Schisandrin B elicits a glutathione antioxidant response and protects against apoptosis via the redox-sensitive ERK/Nrf2 pathway in H9c2 cells

This study investigated the signal transduction pathway involved in the cytoprotective action of (-)schisandrin B [(-)Sch B, a stereoisomer of Sch B]. Using H9c2 cells, the authors examined the effects of (-)Sch B on MAPK and Nrf2 activation, as well as the subsequent eliciting of glutathione response and protection against apoptosis. Pharmacological tools, such as cytochrome P-450 (CYP) inhibitor, antioxidant, MAPK inhibitor, and Nrf2 RNAi, were used to delineate the signaling pathway. (-)Sch B caused a time-dependent activation of MAPK in H9c2 cells, with the degree of ERK activation being much larger than that of p38 or JNK. The MAPK activation was followed by an increase in the level of nuclear Nrf2, an indirect measure of Nrf2 activation, and the eliciting of a glutathione antioxidant response. The activation of MAPK and Nrf2 seemed to involve oxidants generated from a CYP-catalyzed reaction with (-)Sch B. Both ERK inhibition by U0126 and Nrf2 suppression by Nrf2 RNAi transfection largely abolished the cytoprotection against hypoxia/reoxygenation-induced apoptosis in (-)Sch B-pretreated cells. (-)Sch B pretreatment potentiated the reoxygenation-induced ERK activation, whereas both p38 and JNK activations were suppressed. Under the condition of ERK inhibition, Sch B treatment did not protect against ischemia/reperfusion injury in an ex vivo rat heart model. The results indicate that (-)Sch B triggers a redox-sensitive ERK/Nrf2 signaling, which then elicits a cellular glutathione antioxidant response and protects against hypoxia/reoxygenation-induced apoptosis in H9c2 cells. The ERK-mediated signaling is also likely involved in the cardioprotection afforded by Sch B in vivo.     

Suppression of rat Frizzled-2 attenuates hypoxia/reoxygenation-induced Ca2+ accumulation in rat H9c2 cells

Growing evidence suggests that Ca(2+) overload is one of the major contributors of myocardial ischemia/reperfusion-induced injury. Since Frizzled-2 receptor, a seven transmembrane protein, transduces downstream signaling by specialized binding of Wnt5a to increase intracellular Ca(2+) release, this work aimed to investigate the effect of Frizzled-2 on Ca(2+) accumulation in H9c2 cells, which were subjected to hypoxia/reoxygenation to mimic myocardial ischemia/reperfusion. After exposing H9c2 cells to hypoxia/reoxygenation, we observed higher expression of Frizzled-2 and Wnt5a as compared to control group cells. Hypoxia/reoxygenation-induced intracellular Ca(2+) accumulation approached that of cells transfected with frizzled-2 plasmid. In cells treated with RNAi specifically designed against frizzled-2, intracellular Ca(2+) in both hypoxia/reoxygenation-treated cells and plasmid-treated cells were decreased. Rats that underwent ischemia/reperfusion injury exhibited increased intracellular Ca(2+) with high expression levels of Frizzled-2 and Wnt5a as compared to the sham group. Our data indicates that upon binding to Wnt5a, increased Frizzled-2 expression after hypoxia/reoxygenation treatment activated intracellular calcium release in H9c2 cells. Our findings provide a new perspective in understanding calcium overload in myocardial ischemia/reperfusion.     

Mitochondrial permeability transition in rat hepatocytes after anoxia/reoxygenation: role of Ca2+-dependent mitochondrial formation of reactive oxygen species

Onset of the mitochondrial permeability transition (MPT) is the penultimate event leading to lethal cellular ischemia-reperfusion injury, but the mechanisms precipitating the MPT after reperfusion remain unclear. Here, we investigated the role of mitochondrial free Ca(2+) and reactive oxygen species (ROS) in pH- and MPT-dependent reperfusion injury to hepatocytes. Cultured rat hepatocytes were incubated in anoxic Krebs-Ringer-HEPES buffer at pH 6.2 for 4 h and then reoxygenated at pH 7.4 to simulate ischemia-reperfusion. Some cells were loaded with the Ca(2+) chelators, BAPTA/AM and 2-[(2-bis-[carboxymethyl]aono-5-methoxyphenyl)-methyl-6-methoxy-8-bis[carboxymethyl]aminoquinoline, either by a cold loading protocol for intramitochondrial loading or by warm incubation for cytosolic loading. Cell death was assessed by propidium iodide fluorometry and immunoblotting. Mitochondrial Ca(2+), inner membrane permeability, membrane potential, and ROS formation were monitored with Rhod-2, calcein, tetramethylrhodamine methylester, and dihydrodichlorofluorescein, respectively. Necrotic cell death increased after reoxygenation. Necrosis was blocked by 1 μM cyclosporin A, an MPT inhibitor, and by reoxygenation at pH 6.2. Confocal imaging of Rhod-2, calcein, and dichlorofluorescein revealed that an increase of mitochondrial Ca(2+) and ROS preceded onset of the MPT after reoxygenation. Intramitochondrial Ca(2+) chelation, but not cytosolic Ca(2+) chelation, prevented ROS formation and subsequent necrotic and apoptotic cell death. Reoxygenation with the antioxidants, desferal or diphenylphenylenediamine, also suppressed MPT-mediated cell death. However, inhibition of cytosolic ROS by apocynin or diphenyleneiodonium chloride failed to prevent reoxygenation-induced cell death. In conclusion, Ca(2+)-dependent mitochondrial ROS formation is the molecular signal culminating in onset of the MPT after reoxygenation of anoxic hepatocytes, leading to cell death.     

Schisandrin B enhances the glutathione redox cycling and protects against oxidant injury in different types of cultured cells

Tert-butylhydroperoxide (tBHP) challenge caused an initial depletion of cellular reduced glutathione (GSH), which was followed by a gradual restoration of cellular GSH in AML12, H9c2, and differentiated PC12 cells. The time-dependent changes in cellular GSH induced by tBHP were monitored as a measure of GSH recovery capacity (GRC), of which glutathione reductase (GR)-mediated glutathione redox cycling and γ-glutamate cysteine ligase (GCL)-mediated GSH synthesis were found to play an essential role. While glutathione redox cycling sustained the GSH level during the initial tBHP-induced depletion, GSH synthesis restores the GSH level thereafter. The effects of (-)schisandrin B [(-)Sch B] and its analogs (Sch A and Sch C) on GRC were also examined in the cells. (-)Sch B and Sch C, but not Sch A, ameliorated the extent of tBHP-induced GSH depletion, indicative of enhanced glutathione redox cycling. However, the degree of restoration of GSH post-tBHP challenge was not affected or even decreased. Pretreatment with (-)Sch B and Sch C, but not Sch A, protected against oxidant injury in the cells. The (-)Sch B afforded cytoprotection was abolished by N,N'-bis(chloroethyl)-N-nitrosourea pretreatment suggesting the enhancement of glutathione redox cycling is crucially involved in the cytoprotection afforded by (-)Sch B against oxidative stress-induced cell injury.     

Effect of extracellular Mg(2+) on ROS and Ca(2+) accumulation during reoxygenation of rat cardiomyocytes

The effects of Mg(2+) on reactive oxygen species (ROS) and cell Ca(2+) during reoxygenation of hypoxic rat cardiomyocytes were studied. Oxidation of 2',7'-dichlorodihydrofluorescein (DCDHF) to dichlorofluorescein (DCF) and of dihydroethidium (DHE) to ethidium (ETH) within cells were used as markers for intracellular ROS levels and were determined by flow cytometry. DCDHF/DCF is sensitive to H(2)O(2) and nitric oxide (NO), and DHE/ETH is sensitive to the superoxide anion (O(2)(-).), respectively. Rapidly exchangeable cell Ca(2+) was determined by (45)Ca(2+) uptake. Cells were exposed to hypoxia for 1 h and reoxygenation for 2 h. ROS levels, determined as DCF fluorescence, were increased 100-130% during reoxygenation alone and further increased 60% by increasing extracellular Mg(2+) concentration to 5 mM at reoxygenation. ROS levels, measured as ETH fluorescence, were increased 16-24% during reoxygenation but were not affected by Mg(2+). Cell Ca(2+) increased three- to fourfold during reoxygenation. This increase was reduced 40% by 5 mM Mg(2+), 57% by 10 microM 3,4-dichlorobenzamil (DCB) (inhibitor of Na(+)/Ca(2+) exchange), and 75% by combining Mg(2+) and DCB. H(2)O(2) (25 and 500 microM) reduced Ca(2+) accumulation by 38 and 43%, respectively, whereas the NO donor S-nitroso-N-acetyl-penicillamine (1 mM) had no effect. Mg(2+) reduced hypoxia/reoxygenation-induced lactate dehydrogenase (LDH) release by 90%. In conclusion, elevation of extracellular Mg(2+) to 5 mM increased the fluorescence of the H(2)O(2)/NO-sensitive probe DCF without increasing that of the O(2)(-).-sensitive probe ETH, reduced Ca(2+) accumulation, and decreased LDH release during reoxygenation of hypoxic cardiomyocytes. The reduction in LDH release, reflecting the protective effect of Mg(2+), may be linked to the effect of Mg(2+) on Ca(2+) accumulation and/or ROS levels.     

Schisandrin B-induced increase in cellular glutathione level and protection against oxidant injury are mediated by the enhancement of glutathione synthesis and regeneration in AML12 and H9c2 cells

To define the relative role of reduced glutathione (GSH) synthesis and regeneration in schisandrin B (Sch B)-induced increase in cellular GSH level and the associated cytoprotection against oxidative challenge, the effects of L-buthionine-[S,R]-sulfoximine (BSO, a specific inhibitor of gamma-glutamate cysteine ligase (GCL)) and 1,3-bis(2-chloroethyl)-1-nitrourea (BCNU, a specific inhibitor of glutathione reductase (GR)) treatments or their combined treatment were examined in control and Sch B-treated AML12 and H9c2 cells, without and/or with menadione intoxication. Both BSO and BCNU treatments reduced cellular GSH level in AML12 and H9c2 cells, with the effect of BSO being more prominent. The GSH-enhancing effect of Sch B was also suppressed by BSO and BCNU treatments, with the effect of the combined treatment with BSO and BCNU being semi-additive. While Sch B treatment increased the GR but not GCL activity in AML12 and H9c2 cells, it increased the cellular cysteine level. BSO treatment also suppressed the Sch B-induced increase in GR activity. BSO or BCNU treatment per se did not cause any detectable cytotoxic effect, as assessed by lactate dehydrogenase leakage, but the combined treatment with BSO and BCNU was cytotoxic, particularly in H9c2 cells. The cytotoxic effect of BSO and BCNU became more apparent following the menadione challenge. The cytoprotection afforded by Sch B pretreatment was partly suppressed by BSO or BCNU treatment, or completely abrogated by the combined treatment with BSO and BCNU. In conclusion, the results indicate that the cytoprotective action of Sch B is causally related to the increase in cellular GSH level, which is likely mediated by the enhancement of GSH synthesis and regeneration.     

Calcium preconditioning inhibits mitochondrial permeability transition and apoptosis

We tested the hypothesis whether calcium preconditioning (CPC) reduces reoxygenation injury by inhibiting mitochondrial permeability transition (MPT). Cultured myocytes were preconditioned by a brief exposure to 1.5 mM calcium (CPC) and subjected to 3 h of anoxia followed by 2 h of reoxygenation (A-R). Myocytes were also treated with 0.2 microM/l cyclosporin A (CsA), an inhibitor of MPT, before A-R. A significant increase of viable cells and reduced lactate dehydrogenase release was observed both in CPC- and CsA-treated myocytes compared with the A-R group. Cytochrome c release was predominantly observed in the cytoplasm of myocytes in the A-R group in contrast with CPC- or CsA-treated groups, where it was restricted only to mitochondria. Similarly, the cell death by apoptosis was also markedly attenuated in these groups. Electron-dense Ca(2+) deposits in mitochondria were also less frequent. Atractyloside (20 microM/l), an adenine nucleotide translocase inhibitor, caused changes similar to those in the A-R group, suggesting a role of MPT in A-R injury. Protection by inhibition of MPT by CsA and CPC suggests that MPT plays an important role in reoxygenation/reperfusion injury. The data further suggest that preconditioning inhibits MPT by inhibiting Ca(2+) accumulation by mitochondria.     

Thiol protecting agents and antioxidants inhibit the mitochondrial permeability transition promoted by etoposide: implications in the prevention of etoposide-induced apoptosis

Etoposide (VP-16) is known to promote cell apoptosis either in cancer or in normal cells as a side effect. This fact is preceded by the induction of several mitochondrial events, including increase in Bax/Bcl-2 ratio followed by cytochrome c release and consequent activation of caspase-9 and -3, reduction of ATP levels, depolarization of membrane potential (DeltaPsi) and rupture of the outer membrane. These events are apoptotic factors essentially associated with the induction of the mitochondrial permeability transition (MPT). VP-16 has been shown to stimulate the Ca2+-dependent MPT induction similarly to prooxidants and to promote apoptosis by oxidative stress mechanisms, which is prevented by glutathione (GSH) and N-acetylcysteine (NAC). Therefore, the aim of this work was to study the effects of antioxidants and thiol protecting agents on MPT promoted by VP-16, attempting to identify the underlying mechanisms on VP-16-induced apoptosis. The increased sensitivity of isolated mitochondria to Ca2+-induced swelling, Ca2+ release, depolarization of DeltaPsi and uncoupling of respiration promoted by VP-16, which are prevented by cyclosporine A proving that VP-16 induces the MPT, are also efficiently prevented by ascorbate, the primary reductant of the phenoxyl radicals produced by VP-16. The thiol reagents GSH, dithiothreitol and N-ethylmaleimide, which have been reported to prevent the MPT induction, also protect this event promoted by VP-16. The inhibition of the VP-16-induced MPT by antioxidants agrees with the prevention of etoposide-induced apoptosis by GSH and NAC and suggests the generation of oxidant species as a potential mechanism underlying the MPT that may trigger the release of mitochondrial apoptogenic factors responsible for apoptotic cascade activation.     

(-)Schisandrin B ameliorates paraquat-induced oxidative stress by suppressing glutathione depletion and enhancing glutathione recovery in differentiated PC12 cells

Exposure to paraquat (PQ; N,N'-dimethyl-4-4'-bipyridium), a potent herbicide, can lead to neuronal cell death and increased risk of Parkinson's disease because of oxidative stress. In this study, we investigated the effect of (-)schisandrin B [(-)Sch B, a potent enantiomer of schisandrin B] on PQ-induced cell injury in differentiated pheochromocytoma cells (PC12). PQ treatment caused cell injury in PC12 cells, as indicated by the significant increase in lactate dehydrogenase (LDH) leakage. Pretreatment with (-)Sch B (5 μM) protected against PQ-induced toxicity in PC12 cells, as evidenced by the significant decrease in LDH leakage. (-)Sch B induced the cytochrome P-450-mediated reactive oxygen species generation in differentiated PC12 cells. The cytoprotection afforded by (-)Sch B pretreatment was associated with an increase in cellular reduced glutathione (GSH) level as well as the enhancement of γ-glutamylcysteine ligase (GCL) and glutathione reductase (GR) activity in PQ-challenged cells. Both GCL and GR inhibitors abrogated the cytoprotective effect of (-)Sch B in PQ-challenged cells. The biochemical mechanism underlying the GSH-enhancing effect of (-)Sch B was further investigated in PC12 cells subjected to an acute peroxide challenge. Although the initial GSH depletion induced by peroxide was reduced through GR-catalyzed regeneration of GSH in (-)Sch B-pretreated cells, the later enhanced GSH recovery was mainly mediated by GCL-catalyzed GSH synthesis. The results suggest that (-)Sch B treatment may increase the resistance of dopaminergic cells against PQ-induced oxidative stress through reducing the extent of oxidant-induced GSH depletion and enhancing the subsequent GSH recovery.     

Hypoxia-induced alterations in Ca(2+) mobilization in brain microvascular endothelial cells

To investigate the possible cellular mechanisms of the ischemia-induced impairments of cerebral microcirculation, we investigated the effects of hypoxia/reoxygenation on the intracellular Ca(2+) concentration ([Ca(2+)](i)) in bovine brain microvascular endothelial cells (BBEC). In the cells kept in normal air, ATP elicited Ca(2+) oscillations in a concentration-dependent manner. When the cells were exposed to hypoxia for 6 h and subsequent reoxygenation for 45 min, the basal level of [Ca(2+)](i) was increased from 32.4 to 63.3 nM, and ATP did not induce Ca(2+) oscillations. Hypoxia/reoxygenation also inhibited capacitative Ca(2+) entry (CCE), which was evoked by thapsigargin (Delta[Ca(2+)](i-CCE): control, 62.3 +/- 3.1 nM; hypoxia/reoxygenation, 17.0 +/- 1.8 nM). The impairments of Ca(2+) oscillations and CCE, but not basal [Ca(2+)](i), were restored by superoxide dismutase and the inhibitors of mitochondrial electron transport, rotenone and thenoyltrifluoroacetone (TTFA). By using a superoxide anion (O(2)(-))-sensitive luciferin derivative MCLA, we confirmed that the production of O(2)(-) was induced by hypoxia/reoxygenation and was prevented by rotenone and TTFA. These results indicate that hypoxia/reoxygenation generates O(2)(-) at mitochondria and impairs some Ca(2+) mobilizing properties in BBEC.     

A mechanism of virulence: virulent Mycobacterium tuberculosis strain H37Rv, but not attenuated H37Ra, causes significant mitochondrial inner membrane disruption in macrophages leading to necrosis

Infection of human monocyte-derived macrophages with Mycobacterium tuberculosis at low multiplicities of infection leads 48-72 h after the infection to cell death with the characteristics of apoptosis or necrosis. Predominant induction of one or the other cell death modality depends on differences in mitochondrial membrane perturbation induced by attenuated and virulent strains. Infection of macrophages with the attenuated H37Ra or the virulent H37Rv causes mitochondrial outer membrane permeabilization characterized by cytochrome c release from the mitochondrial intermembrane space and apoptosis. Mitochondrial outer membrane permeabilization is transient, peaks 6 h after infection, and requires Ca(2+) flux and B cell chronic lymphocytic leukemia/lymphoma 2-associated protein X translocation into mitochondria. In contrast, only the virulent H37Rv induces significant mitochondrial transmembrane potential (Deltapsi(m)) loss caused by mitochondrial permeability transition. Dissipation of Deltapsi(m) also peaks at 6 h after infection, is transient, is inhibited by the classical mitochondrial permeability transition inhibitor cyclosporine A, has a requirement for mitochondrial Ca(2+) loading, and is independent of B cell chronic lymphocytic leukemia/lymphoma translocation into the mitochondria. Transient dissipation of Deltapsi(m) 6 h after infection is essential for the induction of macrophage necrosis by Mtb, a mechanism that allows further dissemination of the pathogen and development of the disease.     

Effect of hydrogen peroxide on reoxygenation-induced Ca2+ accumulation in rat cardiomyocytes

Reactive oxygen species (ROS) contribute to cell damage during reperfusion of the heart. ROS may exert their effects partly by interfering with Ca(2+) homeostasis of the myocardium. The purpose of this study was to investigate the effects of hydrogen peroxide (H(2)O(2)) on Ca(2+) accumulation during reoxygenation of isolated adult rat cardiomyocytes exposed to 1 h of hypoxia and to relate the effects to possible changes in release of lactate dehydrogenase (LDH), free intracellular Ca(2+) ([Ca(2+)](i)) and Mg(2+)([Mg(2+)](i)), and mitochondrial membrane potential (Deltapsim). Cell Ca(2+) was determined by (45)Ca(2+) uptake. Free [Mg(2+)](i) and [Ca(2+)](i) and Deltapsim were measured by flow cytometry. Reoxygenation-induced Ca(2+) accumulation was attenuated by 23 and 34% by 10 and 25 microM H(2)O(2), respectively, added at reoxygenation. H(2)O(2) at 100 and 250 microM increased cell Ca(2+) by 50 and 83%, respectively, whereas 500 microM H(2)O(2) decreased cell Ca(2+) by 20%. H(2)O(2) at (25 microM) reduced LDH release and [Mg(2+)](i) and increased Deltapsim, indicating cell protection, whereas 250 microM H(2)O(2) increased LDH release and [Mg(2+)](i) and decreased Deltapsim, indicating cell damage. Clonazepam (100 microM) attenuated the increase in Ca(2+) accumulation, the elevation of [Ca(2+)](i), and the decrease in Deltapsim induced by 100 and 250 microM H(2)O(2) during reoxygenation. We report for the first time that 25 microM H(2)O(2) attenuates Ca(2+) accumulation, LDH release, and dissipation of Deltapsim during reoxygenation of hypoxic cardiomyocytes, indicating cell protection.     

p21 WAF1 and hypoxia/reoxygenation-induced premature senescence of H9c2 cardiomyocytes

We have previously reported on hypoxia/reoxygenation-induced premature senescence in neonatal rat cardiomyocytes. In this research, we investigated the effects of p21(WAF1) (p21) in hypoxia/reoxygenation-induced senescence, using H9c2 cells. A plasmid overexpressing wild type p21(WAF1) and a plasmid expressing small hairpin RNA (shRNA) targeting p21(WAF1) were constructed, and transfected into H9c2 cells to control the p21 expression. Hypoxia/reoxygenation conditions were 1% O2 and 5% CO(2), balancing the incubator chamber with N(2) for 6 h (hypoxia 6 h), then 21% oxygen for 8 h (reoxygenation 8 h). Cell cycle was examined using flow cytometry. Senescence was assessed using β-galactosidase staining. The expression of p53, p21, p16(INK4a), and cyclin D1 was assayed using Western blotting. At hypoxia 6 h, cells overexpressing p21 had a larger G1 distribution, stronger β-galactosidase activity, and lower cyclin D1 expression compared to control cells, while the opposite results and higher p53 expression were obtained in p21-knockdown cells. At reoxygenation 8 h, p21-silenced cells had a smaller percentage of G1 cells, weaker β-galactosidase activity and lower 16(INK4a) expression, and higher cyclin D1 expression, but the overexpression group showed no difference. Taken together, this data implies that p21(WAF1) is important for the hypoxia phase, but not the reoxygenation phase, in the H9c2 senescence process.     

Neuroprotective MK801 is associated with nitric oxide synthase during hypoxia/reoxygenation in rat cortical cell cultures.

The neuroprotective effect of MK801 against hypoxia and/or reoxygenation-induced neuronal cell injury and its relationship to neuronal nitric oxide synthetase (nNOS) expression were examined in cultured rat cortical cells. Treatment of cortical neuronal cells with hypoxia (95% N(2)/5% CO(2)) for 2 h followed by reoxygenation for 24 h induced a release of lactate dehydrogenase (LDH) into the medium, and reduced the protein level of MAP-2 as well. MK801 attenuated the release of LDH and the reduction of the MAP-2 protein by hypoxia, suggesting a neuroprotective role of MK801. MK801 also diminished the number of nuclear condensation by hypoxia/reoxygenation. The NOS inhibitors 7-nitroindazole (7-NI) and N (G)-nitro-L-arginine methyl ester (L-NAME), as well as the Ca(2+) channel blocker nimodipine, reduced hypoxia-induced LDH, suggesting that nitric oxide (NO) and calcium homeostasis contribute to hypoxia and/or the reoxygenation-induced cell injury. The levels of nNOS immunoactivities and mRNA by RT-PCR were enhanced by hypoxia with time and, down regulated following 24 h reoxygenation after hypoxia, and were attenuated by MK801. In addition, the reduction of nNOS mRNA levels by hypoxia/reoxygenation was also diminished by MK801. Further delineation of the mechanisms of NO production and nNOS regulation are needed and may lead to additional strategies to protect neuronal cells against hypoxic/reoxygenation insults.     

Hypoxic postconditioning reduces cardiomyocyte loss by inhibiting ROS generation and intracellular Ca2+ overload

We have shown that intermittent interruption of immediate reflow at reperfusion (i.e., postconditioning) reduces infarct size in in vivo models after ischemia. Cardioprotection of postconditioning has been associated with attenuation of neutrophil-related events. However, it is unknown whether postconditioning before reoxygenation after hypoxia in cultured cardiomyocytes in the absence of neutrophils confers protection. This study tested the hypothesis that prevention of cardiomyocyte damage by hypoxic postconditioning (Postcon) is associated with a reduction in the generation of reactive oxygen species (ROS) and intracellular Ca(2+) overload. Primary cultured neonatal rat cardiomyocytes were exposed to 3 h of hypoxia followed by 6 h of reoxygenation. Cardiomyocytes were postconditioned after the 3-h index hypoxia by three cycles of 5 min of reoxygenation and 5 min of rehypoxia applied before 6 h of reoxygenation. Relative to sham control and hypoxia alone, the generation of ROS (increased lucigenin-enhanced chemiluminescence, SOD-inhibitable cytochrome c reduction, and generation of hydrogen peroxide) was significantly augmented after immediate reoxygenation as was the production of malondialdehyde, a product of lipid peroxidation. Concomitant with these changes, intracellular and mitochondrial Ca(2+) concentrations, which were detected by fluorescent fluo-4 AM and X-rhod-1 AM staining, respectively, were elevated. Cell viability assessed by propidium iodide staining was decreased consistent with increased levels of lactate dehydrogenase after reoxygenation. Postcon treatment at the onset of reoxygenation reduced ROS generation and malondialdehyde concentration in media and attenuated cardiomyocyte death assessed by propidium iodide and lactate dehydrogenase. Postcon treatment was associated with a decrease in intracellular and mitochondrial Ca(2+) concentrations. These data suggest that Postcon treatment reduces reoxygenation-induced injury in cardiomyocytes and is potentially mediated by attenuation of ROS generation, lipid peroxidation, and intracellular and mitochondrial Ca(2+) overload.     

N-n-Butyl haloperidol iodide inhibits the augmented Na+/Ca2+ exchanger currents and L-type Ca2+ current induced by hypoxia/reoxygenation or H2O2 in cardiomyocytes

N-n-butyl haloperidol iodide (F(2)), a novel quaternary ammonium salt derivative of haloperidol, was reported to antagonize myocardial ischemia/reperfusion injuries. To investigate its mechanisms, we characterized the effects of F(2) on Na(+)/Ca(2+) exchanger currents (I(NCX)) and the L-type Ca(2+) channel current (I(Ca,L)) of cardiomyocytes during either hypoxia/reoxygenation or exposure to H(2)O(2). Using whole-cell patch-clamp techniques, the I(NCX) and I(Ca,L) were recorded from isolated rat ventricular myocytes. Exposure of cardiomyocytes to hypoxia/reoxygenation or H(2)O(2) enhanced the amplitude of the inward and outward of I(NCX) and I(Ca,L). F(2) especially inhibited the outward current of Na(+)/Ca(2+) exchanger, as well as the I(Ca,L), in a concentration-dependent manner. F(2) inhibits cardiomyocyte I(NCX) and I(Ca,L) after exposure to hypoxia/reoxygenation or H(2)O(2) to antagonize myocardial ischemia/reperfusion injury by inhibiting Ca(2+) overload.     

Phenylarsine oxide induces mitochondrial permeability transition, hypercontracture, and cardiac cell death

The mitochondrial permeability transition (MPT) is implicated in cardiac reperfusion/reoxygenation injury. In isolated ventricular myocytes, the sulfhydryl (SH) group modifier and MPT inducer phenylarsine oxide (PAO) caused MPT, severe hypercontracture, and irreversible membrane injury associated with increased cytoplasmic free [Ca(2+)]. Removal of extracellular Ca(2+) or depletion of nonmitochondrial Ca(2+) pools did not prevent these effects, whereas the MPT inhibitor cyclosporin A was partially protective and the SH-reducing agent dithiothreitol fully protective. In permeabilized myocytes, PAO caused hypercontracture at much lower free [Ca(2+)] than in its absence. Thus PAO induced hypercontracture by both increasing myofibrillar Ca(2+) sensitivity and promoting mitochondrial Ca(2+) efflux during MPT. Hypercontracture did not directly cause irreversible membrane injury because lactate dehydrogenase (LDH) release was not prevented by abolishing hypercontracture with 2,3-butanedione monoxime. However, loading myocytes with the membrane-permeable Ca(2+) chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM) prevented PAO-induced LDH release, thus implicating the PAO-induced rise in cytoplasmic [Ca(2+)] as obligatory for irreversible membrane injury. In conclusion, PAO induces MPT and enhanced susceptibility to hypercontracture in isolated cardiac myocytes, both key features also implicated in cardiac reperfusion and reoxygenation injury.     

Measurements of mitochondrial pH in cultured cortical neurons clarify contribution of mitochondrial pore to the mechanism of glutamate-induced delayed Ca2+ deregulation

To clarify the role of the mitochondrial permeability transition pore (MPT) in the mechanism of the glutamate-induced delayed calcium deregulation (DCD) and mitochondrial depolarization (MD), we studied changes in cytosolic (pH(c)) and mitochondrial pH (pH(m)) induced by glutamate in cultured cortical neurons expressing pH-sensitive fluorescent proteins. We found that DCD and MD were associated with a prominent pH(m) decrease which presumably resulted from MPT opening. This pH(m) decrease occurred with some delay after the onset of DCD and MD. This argued against the hypothesis that MPT opening plays a dominant role in triggering of DCD. This conclusion was also supported by experiments in which Ca(2+) was replaced with antagonist of MPT opening Sr(2+). We found that in Sr(2+)-containing medium glutamate-induced delayed strontium deregulation (DSD), similar to DCD, which was accompanied by a profound MD. Analysis of the changes in pH(c) and pH(m) associated with DSD led us to conclude that MD in Sr(2+)-containing medium occurred without involvement of the pore. In contrast, in Ca(2+)-containing medium such "non-pore mechanism" was responsible only for MD initiation while in the final stages of MD development the MPT played a major role.     

Schisandrin B elicits a glutathione antioxidant response and protects against apoptosis via the redox-sensitive ERK/Nrf2 pathway in AML12 hepatocytes

Abstract This study examined the effects of (-)schisandrin B [(-)Sch B] on MAPK and Nrf2 activation and the subsequent induction of glutathione antioxidant response and cytoprotection against apoptosis in AML12 hepatocytes. Pharmacological tools, such as cytochrome P-450 (CYP) inhibitor, antioxidant, MAPK inhibitors and Nrf2 RNAi, were used to delineate the signalling pathway. (-)Sch B caused a time-dependent activation of MAPK in AML12 cells, particularly the ERK1/2. The MAPK activation was followed by an enhancement in Nrf2 nuclear translocation and the eliciting of a glutathione antioxidant response. Reactive oxygen species arising from a CYP-catalysed reaction with (-)Sch B seemed to be causally related to the activation of MAPK and Nrf2. ERK inhibition by U0126 or Nrf2 suppression by Nrf2 RNAi transfection almost completely abrogated the cytoprotection against menadione-induced apoptosis in (-)Sch B-pre-treated cells. (-)Sch B pre-treatment potentiated the menadione-induced ERK activation, whereas both p38 and JNK activations were suppressed. Under the condition of ERK inhibition, Sch B treatment did not protect against carbon tetrachloride-hepatotoxicity in an in vivo mouse model. In conclusion, (-)Sch B triggers a redox-sensitive ERK/Nrf2 signalling, which then elicits a cellular glutathione antioxidant response and protects against oxidant-induced apoptosis in AML12 cells.     

Inhibition of NHE protects reoxygenated cardiomyocytes independently of anoxic Ca(2+) overload and acidosis

We investigated the question of whether inhibition of the Na(+)/H(+) exchanger (NHE) during ischemia is protective due to reduction of cytosolic Ca(2+) accumulation or enhanced acidosis in cardiomyocytes. Additionally, the role of the Na(+)-HCO(3)(-) symporter (NBS) was investigated. Adult rat cardiomyocytes were exposed to simulated ischemia and reoxygenation. Cytosolic pH [2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)], Ca(2+) (fura 2), Na(+) [sodium-binding benzolfuran isophthatlate (SBFI)], and cell length were measured. NHE was inhibited with 3 micromol/l HOE 642 or 1 micromol/l 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), and NBS was inhibited with HEPES buffer. During anoxia in bicarbonate buffer, cells developed acidosis and intracellular Na and Ca (Na(i) and Ca(i), respectively) overload. During reoxygenation cells underwent hypercontracture (44.0 +/- 4.1% of the preanoxic length). During anoxia in bicarbonate buffer, inhibition of NHE had no effect on changes in intracellular pH (pH(i)), Na(i), and Ca(i), but it significantly reduced the reoxygenation-induced hypercontracture (HOE: 61.0 +/- 1.4%, EIPA: 68.2 +/- 1.8%). The sole inhibition of NBS during anoxia was not protective. We conclude that inhibition of NHE during anoxia protects cardiomyocytes against reoxygenation injury independently of cytosolic acidification and Ca(i) overload.