Caveolae, caveolin, and cavins: potential targets for the treatment of cardiac disease.

Caveolae are omega-shaped membrane invaginations present in essentially all cell types of the cardiovascular system, including endothelial cells, smooth muscle cells, macrophages, cardiac myocytes, and fibroblasts. Numerous functions have been ascribed to this omega-shaped structure. Caveolae are enriched with different signaling molecules and ion channel regulatory proteins and function both in protein trafficking and signal transduction in these cell types. Caveolins are the structural proteins that are necessary for the formation of caveola membrane domains. Mechanistically, caveolins interact with a variety of downstream signaling molecules, as, for example, Src-family tyrosine kinase, p42/44 mitogen-activated protein (MAP) kinase, and endothelial nitric oxide synthase (eNOS) and hold the signal transducers in the inactive condition until activated with proper stimulus. Caveolae are gradually acquiring increasing attention as cellular organelles contributing to the pathogenesis of several structural and functional processes including cardiac hypertrophy, atherosclerosis, and heart failure. At present, very little is known about the role of caveolae in cardiac function and dysfunction, although recent studies with caveolin knock-out mouse have shown that caveolae and caveolins play a pivotal role in various human pathobiological conditions. This review will discuss the possible role and mechanism of action of caveolae and caveolins in different cardiac diseases.

Caveolin induces cardioprotection through epigenetic regulation.

Lipid rafts represent a sub-compartment of the plasma membrane that co-ordinate and regulate varieties of signalling processes, whereas caveolins are the integral membrane protein of the lipid raft. Recent evidence demonstrated the pivotal role of caveolins in cardioprotection against ischaemic injury, although their mechanism of action is not clear. However, new understanding of epigenetic modification during ischaemia reperfusion suggests additional targeted approaches that have not been explored before. To study the role of caveolin on epigenetic regulation, isolated mouse heart was prepared from wild-type (WT) and caveolin-1 knockout (Cav-1 KO) mouse and preconditioned them with four cyclic episodes of ischaemia/reperfusion followed by 30 min. global ischaemia and 120 min. reperfusion. We found that Cav-1 KO mouse abolished the acetylation of histone (H3 and H4) and increased the methylation of histone in the preconditioned heart. The increased histone methylation was significantly correlated with an increased level of histone methyltranferase G9a protein and increased the level of histone decaetylase (HDAC) activity. Cav-1 KO mouse also decreased the translocation of forkhead transcription factor (FOXO3a) to the nucleus and reduced the induction of the expression of SIRT-1 in the preconditioned heart. Cardioprotective property of Cav-1 was further confirmed by reduced ventricular function, increased cardiomyocyte apoptosis, increased expression of junas kinase (JNK) and Bax and decreased expression of phospho-adenosine monophosphate-activated protein kinase (AMPK), phospho-AKT and B cell lymphoma-2 (Bcl-2) in Cav-1 KO preconditioned heart. The results clearly indicate that Cav-1 induces cardioprotection through epigenetic regulation.

Resveratrol and cardiovascular health.

Resveratrol (3,4',5-trihydroxystilbene) is a member of natural, plant-derived chemicals known as polyphenols and is attracting increased attention due to its diverse health benefits especially in case of cardiovascular disease, cancer, diabetes and neurological problems. Despite impressive gains in diagnosis and treatment, cardiovascular disease (CVD) remains a serious clinical problem and threat to public health. Resveratrol possesses potent antioxidant properties and has been shown to decrease low-density lipoprotein-cholesterol oxidation and platelet aggregation. This compound also possesses a range of additional cardioprotective and vasoprotective properties including antiatherosclerotic and vasorelaxation action. Resveratrol also has the capacity to interact with multiple molecular targets, which involve diverse intracellular pathways. Most well-known is the ability of resveratrol to activate sirtuins, a class of NAD(+)-dependent deacetylase that affect multiple transcription factors and other protein targets. Recently, resveratrol was found to induce autophagy and regenerate myocardial ischemic tissue treated with stem cells. Overall observation indicates that resveratrol has a high therapeutic potentials for the treatment of cardiovascular diseases.

Lipid raft in cardiac health and disease.

Lipid rafts are sphingolipid and cholesterol rich micro-domains of the plasma membrane that coordinate and regulate varieties of signaling processes. Lipid rafts are also present in cardiac myocytes and are enriched in signaling molecules and ion channel regulatory proteins. Lipid rafts are receiving increasing attention as cellular organelles contributing to the pathogenesis of several structural and functional processes including cardiac hypertrophy and heart failure. At present, very little is known about the role of lipid rafts in cardiac function and dysfunction. This review will discuss the possible role of lipid rafts in cardiac health and disease.

Does white wine qualify for French paradox? Comparison of the cardioprotective effects of red and white wines and their constituents: resveratrol, tyrosol, and hydroxytyrosol.

It is generally believed that the French paradox is related to the consumption of red wine and not other varieties of wine, including white wine or champagne. Some recent studies have indicated that white wine could also be as cardioprotective as red wine. The present investigation compares the cardioprotective abilities of red wine, white wine, and their principal cardioprotective constituents. Different groups of rats were gavaged with red wine, white wine, resveratrol, tyrosol, and hydroxytyrosol. Red wine and its constituent resveratrol and white wine and its constituents tyrosol and hydroxytyrosol all showed different degrees of cardioprotection as evidenced by their abilities to improve postischemic ventricular performance, reduce myocardial infarct size and cardiomyocyte apoptosis, and reduce peroxide formation. It was discovered in this study that although each of the wines and their components increased the enzymatic activities of the mitochondrial complex (I-IV) and citrate synthase, which play very important roles in oxidative phosphorylation and ATP synthesis, some of the groups were more complex-specific in inducing the activity compared to the other groups. Cardioprotective ability was further confirmed by increased expression of phospho-Akt, Bcl-2, eNOS, iNOS, COX-1, COX-2, Trx-1, Trx-2, and HO-1. The results of this study suggest that white wine can provide cardioprotection similar to red wine if it is rich in tyrosol and hydroxytyrosol.

Caveolin and proteasome in tocotrienol mediated myocardial protection.

The effect of different isomers of tocotrienol was tested on myocardial ischemia reperfusion injury. Although all of the tocotrienol isomers offered some degree of cardioprotection, gamma-tocotrienol was the most protective as evident from the result of myocardial apoptosis. To study the mechanism of tocotrienol mediated cardioprotection, we examined the interaction and/or translocation of different signaling components to caveolins and activity of proteasome. The results suggest that differential interaction of MAP kinases with caveolin 1/3 in conjuncture with proteasome stabilization play a unique role in tocotrienol mediated cardioprotection possibly by altering the availability of pro-survival and anti-survival proteins.

Cardioprotection with alpha-tocopheryl phosphate: amelioration of myocardial ischemia reperfusion injury is linked with its ability to generate a survival signal through Akt activation.

The emerging potential of alpha-tocopheryl phosphate, a phosphoric acid ester of alpha-tocopherol, in health benefits was tested gavaging this compound (5 mg/kg body wt) to a group of rats for a period of thirty days while the control rats were given water only. After thirty days, the rats were sacrificed, the hearts excised, and the isolated hearts were perfused by working mode. Both control and experimental hearts were subjected to 30-min global ischemia followed by 2 h of reperfusion. The tocopheryl phosphate fed rats exhibited significant cardioprotection as evidenced by improved ventricular performance and reduced myocardial infarct size and cardiomyocyte apoptosis. Supplementation with alpha-tocopheryl phosphate converted MAP kinase-induced death signal into a survival signal by enhancing anti-apoptotic p42/44 ERK kinase and p38 MAPKbeta and reducing pro-apoptotic proteins p38 MAPKalpha and JNK. In concert, the phosphorylation of pro-apoptotic c-Src was also reduced. Tocopheryl phosphate increased the DNA binding of the redox-sensitive transcription factor NFkappaB and potentiated the activation of anti-death protein Bcl-2 and survival signaling protein Akt. The results of this study demonstrated for the first time that tocopheryl phosphate could ameliorate myocardial ischemic reperfusion injury by converting ischemia/reperfusion-mediated death signal into a survival signal by modulating MAP kinase signaling.

Essential role of lipid raft in ischemic preconditioning.

Lipid rafts represent a subcompartment of the plasma membrane that coordinate and regulate varieties of signaling processes while caveolins are the integral membrane protein of the lipid raft. To study the role of lipid raft in ischemic preconditioning (PC) of the heart, rat hearts were perfused by working mode and then preconditioned in absence or presence of a lipid raft disintegrator, Methyl-beta-cyclodextrin. As expected, precondition made the heart resistant to ischemia reperfusion (I/R) injury as evident by improved ventricular performance, reduced myocardial infract size and cardiomyocyte apoptosis. Cyclodextrin abolished the cardioprotection. Transmission Electron Microscopy revealed severe degeneration, swelling of mitochondria, chromatin condensation and myofibril disarray in cyclodextrin treated PC heart similar to I/R heart. In the PC hearts, there was an increased association of the proapoptotic p38MAPKalpha with caveolin-1 while there was a reduced association of anti-apoptotic p38MAPKbeta with caveolin-3 indicating that reduced amount of p38MAPKalpha and increased amount of p38MAPKbeta were available to the adapted hearts thereby generating a survival signal. In contrast, there was very weak caveolin-MAP kinase interaction in cyclodextrin treated heart. Myocardial damage was further confirmed by reduced or no expression of anti-apoptotic phospho-AKT, Bcl2, Bcl-xl and increased expression of pro-apoptotic JNK, BAX, and p53 in methyl-beta-cyclodextrin (lipid raft disintegrator) treated heart. These results indicate that lipid raft play a pivotal role in the generation of survival signal in PC or adapted heart and disintegration of lipid raft completely abolish cardioprotection.

Inhibition of mitochondrial permeability transition pore opening by ischemic preconditioning is probably mediated by reduction of oxidative stress rather than mitochondrial protein phosphorylation.

Inhibition of mitochondrial permeability transition pore (MPTP) opening at reperfusion is critical for cardioprotection by ischemic preconditioning (IP). Some studies have implicated mitochondrial protein phosphorylation in this effect. Here we confirm that mitochondria rapidly isolated from preischemic control and IP hearts show no significant difference in calcium-mediated MPTP opening, whereas IP inhibits MPTP opening in mitochondria isolated from IP hearts following 30 minutes of global normothermic ischemia or 3 minutes of reperfusion. Analysis of protein phosphorylation in density-gradient purified mitochondria was performed using both 2D and 1D electrophoresis, with detection of phosphoproteins using Pro-Q Diamond or phospho-amino-specific antibodies. Several phosphoproteins were detected, including voltage-dependent anion channels isoforms 1 and 2, but none showed significant IP-mediated changes either before ischemia or during ischemia and reperfusion, and neither Western blotting nor 2D fluorescence difference gel electrophoresis detected translocation of protein kinase C (alpha, epsilon, or delta isoforms), glycogen synthase kinase 3beta, or Akt to the mitochondria following IP. In freeze-clamped hearts, changes in phosphorylation of GSK3beta, Akt, and AMP-activated protein kinase were detected following ischemia and reperfusion but no IP-mediated changes correlated with MPTP inhibition or cardioprotection. However, measurement of mitochondrial protein carbonylation, a surrogate marker for oxidative stress, suggested that a reduction in mitochondrial oxidative stress at the end of ischemia and during reperfusion may account for IP-mediated inhibition of MPTP. The signaling pathways mediating this effect and maintaining it during reperfusion are discussed.

Molecular mechanism of preconditioning.

During the last 20 years, since the appearance of the first publication on ischemic preconditioning (PC), our knowledge of this phenomenon has increased exponentially. PC is defined as an increased tolerance to ischemia and reperfusion induced by previous sublethal period ischemia. This is the most powerful mechanism known to date for limiting the infract size. This adaptation occurs in a biphasic pattern (i) early preconditioning (lasts for 2-3 h) and (ii) late preconditioning (starting at 24 h lasting until 72-96 h after initial ischemia). Early preconditioning is more potent than delayed preconditioning in reducing infract size. Late preconditioning attenuates myocardial stunning and requires genomic activation with de novo protein synthesis. Early preconditioning depends on adenosine, opioids and to a lesser degree, on bradykinin and prostaglandins, released during ischemia. These molecules activate G-protein-coupled receptor, initiate activation of K(ATP) channel and generate oxygen-free radicals, and stimulate a series of protein kinases, which include protein kinase C, tyrosine kinase, and members of MAP kinase family. Late preconditioning is triggered by a similar sequence of events, but in addition essentially depends on newly synthesized proteins, which comprise iNOS, COX-2, manganese superoxide dismutase, and possibly heat shock proteins. The final mechanism of PC is still not very clear. The present review focuses on the possible role signaling molecules that regulate cardiomyocyte life and death during ischemia and reperfusion.

Cardioprotection with palm oil tocotrienols: comparision of different isomers.

A recent study from our laboratory indicated the cardioprotective ability of the tocotrienol-rich fraction (TRF) from red palm oil. The present study compared cardioprotective abilities of different isomers of tocotrienol against TRF as recently tocotrienol has been found to function as a potent neuroprotective agent against stroke. Rats were randomly assigned to one of the following groups: animals were given, by gavage, either 0.35%, 1%, or 3.5% TRF for two different periods of time (2 or 4 wk) or 0.03, 0.3, and 3 mg/kg body wt of one of the isomers of tocotrienol (alpha, gamma, or delta) for 4 wk; control animals were given, by gavage, vehicle only. After 2 or 4 wk, rats were killed, and their hearts were then subjected to 30 min of global ischemia followed by 2 h of reperfusion. Dose-response and time-response experiments revealed that the optimal concentration for TRF was 3.5% TRF and 0.3 mg/kg body wt of tocotrienol given for 4 wk. TRF as well as all the isomers of tocotrienol used in our study provided cardioprotection, as evidenced by their ability to improve postischemic ventricular function and reduce myocardial infarct size. The gamma-isoform of tocotrienol was the most cardioprotective of all the isomers followed by the alpha- and delta-isoforms. The molecular mechanisms of cardioprotection afforded by tocotrienol isoforms were probed by evaluating their respective abilities to stabilize the proteasome, allowing it to maintain a balance between prodeath and prosurvival signals. Our results demonstrated that tocotrienol isoforms reduced c-Src but increased the phosphorylation of Akt, thus generating a survival signal.

Protective mechanisms of resveratrol against ischemia-reperfusion-induced damage in hearts obtained from Zucker obese rats: the role of GLUT-4 and endothelin.

The resveratrol-induced cardiac protection was studied in Zucker obese rats. Rats were divided into five groups: group 1, lean control; group 2, obese control (OC); group 3, obese rats treated orally with 5 mg kg(-1) day(-1) of resveratrol (OR) for 2 wk; group 4, obese rats received 10% glucose solution ad libitum for 3 wk (OG); and group 5, obese rats received 10% glucose for 3 wk and resveratrol (OGR) during the 2nd and 3rd wk. Body weight, serum glucose, and insulin were measured, and then hearts were isolated and subjected to 30 min of ischemia followed by 120 min of reperfusion. Heart rate, coronary flow, aortic flow, developed pressure, the incidence of reperfusion-induced ventricular fibrillation, and infarct size were measured. Resveratrol reduced body weight and serum glucose in the OR compared with the OC values (414 +/- 10 g and 7.08 +/- 0.41 mmol/l, respectively, to 378 +/- 12 g and 6.11 +/- 0.44 mmol/l), but insulin levels were unchanged. The same results were obtained for the OG vs. OGR group. Resveratrol improved postischemic cardiac function in the presence or absence of glucose intake compared with the resveratrol-free group. The incidence of ventricular fibrillation and infarct size was reduced by 83 and 20% in the OR group, and 67 and 16% in the OGR group, compared with the OC and OG groups, respectively. Resveratrol increased GLUT-4 expression and reduced endothelin expression and cardiac apoptosis in ischemic-reperfused hearts in the presence or absence of glucose intake. Thus the protective effect of resveratrol could be related to its direct effects on the heart.

Caveolin and MAP kinase interaction in angiotensin II preconditioning of the myocardium.

Angiotensin II (Ang II) has been found to exert preconditioning (PC)-like effect in mammalian hearts. The present investigation reported for the first time a unique mitogen activated protein (MAP) kinase signalling in Ang II PC of the heart involving lipid rafts, which generated a survival signal by differentially associating MAP kinases with caveolin. A group of rat hearts was treated with Ang II in the absence or presence of NADPH oxidase inhibitor, apocynin or a cell permeable reactive oxygen species (ROS) scavenger, N-acetyl-cysteine (NAC). Ang II pre-treatment improved post-ischaemic ventricular recovery, myocardial infraction and decreased the number of cardiomyocyte apoptosis indicating PC effect of Ang II. Both apocynin and NAC abolished the PC ability of Ang II. In Ang II treated heart, there was a decreased association of p38MAPKbeta & extracellular-signal regulated kinase (ERK) 1/ 2 (anti-death signalling component) with caveolin while there was an increased association of p38MAPKalpha & Jun N-terminal kinase (JNK) (death signalling component) indicating reduced amount of death signal components and increased amount of anti-death signalling components being available to the Ang II treated heart to generate a survival signal, which was reversed with NAC or apocynin. The survival signal was also demonstrated by increased phosphorylation of serine/threonine-protein kinase B (AKT) and enhanced induction of expression of Bcl-2 during Ang II PC and its reversal with NAC & apocynin treated heart.

Generation of survival signal by differential interaction of p38MAPKalpha and p38MAPKbeta with caveolin-1 and caveolin-3 in the adapted heart.

Sphingomyelin breakdown product ceramide has recently been found to induce an adaptive response and reduce myocardial ischemia/reperfusion injury. Since activation of MAP kinases plays an essential role in myocardial adaptation to ischemic stress and since ceramide is involved in lipid raft formation where MAP kinases can be translocated in response to stress, we reasoned that preconditioning may potentiate the translocation of MAP kinases into the lipid raft. To test the hypothesis, rats were divided into five groups: (i) control, (ii) ischemia/reperfusion (I/R), (iii) I/R+C-2 ceramide, (iv) adapted and (v) adapted+desipramine, an inhibitor of ceramide formation. Isolated hearts were preperfused for 15 min with Krebs Henseleit bicarbonate (KHB) buffer in the absence or presence of 10 microM desipramine followed by adaptation induced by four cyclic episodes of 5 min ischemia and 10 min reperfusion. For myocardial adaptation to ischemia with ceramide, the hearts were perfused with 1 microM C-2 ceramide. All hearts were then subjected to 30 min ischemia and 2 h of reperfusion. As expected, both ischemic adaptation and ceramide adaptation made the heart resistant to I/R injury as evidenced by improved ventricular performance and reduced myocardial infarct size and cardiomyocyte apoptosis, which were significantly blocked with desipramine indicating the involvement of ceramide in ischemic adaptation. Ceramide also participated in the formation of lipid raft, and desipramine disrupted the raft formation. In the adapted hearts, there was an increased association of the proapoptotic p38MAPKalpha with caveolin-1 while there was a reduced association of anti-apoptotic p38MAPKbeta with caveolin-3 indicating reduced amount of p38MAPKalpha and increased amount of p38MAPKbeta were available to the adapted hearts thereby generating a survival signal. Desipramine decreased the association of P38MAPKalpha and C-2 ceramide increased the association of P38MAPKalpha with the lipid raft. The survival signal was further confirmed by increased phosphorylation of AKT and enhanced induction of expression of Bcl-2 during adaptation and its reversal with desipramine. The results indicated a unique ceramide signaling the ischemic and PC hearts involving lipid rafts, which generated a survival signal by differentially associating the p38MAPKalpha and p38MAPKbeta with the caveolin-1 and caveoli-3, respectively.

Mechanism of impairment of cytochrome P450-dependent metabolism in hamster liver during leishmaniasis.

The mechanism of impairment of cytochrome P450 (P450)-dependent metabolism in hamster liver during leishmaniasis is reported. A significant decrease in the level of P450 was observed on the 20th day of infection when the parasite load in the liver was maximum. The decrease in P450 level was accompanied by a significant increase in the level of marker enzymes of liver and degeneration of liver tissue. The impairment was isozyme-specific and concomitant with the induction of nitric oxide synthase. The results of in vitro experiments with generated nitric oxide and with scavengers demonstrated that the impairment is mediated by NO. Treatment of the infected animals with a combination therapy showed reduction in parasite load, reversal of P450 impairment, and recovery of liver enzymes and tissue close to normal.

Ischaemic preconditioning inhibits opening of mitochondrial permeability transition pores in the reperfused rat heart.

Opening of the mitochondrial permeability transition pore (MPTP) is thought to be a critical event in mediating the damage to hearts that accompanies their reperfusion following prolonged ischaemia. Protection from reperfusion injury occurs if the prolonged ischaemic period is preceded by short ischaemic periods followed by recovery. Here we investigate whether such ischaemic preconditioning (IPC) is accompanied by inhibition of MPTP opening. MPTP opening in Langendorff-perfused rat hearts was determined by perfusion with 2-deoxy[3H]glucose ([3H]DOG) and measurement of mitochondrial [3H]DOG entrapment. We demonstrate that IPC inhibits initial MPTP opening in hearts reperfused after 30 min global ischaemia, and subsequently enhances pore closure as hearts recover. However, MPTP opening in mitochondria isolated from IPC hearts occurred more readily than control mitochondria, implying that MPTP inhibition by IPC in situ was secondary to other factors such as decreased calcium overload and oxidative stress. Hearts perfused with cyclosporin A or sanglifehrin A, powerful inhibitors of the MPTP, also recovered better from ischaemia than controls (improved haemodynamic function and less lactate dehydrogenase release). However, the mitochondrial DOG entrapment technique showed these agents to be less effective than IPC at preventing MPTP opening. Our data suggest that protection from reperfusion injury is better achieved by reducing factors that induce MPTP opening than by inhibiting the MPTP directly.

Matrix volume measurements challenge the existence of diazoxide/glibencamide-sensitive KATP channels in rat mitochondria.

A mitochondrial sulphonylurea-sensitive, ATP-sensitive K+ channel (mitoKATP) that is selectively inhibited by 5-hydroxydecanoate (5-HD) and activated by diazoxide has been implicated in ischaemic preconditioning. Here we re-evaluate the evidence for the existence of this mitoKATP by measuring changes in light scattering (A520) in parallel with direct determination of mitochondrial matrix volumes using 3H2O and [14C]sucrose. Incubation of rat liver and heart mitochondria in KCl medium containing Mg2+ and inorganic phosphate caused a decrease in light scattering over 5 min, which was accompanied by a small (15-30 %) increase in matrix volume. The presence of ATP or ADP in the buffer from the start greatly inhibited the decline in A520, whilst addition after a period of incubation (1-5 min) induced a rapid increase in A520, especially in heart mitochondria. Neither response was accompanied by a change in matrix volume, as measured isotopically. However, the effects of ATP and ADP on A520 were abolished by carboxyatractyloside and bongkrekic acid, inhibitors of the adenine nucleotide translocase (ANT) that lock the transporter in two discrete conformations and cause distinct changes in A520 in their own right. These data suggest that rather than matrix volume changes, the effects of ATP and ADP on A520 reflect changes in mitochondrial shape induced by conformational changes in the ANT. Furthermore, we were unable to demonstrate either a decrease in A520 or increase in matrix volume with a range of ATP-sensitive K+ channel openers such as diazoxide. Nor did glibencamide or 5-HD cause any reduction of matrix volume, whereas the K+ ionophore valinomycin (0.2 nM), produced a 10-20 % increase in matrix volume that was readily detectable by both techniques. Our data argue against the existence of a sulphonylurea-inhibitable mitoKATP channel.

The effects of ischaemic preconditioning, diazoxide and 5-hydroxydecanoate on rat heart mitochondrial volume and respiration.

Studies with different ATP-sensitive potassium (K(ATP)) channel openers and blockers have implicated opening of mitochondrial K(ATP) (mitoK(ATP)) channels in ischaemic preconditioning (IPC). It would be predicted that this should increase mitochondrial matrix volume and hence respiratory chain activity. Here we confirm this directly using mitochondria rapidly isolated from Langendorff-perfused hearts. Pre-ischaemic matrix volumes for control and IPC hearts (expressed in microl per mg protein +/- S.E.M., n = 6), determined with (3)H(2)O and [(14)C]sucrose, were 0.67 +/- 0.02 and 0.83 +/- 0.04 (P < 0.01), respectively, increasing to 1.01 +/- 0.05 and 1.18 +/- 0.02 following 30 min ischaemia (P < 0.01) and to 1.21 +/- 0.13 and 1.26 +/- 0.25 after 30 min reperfusion. Rates of ADP-stimulated (State 3) and uncoupled 2-oxoglutarate and succinate oxidation increased in parallel with matrix volume until maximum rates were reached at volumes of 1.1 microl ml(-1) or greater. The mitoK(ATP) channel opener, diazoxide (50 microM), caused a similar increase in matrix volume, but with inhibition rather than activation of succinate and 2-oxoglutarate oxidation. Direct addition of diazoxide (50 microM) to isolated mitochondria also inhibited State 3 succinate and 2-oxoglutarate oxidation by 30 %, but not that of palmitoyl carnitine. Unexpectedly, treatment of hearts with the mitoK(ATP) channel blocker 5-hydroxydecanoate (5HD) at 100 or 300 microM, also increased mitochondrial volume and inhibited respiration. In isolated mitochondria, 5HD was rapidly converted to 5HD-CoA by mitochondrial fatty acyl CoA synthetase and acted as a weak substrate or inhibitor of respiration depending on the conditions employed. These data highlight the dangers of using 5HD and diazoxide as specific modulators of mitoK(ATP) channels in the heart.

Increase in cytosolic Ca2+ levels through the activation of non-selective cation channels induced by oxidative stress causes mitochondrial depolarization leading to apoptosis-like death in Leishmania donovani promastigotes.

Reactive oxygen species are important regulators of protozoal infection. Promastigotes of Leishmania donovani, the causative agent of Kala-azar, undergo an apoptosis-like death upon exposure to H2O2. The present study shows that upon activation of death response by H2O2, a dose- and time-dependent loss of mitochondrial membrane potential occurs. This loss is accompanied by a depletion of cellular glutathione, but cardiolipin content or thiol oxidation status remains unchanged. ATP levels are reduced within the first 60 min of exposure as a result of mitochondrial membrane potential loss. A tight link exists between changes in cytosolic Ca2+ homeostasis and collapse of the mitochondrial membrane potential, but the dissipation of the potential is independent of elevation of cytosolic Na+ and mitochondrial Ca2+. Partial inhibition of cytosolic Ca2+ increase achieved by chelating extracellular or intracellular Ca2+ by the use of appropriate agents resulted in significant rescue of the fall of the mitochondrial membrane potential and apoptosis-like death. It is further demonstrated that the increase in cytosolic Ca2+ is an additive result of release of Ca2+ from intracellular stores as well as by influx of extracellular Ca2+ through flufenamic acid-sensitive non-selective cation channels; contribution of the latter was larger. Mitochondrial changes do not involve opening of the mitochondrial transition pore as cyclosporin A is unable to prevent mitochondrial membrane potential loss. An antioxidant like N-acetylcysteine is able to inhibit the fall of the mitochondrial membrane potential and prevent apoptosis-like death. Together, these findings show the importance of non-selective cation channels in regulating the response of L. donovani promastigotes to oxidative stress that triggers downstream signaling cascades leading to apoptosis-like death.