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Anesthesiology 2002; 97:50–6 © 2002 American Society of Anesthesiologists, Inc. Lippincott Williams & Wilkins, Inc. Differential Modulation of the Cardiac Adenosine Triphosphate–sensitive Potassium Channel by Isoflurane and Halothane Wai-Meng Kwok, Ph.D.,* Anne T. Martinelli, M.D.,† Kazuhiro Fujimoto, M.D., Ph.D.,‡ Akihiro Suzuki, M.D.,‡ Anna Stadnicka, Ph.D.,§ Zeljko J. Bosnjak, Ph.D.ʈ Background: The cardiac adenosine triphosphate–sensitive ics can also be cardioprotective.4–8 This cardioprotec- potassium (KATP) channel is activated during pathophysiologi- tion, termed anesthetic-induced preconditioning, mim- cal episodes such as ischemia and hypoxia and may lead to ics ischemic preconditioning,9 whereby a small ischemic beneficial effects on cardiac function. Studies of volatile anes- episode protects the myocardium from a subsequent, Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/97/1/50/407063/0000542-200207000-00008.pdf by guest on 24 September 2021 thetic interactions with the cardiac KATP channel have been limited. The goal of this study was to investigate the ability of more devastating insult. volatile anesthetics halothane and isoflurane to modulate the The underlying mechanisms involved in anesthetic- cardiac sarcolemmal KATP channel. induced preconditioning have not been elucidated. De- Methods: The K channel current (I ) was monitored ATP KATP spite the potentially numerous targets of volatile anes- using the whole cell configuration of the patch clamp technique from single ventricular cardiac myocytes enzymatically isolated thetics, including ion channels and intracellular second from guinea pig hearts. IKATP was elicited by extracellular ap- messenger systems, the adenosine triphosphate–sensi- plication of the potassium channel openers 2,4-dinitrophenol tive potassium (KATP) channel has been hypothesized to or pinacidil. be one of the major target proteins involved in anesthet- Results: Volatile anesthetics modulated I in an anesthetic- KATP ic-induced cardioprotection.5,10 The sarcolemmal K dependent manner. Isoflurane facilitated the opening of the ATP channel is an attractive target since it acts as a metabolic KATP channel. Following initial activation of IKATP by 2,4-dini- trophenol, isoflurane at 0.5 and 1.3 mM further increased cur- sensor, and its activation leads to shortening of the rent amplitude by 40.4 ؎ 11.1% and 58.4 ؎ 20.6%, respectively. cardiac action potential.11,12 This, in turn, would lead to Similar results of isoflurane were obtained when pinacidil was decreased calcium entry via the voltage-gated calcium used to activate I . However, isoflurane alone was unable to KATP channels and preservation of high-energy phosphates. elicit KATP channel opening. In contrast, halothane inhibited Recent studies have also shown that the K channel on ؎ ؎ IKATP elicited by 2,4-dinitrophenol by 50.6 5.8% and 72.1 ATP 11.6% at 0.4 and 1.0 mM, respectively. When IKATP was activated the inner membrane of mitochondria plays a more piv- by pinacidil, halothane had no significant effect on the current. otal role in cardioprotection, particularly in ischemic 13–15 Conclusions: The cardiac sarcolemmal KATP channel is differ- preconditioning. Volatile anesthetics, isoflurane and entially modulated by volatile anesthetics. Isoflurane can facilitate sevoflurane, were also recently reported to induce a the further opening of the KATP channel following initial channel activation by 2,4-dinitrophenol or pinacidil. The effect of halo- redox-dependent increase in mitochondrial flavoprotein thane was dependent on the method of channel activation, inhib- oxidation, an indicator of mitochondrial KATP channel 16 iting IKATP activated by 2,4-dinitrophenol but not by pinacidil. opening. Consequently, anesthetic-induced and isch- emic preconditioning likely involve complex pathways VOLATILE anesthetics have cardiac depressant effects that may include both the mitochondrial and sarcolem- and inhibit various ion channels in the heart. However, mal KATP channels. multiple effects of volatile anesthetics on the myocar- Evidence for the involvement of the sarcolemmal KATP dium suggest the complexity of the underlying cellular channel in anesthetic-induced preconditioning is de- and molecular mechanisms. Inhibition of cardiac volt- rived from infarct-size studies using whole animal mod- age-gated calcium and sodium channels by volatile anes- els.5 On the other hand, direct studies of volatile anes- 1–3 thetics is well documented and may lead to an in- thetic effects on the KATP channel have been limited. In creased propensity to arrhythmias. However, recent the present study, the effects of two volatile anesthetics, studies have convincingly shown that volatile anesthet- isoflurane and halothane, on the cardiac sarcolemmal KATP channel were investigated using the whole cell configuration of the patch clamp technique. * Assistant Professor, Departments of Anesthesiology and Pharmacology and Toxicology, ‡ Research Fellow, § Assistant Professor, Department of Anesthesi- ology, ʈ Professor, Departments of Anesthesiology and Physiology. † Medical Resident; current address: Department of Obstetrics and Gynecology, Metro- Materials and Methods Health Medical Center, Cleveland, Ohio. Preparation of Isolated Cardiac Ventricular Received from the Department of Anesthesiology, Medical College of Wiscon- sin, Milwaukee, Wisconsin. Submitted for publication December 19, 2001. Myocyte Accepted for publication March 19, 2002. Supported in part by grant Nos. After approval was obtained from the Institutional An- GM-54568 (to Dr. Kwok) and NHLBI-34708 (to Dr. Bosnjak) from the National Institutes of Health, Bethesda, Maryland. imal Care and Use Committee, cardiac myocytes were Address reprint requests to Dr. Kwok: Department of Anesthesiology, Medical enzymatically isolated from guinea pigs weighing 200– College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, Wisconsin 53226. Address electronic mail to: [email protected]. Individual article re- 300 g. The procedure of the cell isolation is a modifica- 17 prints may be purchased through the Journal Web site, www.anesthesiology.org. tion of that of Mitra and Morad and has previously been Anesthesiology, V 97, No 1, Jul 2002 50 DIFFERENTIAL MODULATION OF THE KATP CHANNEL 51 The cell suspension was then filtered, centrifuged, and washed twice in Tyrode solution before the cells were ready for experiments. The cells were stored in Tyrode solution at room temperature (20–25°C) and used within 12 h after isolation. For the patch clamp experi- ments, cells were transferred to a recording chamber mounted on the stage of an inverted microscope. Solutions The isolated myocytes were initially washed in a stan- dard Tyrode solution that contained the following in- Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/97/1/50/407063/0000542-200207000-00008.pdf by guest on 24 September 2021 gredients: 132.0 mM NaCl, 4.8 mM KCl, 1.2 mM MgCl2, 1.0 mM CaCl2, 5.0 mM dextrose, and 10.0 mM HEPES, with pH adjusted to 7.4 with NaOH. After establishing a gigaohm seal, the external Tyrode solution was changed to one appropriate for measurement of potassium chan- nel currents and contained 132.0 mM N-methyl-D-gluca- mine (substitute for sodium), 1.0 mM CaCl2, 2.0 mM MgCl2, 10.0 mM HEPES, and 5.0 mM KCl, with pH ad- justed to 7.4 with HCl. Nisoldipine (200 nM), supplied by Miles Pentex (West Haven, CT), was also added to block the L-type Ca channel current. To elicit activation of the KATP current, 2,4-dinitrophenol or pinacidil, a KATP chan- nel opener, was used. 2,4-Dinitrophenol (Sigma Chemi- cal) was added directly to the external buffer solution to obtain a desired concentration. Pinacidil (Sigma/RBI) was prepared as a 10-mM stock in dimethyl sulfoxide and Fig. 1. Activation of IKATP by 2,4-dinitrophenol (DNP). (Top) Sample whole cell current traces recorded in control, in the diluted to the desired concentration in the external so- presence of 120 ␮M DNP, and in the presence of DNP ؉ gliben- lution. In a specified set of experiments, bimakalim was clamide (Glib, 200 nM). Current was monitored at test potentials used as a potassium channel opener. Bimakalim was sup- .of ؊110, ؊30, and ؉10 mV from a holding potential of ؊40 mV Current at ؊110 mV is the inward-rectifier K current. The out- plied by Garrett Gross, Ph.D. (Professor, Department of ward current sensitive to glibenclamide is IKATP.(Bottom) The Pharmacology and Toxicology, Medical College of Wiscon- corresponding current–voltage relation. Current amplitude was measured at the end of the 100-ms test pulses. sin) and was prepared in dimethyl sulfoxide. The final concentration of dimethyl sulfoxide (0.025%) had no effect on the whole cell K currents. The standard pipette solution 3 reported. In brief, the guinea pigs were anesthetized by contained 60.0 mM K-glutamate, 50.0 mM KCl, 1.0 mM intraperitoneal injection of 180 mg/kg pentobarbital so- CaCl2, 1.0 mM MgCl2, 11.0 mM EGTA, and 0.1–1.0 mM dium and injected with 1,000 U heparin to hinder coag- K2-ATP, with pH adjusted to 7.4 with KOH. ulation. During deep anesthesia, the hearts were quickly The volatile anesthetics, isoflurane (Ohmeda Caribe excised and mounted via the ascending aorta on a Lan- Inc., Liberty Corner, NJ) and halothane (Halocarbon Lab- gendorff-type apparatus. Each heart was perfused retro- oratories, River Edge, NJ), were mixed by adding known gradely at a rate of 6–8 ml/min with Joklik’s medium aliquots of concentrated anesthetics to graduated sy- containing 2.5 U/ml heparin at pH 7.23. After 3–4 min to ringes with the appropriate bath solutions. Isoflurane allow for clearing of blood, the perfusing solution was and halothane superfusions were achieved using a sy- replaced with an enzyme solution containing Joklik’s ringe pump with a constant flow of 1 ml/min. Clinically medium with 0.25 mg/ml collagenase (Gibco Life Tech- relevant concentrations of isoflurane (0.5–1.3 mM, equiv- nologies, Grand Island, NY), 0.13 mg/ml protease (Type alent to 1.048–2.723 vol%) and halothane (0.4–1.0 mM, XIV, Sigma, St.

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