Isoflurane-Induced Facilitation of the Cardiac Sarcolemmal KATP Channel

Anesthesiology 2002; 97:57–65 © 2002 American Society of Anesthesiologists, Inc. Lippincott Williams & Wilkins, Inc. Isoflurane-induced Facilitation of the Cardiac Sarcolemmal KATP Channel Kazuhiro Fujimoto, M.D., Ph.D.,* Zeljko J. Bosnjak, Ph.D.,† Wai-Meng Kwok, Ph.D.‡ Background: Volatile anesthetics have cardioprotective ef- osine triphosphate–sensitive potassium (KATP) channels fects that mimic ischemic preconditioning, including the in- in IPC. Several studies have shown that glibenclamide, a volvement of adenosine triphosphate–sensitive potassium (K ) channels. However, evidence for a direct effect of volatile potent KATP channel blocker, abolishes the beneficial ATP 8 anesthetic on the KATP channel is limited. In this study, the effects of IPC, while KATP channel openers, such as effects of isoflurane on the cardiac sarcolemmal KATP channel bimakalim, pinacidil, and cromakalim, mimicked the car- were investigated. 9,10 dioprotective effects of IPC. Recent studies have also Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/97/1/57/407157/0000542-200207000-00009.pdf by guest on 26 September 2021 Methods: Single ventricular myocytes were enzymatically iso- implicated the contribution of the mitochondrial KATP lated from guinea pig hearts. Whole cell and single-channel 11 configurations, specifically the cell-attached and inside-out channel in IPC. However, the relative contributions of patch mode, of the patch clamp technique were used to monitor the cardiac sarcolemmal and mitochondrial KATP chan- sarcolemmal KATP channel current. nels in IPC are not clear. Recent evidence suggests that Results: In the cell-attached patch configuration, 2,4-dinitro- ␮ the mitochondrial KATP channel may play a more signif- phenol (150 M) opened the sarcolemmal KATP channel. Isoflu- rane (0.5 mM) further increased channel open probability and icant role in cardioprotection than the sarcolemmal 12 the number of active channels in the patch. In contrast, in the channel. Yet the contribution by the sarcolemmal KATP inside-out patch experiments, isoflurane had no significant ef- channel can not be irrefutably discounted. The various fect on the K channel activated by low ATP (0.2–0.5 mM). In ATP signaling pathways that underlie cardioprotection can addition, isoflurane had no effect on the KATP channel when activated by adenosine diphosphate, adenosine ؉ guanosine potentially modulate the mitochondrial and sarcolemmal triphosphate, bimakalim, and 2,4-dinitrophenol under inside- KATP channels. Both channel types may turn out to be 13 out patch configurations. When KATP current was monitored in important in cardioprotection by IPC. the whole cell mode, isoflurane alone was unable to elicit chan- Volatile anesthetics, particularly isoflurane, were re- nel opening. However, during sustained protein kinase C acti- cently found to mimic IPC of the heart.14,15 The cellular vation by 12,13-dibutyrate, isoflurane activated the KATP current that was sensitive to glibenclamide. In contrast, isoflurane had mechanisms underlying anesthetic-induced precondi- no effect on the KATP channel activated by 12,13-dibutyrate in a tioning are not known but may parallel those of IPC. cell-free environment. Since the isoflurane effects were abolished by gliben- Conclusions: Isoflurane facilitated the opening of the sar- clamide, the KATP channels have been implicated. How- colemmal KATP channel in the intact cell, but not in an excised, inside-out patch. The isoflurane effect was not due to a direct ever, direct evidence at the cellular and molecular levels interaction with the KATP channel protein, but required an of the involvement of these channels in anesthetic-in- intracellular component, likely including the translocation of duced cardioprotection is limited.16,17 specific protein kinase C isoforms. This suggests that the sar- Several endogenous factors also modulate the sarcolemmal K channel may have a significant role in anesthet- ATP colemmal K channel. Ischemic and hypoxic factors, ic-induced preconditioning. ATP such as an increase in adenosine diphosphate (ADP),18 production of adenosine,19 and changes in pH20 regulate BRIEF periods of sublethal ischemia reduce the amount K channel openings. Thus, despite the emergence of of myocyte necrosis produced by a subsequent sustained ATP the role of the mitochondrial K channel in cardiopro- period of ischemia.1 This phenomenon, termed ischemic ATP preconditioning (IPC), has been shown to be cardioprotection, effects on the sarcolemmal KATP channel by tective in several mammalian models, including dogs,2,3 these endogenous factors make the channel an attractive rabbits,4 rats,5 pigs,6 and humans.7 Although the under- end effector of cardioprotection. In anesthetic-induced lying mechanism is still unclear, much has been docu- cardioprotection, a direct action of isoflurane on the 16 mented of the involvement of cardiac sarcolemmal aden- cardiac sarcolemmal KATP channel has been reported. Isoflurane decreased channel activity but also dimin- ished the channel’s sensitivity to ATP. Our previous study showed that isoflurane and halothane differentially * Research Fellow, Department of Anesthesiology, † Professor, Departments of 21 Anesthesiology and Physiology, ‡ Assistant Professor, Departments of Anesthesi- modulated the sarcolemmal KATP channel. Under ology and Pharmacology and Toxicology. whole cell conditions, isoflurane potentiated the open- Received from the Department of Anesthesiology, Medical College Wisconsin, Milwaukee, Wisconsin. Submitted for publication December 19, 2001. Accepted ing of 2,4-dinitrophenol– and pinacidil-activated KATP for publication March 19, 2002. Supported in part by grant Nos. GM-54568 (to channel current (IKATP), while halothane inhibited the Dr. Kwok) and NHLBI-34708 (to Dr. Bosnjak) from the National Institutes of Health, Bethesda, Maryland. 2,4-dinitrophenol–activated IKATP and had no effect on Address reprint requests to Dr. Kwok: Department of Anesthesiology, Medical the pinacidil-activated IKATP. College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, Wisconsin 53226. The goal of the present study was to directly investi- Address electronic mail to: [email protected]. Individual article reprints may be purchased through the Journal Web site, www.anesthesiology.org. gate the potentiating effects of isoflurane on the cardiac Anesthesiology, V 97, No 1, Jul 2002 57 58 FUJIMOTO ET AL. sarcolemmal KATP channel at the single-channel level used in the inside-out patch experiments, with intracel- and to determine whether modulators of the channel lular ATP kept at 0.5 mM. affected the anesthetic action. Several modulators of the sarcolemmal KATP channel were used. 2,4-Dinitrophenol, ATP, ADP, adenosine, and guanosine triphosphate (GTP) were all obtained from Materials and Methods Sigma Chemical Co. and dissolved in the appropriate buffer solutions for the patch clamp experiments. The Preparation of Isolated Cardiac Ventricular potassium channel opener, bimakalim, was provided by Myocyte Garrett Gross, Ph.D. (Professor, Department of Pharma- After approval was obtained from the Institutional An- imal Care and Use Committee, single ventricular cells cology and Toxicology, Medical College of Wisconsin). were isolated from enzymatically treated adult guinea Bimakalim was prepared as a 10-mM stock solution in Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/97/1/57/407157/0000542-200207000-00009.pdf by guest on 26 September 2021 pig (200–300 g) hearts. The procedure is a modification dimethyl sulfoxide and diluted to a concentration of ␮ of that of Mitra and Morad,22 which has been previously 5 M in the appropriate buffer before use. described.23 In brief, immediately after thoracotomy, the The volatile anesthetic isoflurane (Ohmeda Caribe heart was rapidly mounted on a Langendorff apparatus Inc., Liberty Corner, NJ) was mixed by adding known and perfused retrogradely through the aorta with warm aliquots of concentrated anesthetics to graduated sy- (37°C) oxygenated buffer containing the Joklik medium ringes with the appropriate bath solutions. Isoflurane and 0.25 mg/ml collagenase (Gibco Life Technologies, superfusion was achieved using a syringe pump with a Grand Island, NY) and 0.13 mg/ml protease (Sigma constant flow of 1 ml/min. Clinically relevant concentra- Chemical Co., St. Louis, MO) for 8–12 min. The isolated tions of isoflurane (0.5 and 1.0 mM, equivalent to 1.048 myocytes were washed and stored in a standard Tyrode and 2.095 vol%, respectively) were used. To determine solution. Only cells with clear borders and well-defined anesthetic concentrations, 1 ml of the superfusate was striations were selected and used for experiments within collected in a metal-capped 2-ml glass vial at the end of 12 h after isolation. each experiment. The superfusate concentration of the anesthetic was then determined by gas chromatography Solutions (head-space analysis) utilizing flame ionization detection The isolated myocytes were initially placed in a stan- Perkin-Elmer Sigma 3B gas chromatograph. dard Tyrode solution that contained the following ingre- dients: 132 mM NaCl, 4.8 mM KCl, 3 mM MgCl2,1mM Recording Procedure and Data Analysis CaCl2,5mM dextrose, and 10 mM HEPES, with pH ad- KATP channel activity was monitored using the whole justed to 7.3 with NaOH. After establishing a gigaohm cell, cell-attached, and excised, inside-out patch config- seal, the external solution was changed to one that was urations of the patch clamp technique. Patch pipettes appropriate for the various patch

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