Neurons set the tone of gap junctional communication in astrocytic networks

A number of studies have contributed to demonstrate that neurons and astrocytes tightly and actively interact. Indeed, the presence of astrocytes in neuronal cultures increases the number of synapses and their efficiency, and thanks to enzymatic and uptake processes, astrocytes play a role in neuroprotection. A typical feature of astrocytes is that they establish cell-cell communication in vitro, as well as in situ, through intercellular channels forming specialized membrane areas defined as gap junctions. These channels are composed of junctional proteins termed connexins (Cxs): in astrocytes connexin 43 (Cx43) and 30 (Cx30) have been shown to prevail. Several recent works indicate that gap junctional communication (GJC) and/or connexin expression in astrocytes are controlled by neurons. Altogether, these observations lead to the concept that neuronal and astrocytic networks interact through mutual setting of their respective mode of communication and that astrocyte gap junctions represent a target in neuroglial interaction.     

Regulation of cell-to-cell communication mediated by astrocytic ATP in the CNS

It has become apparent that glial cells, especially astrocytes, not merely supportive but are integrative, being able to receive inputs, assimilate information and send instructive chemical signals to other neighboring cells including neurons. At first, the excitatory neurotransmitter glutamate was found to be a major extracellular messenger that mediates these communications because it can be released from astrocytes in a Ca(2+)-dependent manner, diffused, and can stimulate extra-synaptic glutamate receptors in adjacent neurons, leading to a dynamic modification of synaptic transmission. However, recently extracellular ATP has come into the limelight as an important extracellular messenger for these communications. Astrocytes express various neurotransmitter receptors including P2 receptors, release ATP in response to various stimuli and respond to extracellular ATP to cause various physiological responses. The intercellular communication "Ca(2+) wave" in astrocytes was found to be mainly mediated by the release of ATP and the activation of P2 receptors, suggesting that ATP is a dominant "gliotransmitter" between astrocytes. Because neurons also express various P2 receptors and synapses are surrounded by astrocytes, astrocytic ATP could affect neuronal activities and even dynamically regulate synaptic transmission in adjacent neurons as if forming a "tripartite synapse". In this review, we summarize the role of astrocytic ATP, as compared with glutamate, in gliotransmission and synaptic transmission in neighboring cells, mainly focusing on the hippocampus. Dynamic communication between astrocytes and neurons mediated by ATP would be a key event in the processing or integration of information in the CNS.     

Diabetes and hyperglycemia impair activation of mitochondrial K(ATP) channels

Hyperglycemia is an important predictor of cardiovascular mortality in patients with diabetes. We investigated the hypothesis that diabetes or acute hyperglycemia attenuates the reduction of myocardial infarct size produced by activation of mitochondrial ATP-regulated potassium (K(ATP)) channels. Acutely instrumented barbiturate-anesthetized dogs were subjected to a 60-min period of coronary artery occlusion and 3 h of reperfusion. Myocardial infarct size (triphenyltetrazolium chloride staining) was 25 +/- 1, 28 +/- 3, and 25 +/- 1% of the area at risk (AAR) for infarction in control, diabetic (3 wk after streptozotocin-alloxan), and hyperglycemic (15% intravenous dextrose) dogs, respectively. Diazoxide (2.5 mg/kg iv) significantly decreased infarct size (10 +/- 1% of AAR, P < 0.05) but did not produce protection in the presence of diabetes (28 +/- 5%) or moderate hyperglycemia (blood glucose 310 +/- 10 mg/dl; 23 +/- 2%). The dose of diazoxide and the degree of hyperglycemia were interactive. Profound (blood glucose 574 +/- 23 mg/dl) but not moderate hyperglycemia blocked the effects of high-dose (5.0 mg/kg) diazoxide [26 +/- 3, 15 +/- 3 (P < 0.05), and 11 +/- 2% (P < 0.05), respectively]. There were no differences in systemic hemodynamics, AAR, or coronary collateral blood flow (by radioactive microspheres) between groups. The results indicate that diabetes or hyperglycemia impairs activation of mitochondrial K(ATP) channels.     

Sarcolemmal and mitochondrial K(ATP) channels and myocardial ischemic preconditioning

Ischemic preconditioning (IPC) is the phenomenon whereby brief periods of ischemia have been shown to protect the myocardium against a sustained ischemic insult. The result of IPC may be manifest as a marked reduction in infarct size, myocardial stunning, or incidence of arrhythmias. While many substances and pathways have been proposed to play a role in the signal transduction mediating the cardioprotective effect of IPC, overwhelming evidence indicates an intimate involvement of the ATP-sensitive potassium channel (K_ATP channel) in this process. Initial hypotheses suggested that the surface or sarcolemmal K_ATP (sarcK_ATP) channel mediated the cardioprotective effects of IPC. However, much research has subsequently supported a major role for the mitochondrial K_ATP channel (mitoK_ATP) as the one involved in IPC-mediated cardioprotection. This review presents evidence to support a role for the sarcK_ATP or the mitoK_ATP channel as either triggers and/or downstream mediators in the phenomenon of IPC.     

Regulation of connexin43 gap junctional communication by phosphatidylinositol 4,5-bisphosphate

Cell-cell communication through connexin43 (Cx43)-based gap junction channels is rapidly inhibited upon activation of various G protein-coupled receptors; however, the mechanism is unknown. We show that Cx43-based cell-cell communication is inhibited by depletion of phosphatidylinositol 4,5-bisphosphate (PtdIns[4,5]P(2)) from the plasma membrane. Knockdown of phospholipase Cbeta3 (PLCbeta3) inhibits PtdIns(4,5)P(2) hydrolysis and keeps Cx43 channels open after receptor activation. Using a translocatable 5-phosphatase, we show that PtdIns(4,5)P(2) depletion is sufficient to close Cx43 channels. When PtdIns(4,5)P(2) is overproduced by PtdIns(4)P 5-kinase, Cx43 channel closure is impaired. We find that the Cx43 binding partner zona occludens 1 (ZO-1) interacts with PLCbeta3 via its third PDZ domain. ZO-1 is essential for PtdIns(4,5)P(2)-hydrolyzing receptors to inhibit cell-cell communication, but not for receptor-PLC coupling. Our results show that PtdIns(4,5)P(2) is a key regulator of Cx43 channel function, with no role for other second messengers, and suggest that ZO-1 assembles PLCbeta3 and Cx43 into a signaling complex to allow regulation of cell-cell communication by localized changes in PtdIns(4,5)P(2).     

Prevention of organochlorine-induced inhibition of gap junctional communication by chaetoglobosin K in astrocytes

Innumerable toxic substances present in the environment inhibit gap junctions, intercellular membrane channels that play fundamental roles in coordinated function of cells and tissues. Included are persistent organochlorine compounds, which pose health risks to humans and animals owing to their widespread use, bioaccumulation, and ability to inhibit gap junction channel-mediated intercellular communication in liver, lung, skin, heart, and brain cells. In this study, the organochlorine xenobiotics dieldrin and endosulfan, at micromolar concentrations, were found to inhibit gap junction-mediated intercellular communication and induce hypophosphorylation of connexin 43 in cultured rat astrocytes, the predominant cell type in the brain coupled through gap junctions. This inhibition of gap junctional communication was substantially reduced by preincubation with chaetoglobosin K (ChK), a bioactive natural produce previously shown to have ras tumor suppressor activity. Chaetoglobosin K also prevented dieldrin and endosulfan-induced hypophosphorylation of connexin 43 and prevented dieldrin-induced connexin 43 plaque dissolution in both astrocytes and cultured liver epithelial cells. The results suggest that stabilization of the native, phosphorylated form of connexin 43 by ChK may contribute to its ability to prevent organochlorine-induced inhibition of gap junction-mediated communication and dissolution of gap junction plaques within the plasma membrane. This revised version was published online in July 2006 with corrections to the Cover Date.     

Cardioprotection by multiple preconditioning cycles does not require mitochondrial K(ATP) channels in pigs

To test whether cardioprotection induced by ischemic preconditioning depends on the opening of mitochondrial ATP-sensitive K(+) (K(ATP)) channels, the effect of channel blockade was studied in barbital-anesthetized open-chest pigs subjected to 30 min of complete occlusion of the left anterior descending coronary artery and 3 h of reflow. Preconditioning was elicited by two cycles of 5-min occlusion plus 10-min reperfusion before the 30-min occlusion period. 5-Hydroxydecanoate (5 mg/kg iv) was injected 15 min before preconditioning or pharmacological preconditioning induced by diazoxide (3.5 mg/kg, 1 ml/min iv). Infarct size (percentage of the area at risk) after 30 min of ischemia was 35.1 +/- 9.9% (n = 7). Preconditioning markedly limited myocardial infarct size (2.7 +/- 1.6%, n = 7), and 5-hydroxydecanoate did not abolish protection (2.4 +/- 0.9%, n = 8). Diazoxide infusion also significantly limited infarct size (14.6 +/- 7.4%, n = 7), and 5-hydroxydecanoate blocked this effect (30.8 +/- 8.0%, n = 7). Thus the opening of mitochondrial K(ATP) channels is cardioprotective in pigs, but these data do not support the hypothesis that opening of mitochondrial K(ATP) channels is required for the endogenous protection afforded by preconditioning.     

Differential role of sarcolemmal and mitochondrial K(ATP) channels in adenosine-enhanced ischemic preconditioning

Adenosine-enhanced ischemic preconditioning (APC) extends the protection afforded by ischemic preconditioning (IPC) by both significantly decreasing infarct size and significantly enhancing postischemic functional recovery. The purpose of this study was to determine whether APC is modulated by ATP-sensitive potassium (K(ATP)) channels and to determine whether this modulation occurs before ischemia or during reperfusion. The role of K(ATP) channels before ischemia (I), during reperfusion (R), or during ischemia and reperfusion (IR) was investigated using the nonspecific K(ATP) blocker glibenclamide (Glb), the mitochondrial (mito) K(ATP) channel blocker 5-hydroxydecanoate (5-HD), and the sarcolemmal (sarc) K(ATP) channel blocker HMR-1883 (HMR). Infarct size was significantly increased (P < 0.05) in APC hearts with Glb-I, Glb-R, and 5-HD-I treatment and partially with 5-HD-R. Glb-I and Glb-R treatment significantly decreased APC functional recovery (P < 0.05 vs. APC), whereas 5-HD-I and 5-HD-R had no effect on APC functional recovery. HMR-IR significantly decreased postischemic functional recovery (P < 0.05 vs. APC) but had no effect on infarct size. These data indicate that APC infarct size reduction is modulated by mitoK(ATP) channels primarily during ischemia and suggest that functional recovery is modulated by sarcK(ATP) channels during ischemia and reperfusion.     

Pharmacological modulation of K(ATP) channels

Pharmacological modulation of ATP-sensitive K+ (K(ATP)) channels is used in the treatment of a number of clinical conditions, including type 2 diabetes and angina. The sulphonylureas and related drugs, which are used to treat type 2 diabetes, stimulate insulin secretion by closing K(ATP) channels in pancreatic beta-cells. Agents used to treat angina, by contrast, act by opening K(ATP) channels in vascular smooth and cardiac muscle. Both the therapeutic K(ATP) channel inhibitors and the K(ATP) channel openers target the sulphonylurea receptor (SUR) subunit of the K(ATP) channel, which exists in several isoforms expressed in different tissues (SUR1 in pancreatic beta-cells, SUR2A in cardiac muscle and SUR2B in vascular smooth muscle). The tissue-specific action of drugs that target the K(ATP) channel is attributed to the properties of these different SUR subtypes. In this review, we discuss the molecular basis of tissue-specific drug action, and its implications for clinical practice.     

Dual roles of mitochondrial K(ATP) channels in diazoxide-mediated protection in isolated rabbit hearts

Whether the mitochondrial ATP-dependent potassium (mK(ATP)) channel is the trigger or the mediator of cardioprotection is controversial. We investigated the critical time sequences of mK(ATP) channel opening for cardioprotection in isolated rabbit hearts. Pretreatment with diazoxide (100 microM), a selective mK(ATP) channel opener, for 5 min followed by 10 min washout before the 30-min ischemia and 2-h reperfusion significantly reduced infarct size (9 +/- 3 vs. 35 +/- 3% in control), indicating a role of mK(ATP) channels as a trigger of protection. The protection was blocked by coadministration of the L-type Ca(2+) channel blockers nifedipine (100 nM) or 5-hydroxydecanoic acid (5-HD; 50 microM) or by the protein kinase C (PKC) inhibitor chelerythrine (5 microM). The protection of diazoxide was not blocked by 50 microM 5-HD but was blocked by 200 microM 5-HD or 10 microM glybenclamide administrated 5 min before and throughout the 30 min of ischemia, indicating a role of mK(ATP) opening as a mediator of protection. Giving diazoxide throughout the 30 min of ischemia also protected the heart, and the protection was not blocked by chelerythrine. Nifedipine did not affect the ability of diazoxide to open mK(ATP) channels assessed by mitochondrial redox state. In electrically stimulated rabbit ventricular myocytes, diazoxide significantly increased Ca(2+) transient but had no effect on L-type Ca(2+) currents. Our results suggest that opening of mK(ATP) channels can trigger cardioprotection. The trigger phase may be induced by elevation of intracellular Ca(2+) and activation of PKC. During the lethal ischemia, mK(ATP) channel opening mediates the protection, independent of PKC, by yet unknown mechanisms.     

Role of mitochondrial and sarcolemmal K(ATP) channels in ischemic preconditioning of the canine heart

We tested whether mitochondrial or sarcolemmal ATP-sensitive K(+) (K(ATP)) channels play a key role in ischemic preconditioning (IP) in canine hearts. In open-chest beagle dogs, the left anterior descending artery was occluded four times for 5 min each with 5-min intervals of reperfusion (IP), occluded for 90 min, and reperfused for 6 h. IP as well as cromakalim and nicorandil (nonspecific K(ATP) channel openers) markedly limited infarct size (6.3 +/- 1.2, 8.9 +/- 1.9, and 7.2 +/- 1.6%, respectively) compared with the control group (40.9 +/- 4.1%). A selective mitochondrial K(ATP) channel blocker, 5-hydroxydecanoate, partially blunted the limitation of infarct size in the animals subjected to IP and those treated with cromakalim and nicorandil (21.6 +/- 3.8, 25.1 +/- 4.6, and 19.8 +/- 5.2%, respectively). A nonspecific K(ATP) channel blocker, glibenclamide, completely abolished the effect of IP (38.5 +/- 6.2%). Intracoronary or intravenous administration of a mitochondria-selective K(ATP) channel opener, diazoxide, at >100 micromol/l could only partially decrease infarct size (19.5 +/- 4.3 and 20.1 +/- 4.4%, respectively). In conclusion, mitochondrial and sarcolemmal K(ATP) channels independently play an important role in the limitation of infarct size by IP in the canine heart.     

K(ATP) channels and insulin secretion disorders

ATP-sensitive potassium (K(ATP)) channels are inhibited by intracellular ATP and activated by ADP. Nutrient oxidation in beta-cells leads to a rise in [ATP]-to-[ADP] ratios, which in turn leads to reduced K(ATP) channel activity, depolarization, voltage-dependent Ca(2+) channel activation, Ca(2+) entry, and exocytosis. Persistent hyperinsulinemic hypoglycemia of infancy (HI) is a genetic disorder characterized by dysregulated insulin secretion and, although rare, causes severe mental retardation and epilepsy if left untreated. The last five or six years have seen rapid advance in understanding the molecular basis of K(ATP) channel activity and the molecular genetics of HI. In the majority of cases for which a genotype has been uncovered, causal HI mutations are found in one or the other of the two genes, SUR1 and Kir6.2, that encode the K(ATP) channel. This article will review studies that have defined the link between channel activity and defective insulin release and will consider implications for future understanding of the mechanisms of control of insulin secretion in normal and diseased states.     

Flow regulates intercellular communication in HAEC by assembling functional Cx40 and Cx37 gap junctional channels

Direct cell-to-cell transfer of ions and small signaling molecules via gap junctions plays a key role in vessel wall homeostasis. Vascular endothelial gap junctional channels are formed by the connexin (Cx) proteins Cx37, Cx40, and Cx43. The mechanisms regulating connexin expression and assembly into functional channels have not been fully identified. We investigated the dynamic regulation of endothelial gap junctional intercellular communication (GJIC) by fluid flow and the participation of each vascular connexin in functional human endothelial gap junctions in vitro. Human aortic endothelial cells (HAEC) were exposed for 5, 16, and 24 h to physiological flows in a parallel-plate flow chamber. Connexin protein expression and localization were evaluated by immunocytochemistry, and functional GJIC was evaluated by dye injection. Connexin-mimetic peptide inhibitors were used to assess the specific connexin composition of functional channels. HAEC monolayers in culture exhibited baseline functional communication at a striking low level despite abundant expression of Cx43 and Cx40 localized at cell-to-cell appositions. Upon exposure to flow, GJIC by dye spread demonstrated a significant time-dependent increase from baseline levels, reaching 7.5-fold in 24 h. Inhibition studies revealed that this response was mediated primarily by Cx40, with lesser contributions of the other two vascular connexins assembled into functional homotypic and/or heterotypic channels. This is the first study to demonstrate that flow simultaneously and differentially regulates expression of the Cx37, Cx40, and Cx43 proteins and their involvement in the augmentation of intercellular communication by dye transfer in human endothelial cells in vitro.     

Sildenafil (Viagra) induces powerful cardioprotective effect via opening of mitochondrial K(ATP) channels in rabbits

Sildenafil citrate (Viagra) is the pharmacological agent used to treat erectile dysfunction in men. Because this drug has a vasodilatory effect, we hypothesized that such an action may induce a preconditioning-like cardioprotective effect via opening of mitochondrial ATP-sensitive K (K(ATP)) channels. Rabbits were treated with sildenafil citrate (0.7 mg/kg iv) either 30 min (acute phase) or 24 h (delayed phase) before 30 min of ischemia and 3 h of reperfusion. Mitochondrial K(ATP) channel blocker 5-hydroxydecanoate (5-HD, 5 mg/kg iv) was given 10 min before ischemia-reperfusion. Infarct size was measured by tetrazolium staining. Sildenafil caused reduction in arterial blood pressure within 2 min of treatment, which returned to nearly baseline levels 3 min later. The infarct size (% risk area, means +/- SE) reduced from 33.8 +/- 1.7 in control rabbits to 10.8 +/- 0.9 during the acute phase (68% reduction, P < 0.05) and 19.9 +/- 2.0 during the delayed phase (41% reduction, P < 0.05). 5-HD abolished protection with an increase in infarct size to 35.6 +/- 0.4% and 36.8 +/- 1.6% during the acute and delayed phase, respectively (P < 0.05). Similar acute and delayed cardioprotective effects were observed when sildenafil was administered orally. Systemic hemodynamics also decreased after oral administration of the drug. However, these changes were mild and occurred slowly. For the first time, we demonstrate that sildenafil induces acute and delayed protective effects against ischemia-reperfusion injury, which are mediated by opening of mitochondrial K(ATP) channels.     

ATP consumption by uncoupled mitochondria activates sarcolemmal K(ATP) channels in cardiac myocytes

We tested whether close coupling exists between mitochondria and sarcolemma by monitoring whole cell ATP-sensitive K(+) (K(ATP)) current (I(K,ATP)) as an index of subsarcolemmal energy state during mitochondrial perturbation. In rabbit ventricular myocytes, either pinacidil or the mitochondrial uncoupler dinitrophenol (DNP), which rapidly switches mitochondria from net ATP synthesis to net ATP hydrolysis, had little immediate effect on I(K,ATP). In contrast, in the presence of pinacidil, exposure to 100 microM DNP rapidly activated I(K,ATP) with complex kinetics consisting of a quick rise [time constant of I(K,ATP) increase (tau) = 0.13 +/- 0.01 min], an early partial recovery (tau = 0.43 +/- 0.04 min), and then a more gradual increase. This DNP-induced activation of I(K,ATP) was reversible and accompanied by mitochondrial flavoprotein oxidation. The F(1)F(0)-ATPase inhibitor oligomycin abolished the DNP-induced activation of I(K,ATP). The initial rapid rise in I(K,ATP) was blunted by atractyloside (an adenine nucleotide translocator inhibitor), leaving only a slow increase (tau = 0.66 +/- 0.17 min, P < 0.01). 2,4-Dinitrofluorobenzene (a creatine kinase inhibitor) slowed both the rapid rise (tau = 0.20 +/- 0.01 min, P < 0.05) and the subsequent declining phase (tau = 0.88 +/- 0.19 min, P < 0.05). From single K(ATP) channel recordings, we excluded a direct effect of DNP on K(ATP) channels. Taken together, these results indicate that rapid changes in F(1)F(0)-ATPase function dramatically alter subsarcolemmal energy charge, as reported by pinacidil-primed K(ATP) channel activity, revealing cross-talk between mitochondria and sarcolemma. The effects of mitochondrial ATP hydrolysis on sarcolemmal K(ATP) channels can be rationalized by reversal of F(1)F(0)-ATPase and the facilitation of coupling by the creatine kinase system.     

Oxidative stress inhibits vascular K(ATP) channels by S-glutathionylation

The K(ATP) channel is an important player in vascular tone regulation. Its opening and closure lead to vasodilation and vasoconstriction, respectively. Such functions may be disrupted in oxidative stress seen in a variety of cardiovascular diseases, while the underlying mechanism remains unclear. Here, we demonstrated that S-glutathionylation was a modulation mechanism underlying oxidant-mediated vascular K(ATP) channel regulation. An exposure of isolated mesenteric rings to hydrogen peroxide (H(2)O(2)) impaired the K(ATP) channel-mediated vascular dilation. In whole-cell recordings and inside-out patches, H(2)O(2) or diamide caused a strong inhibition of the vascular K(ATP) channel (Kir6.1/SUR2B) in the presence, but not in the absence, of glutathione (GSH). Similar channel inhibition was seen with oxidized glutathione (GSSG) and thiol-modulating reagents. The oxidant-mediated channel inhibition was reversed by the reducing agent dithiothreitol (DTT) and the specific deglutathionylation reagent glutaredoxin-1 (Grx1). Consistent with S-glutathionylation, streptavidin pull-down assays with biotinylated glutathione ethyl ester (BioGEE) showed incorporation of GSH to the Kir6.1 subunit in the presence of H(2)O(2). These results suggest that S-glutathionylation is an important mechanism for the vascular K(ATP) channel modulation in oxidative stress.     

Carvedilol in heart mitochondria: protonophore or opener of the mitochondrial K(ATP) channels?

Carvedilol ([1-[carbazolyl-(4)-oxy]-3-[2-methoxyphenoxyethyl) amino]-propanol-(2)]) has been shown to protect cardiac mitochondria from oxidative stress. In this work we examined the mechanisms responsible for an observed depressive effect in the mitochondrial transmembrane potential (delta psi). Two possible mechanisms were considered: a protonophoretic activity and the opening of mitochondrial ATP-sensitive potassium channels. We show that carvedilol increases mitochondrial inner membrane permeability to protons, but not to potassium, causing an increase in state IV respiration in the presence and absence of oligomycin. By contrast, a K(ATP)-channel inhibitor, 5-hydroxydecanoic acid, did not affect carvedilol-induced depolarizations. Hence, our results suggest that carvedilol depresses mitochondrial delta psi by a weak protonophoretic mechanism.     

Delayed cardioprotection by isoflurane: role of K(ATP) channels

Isoflurane mimics the cardioprotective effect of acute ischemic preconditioning with an acute memory phase. We determined whether isoflurane can induce delayed cardioprotection, the involvement of ATP-sensitive potassium (K(ATP)) channels, and cellular location of the channels. Neonatal New Zealand White rabbits at 7-10 days of age (n = 5-16/group) were exposed to 1% isoflurane-100% oxygen for 2 h. Hearts exposed 2 h to 100% oxygen served as untreated controls. Twenty-four hours later resistance to myocardial ischemia was determined using an isolated perfused heart model. Isoflurane significantly reduced infarct size/area at risk (means +/- SD) by 50% (10 +/- 5%) versus untreated controls (20 +/- 6%). Isoflurane increased recovery of preischemic left ventricular developed pressure by 28% (69 +/- 4%) versus untreated controls (54 +/- 6%). The mitochondrial K(ATP) channel blocker 5-hydroxydecanoate (5-HD) completely (55 +/- 3%) and the sarcolemmal K(ATP) channel blocker HMR 1098 partially (62 +/- 3%) attenuated the cardioprotective effects of isoflurane. The combination of 5-HD and HMR-1098 completely abolished the cardioprotective effect of isoflurane (56 +/- 5%). We conclude that both mitochondrial and sarcolemmal K(ATP) channels contribute to isoflurane-induced delayed cardioprotection.     

Opening of mitochondrial K+ channels increases ischemic ATP levels by preventing hydrolysis

Mitochondrial ATP-sensitive K⁺ channels (mitoK_ATP) have been proposed to mediate protection against ischemic injury by increasing high-energy intermediate levels. This study was designed to verify if mitochondria are an important factor in the loss of cardiac ATP associated to ischemia, and determine the possible role of mitoK_ATP in the control of ischemic ATP loss. Langendorff-perfused rat hearts subjected to ischemia were found to have significantly higher ATP contents when pretreated with oligomycin or atractyloside, indicating that mitochondrial ATP hydrolysis contributes toward ischemic ATP depletion. MitoK_ATP opening induced by diazoxide promoted a similar protection against ATP loss. Diazoxide also inhibited ATP hydrolysis in isolated, nonrespiring mitochondria, an effect accompanied by a drop in the membrane potential and Ca²⁺ uptake. In hearts subjected to ischemia followed by reperfusion, myocardial injury was prevented by diazoxide, but not atractyloside or oligomycin, which, unlike diazoxide, decreased reperfusion ATP levels. Our results suggest that mitoK_ATP-mediated protection occurs due to selective inhibition of mitochondrial ATP hydrolysis during ischemia, without affecting ATP synthesis after reperfusion.