mitoKATP Channel Research Articles

Modulation of Myocardial Mitochondrial VDAC/ MitoKATP Channels in Sepsis

In the present study, we tested the hypothesis that norepinephrine by modulating myocardial voltage‐dependent anion channel (VDAC)/mitoKATP channels can affect the release of cytochrome C and regulate intrinsic apoptotic cascade. Male Sprague‐Dawley rats (350‐400g) were made septic using 200mg/kg cecal inoculum, ip. Sham animals received 5% dextrose water, ip. VDAC mRNA and protein expression was determined in the myocardium and isolated single adult rat ventricular myocytes (ARVMs) obtained from sham and septic rats. In addition, mitochondria and cytosolic fractions were isolated from ARVM treated with norepinephrine (NE, 10ìmoles) in the presence and absence of 5‐Hydroxydecanoic acid (5HD; 100ìmoles), a selective mitoKATP channel blocker. Sepsis produced a significant down‐regulation of VDAC mRNA and protein along with upregulation of Bax in both myocardium and ARVMs. Pretreatment of septic ARVMs with 5HD blocked NE‐induced decrease in VDAC expression and the release of cytochrome C. These results suggest that sepsis downregulates myocardial VDAC expression. It appears that NE modulates mitoKATP channels and increases the release of cytochrome C, which may play a crucial role in sepsis‐induced cardiomyocyte dysfunction.

Inhibition of hypothermic preservation‐induced Smac/DIABLO protein expression by diazoxide in donor rat myocardium

AIMTo investigate the effect of a mitochondrial ATP‐sensitive potassium channel (mitoKATP) opener diazoxide (DE) on Smac/DIABLO protein expressions in rat heart suffered from different duration of hypothermic preservation.METHODSThe Langendorff model of isolated rat heart was used. After stored in 4ºC Celsior solution with or without DE (30 μmol/L) for different time (0, 3, 6, 9 or 12 h). Cell apoptosis was detected by TUNEL technique. The expression of Smac/ DIABLO protein was also analyzed by Western blotting.RESULTS(1) Compared with the hypothermic preservation groups, DE reduced the percentage of apoptotic cells. (2) The peak level of Smac/DIABLO protein expression appeared at 6 h after hypothermic preservation, which was postponed to 9 h by DE. (3) The above effects of DE were attenuated by a mitoKATP channel inhibitor 5‐hydroxydecanoate (5‐HD).CONCLUSIONSThe findings indicate that in the isolated rat heart DE protects myocardium against different duration of hypothermic preservation injury via opening of mitoKATP channel and inhibition of Smac/DIABLO protein expression.

Regulation of Neuronal KATP Channels by Signaling Elicited by cGMP-Dependent Protein Kinase Activation

The ATP-sensitive potassium (KATP) channel couples intracellular metabolic state to cell excitability. Recently, we have demonstrated that activation of the nitric oxide (NO)/cGMP/cGMP-dependent protein kinase (PKG) signaling cascade results in stimulation of Kir6.2/SUR1 (i.e. the neuronal-type KATP) channels. To understand how PKG activation induces plasma-membrane KATP channel stimulation, in the present study we investigated the potential involvement of the mitochondrial KATP (mitoKATP) channel and reactive oxygen species (ROS) in signal transduction. By performing single-channel recordings in transfected human embryonic kidney (HEK) 293 cells and neuroblastoma SH-SY5Y cells, we found that the enhancement of Kir6.2/SUR1 channel currents by PKG activation observed in cell-attached patches was diminished by the selective mitoKATP channel inhibitor 5-hydroxydecanoic acid (5-HD), ROS scavengers, and catalase, an enzyme that decomposes hydrogen peroxide (H2O2). 5-HD, ROS scavengers and catalase also significantly attenuated Kir6.2/SUR1 channel stimulation induced by NO donors. Moreover, bath application of H2O2 increased the activity of Kir6.2/SUR1 channels in cell-attached but not inside-out patches, and the stimulatory effect was not affected by 5-HD, excluding ROS as a signal upstream of the mitoKATP channel to mediate Kir6.2/SUR1 channel stimulation. In addition, H2O2 failed to stimulate tetrameric Kir6.2LRKR368/369/370/371AAAA channels expressed without the SUR subunit in intact cells. Altogether, these novel findings suggest that PKG stimulates neuronal KATP channels via opening of mitoKATP channels and ROS generation in a SUR1 subunit-dependent manner, implicating functional coupling between mitoKATP and plasma-membrane KATP channels upon PKG activation. The NO/cGMP/PKG/mitoKATP/ROS signaling cascade may contribute to neuroprotection under ischemic conditions by enhancing the function of plasma-membrane KATP channels whose activation reduces cell excitability.

Specific Inhibition of Protein Kinase B Protects Against Ischemic Myocardial Injury in Rat and Man

It is believed, but not without dispute, that activation of PKB is essential to obtain cardioprotection by ischemic preconditioning (IP). Here we have investigated the role of PKB activity in ischemic myocardial injury and IP using novel specific PKB inhibitors, examined whether any effect is species-dependent and determined its location in the transduction pathway. The specific PKB inhibitors VIII (0.05, 0.5 and 5μM) and XI (0.1, 1 and 10μM) were co-incubated with rat ventricular myocardium for 20min prior to 90min ischemia/120min reoxygenation at 37°C (n=6/group). CK release and cell necrosis and apoptosis (% of nuclei) were significantly decreased by more than 60% at all concentrations of both inhibitors. Similar protection was obtained with IP, results that were unaffected by PKB inhibitors. The PI-3K inhibitors LY294002 (10μM) and wortmanin (0.1μM) administered for 20min prior to ischemia induced identical results to those seen with PKB inhibitors. The protection afforded by PKB inhibitor XI was unaffected by the presumed mitoKATP channel blocker 5-HD (10μM) but was abrogated by the p38MAPK inhibitor SB203580 (10μM). Western Blot and Proteome Profiler studies confirmed a decrease in PKB phosphorylation in myocardium exposed to IP, wortmanin and PKB inhibitor XI. Studies using human myocardium also showed that both PKB inhibitor XI (1μM) and PI-3K inhibitor wortmanin (0.1μM) equally reduced CK release and cell necrosis and apoptosis. The diabetic myocardium, that could not be protected by IP or diazoxide (100μM), was however protected by PKB inhibitor XI and wortmanin, further suggesting that PKB is located beyond the mitochondria. In conclusion, inhibition of PKB activity is protective against ischemic injury of the rat and human myocardium and is as potent as IP. Importantly, PKB is downstream of the 5-HD target but upstream of p38MAPK.

Large Conductance Potassium Channel In Mitochondria of Endothelial Cell

It is well established that endothelial dysfunction contributes to ischemia-reperfusion injury of cardiovascular system. This phenomenon can be limited by the ischemic preconditioning. Recently, it was shown that mitochondrial ATP regulated potassium channel activation induced ischemic preconditiong of the endothelium in humans in vivo.In our study a single channel activity was measured after patch-clamp of the mitoplasts isolated from endothelial cell line (EA.hy926). Mitoplast samples were prepared by addition to a hypotonic solution causing the cristae of the inner membrane to unfold and breaking of the outer membrane. Isotonicity was restored by adding of hypertonic solution. A potassium selective current was recorded with mean conductance 270 ± 10 pS in symmetrical 150 mM KCl solution. Patch-clamp single channel studies showed properties of the large conductance Ca2+−regulated potassium channel (BKCa channel): it was activated by calcium and NS1619 an activator of BKCa channel at micromolar concentration range. These effects were blocked irreversible by iberiotoxin (IbTx), an inhibitor of BKCa channel. Additionally, we showed that inhibitor of mitoKATP channel (ATP/Mg2+ complex) have no effects on observed activity of ion channel.We conclude that large conductance Ca2+−regulated potassium channels are present in mitochondria isolated from endothelial cell line.This work was supported by Polish Mitochondrial Network MitoNet.pl and grant N30105331/1676

New Aspects for the Treatment of Cardiac Diseases Based on the Diversity of Functional Controls on Cardiac Muscles: Mitochondrial Ion Channels and Cardioprotection

Mitochondrial ATP-sensitive K+ (mitoKATP) and Ca2+-activated K+ (mitoKCa) channels exist in cardiac myocytes, and they play key roles in cardioprotection. We have recently reported that K+ influx through mitoKATP or mitoKCa channels occurs independently of each other and confers cardioprotection in a similar manner. Activation of mitoKATP channel is augmented by protein kinase C (PKC), whereas mitoKCa channel is activated by protein kinase A (PKA). However, phosphatidylinositol 3-kinase (PI3-K) is linked to neither mitoKATP nor mitoKCa channels. We have demonstrated that bioactive substances modulate the opening of mitoKATP channels via a PKC-dependent pathway or opening of mitoKCa channels via a PKA-dependent pathway and thereby protecting the heart from ischemia/reperfusion injury. Several endogenous substances such as adenosine and bradykinin can reduce infarct size by activation of mitoKATP channels in a PKC-dependent manner. Adrenomedullin, a potent vasodilator peptide, potentiates the opening of mitoKCa channels by PKA activation. Treatment with adrenomedullin prior to ischemia results in the reduction of infarct size via a PKA-mediated activation of mitoKCa channels. Thus, some endogenous substances confer cardioprotection via PKA- or PKC-mediated activation of mitoKATP or mitoKCa channels.

Blockade of the erbB2 Receptor Induces Cardiomyocyte Death through Mitochondrial and Reactive Oxygen Species-dependent Pathways

Overexpression of the receptor tyrosine kinase erbB2 (Her2 in humans) is correlated with a poor prognosis in breast and ovarian cancers. Treatment with trastuzumab (a monoclonal antibody against erbB2) improves survival; however, it also causes cardiomyopathy. We hypothesized that blockade of the erbB2 receptor induces cardiomyocyte death through a mitochondrial pathway that is dependent on the production of reactive oxygen species (ROS). We first showed that levels of erbB2 receptor are significantly decreased in an animal model of ischemic heart disease and in human ischemic cardiomyopathy. We treated neonatal rat cardiomyocytes with an inhibitory erbB2 antibody to study the mechanism behind the deleterious effects of erbB2 blockade. These cells displayed a dose-dependent increase in ROS production and cell death compared with control IgG-treated cells; these processes were reversed by the antioxidant, N-acetylcysteine. The effects of erbB2 antibody on both cell death and ROS production were also reversed by cyclosporine A and diazoxide, chemicals that regulate the pro- and anti-apoptotic channels in the mitochondria, respectively. Furthermore, mouse embryonic fibroblasts lacking Bax and Bak (proteins that mediate cell death through a mitochondrial pathway) were resistant to the deleterious effects of erbB2 antibody. These effects of erbB2 blockade appear to occur through a pathway involving AKT and PKC-alpha. Our results suggest that erbB2 plays a role in cardiomyocyte survival, and that the deleterious effects of trastuzumab on the heart occur through a mitochondrial pathway and is mediated by ROS production. Manipulation of redox signaling may be beneficial in cancer patients receiving trastuzumab.

Abstract 241: Intermittent Hypoxia Protects Cardiomyocytes Against Ischemia/Reperfusion-Induced Ca 2+ Overload and Contraction Suppression via Activation of α 1b -adrenoceptors

We have demonstrated that long-term training to intermittent high altitude hypoxia (IH) protects hearts against ischemia-reperfusion (I/R)-induced Ca 2+ overload and improved postischemic myocardial contractility. This cardioprotection is non-invasive and maintains much longer time compared with that induced by ischemic preconditioning, thereby, it may have great clinical potential. However, its precise signaling pathways are not fully clarified. Here we hypothesized that IH may attenuate I/R-induced cytosolic and mitochondrial Ca 2+ overload via activation of α 1 -adrenoceptor (α 1 -ARs) pathways. To test it, dynamics of cytosolic free Ca 2+ concentration ([Ca 2+ ] c ), mitochondrial [Ca 2+ ] ([Ca 2+ ] m ) and cell contraction were examined during preischemia and I/R in ventricular cardiomyocytes from normoxic and IH rats. IH significantly attenuated I/R-induced [Ca 2+ ] c and [Ca 2+ ] m overload by 63.5 ± 3.6% and 57.0 ± 4.5% at 30 min of reperfusion (R30), respectively. Consistently, I/R-induced depression on Ca 2+ transients and cell shortening was markedly attenuated by IH, with decreases in mitochondrial cytochrome c release and ATP reduction during reperfusion. Meanwhile, the α 1B -AR and α 1D -AR density obviously decreased during ischemia, while α 1A -AR density remained unchanged. IH did not change α 1A -AR density, but increased α 1B -AR density by ~50% and decreased α 1D -AR density further by 30% during I/R. Moreover, IH-induced protections were abolished by α 1B -AR antagonist chloroethylclonidine but not α 1A - or α 1D -AR antagonist. Furthermore, IH-afforded protection was lost when epsilon isoform of protein kinase C (PKCϵ) was inhibited with PKCϵ V1–2 and mitochondrial ATP sensitive potassium (mitoK ATP ) channels with 5-hydroxydecanoate, a downstream signaling pathway and effector of α 1 -ARs. Our findings indicate that α 1B -AR subtype might be one of the important mediators in IH protects myocytes against I/R injury via maintaining intracellular Ca 2+ homeostasis and protecting the ability of mitochondria through activation of the α 1B -ARs and PKCϵ pathway as well as mitoK ATP channel.. by via translocation of PKCϵ and opening of mitoK ATP channels. Mitochondrial function appears to be a determinant of cardioprotection induced by IH.

Spirocyclic Benzopyran-Based Derivatives as New Anti-ischemic Activators of Mitochondrial ATP-Sensitive Potassium Channel

Heart mitochondrial ATP-sensitive potassium channels (mito-K ATP channels) are deeply implicated in the self-defense mechanism of ischemic preconditioning. Therefore, exogenous molecules activating these channels are considered as a promising pharmacological tool to reduce the myocardial injury deriving from ischemia/reperfusion events. This paper reports the synthesis and pharmacological evaluation of original spiromorpholine- and spiromorpholone-benzopyran derivatives, with the aim to obtain selective activators of mito-K ATP channels. Some compounds of this series showed appreciable cardioprotective effects on rat isolated and perfused hearts, submitted to ischemia/reperfusion cycles. The selective mito-K ATP channel blocker 5-hydroxydecanoic acid antagonized the anti-ischemic activity, indicating a clear implication of this pharmacological target. Furthermore, these effects were not associated with significant hypotensive and vasorelaxing properties, which represent one of the main limiting factors for the clinical use of nonselective K ATP-openers against myocardial ischemia.

Ghrelin activates mitochondrial but not sarcolemmal ATP-sensitive K+ channels in rabbit ventricular cells

Mitochondrial ATP-sensitive K+ (mitoKATP) channels are activated by protein kinase C (PKC) and implicated in the mechanism of cardioprotection. Ghrelin, a peptide of 28 amino acids, was first identified in 1999 as an endogenous ligand for the growth hormone secretagogue receptor type 1a (GHSR-1a, i.e., ghrelin receptor). The GHSR-1a belongs to the family of receptors coupling to the Gq-phospholipase C pathway, yielding diacylglycerol which activates PKC. Recently, ghrelin has been suggested to be useful for the treatment of heart failure and ischemic heart disease.

Involvement of mitoK ATP channel in protective mechanisms of cerebral ischemic tolerance

Little work has been performed to determine roles of mitochondrial ATP-sensitive potassium channels (mitoK ATP) in ischemic preconditioning (IPC) in brain. To investigate the role on cerebral IPC, we examined effect of 5-hydroxydecanoate (5-HD), a selective mitoK ATP blocker, and diazoxide (DZX), a selective mitoK ATP opener on various IPC models. An IPC model with gerbil: 2 min bilateral common carotid arteries occlusion (BLCO) + 24 h recovery + 5 min BLCO. 5-HD, DZX, vehicle was administered 30 min before 5 min BLCO. Seven days later, surviving CA1 neurons were counted. A focal IPC model with rat: 15 min middle cerebral artery occlusion (MCAO) + 48 h recovery + 90 min MCAO. Twenty-four hours before 90 min MCAO, 5-HD, DZX, or vehicle was administered. One day after 90 min MCAO, neurological symptoms and infarct volumes were evaluated. An in vitro IPC model with primary neuronal cultures: 8 min oxygen–glucose deprivation (OGD) + 24 h recovery + 70 min OGD. Thirty minutes before 70 min OGD, 5-HD or DZX were added. One day later, surviving neurons were counted. Mitochondrial membrane potential was also monitored. 5-HD significantly attenuated the protective effect of IPC in gerbil model, rat model, and in vitro OGD model. DZX significantly facilitated the protective effect of IPC in gerbil and rat model. The mitochondrial membranes were depolarized with IPC, and 5-HD treatment significantly reduced this effect. These results strongly suggest that mitoK ATP channel activation plays a key role in development of a protective mechanism of cerebral IPC.

Signalosomes: delivering cardioprotective signals from GPCRs to mitochondria

Signalosomes: delivering cardioprotective signals from GPCRs to mitochondria

Conditioning the heart induces formation of signalosomes that interact with mitochondria to open mitoKATPchannels

Perfusion of the heart with bradykinin triggers cellular signaling events that ultimately cause opening of mitochondrial ATP-sensitive K+ (mitoKATP) channels, increased H2O2 production, inhibition of the mitochondrial permeability transition (MPT), and cardioprotection. We hypothesized that the interaction of bradykinin with its receptor induces the assembly of a caveolar signaling platform (signalosome) that contains the enzymes of the signaling pathway and that migrates to mitochondria to induce mitoKATP channel opening. We developed a novel method for isolating and purifying signalosomes from Langendorff-perfused rat hearts treated with bradykinin. Fractions containing the signalosomes were found to open mitoKATP channels in mitochondria isolated from untreated hearts via the activation of mitochondrial PKC-epsilon. mitoKATP channel opening required signalosome-dependent phosphorylation of an outer membrane protein. Immunodetection analysis revealed the presence of the bradykinin B2 receptor only in the fraction isolated from bradykinin-treated hearts. Immunodetection and immunogold labeling of caveolin-3, as well as sensitivity to cholesterol depletion and resistance to Triton X-100, attested to the caveolar nature of the signalosomes. Ischemic preconditioning, ischemic postconditioning, and perfusion with ouabain also led to active signalosome fractions that opened mitoKATP channels in mitochondria from untreated hearts. These results provide initial support for a novel mechanism for signal transmission from a plasma membrane receptor to mitoKATP channels.

Novel biologic mechanisms of levosimendan and its effect on the failing heart

Background: Calcium sensitizers belong to a new class of cardiac enhancers that stimulate cardiac contractility without causing intracellular calcium overload or increasing myocardial oxygen demand. Levosimendan, the most well-studied calcium sensitizer in the real clinical practice, produces greater hemodynamic and symptomatic improvement in patients with acute heart failure than traditional inotropes. Objective: To review the recent experimental and clinical evidence on novel biologic mechanisms explaining the pleiotropic effects of levosimendan on the failing heart. Methods: A systematic search of peer-reviewed publications was performed on Medline and EMBASE from January 1995 to December 2007. The results of unpublished trials were obtained from presentations at national and international meetings. Results: Levosimendan has a unique dual mechanism of action by enhancing cardiac contractility and causing peripheral vasodilatation. Immunomodulatory and antiapoptotic properties of levosimendan may be an additional biologic mechanism that prevents further cytotoxic and hemodynamic consequences of abnormal immune and neurohormonal responses in acute heart failure, leads to cardioprotection, and beneficially intervenes in the progression of syndrome. Experimental data show that levosimendan exerts its cardioprotective effects through its antioxidant properties and seems to be a potent inhibitor of H2O2-induced cardiomyocyte apoptotic cell death. Clinical data demonstrate that levosimendan does not increase markers of oxidative and nitrosative stress, in contrast to placebo treatment, in advanced chronic heart failure patients. Levosimendan has also been shown to activate mitoKATP channels which are important mediators of ischemic preconditioning. Pharmacological modulation of KATP channels may prove beneficial in patients at risk of myocardial ischemia, particularly those requiring inotropic support. Conclusion: Pleiotropic effects of levosimendan appear to have important clinical and prognostic implications in acute heart failure syndromes and ischemic heart disease.

TNF-α gene promoter polymorphism at nucleotide −308 and the inflammatory response and oxidative stress induced by cardiac surgery: role of heart failure and medical treatment☆

Increased TNF-alpha during cardiac surgery is thought to be responsible for perioperative complications. The TNF-alpha gene promoter polymorphism G/A at position -308 has been associated with enhanced TNF-alpha secretion, as has been heart failure. Therefore, the aims of this study were to investigate: (i) whether the TNF-alpha G/A polymorphism is associated with exacerbation of TNF-alpha plasma levels during cardiac surgery; (ii) whether TNF-alpha production is further increased by heart failure and influenced by medical treatment; and (iii) whether this polymorphism is associated with increased oxidative stress and perioperative complications. The TNF-alpha gene promoter polymorphism was studied in 100 consecutive patients undergoing cardiac surgery. Of them, 65 were identified with the common allele G/G, whereas 34 patients were with the G/A polymorphism and 1 was A/A. TNF-alpha plasma levels (ELISA) and peroxynitrite content in peripheral blood lymphocytes (flow cytometry) were measured before surgery, before cardiopulmonary bypass (CPB), and 30 min, 4 and 24h after initiation of CPB. The changes observed in TNF-alpha plasma levels during cardiac surgery were unaffected by the G/A polymorphism. TNF-alpha values were elevated before surgery in patients with more advanced NYHA class (1.66+/-0.14, 2.29+/-0.06 and 2.57+/-0.11 ln(mmol/l+1), for NYHA I, II and III; p=0.004) but again they were not correlated with the G/A polymorphism. Peroxynitrite content in lymphocytes was similar upon the initiation of surgery in the G/A and G/G groups and also in all NYHA class groups, and thereafter levels were similarly increased by surgery in all groups. However, analysis of the effect of preoperative medication showed that the mitoK(ATP) channel opener nicorandil reduced TNF-alpha values before surgery and blunted the increase in peroxynitrite caused by surgery. Perioperative complications were not related to either TNF-alpha polymorphism or TNF-alpha and peroxynitrite levels. The TNF-alpha gene promoter polymorphism G/A at position -308 does not influence TNF-alpha plasma levels during cardiac surgery, is not associated with greater oxidative stress, and does not result in a greater incidence of perioperative complications. However, importantly, treatment with the mitoK(ATP) channel opener nicorandil prior to surgery significantly reduced basal TNF-alpha values and also the oxidative stress induced by surgery.

Determination of the rate of K + movement through potassium channels in isolated rat heart and liver mitochondria

Both ATP-regulated (mitoK ATP) and large conductance calcium-activated (mitoBK Ca) potassium channels have been proposed to regulate mitochondrial K + influx and matrix volume and to mediate cardiac ischaemic preconditioning (IP). However, the specificity of the pharmacological agents used in these studies and the mechanisms underlying their effects on IP remain controversial. Here we used increasing concentrations of K +-ionophore (valinomycin) to stimulate respiration by rat liver and heart mitochondria in the presence of the K +/H + exchanger nigericin. This allowed rates of valinomycin-induced K + influx to be determined whilst parallel measurements of light scattering (A 520) and matrix volume ( 3H 2O and [ 14C]-sucrose) enabled rates of K + influx to be correlated with increases in matrix volume. Light scattering readily detected an increase in K + influx of < 5 nmol K + min − 1 per mg protein corresponding to < 2% mitochondrial matrix volume increase. In agreement with earlier data no light-scattering changes were observed in response to any mitoK ATP channel openers or blockers. However, the mitoBK Ca opener NS1619 (10–50 µM) did decrease light scattering slightly, but this was also seen in K +-free medium and was accompanied by uncoupling. Contrary to prediction, the mitoBK Ca blocker paxilline (10–50 µM) decreased rather than increased light scattering, and it also slightly uncoupled respiration. Our data argue against the presence of significant activities of either the mitoK ATP or the mitoBK Ca channel in rat liver and heart mitochondria and provide further evidence that preconditioning induced by pharmacological openers of these channels is more likely to involve alternative mechanisms.

Modulation of the c-Jun N-terminal kinase activity in the embryonic heart in response to anoxia-reoxygenation: involvement of the Ca2+ and mitoKATP channels

Whether the response of the fetal heart to ischemia-reperfusion is associated with activation of the c-Jun N-terminal kinase (JNK) pathway is not known. In contrast, involvement of the sarcolemmal L-type Ca2+ channel (LCC) and the mitochondrial KATP (mitoKATP) channel has been established. This work aimed at investigating the profile of JNK activity during anoxia-reoxygenation and its modulation by LCC and mitoK(ATP) channel. Hearts isolated from 4-day-old chick embryos were submitted to anoxia (30 min) and reoxygenation (60 min). Using the kinase assay method, the profile of JNK activity in the ventricle was determined every 10 min throughout anoxia-reoxygenation. Effects on JNK activity of the LCC blocker verapamil (10 nM), the mitoK(ATP) channel opener diazoxide (50 microM) and the blocker 5-hydroxydecanoate (5-HD, 500 microM), the mitochondrial Ca2+ uniporter (MCU) inhibitor Ru360 (10 microM), and the antioxidant N-(2-mercaptopropionyl) glycine (MPG, 1 mM) were determined. In untreated hearts, JNK activity was increased by 40% during anoxia and peaked fivefold relative to basal level after 30-40 min reoxygenation. This peak value was reduced by half by diazoxide and was tripled by 5-HD. Furthermore, the 5-HD-mediated stimulation of JNK activity during reoxygenation was abolished by diazoxide, verapamil or Ru360. MPG had no effect on JNK activity, whatever the conditions. None of the tested pharmacological agents altered JNK activity under basal normoxic conditions. Thus, in the embryonic heart, JNK activity exhibits a characteristic pattern during anoxia and reoxygenation and the respective open-state of LCC, MCU and mitoKATP channel can be a major determinant of JNK activity in a ROS-independent manner.

Design and synthesis of nitrate esters of aromatic heterocyclic compounds as pharmacological preconditioning agents

Ischemic preconditioning (IPC) constitutes an endogenous protective mechanism in which one or more brief periods of myocardial ischemia and reperfusion render the myocardium resistant to a subsequent more-sustained ischemic insult. Pharmacological preconditioning represents an ideal alternative of IPC. We now describe the design and synthesis of indole, quinoline, and purine systems with an attached pharmacophoric nitrate ester group. The indole and quinoline derivatives 4 and 5 possess structural features of the nitrate containing K ATP channel openers. Purine analogues 11 and 12, substituted at the position 6 by a piperidine moiety and at position 9 by an alkyl nitrate, could combine the effects of the nitrate containing K ATP channel openers and those of adenosine. Compound 13 bears the nicotinamide moiety of nicorandil instead of nitrate ester. Compounds 4, 5, and 11 reduced infarction and the levels of malondialdehyde (MDA) at reperfusion in anesthetized rabbits. Compounds 12 and 13 did not significantly reduce the infarct size. Analogues 4 and 5 increased cGMP and MDA during ischemia, while combined analogue 4 and mitoK ATP blocker 5-hydroxydecanoic acid (5-HD) abrogated this benefit suggesting an action through mitoK ATP channel opening. Treatment with derivative 11 combined with 5-HD as well as treatment with 11 and adenosine receptor blocker 8-( p-sulfophenyl)theophylline (SPT) did not abrogate cardioprotection. Compound 11 is a lead molecule for the synthesis of novel analogues possessing a dual mode of action through cGMP–mitoK ATP channel opening-free radicals and through adenosine receptors.

ROS-Independent Preconditioning in Neurons via Activation of mitoKATP Channels by BMS-191095

Previously, we have shown that the selective mitochondrial ATP-sensitive potassium (mitoK(ATP)) channel opener BMS-191095 (BMS) induces neuronal preconditioning (PC); however, the exact mechanism of BMS-induced neuroprotection remains unclear. In this study, we have identified key components of the cascade resulting in delayed neuronal PC with BMS using isolated rat brain mitochondria and primary cultures of rat cortical neurons. BMS depolarized isolated mitochondria without an increase in reactive oxygen species (ROS) generation and induced rapid phosphorylation of Akt and glycogen synthase kinase-3beta. Long-term (3 days) treatment of neurons with BMS resulted in sustained mitochondrial depolarization, decreased basal ROS generation, and elevated ATP levels. This treatment also elicited almost complete protection against glutamate excitotoxicity, which could be abolished using the phosphoinositide 3-kinase (PI3K) inhibitor wortmannin, but not with the superoxide dismutase (SOD) mimetic M40401. Long-term BMS treatment induced a PI3K-dependent increase in the expression and activity of catalase without affecting manganese SOD and copper/zinc-dependent SOD. Finally, the catalase inhibitor 3-aminotriazole dose-dependently antagonized the neuroprotective effect of BMS-induced PC. In summary, BMS depolarizes mitochondria without ROS generation, activates the PI3K-Akt pathway, improves ATP content, and increases catalase expression. These mechanisms appear to play important roles in the neuroprotective effect of BMS.

One potential new target of cancer therapy, mitochondrial ATP-sensitive potassium channels

Plenty of evidence strongly suggests that mitoK ATP channels played an important role in cellular death and survival in many tissues, but it is still unknown whether mitoK ATP channels can treat cancer via inducing cellular apoptosis.