The K+ channel openers diazoxide and NS1619 induce depolarization of mitochondria and have differential effects on cell Ca2+ in CD34+ cell line KG-1a (original) (raw)
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Circulation Research, 1997
Previous studies showed a poor correlation between sarcolemmal K currents and cardioprotection for ATP-sensitive K channel (K ATP ) openers. Diazoxide is a weak cardiac sarcolemmal K ATP opener, but it is a potent opener of mitochondrial K ATP , making it a useful tool for determining the importance of this mitochondrial site. In reconstituted bovine heart K ATP , diazoxide opened mitochondrial K ATP with a K 1/2 of 0.8 μmol/L while being 1000-fold less potent at opening sarcolemmal K ATP . To compare cardioprotective potency, diazoxide or cromakalim was given to isolated rat hearts subjected to 25 minutes of global ischemia and 30 minutes of reperfusion. Diazoxide and cromakalim increased the time to onset of contracture with a similar potency (EC 25 , 11.0 and 8.8 μmol/L, respectively) and improved postischemic functional recovery in a glibenclamide (glyburide)-reversible manner. In addition, sodium 5-hydroxydecanoic acid completely abolished the protective effect of diazoxide. Whole-myocyte studies showed that diazoxide was significantly less potent than cromakalim in increasing sarcolemmal K currents. Diazoxide shortened ischemic action potential duration significantly less than cromakalim at equicardioprotective concentrations. We also determined the effects of cromakalim and diazoxide on reconstituted rat mitochondrial cardiac K ATP activity. Cromakalim and diazoxide were both potent activators of K flux in this preparation (K 1/2 values, 1.1±0.1 and 0.49±0.05 μmol/L, respectively). Both glibenclamide and sodium 5-hydroxydecanoic acid inhibited K flux through the diazoxide-opened mitochondrial K ATP . The profile of activity of diazoxide (and perhaps K ATP openers in general) suggests that they protect ischemic hearts in a manner that is consistent with an interaction with mitochondrial K ATP .
Circulation Research, 1999
Growing evidence exists that ATP-sensitive mitochondrial potassium channels (MitoK ATP channel) are a major contributor to the cardiac protection against ischemia. Given the importance of mitochondria in the cardiac cell, we tested whether the potent and specific opener of the MitoK ATP channel diazoxide attenuates the lethal injury associated with Ca 2ϩ overload. The specific aims of this study were to test whether protection by diazoxide is mediated by MitoK ATP channels; whether diazoxide mimics the effects of Ca 2ϩ preconditioning; and whether diazoxide reduces Ca 2ϩ paradox (PD) injury via protein kinase C (PKC) signaling pathways. Langendorff-perfused rat hearts were subjected to the Ca 2ϩ PD (10 minutes of Ca 2ϩ depletion followed by 10 minutes of Ca 2ϩ repletion). The effects of the MitoK ATP channel and other interventions on functional, biochemical, and pathological changes in hearts subjected to Ca 2ϩ PD were assessed. In hearts treated with 80 mol/L diazoxide, left ventricular end-diastolic pressure and coronary flow were significantly preserved after Ca 2ϩ PD; peak lactate dehydrogenase release was also significantly decreased, although ATP content was less depleted. The cellular structures were well preserved, including mitochondria and intercalated disks in diazoxidetreated hearts compared with nontreated Ca 2ϩ PD hearts. The salutary effects of diazoxide on the Ca 2ϩ PD injury were similar to those in hearts that underwent Ca 2ϩ preconditioning or pretreatment with phorbol 12-myristate 13-acetate before Ca 2ϩ PD. The addition of sodium 5-hydroxydecanoate, a specific MitoK ATP channel inhibitor, or chelerythrine chloride, a PKC inhibitor, during diazoxide pretreatment completely abolished the beneficial effects of diazoxide on the Ca 2ϩ PD. Blockade of Ca 2ϩ entry during diazoxide treatment by inhibiting L-type Ca 2ϩ channel with verapamil or nifedipine also completely reversed the beneficial effects of diazoxide on the Ca 2ϩ PD. PKC-␦ was translocated to the mitochondria, intercalated disks, and nuclei of myocytes in diazoxide-pretreated hearts, and PKC-␣ and PKC-⑀ were translocated to sarcolemma and intercalated disks, respectively. This study suggests that the effect of the MitoK ATP channel is mediated by PKC-mediated signaling pathway.
Cellular Physiology and Biochemistry, 2010
K ATP channel openers protect ischemic-reperfused myocardium by mimicking ischemic preconditioning, however, the protection mechanisms have not been fully clarified yet. Since the skinned fibers technique gives an opportunity to investigate an entire population of mitochondria in their native milieu, in this study we have investigated the effects of K ATP channel openers pinacidil and diazoxide on the respiration rate of rat heart mitochondria in situ, oxidizing physiological substrates pyruvate and malate (6+6 mM). Respiration rates were recorded by the means of Clark-type oxygen electrode in the physiological salt solution (37°C). Our results showed that both pinacidil and diazoxide (60-1250 µM) in a concentrationdependent manner increased pyruvate-malate supported State 2 respiration rate of skinned cardiac fibers (59.1 ± 5.1 nmol O/min/mg fiber dry weight, RCI 2.6 ± 0.2, n=4) by 15-120%. Moreover, diazoxide did not affect, whereas pinacidil (60-1250 µM) decreased the State 3 respiration rate of skinned cardiac fibers (116.6 ± 13.6 nmol O/min/mg fiber dry weight, RCI 2.3 ± 0.2, n=4) by 4-27%. Thus, common effect for both K ATP channel openers is uncoupling of pyruvate and malate oxidizing mitochondria in skinned cardiac fibers, whereas pinacidil under same conditions also inhibits mitochondrial respiratory chain. Since mitochondria in situ resemble to the great extent mitochondria in vivo, our results suggest that uncoupling and/or respiratory chain inhibition could play a role in the cardioprotection by K ATP channel openers.
Bioenergetic consequences of opening the ATP-sensitive K(+) channel of heart mitochondria
American journal of physiology. Heart and circulatory physiology, 2001
There is an emerging consensus that pharmacological opening of the mitochondrial ATP-sensitive K(+) (K(ATP)) channel protects the heart against ischemia-reperfusion damage; however, there are widely divergent views on the effects of openers on isolated heart mitochondria. We have examined the effects of diazoxide and pinacidil on the bioenergetic properties of rat heart mitochondria. As expected of hydrophobic compounds, these drugs have toxic, as well as pharmacological, effects on mitochondria. Both drugs inhibit respiration and increase membrane proton permeability as a function of concentration, causing a decrease in mitochondrial membrane potential and a consequent decrease in Ca(2+) uptake, but these effects are not caused by opening mitochondrial K(ATP) channels. In pharmacological doses (<50 microM), both drugs open mitochondrial K(ATP) channels, and resulting changes in membrane potential and respiration are minimal. The increased K(+) influx associated with mitochondria...
Objective: Mitochondrial calcium-activated K + (mitoK Ca ) channels have been described as channels that are activated by Ca 2+ , inner mitochondrial membrane depolarization and drugs such as NS-1619. NS-1619 is cardioprotective, leading to the assumption that this effect is related to the opening of mitoK Ca channels. Here, we show several weaknesses in this hypothesis. Methods: Isolated mitochondria from rat hearts were tested for evidence of mitoK Ca activity by analyzing functional parameters in K + -rich and K + -free media. Results: NS-1619 promoted mitochondrial depolarization both in K + -rich and K + -free media. Respiratory rate increments were also seen in the presence of NS-1619 for both media. In parallel, NS-1619 promoted respiratory inhibition, as evidenced by respiratory measurements in state 3. Mitochondrial volume measurements conducted using light scattering showed that NS-1619 led to swelling, in a manner unaltered by inhibitors of mitoK Ca channels, antagonists of adenosine triphosphate-sensitive potassium channels or inhibitors of the permeability transition. Swelling was also maintained when K + in the media was substituted with tetraethylammonium (TEA + ), which is not transported by any known K + carrier. Electron microscopy experiments gave support to the idea that NS-1619-induced mitochondrial swelling took place in the absence of K + . In addition to testing the pharmacological effects of NS-1619, we attempted, unsuccessfully, to promote mitoK Ca activity by altering Ca 2+ concentrations in the medium and inducing mitochondrial uncoupling. Conclusion: Our data indicate that NS-1619 promotes non-selective permeabilization of the inner mitochondrial membrane to ions, in addition to partial respiratory inhibition. Furthermore, we found no specific K + transport in isolated heart mitochondria compatible with mitoK Ca opening, whether by pharmacological or physiological stimuli. Our results indicate that NS-1619 has extensive mitochondrial effects unrelated to mitoK Ca and suggest that tissue protection mediated by NS-1619 may occur through mechanisms other than activation of these channels.
Proceedings of the National Academy of Sciences, 2002
Mitochondrial ATP-sensitive K (mitoK(ATP)) channels play a central role in protecting the heart from injury in ischemic preconditioning. In isolated mitochondria exposed to elevated extramitochondrial Ca, P(i), and anoxia to simulate ischemic conditions, the selective mitoK(ATP) channel agonist diazoxide (25-50 microM) potently reduced mitochondrial injury by preventing both the mitochondrial permeability transition (MPT) and cytochrome c loss from the intermembrane space. Both effects were blocked completely by the selective mitoK(ATP) antagonist 5-hydroxydecanoate. The protective effect against Ca-induced MPT was most evident under conditions in which the ability of electron transport to support membrane potential (Deltapsi(m)) was decreased and inner membrane leakiness was increased moderately. Under these conditions, mitoK(ATP) channel activity strongly regulated Deltapsi(m), and diazoxide prevented MPT by inhibiting the driving force for Ca uptake. Phorbol 12-myristate 13-acetate mimicked the protective effects of diazoxide, unless 5-hydroxydecanoate was present, indicating that protein kinase C activation also protects mitochondria by activating mitoK(ATP) channels. Because Deltapsi(m) recovery ultimately is required for heart functional recovery, these results may explain how mitoK(ATP) channel activation mimics ischemic preconditioning by protecting mitochondria as they pass through a critical vulnerability window during ischemia/reperfusion.
Pharmacological preconditioning by diazoxide downregulates cardiac L-type Ca2+ channels
British Journal of Pharmacology, 2010
BACKGROUND AND PURPOSE Pharmacological preconditioning (PPC) with mitochondrial ATP-sensitive K + (mitoKATP) channel openers such as diazoxide, leads to cardioprotection against ischaemia. However, effects on Ca 2+ homeostasis during PPC, particularly changes in Ca 2+ channel activity, are poorly understood. We investigated the effects of PPC on cardiac L-type Ca 2+ channels. EXPERIMENTAL APPROACH PPC was induced in isolated hearts and enzymatically dissociated cardiomyocytes from adult rats by preincubation with diazoxide. We measured reactive oxygen species (ROS) production and Ca 2+ signals associated with action potentials using fluorescent probes, and L-type currents using a whole-cell patch-clamp technique. Levels of the a1c subunit of L-type channels in the cellular membrane were measured by Western blot. KEY RESULTS PPC was accompanied by a 50% reduction in a1c subunit levels, and by a reversible fall in L-type current amplitude and Ca 2+ transients. These effects were prevented by the ROS scavenger N-acetyl-L-cysteine (NAC), or by the mitoKATP channel blocker 5-hydroxydecanoate (5-HD). PPC signficantly reduced infarct size, an effect blocked by NAC and 5-HD. Nifedipine also conferred protection against infarction when applied during the reperfusion period. Downregulation of the a1c subunit and Ca 2+ channel function were prevented in part by the protease inhibitor leupeptin. CONCLUSIONS AND IMPLICATIONS PPC downregulated the a1c subunit, possibly through ROS. Downregulation involved increased degradation of the Ca 2+ channel, which in turn reduced Ca 2+ influx, which may attenuate Ca 2+ overload during reperfusion.