane is not a specific phenomenon in aortic tissues but a common underlying mechanism in other vascular tissues. In contrast, some studies that have monitored [Ca2+]iin A7r5, a cultured smooth muscle cell line established from rat aorta, have shown that halothane suppressed rather than stimulated vasopressin-activated influx of Ca2+. 35,36The discrepancy might be attributable to the absence of VOCC blockers in their studies in addition to the differences in the cell type, vasoconstrictor, or technique used. The current study appears to be the first to demonstrate directly the activation of the VICCs by halothane in vascular smooth muscle.
The effect of halothane on influx of Ca2+was similar to that of isoflurane shown in our previous study. 7Isoflurane induced influx of Ca2+resistant to nifedipine and verapamil, and its effect was not linearly dose-dependent, showing a maximal effect at 2.3%. Most anesthetic agents may exert their effects on VICCs through common mechanisms. The distinct feature of contraction by halothane is that release of Ca2+also contributed to its effect, unlike with isoflurane. In contrast, sevoflurane seems completely devoid of these effects within a clinically relevant concentration range (1–4%). Although sevoflurane has been shown to suppress KCl-induced contraction in coronary arteries, 18vasodilating action was abolished by verapamil in this study. Therefore, it can be assumed that the direct vascular effect of sevoflurane is mediated mainly by suppression of VOCCs and that it is inactive with the other contractile mechanisms found for halothane or isoflurane.
Halothane is suggested to have dual contractile effects on verapamil-treated precontracted rat aorta, with sustained contraction at up to 2% attributable mainly to influx of Ca2+via VICCs and transient contraction at 4% attributable mainly to release of Ca2+from the SR. In contrast, sevoflurane had no contractile effect. Although these anesthetic agents have VOCC-blocking action in common, they are different in exerting contractile effects. The contractile effect of halothane is weak in rat aorta, hardly visible in normal conditions. The overall consequence of this effect on the other vascular tissues and the systemic circulation may be more profound, however.