Cardiac KATP Research Articles

Pharmacological and Histochemical Distinctions Between Molecularly Defined Sarcolemmal KATPChannels and Native Cardiac Mitochondrial KATPChannels

A variety of direct and indirect techniques have revealed the existence of ATP-sensitive potassium (KATP) channels in the inner membranes of mitochondria. The molecular identity of these mitochondrial KATP (mitoKATP) channels remains unclear. We used a pharmacological approach to distinguish mitoKATP channels from classical, molecularly defined cardiac sarcolemmal KATP (surfaceKATP) channels encoded by the sulfonylurea receptor SUR2A and the pore-forming subunit Kir6.2. SUR2A and Kir6.2 were expressed in human embryonic kidney (HEK)293 cells, and their activities were measured by patch-clamp recordings of membrane current. SurfaceKATP channels are activated potently by 100 microM pinacidil but only weakly by 100 microM diazoxide; in addition, they are blocked by 10 microM glibenclamide, but are insensitive to 500 microM 5-hydroxydecanoate. This pharmacology, which was confirmed with patch-clamp recordings in intact rabbit ventricular myocytes, contrasts with that of mitoKATP channels as indexed by flavoprotein oxidation. MitoKATP channels in myocytes are activated equally by 100 microM diazoxide and 100 microM pinacidil. In contrast to its lack of effect on surfaceKATP channels, 5-hydroxydecanoate is an effective blocker of mitoKATP channels. Glibenclamide's effects on mitoKATP channels are difficult to assess, because it independently activates flavoprotein fluorescence, consistent with a previously described primary uncoupling effect. Confocal imaging of the subcellular distribution of expressed fluorescent Kir6.2 in HEK cells and in myocytes revealed no targeting of mitochondrial membranes. The differences in drug sensitivity and subcellular localization indicate that mitoKATP channels are distinct from surface KATP channels at a molecular level.

Effect of bupivacaine on ATP-dependent potassium channels in rat cardiomyocytes

Bupivacaine induces fatal arrhythmia when accidentally injected i.v. or overdosed, whereas lidocaine is used as an anti-arrhythmic agent. We have suggested recently that the anti-arrhythmic effect of lidocaine may be explained by suppression of ATP-sensitive potassium (KATP) channels. Therefore, it could be argued that different sensitivities of KATP channels to both drugs could be a reason for their different arrhythmic and anti-arrhythmic properties. In this study, we have investigated the direct action of bupivacaine on KATP channels in cardiomyocytes. The effects of bupivacaine on the cardiac KATP channel were investigated using the patch-clamp technique on enzymatically dissociated cardiomyocytes of adult rats. Bupivacaine was applied to the outer side of excised membrane patches using a multiple-barrel perfusion system. Concentration-response curves indicated that bupivacaine blocked the mean current of the KATP channels at a half-maximum inhibiting concentration (IC50) of 29 mumol litre-1, similar to that reported for lidocaine (43 mumol litre-1). Binding of bupivacaine influenced the gating of this channel, but did not reduce the conductance of the open channel. Bupivacaine and lidocaine were equipotent in blocking KATP channels. However, because of its excessive block of the sodium channel in the inactivated state, block of KATP channels by bupivacaine will only enhance its cardiotoxicity.

Recombinant cardiac ATP-sensitive K+ channel subunits confer resistance to chemical hypoxia-reoxygenation injury.

Opening of cardiac ATP-sensitive K+ (KATP) channels has emerged as a promising but still controversial cardioprotective mechanism. Defining KATP channel function at the level of recombinant channel proteins is a necessary step toward further evaluation of the cardioprotective significance of this ion conductance. KATP channel deficient COS-7 cells were found to be vulnerable to chemical hypoxia-reoxygenation injury that induced significant cytosolic Ca2+ loading (from 97+/-3 to 236+/-11 nmol/L). In these cells, the potassium channel opener pinacidil (10 micromol/L) did not prevent Ca2+ loading (from 96+/-3 nmol/L before to 233+/-12 nmol/L after reoxygenation) or evoked membrane current. Cotransfection with Kir6.2/SUR2A genes, which encode cardiac KATP channel subunits, resulted in a cellular phenotype that, in the presence of pinacidil (10 micromol/L), expressed K+ current and gained resistance to hypoxia-reoxygenation (Ca2+ concentration from 99+/-7 to 127+/-11 nmol/L; P>0.05). Both properties were abolished by the KATP channel blocker glyburide (1 micromol/L). In COS-7 cells transfected with individual channel subunits Kir6.2 or SUR2A, which alone do not form functional cardiac KATP channels, pinacidil did not protect against hypoxia-reoxygenation. The fact that transfer of cardiac KATP channel subunits protected natively KATP channel deficient cells provides direct evidence that the cardiac KATP channel protein complex harbors intrinsic cytoprotective properties. These findings validate the concept that targeting cardiac KATP channels should be considered a valuable approach to protect the myocardium against injury.

Changes in ventricular repolarization during acidosis and low-flow ischemia.

Myocardial ischemia, primarily a metabolic insult, is also defined by altered cardiac mechanical and electrical activity. We have investigated the metabolic contributions to the electrophysiological changes during low-flow ischemia (7.5% of the control flow) using 31P NMR spectroscopy to monitor metabolic parameters, suction electrodes to study epicardial monophasic action potentials, and 86Rb as a tracer for K+-equivalent efflux during low-flow ischemia in the Langendorff-perfused ferret heart. Shortening of the action potential duration at 90% repolarization (APD90) was most marked between 1 and 5 min after induction of ischemia, at which time it shortened from 261 +/- 4 to 213 +/- 8 ms. The period of marked APD90 shortening was accompanied by a fivefold increase in the rate of 86Rb efflux, both of which were inhibited by the ATP-sensitive K+ (KATP)-channel blockers glibenclamide and 5-hydroxydecanoate (5-HD), as well as by a significant fall in intracellular pH (pHi) from 7.14 +/- 0.02 to 6.83 +/- 0.03 but no change in intracellular ATP concentration ([ATP]i). We therefore investigated whether a fall in pHi could be the metabolic change responsible for modulating cardiac KATP channel activity in the intact heart during ischemia. Both metabolic (30 mM lactate added to extracellular solution) and respiratory (PCO2 increased to 15%) acidosis caused an initial lengthening of APD90 to 112 +/- 1.5 and 113 +/- 0.9%, respectively, followed by shortening during continued acidosis to 106 +/- 1.2 and 106 +/- 1.4%, respectively. The shortening of APD90 during continued acidosis was inhibited by glibenclamide, consistent with acidosis causing activation of KATP channels at normal [ATP]i. The similar responses to metabolic (induced by adding either l- or d-lactate) and respiratory acidosis suggest that lactate has no independent metabolic effect on action potential repolarization.

Ligand-insensitive state of cardiac ATP-sensitive K+ channels. Basis for channel opening.

The mechanism by which ATP-sensitive K+ (KATP) channels open in the presence of inhibitory concentrations of ATP remains unknown. Herein, using a four-state kinetic model, we found that the nucleotide diphosphate UDP directed cardiac KATP channels to operate within intraburst transitions. These transitions are not targeted by ATP, nor the structurally unrelated sulfonylurea glyburide, which inhibit channel opening by acting on interburst transitions. Therefore, the channel remained insensitive to ATP and glyburide in the presence of UDP. "Rundown" of channel activity decreased the efficacy with which UDP could direct and maintain the channel to operate within intraburst transitions. Under this condition, the channel was sensitive to inhibition by ATP and glyburide despite the presence of UDP. This behavior of the KATP channel could be accounted for by an allosteric model of ligand-channel interaction. Thus, the response of cardiac KATP channels towards inhibitory ligands is determined by the relative lifetime the channel spends in a ligand-sensitive versus -insensitive state. Interconversion between these two conformational states represents a novel basis for KATP channel opening in the presence of inhibitory concentrations of ATP in a cardiac cell.

Myocardial ischemia induces differential regulation of KATP channel gene expression in rat hearts.

The cardiac ATP-sensitive potassium (KATP) channel is thought to be a complex composed of an inward rectifier potassium channel (Kir6.1 and/or Kir6.2) subunit and the sulfonylurea receptor (SUR2). This channel is activated during myocardial ischemia and protects the heart from ischemic injury. We examined the transcriptional expression of these genes in rats with myocardial ischemia. 60 min of myocardial regional ischemia followed by 24-72 h, but not 3-6 h, of reperfusion specifically upregulated Kir6.1 mRNA not only in the ischemic (approximately 2.7-3.1-fold) but also in the nonischemic (approximately 2.0-2.6-fold) region of the left ventricle. 24 h of continuous ischemia without reperfusion also induced an increase in Kir6.1 mRNA in both regions, whereas 15-30 min of ischemia followed by 24 h of reperfusion did not induce such expression. In contrast, mRNAs for Kir6.2 and SUR2 remained unchanged under these ischemic procedures. Western blotting demonstrated similar increases in the Kir6.1 protein level both in the ischemic (2.4-fold) and the nonischemic (2.2-fold) region of rat hearts subjected to 60 min of ischemia followed by 24 h of reperfusion. Thus, prolonged myocardial ischemia rather than reperfusion induces delayed and differential regulation of cardiac KATP channel gene expression.

Opening of cardiac sarcolemmal KATP channels by dinitrophenol separate from metabolic inhibition.

Opening of ATP-sensitive K+ (KATP) channels by the uncoupler of oxidative phosphorylation, 2,4 dinitrophenol (DNP), has been assumed to be secondary to metabolic inhibition and reduced intracellular ATP levels. Herein, we present data which show that DNP (200 microM) can induce opening of cardiac KATP channels, under whole-cell and inside-out conditions, despite millimolar concentrations of ATP (1-2. 5 mm). DNP-induced currents had a single channel conductance (71 pS), inward rectification, reversal potential, and intraburst kinetic properties (open time constant, tauopen: 4.8 msec; fast closed time constant, tauclosed(f): 0.33 msec) characteristic of KATP channels suggesting that DNP did not affect the pore region of the channel, but may have altered the functional coupling of the ATP-dependent channel gating. A DNP analogue, with the pH-titrable hydroxyl replaced by a methyl group, could not open KATP channels. The pH-dependence of the effect of DNP on channel opening under whole-cell, cell-attached, and inside-out conditions suggested that transfer of protonated DNP across the sarcolemma is essential for activation of KATP channels in the presence of ATP. We conclude that the use of DNP for metabolic stress-induced KATP channel opening should be reevaluated.

Role of Cardiac KATP Channels During Anoxia and Ischemia

In isolated heart cells, maintained anoxia causes a transient opening of KATP channels. In the ischemic myocardium, this extra K+ conductance results in a decreased contractility and may be arrhythmogenic. Recent studies provide further evidence that the transient activity of KATP channels during anoxia is correlated with the time course of extracellular K+ accumulation during ischemia.

A disrupter of actin microfilaments impairs sulfonylurea-inhibitory gating of cardiac KATP channels.

The efficacy with which sulfonylurea drugs inhibit cardiac ATP-sensitive K+ (KATP) channels is reduced during metabolic compromise and cellular contracture. Disruption of the actin microfilament network, which occurs under similar conditions, reduces the sensitivity of the channel toward intracellular ATP. To investigate whether a disrupter of actin microfilaments could also affect the responsiveness of the KATP channel to sulfonylurea drugs, single-channel currents were measured in the inside-out configuration of excised patches from guinea pig ventricular myocytes. Treatment of the internal side of patches with deoxyribonuclease (DNase) I (100 micrograms/ml), which forms complexes with G actin and prevents actin filament formation, antagonized sulfonylurea-induced inhibition of KATP channels that was coupled with a loss of sensitivity to ATP. The apparent dissociation constant and Hill coefficient for the inhibitory effect of glyburide, a prototype sulfonylurea, on KATP-channel opening were, respectively, 0.13 microM and 0.95 before and 2.7 microM and 0.98 after DNase treatment. DNase did not alter intraburst kinetic properties of the channel. When DNase was denatured or coincubated with purified actin (200 micrograms/ml), it no longer decreased glyburide-induced channel inhibition. This suggests that sulfonylurea-inhibitory gating of cardiac KATP channels may also be regulated through a mechanism involving subsarcolemmal actin microfilament networks.

Cloning, tissue expression, and chromosomal localization of SUR2, the putative drug-binding subunit of cardiac, skeletal muscle, and vascular KATP channels

Cloning, tissue expression, and chromosomal localization of SUR2, the putative drug-binding subunit of cardiac, skeletal muscle, and vascular KATP channels

Openings of cardiac KATP channel by oxygen free radicals produced by xanthine oxidase reaction.

We examined the effects of oxygen free radicals (OFRs) on action potentials and membrane currents of guinea pig ventricular myocytes. OFRs produced biphasic changes in the action potential duration, initial lengthening (30 s after exposure to OFRs) and subsequent shortening (within 5 min). In voltage-clamp experiments, OFRs suppressed the L-type calcium current, the delayed rectifier K+ current, and the inward rectifier K+ current. In addition, OFRs increased the time-independent outward current (I(term)) at potentials greater than -30 mV. The increases in I(term) reflected activation of the ATP-sensitive K+ (KATP) channels, as glibenclamide (1 microM) blocked this current. In inside-out patches, OFRs significantly increased the open probability of the channel at a relatively narrow range of ATP concentrations (0.2-2 mM), and this effect was enhanced in the presence of ADP (0.1 mM) and abolished in the presence of either free radical scavengers or gliben-clamide. These findings are compatible with the notion that OFRs activate KATP channels by modulating ATP binding sites of the KATP channels, without affecting ADP binding or glibenclamide binding sites.

Diadenosine polyphosphate-induced inhibition of cardiac KATP channels: operative state-dependent regulation by a nucleoside diphosphate.

It has been proposed that the regulatory action of mononucleotides, such as ATP and UDP, on cardiac ATP-sensitive K+ (KATP) channels is determined by the state of the channel. Recently, dinucleotides, such as diadenosine tetraphosphate (Ap4A) and diadenosine pentaphosphate (Ap5A), have been recognized as novel intracellular ligands of cardiac KATP channels. However, it is not known whether the state of KATP channels also determines the response of the channel to dinucleotides. Therefore, we examined the action of diadenosine polyphosphates on KATP channel activity during different operative channel states, using the inside-out patch clamp technique applied to patches excised from guinea-pig ventricular myocytes. Spontaneous openings of KATP channels (or operative condition 1) were inhibited by Ap4A and Ap5A. Addition of UDP, which on its own did not affect spontaneous openings of KATP channels, prevented Ap4A and Ap5A to inhibit spontaneous KATP channel activity. In contrast, following "run-down" of spontaneous channel activity, UDP induced KATP channel openings (or operative condition 2), but could no longer antagonize the inhibitory effect of Ap4A and Ap5A. We conclude that the inhibitory action of diadenosine polyphosphates on KATP channels can be blocked by UDP only when KATP channels are in operative condition 1, but not in operative condition 2. Thus, the operative condition of KATP channels determines the UDP-mediated regulation of the diadenosine polyphosphate-dependent inhibitory channel gating. This finding further suggests that the operative state of the cardiac KATP channel protein is a critical determinant of the regulatory action of an intracellular ligand.

Functional linkage of the cardiac ATP-sensitive K+ channel to the actin cytoskeleton

The role of the cytoskeleton in the rundown and reactivation of adenosine triphosphate (ATP) sensitive K+ channels (KATP channels) was examined by perturbing selectively the intracellular surface of inside-out membrane patches excised from guinea-pig ventricular myocytes. Actin filament-depolymerizing agents (cytochalasins and desoxyribonuclease I) accelerated channel rundown, while actin filament stabilizer (phalloidin) or phosphatidylinositol biphosphate (PIP2; inhibitor of F-actin-severing proteins) inhibited spontaneous and/or Ca2+-induced rundown. When rundown was induced by cytochalasin D or by long exposure to high Ca2+, channel activity could not be restored by exposure to MgATP, but application of F-actin with MgATP could reinstitute channel activity. The processes of rundown and reactivation of cardiac KATP channels may thus be influenced by the assembly and disassembly of the actin cytoskeletal network, which provides a novel regulatory mechanism of this channel.

Cardiac ATP-sensitive K+ channel: a target for diadenosine 5?,5?-P1,P5-pentaphosphate

In numerous studies the intracellular mononucleotide-dependent gating of ATP-sensitive K+ (KATP) channels has been demonstrated. However, it is not known whether dinucleotide polyphosphates, a family of endogenous compounds structurally-related to ATP, could also modulate this ion conductance. Therefore, in the present study we assessed the direct effect of diadenosine 5',5''-P1,P5-pentaphosphate (Ap5A) on cardiac KATP channel activity using the inside-out configuration of the patch-clamp technique. Addition of Ap5A (50 microM) to the internal side of membrane patches, excised from guinea-pig ventricular cells, strongly inhibited KATP channel activity. The estimated NPO (where N is the number of channels in the patch and PO the open probability of each channel) was 4.16 +/- 0.50 in the absence and 0.85 +/- 0.30 in the presence of Ap5A (50 microM). This effect of Ap5A was partially reversible, and the NP0 was 2.26 +/- 0.60 after washout of Ap5A. Exposure of KATP channels to increasing concentrations of Ap5A revealed that the Ap5A-induced inhibition is concentration-dependent with the half-maximal effective concentration of 16 microM (Hill coefficient: 1.6). On the basis of these results, we conclude that Ap5A is a potent antagonist of the KATP channel activity. This represents a previously unrecognized property of Ap5A, as well as the discovery of a potentially novel endogenous ligand of myocardial KATP channels.

Regulation of adenosine triphosphate-sensitive potassium channels from rabbit ventricular myocytes by protein kinase C and type 2A protein phosphatase

Myocytes from rabbit ventricle were enzymatically dissociated and the effects of protein kinase C (PKC) on the properties of single ATP-sensitive (KATP) channels were studied using excised inside-out membrane patches. Application of a purified, constitutively active form of PKC (20 nM) to the intracellular surface of inside-out patches caused a 48% +/- 4% (n = 18) reduction in the open probability of single KATP channels. In the presence of the PKC inhibitors peptide PKC(19-31) or chelerythrine chloride, PKC had no effect on KATP channel properties. Heat-inactivated PKC had no effect on channel properties. KATP channel activity returned spontaneously after removal of PKC. However, application of okadaic acid, at a concentration (5 nM) appropriate for specific inhibition of type 2A protein phosphatase (PP-2A), after removal of PKC, prevented spontaneous recovery of channel activity. Treatment with purified PP-2A during the PKC-mediated inhibition of KATP channel activity caused a partial or full restoration of activity. The Hill coefficient for ATP binding was reduced from 2.2 (control) to 1.2 in the presence of PKC. The apparent inhibition constant (Ki) for ATP was unaffected by PKC [Ki(control) = 21 microM; Ki(PKC) = 20 microM]. PKC is, therefore, capable of inhibiting cardiac KATP channel activity, and the extent to which the channels remain phosphorylated appears to be dependent on membrane-associated PP-2A activity. These enzymes may, therefore, be involved in signal transduction mechanisms which serve to regulate the activity of cardiac KATP channels.

Voltage dependent inhibition of ATP sensitive potassium channels by flecainide in guinea pig ventricular cells

The aim was to examine the effects of flecainide, a class Ic antiarrhythmic drug, on the ATP sensitive potassium channel (KATP channel) current in guinea pig ventricular cells, using the inside-out patch clamp technique. KATP channel activities were recorded from inside-out membrane patches with 140 mM KCl solution bathing both the external and internal surfaces of the membrane (20-22 degrees C). Flecainide was added to the intracellular medium (ATP-free, pH = 7.3). Flecainide (1-300 microM) inhibited the outward KATP channel current evoked at the holding potential of +40 mV, in a concentration dependent manner. The flecainide concentration for half maximum inhibition of the channel activity (IC50) and Hill coefficient of the flecainide inhibition were estimated to be 17.3 microM and 1.1, respectively. However, flecainide did not affect the inwardly directed KATP channel current measured at the potential of -40 mV. When the inhibitory effects of flecainide on the outward current were examined under conditions in which pH was decreased from 7.3 (control) to 6.8, the IC50 and Hill coefficient became 27.3 microM and 1.2, respectively. Furthermore, in the presence of 0.1 mM ADP on the cytosolic side of the membrane (pH = 7.3), flecainide blocked the outward currents with the IC50 of 47.0 microM and a Hill coefficient of 0.9. After 1 min exposure of the cytoplasmic side of the membrane to trypsin (1 mg.ml-1), glibenclamide (2 microM) did not inhibit the KATP channel currents, while flecainide (30 microM) reversibly inhibited this trypsin enhanced KATP channel activity. Flecainide at relatively high concentrations blocks the cardiac KATP channels only when the currents are directed outward, and in a concentration dependent manner. The potency of flecainide in blocking KATP channels decreased under conditions of increased H+ or ADP concentrations on the cytosolic side of the membrane, as may occur in myocardial ischaemia or hypoxia.

Cloning and functional expression of a rat heart KATP channel.

Potassium channels that are ATP-sensitive (KATP) couple membrane potential to the metabolic status of the cell. KATP channels are inhibited by intracellular ATP and are stimulated by intracellular nucleotide diphosphates. KATP channels are important regulators of secretory processes and muscle contraction, and are targets for therapeutic treatment of type II diabetes by the inhibitory sulphonylureas and for hypertension by activators such as pinacidil. In cardiac tissue, KATP channels are central regulators of post-ischaemic cardioprotection. Electrophysiological and pharmacological characteristics vary among KATP channels recorded from diverse tissues suggesting extensive molecular heterogeneity. A complementary DNA encoding a KATP channel was isolated from rat heart using the polymerase chain reaction. We report here that the expressed channels possess all of the essential features of native cardiac KATP channels, including sensitivity to intracellular nucleotides. In addition the cloned channels are activated by the potassium channel opener, pinacidil, but are not inhibited by the sulphonylurea, glibenclamide.

The ATP-Dependent Potassium Channel

The major objectives of the present study were to examine the effects of ischemic preconditioning and two endogenous substances, adenosine and acetylcholine (ACh), on myocardial infarct size to determine the role of the cardiac KATP channel in mediating these effects. Barbital-anesthetized open-chest dogs subjected to 60 min of left anterior descending coronary artery (LAD) occlusion followed by 4 h of reperfusion were used. Preconditioning elicited by 10 min of LAD occlusion or intracoronary infusion of adenosine (400 micrograms/min) or ACh (10 micrograms/min) for 10 min, followed by 10 min of reperfusion or drug-free period before the 60-min occlusion period, markedly reduced myocardial infarct size to a similar extent compared to that in control dogs that were infused with an equivalent volume of saline into the LAD for 10 min followed by a 10-min saline-free period before the 60-min ischemic insult. The infarct size-limiting effects of these three endogenous interventions were totally abolished by the specific KATP-channel blockers glibenclamide or 5-hydroxydecanoate. These results indicate that cardiac KATP-channel activation is an important endogenous protectant against ischemia-reperfusion injury.

The ATP-Dependent Potassium Channel

Summary: The major objectives of the present study were to examine the effects of ischemic preconditioning and two endogenous substances, adenosine and acetylcholine (ACh), on myocardial infarct size to determine the role of the cardiac KATP channel in mediating these effects. Barbital-anesthetized open-chest dogs subjected to 60 min of left anterior descending coronary artery (LAD) occlusion followed by 4 h of reperfusion were used. Preconditioning elicited by 10 min of LAD occlusion or intracoronary infusion of adenosine (400 μg/min) or ACh (10 μg/min) for 10 min, followed by 10 min of reperfusion or drug-free period before the 60-min occlusion period, markedly reduced myocardial infarct size to a similar extent compared to that in control dogs that were infused with an equivalent volume of saline into the LAD for 10 min followed by a 10-min saline-free period before the 60-min ischemic insult. The infarct sizelimiting effects of these three endogenous interventions were totally abolished by the specific KATP-channel blockers glibenclamide or 5-hydroxydecanoate. These results indicate that cardiac KATP-channel activation is an important endogenous protectant against ischemia-reperfusion injury.

Acetylcholine mimics ischemic preconditioning via a glibenclamide-sensitive mechanism in dogs.

The major objectives of the present study were to examine the ability of acetylcholine (ACh) to mimic ischemic preconditioning in dogs and to determine the role of cardiac ATP-sensitive potassium (KATP) channels in mediating its effects. Barbital-anesthetized open-chest dogs were subjected to 60 min of left anterior descending coronary artery (LAD) occlusion followed by 4 h of reperfusion. Preconditioning was elicited by 10 min of LAD occlusion followed by 10 min of reperfusion before the 60-min occlusion period. ACh (10 micrograms/min) or an equivalent volume of saline were infused into the LAD for 10 min followed by a 10-min drug-free period before the 60-min ischemic insult. In another group, the specific KATP channel blocker glibenclamide (0.3 mg/kg iv) was given 15 min before ACh administration. Transmural myocardial blood flow was measured at 30 min of occlusion, and infarct size (IS) was determined by triphenyltetrazolium staining and expressed as a percentage of the anatomic area at risk (AAR). There were no significant differences in hemodynamics, collateral blood flow, or AAR between groups. Preconditioning produced a marked reduction (P < 0.05) in IS (5.3 +/- 3.0 vs. 23.7 +/- 5.9% in the controls). ACh, similar to preconditioning, resulted in a dramatic decrease in IS (10.0 +/- 2.9%), whereas glibenclamide completely abolished its protective effects (20.9 +/- 4.8%). These results are the first to indicate that ACh mimics ischemic preconditioning via a cardiac KATP channel-sensitive mechanism in dogs.