Potassium Channel Opening and Coronary Vasodilation by Halothane

Potassium Channel Opening and Coronary Vasodilation by Halothane D. R. Larach, H. G. Schuler, K. A. Zangari, and R. L. McCann Department of Anesthesia, College of Medicine The Pennsylvaniu State University, Hershey, Pennsylvania 17033

I. Introduction Volatile anesthetics such as halothane and isoflurane dilate the coronary circulation in part by directly relaxing arterial vascular smooth muscle (VSM) (1-4). The ion-conducting state of membrane K + channels constitutes an important contractile regulatory mechanism in VSM ; outward K+ currents hyperpolarize the sarcolemma, reducing Ca'+ influx through voltage-sensitive Ca2+ channels. The resulting decrease in intracellular [Ca"] relaxes VSM ( 5 ) . One K' channel subtype in VSM is the ATPsensitive K t channel (KATp channel); normally it is closed by millirnolar intracellular ATP concentrations. Tissue hypoxia, hormones (e.g., endothelium-derived hyperpolarizing factor, vasoactive intestinal peptide), and drugs (e.g., diazoxide, crornakalim) vasodilate primarily by opening K,,, channels (6). Because halothane hyperpolarizes intact noncerebral VSM cells (7), and volatile anesthetics hyperpolarize neurons by opening membrane K' channels (8,9), we postulated that halothane may dilate coronary vessels by opening VSM K t channels. We reasoned that if KATp channel opening is an important mechanism of halothane vasodilation, then channel blockade ought to decrease the effect of halothane. Previously, we reported that K,,, channel blockade with glyburide, but not Kca channel Aduonct..\ in P / ~ a r - n i a ~ o k ~\'o/unw pv, 31 Copyright Q 1994 hy Academic Press. Inc. ,411 righl\ of rcproduction i n any form reserved.

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blockade with tetraethylammonium ion (TEA' ), attenuates halothane vasodilation in rat coronary resistance vessels (10). In contrast, halothane depolarizes cerebral arterial VSM cells (1 I), and halothane depression of myocardial contractility is not affected by K+ channel blockade with 4-aminopyridine (4-AP), TEA', or glyburide (12). More recently, electrophysiological studies have not revealed evidence of K+ channel opening by halothane. Thus, Buljubasic et al. have shown that halothane reduces whole-cell Kf currents in isolated coronary VSM cells (13), and Marijic et al. reported that K,, channel blockade potentiates volatile anesthetic vasodilation (14). In addition, in cerebral VSM Eskinder et a / . report that halothane decreases the activity of a 4-AP-sensitive K'-channel (15). Thus, there are conflicting data regarding the role of K' channels in mediating anesthetic vasodilation. To establish whether halothane relaxes intact epicardial coronary arteries by opening VSM Kt channels, we performed studies of halothane vasodilation in the presence and absence of glyburide or 5hydroxydecanoate (5-HD), pharmacological blockers of KATp channels, ( 1 6 ~ 7 )In . particular, we questioned whether the differences we observed previously between rat and porcine coronary tissues were due to differences in the source of the baseline tone in the blood vessel prior to inducing anesthetic vasodilation. In the arrested perfused heart with intact endothelium, one source of the high coronary vascular resistance may be the endothelial-derived vasoconstrictor peptide endothelin. Because endothelin is associated with the closure of KATp channels, we examined whether precontracting porcine conducting coronary artery segments in vitro with endothelin and other agonists would unmask a KATp channelmediated mechanism of halothane vasodilation in this tissue, similar to that observed in the rat coronary resistance vessels in situ. Also, we examined the effects of various transmembrane K+ gradients on halothane responses to further elucidate the importance of K' channel opening in anesthetic vasodilation. This article presents results showing that halothane does vasodilate porcine conducting coronary arteries by opening KATp channels when tone is provided by endothelin but not methacholine (MCh), demonstrating that halothane vasodilation of coronary VSM by opening KATp channels is specific for certain contractile stimuli.

II. Isolated Coronary Vessel Preparation Coronary ring segments were dissected from fresh porcine hearts (87 rings from n = 11 hearts), obtained from a slaughterhouse, the endothelium was denuded, and 8 rings from each heart were mounted to monitor

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Potassium Channel Opening and Vasodilation by Halothane

isometric force in adjacent tissue baths containing buffer. Rings were conditioned and individually stretched to their optimal length for force development ( L o )as described by us previously (10). The developed force induced by isotonic 50 m M K' at Lo following conditioning, and in the absence of inhibitors or anesthetics, was termed the "initial K + force"; this force for each ring (137 ? 65 mN, average t SD) was used as the reference value ( 100%) to normalize the subsequent contractile responses of that ring. (Note: A 1-g mass exerts a force of 9.8 mN at sea level.) The rings weighed 19.5 t 10 mg, and the baseline passive force measured 14 mN. when rings were stretched to Lo was 22.5 Each of the 8 rings was randomly assigned to a different experimental group (Fig. I ) based on the presence or absence of the following three reagents: indomethacin (10 kuM) to block prostanoid generation, glyburide (100 n M ) to block K,,, channels, and halothane (0.0175 atm, equivalent to 2.5 MAC, where MAC is the median effective concentration for general anesthesia in the pig). Thus, each heart provided all necessary experimental and control rings for each condition. Glyburide, indomethacin, and anesthetic were allowed to equilibrate with the tissue for 45, 30, and I5 min, respectively before agonist stimulation began. The concentration of halothane in the gas mixture ventilating the baths was monitored by

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Raman or mass spectrometry during each experiment; prior experiments show excellent correlation between gas-phase halothane concentrations and buffer-phase values by gas chromatography. Figure 1 also shows the experimental protocol. After blocker and/or halothane pretreatment, cumulative endothelin-l (ET) concentrationresponse curves (0.1 to 100 nM) were generated for each bath. Finally, during a stable contraction at the highest [ET] after halothane washout, the K+ channel opener cromakalim (1 p M ) was used to test for glyburide efficacy. Endothelial removal was confirmed using substance P ( I p M ) or ATP (10 p M ) after vessel conditioning during K+ contraction: the average response was a small contraction (1.0 ? 0.3% change). The irreversible contraction induced by ET prevented us from comparing the force developed by high-K+ exposure between the beginning and end of an experiment, which we usually use as an index of tissue viability. However, the absolute force generated by high K+ in the presence of 100 nM ET at the end of an experiment was always at least 1 11% of that achieved by high Kt alone at the start of an experiment (mean 138 t 3% increase); this indicates no gross deterioration of contractile function during these studies. Thirty-two porcine coronary rings (from n = 4 hearts) were denuded of endothelium and prepared as described above, except three conditioning exposures to 3 p M methacholine (MCh) were required for development of a stable MCh contraction. Figure 2 shows the experimental protocol. After a baseline MCh contraction, two rings from each heart received glyburide (100 nM), two received 5-HD (100 yM),two received both blockers, and two control rings received neither blocker. One of each pair of rings received halothane (1.0, 1.5, and 2.5 MAC, in sequence) for 10 min before being reexposed to MCh, the other serving as a simultaneous control. These rings showed minimal relaxation or contraction to 1 p M substance P as evidence of successful endothelium removal; the passive force at Lo was 27 t 4 mN (mean ? SEM), and the initial K + force was 124 2 17 mN. An additional series of 24 rings (from n = 3 hearts) were studied with the identical protocol, except the endothelium was preserved in these rings (1 p M substance P produced an average 23 -+ 1% relaxation); the passive force at Lo was 28 t 3 mN, and the initial Kt force was 113 t 27 mN. Porcine coronary artery rings from four hearts were prepared as described above, and each ring was exposed sequentially to isotonic buffer containing 10, 20, 30, 50, and 80 mM K+. Four rings from each heart received halothane, glyburide, and indomethacin prior to the Ktexposure, using an experimental design and drug concentrations analogous to that of Fig. 1. These rings (weight 21 t 6 mg, mean 2 SD) had a passive force

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Potassium Channel Opening and Vasodilation by Halothane

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Fig. 2 Experimental design and protocol for the halothane vasodilation experiments with porcine coronary artery rings stimulated by methacholine (MCh).

of 36.0 ? 14.4 mN at Lo, and the initial K+ force averaged 127 k 51 mN. The endothelium was denuded, as confirmed by the lack of vasorelaxation in response to substance P. To facilitate the analysis of the effect of glyburide on the magnitude of halothane-induced relaxation, we calculated the absolute difference in developed tension between the halothane and no-halothane rings (normalized as a percentage of the initial K+ force) for each experimental condition (“halothane relaxation”). To determine overall effects, data were analyzed by analysis of variance using the area under the force curve for each ring as the response variable. A two-way analysis of variance, random effects model was used for the ET concentration analyses. The MCh and KCI experiments were analyzed using repeated-measures analysis of variance with appropriate correction for multiple inference. Statistical significance was defined at the a = 0.05 level. Values are presented as means k SEM except where stated otherwise. The composition of modified Krebs-Henseleit buffer was as follows (in mM): NaCl, 118; KCl, 4.7, EDTA, 0.5; KH,PO,, 1.2; MgSO,, 1.2; CaCI,, 3.0; NaHCO,, 25.0; and glucose, 10.0. The buffer was adjusted to pH 7.4 and bubbled with a mixture of O2 and CO, ( 9 5 5 , v/v). High-K+ buffer was isotonic: the “a+] was decreased by an equimolar amount to the elevation in [K’]; thus, when KCl was increased to 50 mM, NaCl was

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decreased to 72.7 mM. Halothane (thymol-free) was a gift from Halocarbon Laboratories (Hackensack, NJ), and cromakalim was a gift of Beecham Pharmaceuticals (Surrey, UK). Endothelin- 1 was purchased from Sigma (St. Louis, MO), and 5-HD from Research Biochemicals Inc. (Natick, MA). Unless specified, all chemicals were supplied by Fisher, Sigma, or Baker, at the highest available purity. Glyburide was dissolved in 0.1 N NaOH and diluted 1: 1000 in buffer before use; all other drugs were dissolved in water and diluted in buffer before use.

111. Effects of Endothelin Figure 3 shows the ET concentration versus developed force responses in coronary rings without (Fig. 3A) and with indomethacin pretreatment (Fig. 3B). The highest [ET] studied was on the steep portion of the concentration-response curve. Pretreatment with glyburide alone did not signifi-

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cantly affect ET contraction, either in the absence or presence of indomethacin. Halothane alone significantly attenuated ET contraction only in rings without indomethacin (see below). To facilitate the analysis of the effect of glyburide on the magnitude of halothane-induced relaxation, we calculated the absolute difference in developed tension between the halothane and no-halothane rings (normalized as a percentage of the initial K f contraction) for each experimental condition. This difference, called halothane relaxation, is plotted in Figs. 4 and 5 as a function of [ET]. In the absence of indomethacin (Fig. 4), the overall effect of glyburide pretreatment was to attenuate halothane relaxation significantly ( p = 0.038 compared with zero) over the ET range of 0.1 to 30 nM. A sigmoid relationship between [ET] and halothane relaxation existed in vehicletreated (without glyburide) rings, except for 100 nM; unless stated otherwise, analyses included only the sigmoid portion of the responses. Inclusion of the 100 nM ET data resulted in loss of statistical significance. Analysis of variance revealed that animal, glyburide pretreatment, and ET concentration each significantly affected the halothane relaxation response ( p < 0.001), but glyburide and [ET] showed n o interaction (i.e., the vehicle and glyburide curves were significantly offset vertically but had similar

[Endothelin-I] (M) Fig. 4 Data from Fig. 3A replotted as the halothane relaxation response (see text) normalized to the initial Kt force. Glyburide significantly attenuates the halothane relaxation response ( p = 0.038). At the [ET] associated with peak halothane relaxation. glyburide pretreatment decreased halothane relaxation by 47% (percent change).

0.R. Lorach et a/.

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slopes). Thus, glyburide attenuated halothane relaxation by 20.8 4.6% (from 24.6 k 4.6% with vehicle to 3.8 0.08% with glyburide) of the initial K+ contraction averaged over all ET concentrations from 0.1 to 30 nM. At the [ET] associated with peak halothane relaxation, glyburide pretreatment decreased halothane relaxation by 47% (percent change). Indomethacin pretreatment (Fig. 5 ) abolished the halothane relaxation response, both in the absence and presence of glyburide. Cromakalim caused a 5.3 ? 1.1% relaxation in vehicle-treated rings, but glyburide pretreatment abolished cromakalim relaxation (0.2 ? 0.6% contraction). These data indicate that glyburide treatment was effective in blocking the K,, channels in VSM.

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IV. Effects of Methacholine A. Endothelium-Denuded Rings In the absence of halothane, following MCh preconditioning, repeated exposures to 3 pM MCh consistently contracted the rings by 39 3% of the initial Kf response. In control rings without halothane, the magnitude

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Potassium Channel Opening and Vasodilation by Halothane

of repeated MCh contractions were unaffected by time or treatment with glyburide. Halothane administration caused a concentration-dependent attenuation of MCh vasoconstriction ( p < 0.005, Fig. 6). The highest halothane dose decreased the MCh response by 44% (percent change). However, the halothane vasodilation effect was not affected by treatment with either glyburide o r 5-HD (Fig. 6). The MCh-induced force was significantly greater with, than without, 5-HD treatment in rings receiving halothane, indicating a 5-HD potentiating effect on MCh vasoconstriction ( p < 0.01). Cromakalim at 1 F M caused a43 2 4% relaxation ofthe rings, and this response was significantly attenuated by glyburide treatment to 18 -+ 4% ( p < 0.005); however, 5-HD did not affect cromakalim vasodilation. When considering cromakalim vasodilation, there was no interaction between the effect of glyburide and either 5-HD or prior halothane treatment.

B. Endothelium-Intact Rings Methacholine without halothane contracted the rings by 51 2 4% of the initial K' response in a stable manner that was unaffected by glyburide or 5-HD (mean of all four blocker treatment groups). Halothane caused 80%

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Fig. 6 Halothane concentration-response data in coronary rings repeatedly contracted with methacholine (MCh). The ordinate shows developed force to MCh as a percentage of the initial K f force. MCh responses without halothane averaged 39 k 3% of the initial K' force, and halothane (2.5 MAC) attenuated the MCh contraction by 44%. Neither glyburide nor 5-HD pretreatment had any significant effect on halothane attenuation of MCh-induced force or on the slope of [halothanel-force relation. Data are from n = 4 hearts.

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a significant dose-dependent attenuation of MCh-induced force development (developed force was 55 t 9, 49 8 , 4 0 % 7, and 36 & 6% of the initial K' response at 0, 1.0, 1.5, and 2.5 MAC halothane, respectively; p < 0.005, data not shown). Similar to the case in endothelium-denuded rings, halothane vasodilation was not significantly altered by the presence of glyburide or 5-HD alone or in combination. +_

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Fig. 7 Potassium vasoconstriction concentration-response curves in epicardial coronary arteries unstimulated by any receptor agonists. Halothane significantly attenuates the K+induced force (p = 0.013); however, the slopes of the curves are equivalent in both the presence and absence of halothane (A), which is not consistent with a K+ channel opening action by the anesthetic. The action of halothane is unaffected by glyburide treatment (B). Data are from n = 4 hearts.

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Fig. 8 Potassium concentration-force responses in the presence of indornethacin (see Fig. 7). Halothane relaxation was abolished by the cyclooxygenase blockade. Data are from n = 4 hearts.

V. Effects of Potassium Figure 7 shows that graded K + depolarization caused a concentrationdependent vasoconstriction ( p = 0.001). Halothane-treated rings developed significantly less force than control rings ( p = 0.0131, but the slope of the [K+J-force curve was not affected by halothane (interaction p > 0.8). Halothane produced a similar relaxation response in glyburide-treated rings without changing the slope (halothane main effect p = 0.01 ; interaction p > 0.7). Indomethacin treatment abolished the halothane vasodilator response ( p = 0.14) during K+-induced contraction (Fig. 8). Likewise, the [K+]-force relationship was not altered by glyburide and/or halothane in the presence of indomethacin ( p > 0.7, data not shown).

VI. Discussion In porcine coronary conducting arteries contracted with endothelin- 1, we have shown that KATPchannel blockade with glyburide caused up to a 47% reduction in halothane vasorelaxation. This finding confirms and extends our observation of a large glyburide-sensitive component of halothane vasodilation in rat coronary resistance vessels (10). Thus, the data

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from both rat resistance and ET-contracted porcine conducting coronary preparations are consistent with an effect of halothane to open KATp channels as a part of its mechanism of action. This article describes the first demonstration of KATp channel involvement in anesthetic vasodilation within a macroscopic blood vessel and may be important for understanding volatile anesthetic actions at sites of coronary spasm and eccentric coronary atherosclerotic lesions subject to dynamic stenosis. Furthermore, the availability of a large-vessel model of anesthetic KATp channel activation will facilitate further studies into the cellular mechanisms of anesthetic action. Halothane is known to be a less effective vasodilator during high-K+ coronary contraction than with receptor agonist-induced precontraction (1 ), and previously we reported that resistance vessel coronary VSM depolarization with high-K+ buffer markedly attenuates halothane vasodilation in the rat heart (10). These findings are consistent with halothane possessing a K+ channel opening action, because high external K+ lowers the transsarcolemmal K+ gradient, preventing channel-mediated hyperpolarization. Alternatively, halothane could directly decrease CaZf influx through Ca2+channels. In the current study, we extended our observations by testing the vasodilator action of halothane on the contractions induced by varying concentrations of external K + in epicardial arteries. If the vasodilator mechanism of halothane involves K+ channel opening, then one would expect the magnitude of vasodilation to decrease as the exernal [K'] increases; that is, the two lines in Fig. 7A would converge toward the right. If the KATp channel were the responsible K+ channel, then one would expect pretreatment with glyburide to reverse the halothane vasodilation, particularly at low external [K+], causing the lines of Fig. 7B to converge at the left-hand side. Surprisingly, we observed neither of these responses to halothane or glyburide (Fig. 7), suggesting that halothane vasodilation of K+-contracted epicardial coronary VSM, in the absence of agonists or other stimuli, does not involve processes sensitive to the transsarcolemmal K+ gradient or membrane potential. This finding is not consistent with halothane possessing either a K+ channel- or a voltage-sensitive Ca2+channel-dependent mechanism, and it implies that the vasodilator effect of halothane under these conditions may be mediated by other intracellular processes, such as the inositol trisphosphate (IP,)-regulated sarcoplasmic reticulum (SR) Ca2+release channel or non-Ca2+-dependentprocesses that regulate myosin light chain phosphorylation and contraction, such as protein kinase C. It would be interesting to see if the coadministration of a K+ channelclosing agonist such as ET would affect the halothane versus [K'] response curves and cause the epicardial coronary data to resemble the coronary

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resistance vessel responses to K + , in which halothane vasodilation is attenuated at high external K + . The glyburide-sensitive nature of halothane vasodilation in the ETcontracted arteries seen in this study stands in marked contrast to our previous results with prostaglandin F,, (PGF,,)-contracted coronary rings, in which neither glyburide nor TEA' significantly inhibited halothane vasodilation. Similar to our prior report, the current study suggests a lack of K A T p channel involvement in the methacholine (MCh)-contracted arteries and in the K + depolarized vessels receiving no agonist. Thus, the K A T p channel-opening action of halothane appears to depend on the nature of the underlying vascular tone. Despite the sensitivity of K A T p channels to closure by normal [ATP],, there is evidence that K A T p channels in certain VSM preparations may have a relatively high open-state probability (18,191. We hypothesize that the K A T p channel involvement in our perfused arrested rat heart studies was explained by the presence of autologous tonic vasoconstrictor substances that act by closing VSM K,,, channels. Indeed, Miyoshi et ul. have reported that ET possesses a powerful K A T p channel-closing action (18). It may be tempting to speculate that the presence of a large population of open-state K A T p channels may explain the lack of halothane effect on K A T p channels when epicardial VSM is contracted with K' , PGF2cl,or MCh, because the K A T p channel-opening effect of halothane would be masked when these channels are already largely open. However, such a speculation is not supported by the lack of a vasoconstrictor effect of glyburide alone (in concentrations documented to block cromakalim-induced K A T p channel opening) in each of the preparations we have studied. Obviously, patch clamp techniques need to be applied to further extend our pharmacological studies t o the channel-population and single-channel level, as well as to understand the nature of the interactions among halothane, glyburide, and ET compared with other agonists. Of interest is the observation that cyclooxygenase blockade with indomethacin abolishes halothane vasodilation, both in the presence and absence of glyburide. This suggests that generation of a prosta..oid intermediate may be a necessary condition for halothane relaxation of ETcontracted coronary VSM. However, studies with indomethacin in rat aorta (20) and rat myocardium (12) do not show cyclooxygenase dependence of halothane action, unlike our finding in coronary arteries. It is tempting to speculate that halothane induces the synthesis of a cyclooxygenase-dependent vasodilating prostanoid such as prostacyclin, which in turn opens K A T p channels in coronary vessels (21 ). Formal testing of this hypothesis will be required. Other notable findings from this study include the observation that

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100 p M 5-HD, which is reported to completely inhibit KATp channels of cardiomyocytes ( 1 7), did not attenuate cromakalim vasodilation. This suggests that 5-HD is not an effective blocker of KATp channels in VSM preparations. In addition, 5-HD, but not glyburide, potentiated MCh vasoconstriction, suggesting that KATp channels are not fully closed during MCh exposure. In conclusion, data from ET-contracted porcine epicardial coronary arteries and rat resistance coronary vessels suggest that approximately 50% of the halothane vasodilation response is caused by opening of glyburide-sensitive KA-p channels by the anesthetic. In support of this mechanism, we have also reported in rat coronary resistance vessels that high-K+ depolarization attenuates halothane vasodilation and that halothane treatment nearly obliterates cromakalim vasodilation, which is consistent with halothane and cromakalim both opening the same (KATp) channel (10). The fact that such a KATp channel-opening mechanism cannot be detected in arteries precontracted with other agonists, or in K+-contracted vessels without agonist stimulation, implies that other factors related to the specific cellular mechanisms activated by the various agonists are important in regulating or unmasking the direct anesthetic action. Future studies should address the nature of these agonist-dependent factors. Patch clamp studies of isolated K,, channels and molecular techniques for identifying K' channel subtypes will be useful for defining these cellular actions of halothane. Further research also is needed to define the role of vasodilating prostanoid intermediates in halothane coronary relaxation.

Acknowledgments The authors thank the Department of Anesthesia. Pennsylvania State University, for supporting this research, Julie Martel for statistical consultation, David M. Fehr for helpful discussions, and Marilyn Green Larach for reviewing the manuscript.

References 1. Bollen, B. A,, Tinker, J. H., and Hermsmeyer, K. (1987). Halothane relaxes previously

constricted isolated porcine coronary artery segments more than isoflurane. Anesthesiology 66, 748-752. 2 . Sill, J. C., Bove, A. A., Nugent, M., Blake, G. A., Dewey, J. D., and Grabau, C. (1987). Effects of isoflurane on coronary arteries and coronary arterioles in the intact dog. Anesthesiology 66, 273-279. 3. Larach, D. R.,Schuler, H. G., Skeehan, T. M., and Peterson, C. J. (1990). Direct effects of myocardial depressant drugs on coronary vascular tone: Anesthetic vasodilation by halothane and isoflurane. J . Pharmacol. Exp. Ther. 254, 58-64. 4. Larach, D. R., and Schuler, H. G. (1991). Direct vasodilation by sevoflurane. isoflurane, and halothane alters coronary flow reserve in the isolated rat heart. Anesthesiology 7 5 , 268-278.

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