NO directly activates mitoK(ATP) channels and potentiates the ability of diazoxide to open these channels. These results provide novel mechanistic links between NO-induced cardioprotection and mitoK(ATP) channels.

Background —Pharmacological evidence has implicated ATP-sensitive K + (K ATP ) channels as the effectors of cardioprotection, but the relative roles of mitochondrial (mitoK ATP ) and sarcolemmal (surfaceK ATP ) channels remain controversial. Methods and Results —We examined the effects of the K ATP channel blocker HMR1098 and the K ATP channel opener P-1075 on surfaceK ATP and mitoK ATP channels in rabbit ventricular myocytes. HMR1098 (30 μmol/L) inhibited the surfaceK ATP current activated by metabolic inhibition, whereas the drug did not blunt diazoxide (100 μmol/L)-induced flavoprotein oxidation, an index of mitoK ATP channel activity. P-1075 (30 μmol/L) did not increase flavoprotein oxidation but did elicit a robust surfaceK ATP current that was completely inhibited by HMR1098. These results indicate that HMR1098 selectively inhibits surfaceK ATP channels, whereas P-1075 selectively activates surface K ATP channels. In a cellular model of simulated ischemia, the mitoK ATP channel opener diazoxide (100 μmol/L), but not P-1075, blunted cellular injury. The cardioprotection afforded by diazoxide or by preconditioning was prevented by the mitoK ATP channel blocker 5-hydroxydecanoate (500 μmol/L) but not by the surfaceK ATP channel blocker HMR1098 (30 μmol/L). Conclusions —The cellular effects of mitochondria- or surface-selective agents provide further support for the emerging consensus that mitoK ATP channels rather than surfaceK ATP channels are the likely effectors of cardioprotection.

IntroductionBrief periods of ischemia that precede sustained ischemia lead to a reduction of infarct size (1-4). This phenomenon is known as ischemic preconditioning (IPC) and the mechanisms underlying it remain unclear, despite extensive study (5-7). Sarcolemmal ATP-sensitive potassium (sarcK ATP ) channels were proposed to play an important role in the cardioprotective effect, because potassium channel openers (KCOs) mimicked the cardioprotection and the K ATP channel blocker glibenclamide abolished the IPC (8-13). Since the discovery of sarcK ATP channels (14), it has been recognized that activation of these channels may serve an endogenous cardioprotective mechanism: action potential shortening due to K ATP channel activation is expected to reduce Ca 2+ influx in ischemic heart cells, producing a cardioplegic effect. However, recent studies indicate that mitochondria harbor another type of K ATP channel (15,16), and activation of the mitochondrial K ATP (mitoK ATP ) channel rather than the sarcK ATP channel has been proposed to underlie the cardioprotective effect of IPC or . Because the mitoK ATP channel has yet to be identified at a molecular level, the existing evidence is almost entirely based on pharmacological studies.The molecular structure of sarcK ATP channels has been clarified by cloning members of the inwardly rectifying K + channel subfamily Kir6.0 (Kir6.1 and Kir6.2) and the receptors for sulfonylureas (SUR1, SUR2A, and SUR2B) (21,22). Accumulating evidence indicates that native sarcK ATP channels are composed of these two structurally distinct subunits. Various combinations of Kir6.0 and SUR convey the heterogeneity in channel properties observed in native cells of various tissues, such as heart, pancreatic β cells, skeletal and smooth muscles, and neurons (22). Cardiac sarcK ATP channels have been suggested in a reconstitution study to comprise SUR2A and Kir6.2 (23). Our recent functional study using Kir6.2-deficient mice has provided direct evidence that Kir6.2 forms the pore region of cardiac sarcK ATP channels but not of vascular sarcK ATP channels (24). Although the molecular identity of mitoK ATP channel remains unclear, experiments using dominant negative gene transfer have indicated that neither Kir6.1 nor Kir6.2 is a functionally important part of the mitoK ATP channel in native heart cells (25). Despite the paucity of molecular tools available to probe the role of mitoK ATP channels, Kir6.2-deficient mice are potentially useful to define the pathophysiological significance Recently it has been postulated that mitochondrial ATP-sensitive K + (mitoK ATP ) channels rather than sarcolemmal K ATP (sarcK ATP ) channels are important as end effectors and/or triggers of ischemic preconditioning (IPC). To define the pathophysiological significance of sarcK ATP channels, we conducted functional experiments using Kir6.2-deficient (KO) mice. Metabolic inhibition with glucose-free, dinitrophenol-containing solution activated sarcK ATP current and shortened the action potential duration in ventric...

IntroductionBrief periods of ischemia that precede sustained ischemia lead to a reduction of infarct size (1-4). This phenomenon is known as ischemic preconditioning (IPC) and the mechanisms underlying it remain unclear, despite extensive study (5-7). Sarcolemmal ATP-sensitive potassium (sarcK ATP ) channels were proposed to play an important role in the cardioprotective effect, because potassium channel openers (KCOs) mimicked the cardioprotection and the K ATP channel blocker glibenclamide abolished the IPC (8-13). Since the discovery of sarcK ATP channels (14), it has been recognized that activation of these channels may serve an endogenous cardioprotective mechanism: action potential shortening due to K ATP channel activation is expected to reduce Ca 2+ influx in ischemic heart cells, producing a cardioplegic effect. However, recent studies indicate that mitochondria harbor another type of K ATP channel (15,16), and activation of the mitochondrial K ATP (mitoK ATP ) channel rather than the sarcK ATP channel has been proposed to underlie the cardioprotective effect of IPC or . Because the mitoK ATP channel has yet to be identified at a molecular level, the existing evidence is almost entirely based on pharmacological studies.The molecular structure of sarcK ATP channels has been clarified by cloning members of the inwardly rectifying K + channel subfamily Kir6.0 (Kir6.1 and Kir6.2) and the receptors for sulfonylureas (SUR1, SUR2A, and SUR2B) (21,22). Accumulating evidence indicates that native sarcK ATP channels are composed of these two structurally distinct subunits. Various combinations of Kir6.0 and SUR convey the heterogeneity in channel properties observed in native cells of various tissues, such as heart, pancreatic β cells, skeletal and smooth muscles, and neurons (22). Cardiac sarcK ATP channels have been suggested in a reconstitution study to comprise SUR2A and Kir6.2 (23). Our recent functional study using Kir6.2-deficient mice has provided direct evidence that Kir6.2 forms the pore region of cardiac sarcK ATP channels but not of vascular sarcK ATP channels (24). Although the molecular identity of mitoK ATP channel remains unclear, experiments using dominant negative gene transfer have indicated that neither Kir6.1 nor Kir6.2 is a functionally important part of the mitoK ATP channel in native heart cells (25). Despite the paucity of molecular tools available to probe the role of mitoK ATP channels, Kir6.2-deficient mice are potentially useful to define the pathophysiological significance Recently it has been postulated that mitochondrial ATP-sensitive K + (mitoK ATP ) channels rather than sarcolemmal K ATP (sarcK ATP ) channels are important as end effectors and/or triggers of ischemic preconditioning (IPC). To define the pathophysiological significance of sarcK ATP channels, we conducted functional experiments using Kir6.2-deficient (KO) mice. Metabolic inhibition with glucose-free, dinitrophenol-containing solution activated sarcK ATP current and shortened the action potential duration in ventric...

Nicorandil exerts a direct cardioprotective effect on heart muscle cells, an effect mediated by selective activation of mitoK(ATP) channels.

To investigate the membrane current changes induced by membrane stretching, single guinea pig ventricular myocytes were superfused with solutions of various osmolarities, and the whole-cell current was recorded by the patchclamp technique. The application of 70% and 130% osmolar bath solutions increased and decreased the amplitude of delayed rectifier K' current (IK), respectively, whereas no obvious change was observed in the L-type Ca2' current or the inward rectifier K' current. When the Na+-K+ pump current (IpUmp) was recorded by the use of high-Na+ (>35 mmol/L) pipette solutions, 'pump was also increased and decreased by the superfusion of hypotonic and hypertonic solutions, respectively, in approximately half of the cells. An increase of the IpUmp was also observed in the absence of external Na+, excluding a possibility that the enhancement of IpUmp was secondary to an elevation of cytosolic Na+. In most cells that did not show the increase of 1pUmp. the hypotonic superfusion induced a gradual activation of Cl-current. The hypertonic superfusion did not cause any consistent change in the membrane Cl-conductance. Since the response of 'K was observed in all experiments, its mechanism was studied. We failed to observe marked changes in the kinetic and conductance properties of 'K in the hypotonic solution. The involvements of either the protein kinases or Ca2' were also ruled out as major mechanisms underlying the IK response. (Circ Res. 1994;75: 887-895.) Key Words * cardiac myocytes * cell swelling * delayed rectifier K+ current * Na+-K+ pump current T he heart rate increases in response to an increase of venous return, and an acceleration of the spontaneous rhythm was demonstrated by stretching the isolated cardiac pacemaker tissue.12 Contraction of the ventricle increases under conditions of volume overload.3 These autoregulatory responses to mechanical stimuli are considered to be mediated at least in part by changes in the membrane current. Voltage-clamp analysis, however, is made difficult by a mechanical dislodgment of the intracellular electrode during stretch of the myocyte. To circumvent this difficulty, cell swelling by superfusing hypotonic solutions4 or by applying positive pressure into the whole-cell patch-clamp electrode5 was used to study the ©) 1994 American Heart Association, Inc. stretch applied to the longitudinal axis of the cell, however, occasionally increased the amplitude of a time-independent current, which was most probably mediated by an increase in the intracellular Ca`. It was suggested that cell swelling in the hypotonic solution might reflect the effect of stretching the cell membrane on ionic channels and that the mechanical stretch along the cellular longitudinal axis failed to apply effective stress to the surface membrane.The present study aims at clarifying changes in the membrane current during superfusion of the ventricular myocytes with hypotonic solutions. It was observed that the amplitude of IK, the Na+-K+ pump current (IpUmp), and a time-independen...

Our results support the hypothesis that adenosine receptor activation primes the opening of mitochondrial K(ATP) channels in a protein kinase C-dependent manner. The findings provide tangible links among various key elements in the preconditioning cascade.

IntroductionBrief periods of ischemia that precede sustained ischemia lead to a reduction of infarct size (1-4). This phenomenon is known as ischemic preconditioning (IPC) and the mechanisms underlying it remain unclear, despite extensive study (5-7). Sarcolemmal ATP-sensitive potassium (sarcK ATP ) channels were proposed to play an important role in the cardioprotective effect, because potassium channel openers (KCOs) mimicked the cardioprotection and the K ATP channel blocker glibenclamide abolished the IPC (8-13). Since the discovery of sarcK ATP channels (14), it has been recognized that activation of these channels may serve an endogenous cardioprotective mechanism: action potential shortening due to K ATP channel activation is expected to reduce Ca 2+ influx in ischemic heart cells, producing a cardioplegic effect. However, recent studies indicate that mitochondria harbor another type of K ATP channel (15,16), and activation of the mitochondrial K ATP (mitoK ATP ) channel rather than the sarcK ATP channel has been proposed to underlie the cardioprotective effect of IPC or . Because the mitoK ATP channel has yet to be identified at a molecular level, the existing evidence is almost entirely based on pharmacological studies.The molecular structure of sarcK ATP channels has been clarified by cloning members of the inwardly rectifying K + channel subfamily Kir6.0 (Kir6.1 and Kir6.2) and the receptors for sulfonylureas (SUR1, SUR2A, and SUR2B) (21,22). Accumulating evidence indicates that native sarcK ATP channels are composed of these two structurally distinct subunits. Various combinations of Kir6.0 and SUR convey the heterogeneity in channel properties observed in native cells of various tissues, such as heart, pancreatic β cells, skeletal and smooth muscles, and neurons (22). Cardiac sarcK ATP channels have been suggested in a reconstitution study to comprise SUR2A and Kir6.2 (23). Our recent functional study using Kir6.2-deficient mice has provided direct evidence that Kir6.2 forms the pore region of cardiac sarcK ATP channels but not of vascular sarcK ATP channels (24). Although the molecular identity of mitoK ATP channel remains unclear, experiments using dominant negative gene transfer have indicated that neither Kir6.1 nor Kir6.2 is a functionally important part of the mitoK ATP channel in native heart cells (25). Despite the paucity of molecular tools available to probe the role of mitoK ATP channels, Kir6.2-deficient mice are potentially useful to define the pathophysiological significance Recently it has been postulated that mitochondrial ATP-sensitive K + (mitoK ATP ) channels rather than sarcolemmal K ATP (sarcK ATP ) channels are important as end effectors and/or triggers of ischemic preconditioning (IPC). To define the pathophysiological significance of sarcK ATP channels, we conducted functional experiments using Kir6.2-deficient (KO) mice. Metabolic inhibition with glucose-free, dinitrophenol-containing solution activated sarcK ATP current and shortened the action potential duration in ventric...