BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 229, 852–856 (1996)ARTICLE NO. 1891

Effects of Volatile Anesthetic Isoflurane on ATP-Sensitive K/ Channelsin Rabbit Ventricular Myocytes

Jin Han, Euiyong Kim, Won Kyung Ho,* and Yung E. Earm*,1

Department of Physiology and Biophysics, College of Medicine, Inje University, Pusan, Korea; and *Departmentof Physiology and Biophysics, Seoul National University College of Medicine, Seoul 110-799, Korea

Received November 7, 1996

It has been suggested that volatile anesthetic, isoflurane mediates cardioprotective effects via activationof the ATP-sensitive K/ (KATP) channels. However, no direct evidence has been provided to define whetherisoflurane activates cardiac KATP channels using patch-clamp technique. We examined the effects of isoflur-ane on the KATP channels in rabbit ventricular myocytes by use of patch-clamp technique. Contrary to theresults of the in vivo experiments, isoflurane inhibited the channel activity without a change in the single-channel conductance. Isoflurane decreased the channel activity by a decrease in burst duration and anincrease in the inter-burst duration. On the other hand, isoflurane diminished the ATP sensitivity of KATP

channels, indicating an increased probability of KATP channel opening for a given concentration of ATPafter isoflurane anesthesia. The result supports, at least in part, the hypothesis that isoflurane mediatescardioprotective effects via KATP channel activation. q 1996 Academic Press

Activation of ATP-sensitive K/ (KATP) channels in cardiac muscle produce cardioprotectiveeffects during myocardial ischemia (1,2). KATP channel agonists protect against infarction ina manner similar to that caused by ischemic preconditioning (3). Previous experimental evi-dence also indicates that volatile anesthetics exert beneficial actions in ischemic myocardium(4) and enhance functional recovery of stunned myocardium (5). More recently, isoflurane hasbeen demonstrated to produce cardioprotective effects in stunned myocardium in vivo, andthese effects are blocked by the glyburide, a KATP channel antagonist (6). This finding suggeststhat KATP channel activation by isoflurane may mediate antiischemic effects. However, thusfar, there has been little direct evidence to determine the nature of the interaction betweenvolatile anesthetics and KATP channels using patch-clamp technique. The present investigationwas therefore undertaken to see whether isoflurane, a volatile anesthetic, could affect KATP

channel activity by using the excised membrane patch-clamp technique in rabbit ventricularmyocytes. We report herein that isoflurane does inhibit cardiac KATP channel activity anddiminish the sensitivity of the cardiac KATP channel to ATP.

MATERIALS AND METHODSCell preparation. Rabbit ventricular myocytes were isolated by using a Langendorff column at 377C for coronary

perfusion and collagenase (5 mg/50cc, Yakult, Japan) for enzymatic dispersion previously (7). Isolated cells werestored in a high K/, low Cl0 storage medium (8) (composition in mM: taurine 10, glutamic acid 70, KCl 25, KH2PO4

10, glucose 22, EGTA 0.5, pH 7.4 with KOH).Single channel recordings. Single-channel recordings were measured at room temperature (25 { 37C) in excised

membrane patch configurations of the patch-clamp technique (9) with a patch-clamp amplifier (Axopatch-1D, AxonInstruments, Foster City, CA, USA). Open probability (P0) was calculated by use of the pClamp software (AxonInstruments, Foster City, CA, USA) as described previously (10). The channel activity was expressed as P0 . Thesolution facing the outside of the patch membrane contained (in mM): KCl 140, CaCl2 2, MgCl2 1, glucose 10,

1 Correspondence to Department of Physiology & Biophysics, Seoul National University College of Medicine,Yonkeun-Dong, Chongno-Ku, Seoul 110-799, Korea. Fax: /82-2-763-9667.

0006-291X/96 $18.00Copyright q 1996 by Academic PressAll rights of reproduction in any form reserved.

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FIG. 1. Effect of isoflurane on ATP-sensitive K/ (KATP) channel activity. A. Single-channel recordings from inside-out patches. The solution exchange protocol for ATP and isoflurane is shown above current traces. c, closed level.Patch membrane potentials were held at 070 mV. Low-pass filter, 2 kHz. B. Concentration–response relationshipfor the inhibitory action of isoflurane. The equation for the linear regression line is: relative channel activity Å 1.010 (0.19 1 [isoflurane]i) (R Å 00.988, P Å 0.0002; standard deviation of slope Å 0.061).

HEPES 10, pH 7.4 with KOH. The solution facing the inside of the patch membrane contained (in mM): KCl 127,KOH 13, MgCl2 1, EGTA 5, glucose 10, HEPES 10, pH 7.4 with KOH. Isoflurane was obtained from IlsungPharmaceutical Co. Ltd. (Seoul, Korea). All other drugs were obtained from Sigma (St. Louis, MO, USA).

Isoflurane administration. The perfusate was equilibrated with isoflurane by passing 100% O2 gas (flow rate: 1 l/min) through a vaporizer (Cyprane, England) before bubbling the solution. The isoflurane concentration of the perfusatein the recording chamber was analyzed by gas chromatography (G-3000, Hitachi, Tokyo, Japan). At 257C, the perfusatecontained 0.72, 1.71 and 2.54 mM isoflurane when equilibrated with 1 vol%, 3 vol% and 5 vol% isoflurane, respectively.

RESULTS AND DISCUSSION

To determine whether isoflurane is involved in the modulation of KATP channel activity, weused the inside-out patch configuration. The effects of isoflurane on KATP channel activity isshown in Fig. 1. When inside-out patches were formed in ATP-free solution, the activationof the KATP channel was observed. KATP channel activity was rapidly and reversibly inhibitedby ATP (500 mM or 2 mM). In the concentration range 1 vol% to 5 vol%, the application ofisoflurane to the bath induced a marked and reversible decrease in KATP channel activity (Fig.1A). Isoflurane produced a concentration-dependent inhibition of KATP channel activity (n Å23 patches). To obtain the concentration of isoflurane ([isoflurane]i)-KATP channel activityrelationship, we determined the effect of one concentration of isoflurane from each inside-outpatch (Fig. 1B). A plot of relative channel activity as a function of [isoflurane]i , using dataobtained from a total of 5 patches, resulted in data points well fitted by a straight line (thedecrease in relative channel activity, 0.19 { 0.01 per the increase in [isoflurane]i , 1 vol%).Similar data were obtained with outside-out membrane patches where isoflurane was appliedto the extracellular aspect of the membrane (n Å 2 patches, not shown).

The current-voltage (I-V) relationship obtained from a total of 8 patches before and afterthe application of isoflurane is shown in Fig. 2A. The I-V curves before and after the applicationof isoflurane (3 and 5 vol%) at negative membrane potentials displayed a linear relationshipwith a single channel conductance of 77.3 { 1.7 (before isoflurane, O), 76.8 { 2.2 (3 vol%,D) and 76.1 { 2.5 (5 vol%, Ç) pS, respectively. The open and closed time distributions wereanalyzed to determine the effect of isoflurane on the kinetic properties of the channel. Probabil-

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FIG. 2. A. Effect of isoflurane on the conductance of the KATP channel. Upper panel, current traces recorded froma single patch. Lower panel, current–voltage relationship and the effect of isoflurane on it. B. Effects of isofluraneon the kinetic properties of the KATP channel. The histograms of the open (upper left panel) and closed (upper rightpanel) time within bursts were analyzed from the current records at 10 kHz. The histograms of the burst (lower leftpanel) and inter-burst (lower right panel) duration were analyzed from the current records at 0.1 kHz. *Isofluranedecreased the burst duration and increased the inter-burst duration.

ity density histograms of the open and closed times measured at 050 mV are shown in Fig.2B. Isoflurane had no effect on the fast open and closed kinetics within burst (Fig. 2B, upperpanels). On the other hand, isoflurane decreased the burst duration and increased the inter-burst duration. The time constant of the burst duration (tb) was markedly decreased from 41.1to 15.6 ms by isoflurane (Fig. 2B, left lower panel). tC2 represented the time constant of theinter-burst duration (Fig. 2C, right lower panel). The value of tC2 was increased from 72 to213 ms by isoflurane.

Interestingly, we also found that two successive applications of the same concentration ofATP (2 mM) after exposure to isoflurane produced much less block (Fig. 1A, middle andlower panels). This suggests that the ATP sensitivity of the channel may have been changedby isoflurane. To determine the effects of isoflurane on ATP sensitivity of KATP channels,different concentrations of ATP (2 mM to 2 mM) were subsequently applied (Fig. 3). Afterthe application of isoflurane (3 vol%), ATP produced much less block than before (Fig. 3A).In Fig. 3B, the graph shows the dose-response relationship for inhibition of KATP channelactivity by ATP before (s) and after (l) the application isoflurane. The continuous lines inthe graph were the fitted curves to the Hill equation using the least-squares method as describedpreviously (10, 11). Isoflurane changed the ATP concentration at the half-maximal inhibition(Ki) from 67.6 { 19.8 to 163.2 { 28.5 mM (n Å 7 patches).

Our results demonstrate that isoflurane inhibits KATP channel activity in rabbit ventricularmyocytes. In a different set of experiments, we also observed that both volatile anesthetic,halothane and intravenous anesthetic, pentobarbitone inhibited the channel activity in a mannersimilar to isoflurane (not shown). As the concentration of the anesthetics is important fordetermining the degree of KATP channel inhibition, it is possible that they act by a non-specificmembrane interaction or by an action on specific hydrophobic protein region. Alternatively,isoflurane may interact with the ATP-binding site of the channel directly as isoflurane dimin-

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FIG. 3. Isoflurane diminishes the sensitivity of the KATP channels to ATP. A. An original data trace when differentconcentrations of ATP were sequentially applied to a membrane patch. B. Concentration–response relationshipsbetween the relative channel activity and the concentration of ATP before (s) and after (l) the application ofisoflurane.

ished the sensitivity of the KATP channel to ATP. However, our results cannot discriminatewhether isoflurane act directly on the KATP channel protein or indirectly via an alteration ofthe physical characteristics of the membrane lipid. To discriminate between these possibilities,the molecular mechanism of the anesthetics-induced KATP channel inhibition remains to befurther elucidated. To our knowledge, these are the first data obtained with single channeltechniques which relate to the effects of volatile anesthetic, isoflurane on the KATP channel.Recently, isoflurane has been demonstrated to exert antiischemic effects via activation of KATP

channels in the in vivo experiments (6,12). Contrary to these results, we did not find theactivation of KATP channel elicited by isoflurane in rabbit ventricular myocytes. One possibleexplanation for the discrepant data for isoflurane and its effect on KATP channel activity maybe a possible competition between ATP and isoflurane for the ATP-binding site. Our resultsupports this possibility as isoflurane diminishes ATP sensitivity, indicating an increasedlikelihood of KATP channel opening for a given concentration of ATP after isoflurane anesthesia.The result supports, at least in part, the hypothesis that isoflurane mediates cardioprotectiveeffects via KATP channel activation. It is also possible that isoflurane specifically activates theKATP channel in coronary vascular smooth muscle cells although isoflurane inhibits KATP

channel activity in cardiac myocytes. Therefore, whether volatile anesthetics specifically acti-vate the KATP channels in coronary vascular smooth muscle cells as demonstrated in in vivo(12) and in vitro (13) experiments remains to be assessed by using single channel techniques.

ACKNOWLEDGMENTSThe authors thank Professor D. H. Moon, and Dr. Y. S. Han, in the Department of Preventive Medicine, Inje

University, and Professor D. H. Park, in the Department of Chemistry, Inje University, for performing assays ofisoflurane bath concentration with the use of gas chromatography; Professor C. M. Shin, and Y. K. Choi, in theDepartment of Anesthesiology, Inje University, for supplying a vaporizer and valuable comments.

REFERENCES1. Noma, A. (1983) Nature 305, 147–148.2. Auchampach, J. A., Maruyama, M., Cavero, I., and Gross, G. J. (1992) Circulation 86, 311–319.

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3. Gross, G. J., and Auchampach, J. A. (1992) Circ. Res. 70, 223–233.4. Kanaya, N., and Fujita, S. (1994) Anesth. Analg. 79, 447–454.5. Warltier, D. C., Al-Wathiqui, M. H., Kampine, J. P., and Schmeling, W. T. (1988) Anesthesiology 69, 552–565.6. Kersten, J. R., Lowe, D., Hettrick, D. A., Pagel, P. S., Gross, G. J., and Warltier, D. C. (1996) Anes. Analg. 83,

27–33.7. Han, J., Leem, C. H., So, I., Kim, E., Hong, S., Ho, W., Sung, H., and Earm, Y. E. (1994) J. Mol. Cell. Cardiol.

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10. Han, J., So, I., Kim, E. Y., and Earm, Y. E. (1993) Pflugers. Arch. 425, 546–548.11. Han, J., Kim, E., Ho, W. K., and Earm, Y. E. (1995) Heart. Vessels. 9, 94–96.12. Cason, B. A., Shubayev, I., and Hicky, R. F. (1994) Anesthesiology 81, 1245–1255.13. Larach, D. R., and Schuler, H. G. (1993) J. Pharmacol. Exp. Ther. 267, 72–81.

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