Mei-Lin Wu

Activation of the transient receptor potential M2 channel and poly(ADP-ribose) polymerase is involved in oxidative stress-induced cardiomyocyte death

Overproduction of reactive oxygen species is one of the major causes of cell death in ischemic–reperfusion (I/R) injury. In I/R animal models, electron microscopy (EM) has shown mixed apoptotic and necrotic characteristics in the same cardiomyocyte. The present study shows that H2O2 activates both apoptotic and necrotic machineries in the same myocyte and that the ultrastructure seen using EM is very similar to that in I/R animal studies. The apoptotic component is caused by the activation of clotrimazole-sensitive, NAD+/ADP ribose/poly(ADP-ribose) polymerase (PARP)-dependent transient receptor potential M2 (TRPM2) channels, which induces mitochondrial [Na+]m (and [Ca2+]m) overload, resulting in mitochondrial membrane disruption, cytochrome c release, and caspase 3-dependent chromatin condensation/fragmentation. The necrotic component is caspase 3-independent and is caused by PARP-induced [ATP]i/NAD+ depletion, resulting in membrane permeabilization. Inhibition of either TRPM2 or PARP activity only partially inhibits cell death, while inhibition of both completely prevents the ultrastructural changes and myocyte death.

Mechanism of Hydrogen Peroxide and Hydroxyl Free Radical–Induced Intracellular Acidification in Cultured Rat Cardiac Myoblasts

After a transient ischemic attack of the cardiac vascular system, reactive oxygen-derived free radicals, including the superoxide (O2-.) and hydroxyl (.OH) radicals can be easily produced during reperfusion. These free radicals have been suggested to be responsible for reperfusion-induced cardiac stunning and reperfusion-induced arrhythmia. Hydrogen peroxide (H2O2) is often used as an experimental source of oxygen-derived free radicals. Using freshly dissociated single rat cardiac myocytes and the rat cardiac myoblast cell line, H9c2, we have shown, for the first time, that an intriguing pHiota acidification (approximately 0.24 pH unit) is induced by the addition of 100 micromol/L H2O2 and that this dose is without effect on the intracellular free Ca2+ levels or viability of the cells. Using H9c2 as a model cardiac cell, we have shown that it is the intracellular production of .OH, and not O2-. or H2O2, that results in this acidification. We have excluded any involvement of (1) the three known cardiac pHi regulators (the Na+-H+ exchanger, the Cl--HCO3 exchanger, and the Na+-HCO3 co-transporter), (2) a rise in intracellular Ca2+ levels, and (3) inhibition of oxidative phosphorylation. However, we have found that H2O2-induced acidosis is due to inhibition of the glycolytic pathway, with hydrolysis of intracellular ATP and the resultant intracellular acidification. In cardiac muscle and in skinned cardiac muscle fiber, it has been shown that a small intracellular acidification may severely inhibit contractility. Therefore, the sustained pHi decrease caused by hydroxyl radicals may contribute, in some part, to the well-documented impairment of cardiac mechanical function (ie, reperfusion cardiac stunning) seen during reperfusion ischemia.

Arachidonic acid induces both Na+ and Ca2+ entry resulting in apoptosis

Marked accumulation of arachidonic acid (AA) and intracellular Ca2+ and Na+ overloads are seen during brain ischemia. In this study, we show that, in neurons, AA induces cytosolic Na+ ([Na+]cyt) and Ca2+ ([Ca2+]cyt) overload via a non‐selective cation conductance (NSCC), resulting in mitochondrial [Na+]m and [Ca2+]m overload. Another two types of free fatty acids, including oleic acid and eicosapentaenoic acid, induced a smaller increase in the [Ca2+]i and [Na+]i. RU360, a selective inhibitor of the mitochondrial Ca2+ uniporter, inhibited the AA‐induced [Ca2+]m and [Na+]m overload, but not the [Ca2+]cyt and [Na+]cyt overload. The [Na+]m overload was also markedly inhibited by either Ca2+‐free medium or CGP3715, a selective inhibitor of the mitochondrial Na+cyt‐Ca2+m exchanger. Moreover, RU360, Ca2+‐free medium, Na+‐free medium, or cyclosporin A (CsA) largely prevented AA‐induced opening of the mitochondrial permeability transition pore, cytochrome c release, and caspase 3‐dependent neuronal apoptosis. Importantly, Na+‐ionophore/Ca2+‐free medium, which induced [Na+]m overload, but not [Ca2+]m overload, also caused cyclosporin A‐sensitive mitochondrial permeability transition pore opening, resulting in caspase 3‐dependent apoptosis, indicating that [Na+]m overload per se induced apoptosis. Our results therefore suggest that AA‐induced [Na+]m overload, acting via activation of the NSCC, is an important upstream signal in the mitochondrial‐mediated apoptotic pathway. The NSCC may therefore act as a potential neuronal death pore which is activated by AA accumulation under pathological conditions.

The modulatory effects of endothelin-1, carbachol and isoprenaline upon Na(+)-H+ exchange in dog cardiac Purkinje fibres.

1. The modulatory effects of carbachol, endothelin‐1 and isoprenaline upon Na(+)‐H+ exchange were examined in dog cardiac Purkinje fibres. Intracellular pH (pHi) and intracellular sodium activity (aiNa) were recorded using pH and Na(+)‐selective microelectrodes. Acid extrusion via Na(+)‐H+ exchange was estimated from the pHi recovery rate (multiplied by intrinsic buffering power (beta i) and adding mean background acid load) in response to an internal acid load induced by the removal of 20 mM NH4Cl. All experiments in this work were performed in Hepes‐buffered solutions at 37 degrees C. 2. beta i was estimated at various values of pHi in the range of 7.4‐6.4 and was calculated from the fall of pHi induced by the addition and removal of NH4Cl. Experiments were performed when Na(+)‐H+ exchange was blocked. The values of beta i in this tissue were only slightly dependent on pHi in the range of 7.4‐6.4 with an empirical relationship: beta i = ‐4.69 pHi + 64.59. 3. Endothelin‐1 (10(‐8) M) alkalinized the resting pHi by approximately 0.1 pH unit and accelerated acid extrusion, by approximately 96%, at pHi approximately 6.9. A reduction of background acid loading within the cell cannot account for the augmentation of pHi recovery, since the rate of acid extrusion was not changed either at resting pHi or at internal acidification in Na(+)‐free solution (a measure of background loading) by the addition of endothelin‐1. The protein kinase C inhibitors staurosporin (10(‐6) M) and 1‐(5‐isoquinolinylsulphonyl)‐2‐methyl‐piperazine (H‐7, 50 microM) could not block the effect of endothelin‐1 on the antiporter. 4. At pHi approximately 6.8, carbachol (7.5 x 10(‐4) M) accelerated pHi recovery by approximately 68% and alkalinized the resting pHi by approximately 0.1 pH unit. This stimulatory effect of carbachol was completely blocked by pretreatment with atropine (10(‐4) M) and staurosporine 10(‐6) M. The background acid load was not reduced by adding carbachol, since the acid extrusion during pHi recovery or at the resting state was not affected by the addition of carbachol to a sodium‐free solution. 5. Isoprenaline (10(‐6) M) slowed pHi recovery by approximately 45% measured at pHi 6.9 with no change in resting pHi. A rise in background acid loading could not account for the reduction of acid extrusion. Pretreated with atenolol (10(‐6) M), a beta 1‐selective antagonist, completely blocked the effect of isoprenaline.(ABSTRACT TRUNCATED AT 400 WORDS)

Free fatty acids act as endogenous ionophores, resulting in Na+ and Ca2+ influx and myocyte apoptosis

AIMS Disturbances in lipid metabolism have been suggested to play an important role in myocardial damage. Marked accumulation of free fatty acids (FFAs), including arachidonic acid (AA), palmitic acid, oleic acid, and linoleic acid, occurs during post-ischaemia and reperfusion (post-I/R). Possible cellular mechanisms of AA/FFAs-induced myocyte apoptosis were investigated. METHODS AND RESULTS In neonatal rat ventricular myocytes, AA/FFAs activate a novel non-selective cation conductance (NSCC), resulting in both intracellular Ca(2+) and Na(+) overload. AA caused sustained cytosolic [Na(+)](cyt) and [Ca(2+)](cyt) overload, resulting in mitochondrial [Na(+)](m) and [Ca(2+)](m) overload, which induced caspase-3-mediated apoptosis. Similar apoptotic effects were seen using Na(+) ionophore cocktail/Ca(2+)-free medium, which induced [Na(+)](cyt) and [Na(+)](m), but not [Ca(2+)](cyt) and [Ca(2+)](m) overload. Electron microscopy showed that inhibition of [Na(+)](m) overload prevented disruption of the mitochondrial membrane, showing that [Na(+)](m) overload is an important upstream signal in AA- and FFA-induced myocyte apoptosis. CONCLUSION AA and FFAs, which accumulate in the myocardium during post-I/R, may therefore act as naturally occurring endogenous ionophores and contribute to the myocyte death seen during post-I/R.

Early metabolic inhibition-induced intracellular sodium and calcium increase in rat cerebellar granule cells

1 Possible mechanisms responsible for the increases in intracellular calcium ([Ca2+]i) and sodium ([Na+]i) levels seen during metabolic inhibition were investigated by continuous [Ca2+]i and [Na+]i measurement in cultured rat cerebellar granule cells. An initial small mitochondrial Ca2+ release was seen, followed by a large influx of extracellular Ca2+. A large influx of extracellular Na+ was also seen. 2 The large [Ca2+]i increase was not due to opening of voltage‐dependent or voltage‐independent calcium channels, activation of NMDA/non‐NMDA channels, activation of the Na+i‐Ca2+o exchanger, or inability of plasmalemmal Ca2+‐ATPase to extrude, or mitochondria to take up, calcium. 3 The large [Na+]i increase was not due to activation of the TTX‐sensitive Na+ channel, the Na+i‐Ca2+o exchanger, the Na+‐H+ exchanger, or the Na+‐K+‐2Cl− cotransporter, or an inability of Na+‐K+‐ATPase to extrude the intracellular sodium. 4 Phospholipase A2 (PLA2) activation may be involved in the large influx, since both were completely inhibited by PLA2 inhibitors. Moreover, melittin (a PLA2 activator) or lysophosphatidylcholine or arachidonic acid (both PLA2 activation products) caused similar responses. Inhibition of PLA2 activity may help prevent the influx of these ions that may result in serious brain injury and oedema during hypoxia/ischaemia.

Intracellular zinc release-activated ERK-dependent GSK-3β–p53 and Noxa–Mcl-1 signaling are both involved in cardiac ischemic-reperfusion injury.

Oxidative stress and nitrosative stress are both suggested to be involved in cardiac ischemia-reperfusion (I/R) injury. Using time-lapse confocal microscopy of cardiomyocytes and high-affinity O2−• and Zn2+ probes, this study is the first to show that I/R, reactive oxygen species (ROS), and reactive nitrogen species (RNS) all cause a marked increase in the [O2−•]i, resulting in cytosolic and mitochondrial Zn2+ release. Exposure to a cell-penetrating, high-affinity Zn2+i chelator, TPEN, largely abolished the Zn2+i release and markedly protected myocytes from I/R-, ROS-, RNS-, or Zn2+/K+ (Zn2+i supplementation)-induced myocyte apoptosis for at least 24 h after TPEN removal. Flavonoids and U0126 (a MEK1/2 inhibitor) largely inhibited the myocyte apoptosis and the TPEN-sensitive I/R- or Zn2+i supplement-induced persistent extracellular signal-regulated kinase 1 and 2 (ERK1/2) phosphorylation, dephosphorylation of p-Ser9 on glycogen synthase kinase 3β (GSK-3β), and the translocation into and accumulation of p-Tyr216 GSK-3β and p53 in, the nucleus. Silencing of GSK-3β or p53 expression was cardioprotective, indicating that activation of the ERK–GSK-3β–p53 signaling pathway is involved in Zn2+-sensitive myocyte death. Moreover, the ERK-dependent Noxa–myeloid cell leukemia-1 (Mcl-1) pathway is also involved, as silencing of Noxa expression was cardioprotective and U0126 abolished both the increase in Noxa expression and in Mcl-1 degradation. Thus, acute upstream Zn2+i chelation at the start of reperfusion and the use of natural products, that is, flavonoids, may be beneficial in the treatment of cardiac I/R injury.

Mitochondrial Na+ overload is caused by oxidative stress and leads to activation of the caspase 3- dependent apoptotic machinery

Oxidative stress is one of the major causes of cell death. Using time‐lapse confocal recording of live cardiomyocytes, we showed that H2O2 (OH•) caused a marked increase in Na+ and Ca2+ levels in both the cytosol ([Na]cyt, [Ca]cyt) and mitochondria ([Na]m, [Ca]m). The H2O2‐induced intracellular Na+ ([Na]i) overload contributed to the H2O2‐induced [Ca]cyt/[Ca]m overload via activation of the reverse mode of the Na‐Ca exchanger. When myocytes were treated for 40 min with 100 µM H2O2 in normal medium, then returned to H2O2‐free medium, the percentage of apoptotic cells increased from 4% at 0 h to 55 and 85% at 4.5 and 16 h, respectively. H2O2‐induced apoptosis was completely prevented by using Na‐free, but not Ca‐free, medium. When a Na+ ionophore cocktail in Ca‐free medium was used instead of H2O2 to increase the [Na]i by more than 30 mM without any change in the [Ca]i, cytochrome c release and caspase 3‐dependent apoptosis occurred, showing that [Na]i overload per se induced apoptosis. We also showed that the increase in the mitochondrial, but not the cytosolic, Na+ levels resulted in the opening of the permeation transition pore, followed by cytochrome c release. Our findings therefore suggest that H2O2‐induced [Na]m overload is an important upstream signal for the apoptotic machinery, and the prevention of [Na]m overload thus represents a particularly attractive target for strategies aimed at preventing oxidative stress‐induced cell death.

Regulation of acetylcholine release by intracellular acidification of developing motoneurons in Xenopus cell cultures

1 The effects of intracellular pH changes on the acetylcholine (ACh) release and cytoplasmic Ca2+ concentration at developing neuromuscular synapses were studied in Xenopus nerve‐muscle co‐cultures. 2 Spontaneous and evoked ACh release of motoneurons was monitored by using whole‐cell voltage‐clamped myocytes. Intracellular alkalinization with 15 mm NH4Cl slightly reduced the frequency of spontaneous synaptic currents (SSCs). However, cytosolic acidification following withdrawal of extracellular NH4Cl caused a marked and transient increase in spontaneous ACh release. 3 Another method of cytosolic acidification was used in which NaCl in Ringer solution was replaced with weak organic acids. The increase in spontaneous ACh release paralleled the level of intracellular acidification resulting from addition of these organic acids. Acetate and propionate but not isethionate, methylsulphate and glucuronate, caused an increase in intracellular pH and a marked increase in spontaneous ACh release. 4 Impulse‐evoked ACh release was slightly augmented by intracellular alkalinization and inhibited by cytosolic acidification. 5 Cytosolic acidification was accompanied by an elevation in the cytoplasmic Ca2+ concentration ([Ca2+]i), resulting from both external Ca2+ influx and intracellular Ca2+ mobilization. In contrast, the increase in [Ca2+]i induced by high K+ was inhibited by cytosolic acidification. 6 We conclude that cytosolic acidification regulates spontaneous and evoked ACh release differentially in Xenopus motoneurons, increasing spontaneous ACh release but inhibiting evoked ACh release.

Arachidonic acid-induced H+ and Ca2+ increases in both the cytoplasm and nucleoplasm of rat cerebellar granule cells

1 Arachidonic acid (AA) exerts multiple physiological and pathophysiological effects in the brain. By continuously measuring the intracellular pH (pHi) and Ca2+ levels ([Ca2+]i) in primary cultured rat cerebellar granule cells, we have found, for the first time, that 20 min treatment with 10 μm AA resulted in marked increases in Ca2+ and H+ levels in both the cytosol and nucleus. 2 A much higher concentration (40 mm) of another weak acid, propionic acid, was needed to induce a similar change in pHi. The [Ca2+]i increase was probably caused by AA‐induced activation of Ni2+‐sensitive cationic channels, but did not involve NMDA channels or the Na+‐Ca2+ exchanger. 3 AA‐induced acidosis occurs by a different mechanism involving predominantly the passive diffusion of the un‐ionized form of AA, rather than a protein carrier, as proposed by Kamp & Hamilton for fatty acids (FAs) in artificial phospholipid bilayers (the ‘flip‐flop’ model). The following results, which are similar to those observed in lipid bilayers, support this conclusion: (1) FAs containing a ‐COOH group (AA, linoleic acid, α‐linolenic acid, and docosahexaenoic acid) induced intracellular acidosis, whereas a FA with a ‐COOCH3 group (AA methyl ester) had little effect on pHi, (2) a FA amine, tetradecylamine, induced intracellular alkalosis, and (3) the AA‐/FA‐induced pHi changes were reversed by bovine serum albumin. 4 Further evidence in support of a passive diffusion model, rather than a membrane protein carrier, is that: (1) there was a linear relationship between the initial rate of acid flux and the concentration of AA (2‐100 μm), (2) acidosis was not inhibited by 4,4′‐diisothiocyanatostilbene‐2,2′‐disulphonic acid, a potent inhibitor of the plasma membrane FA carrier protein, and (3) the involvement of most known H+‐related membrane carriers and H+ conductance has been ruled out. 5 Since AA can be released under both physiological and pathophysiological conditions, the possible significance of the AA‐evoked increases in H+ and Ca2+ in both the cytoplasm and nucleoplasm is discussed.