H2O2-induced changes in mitochondrial activity in isolated mouse pancreatic acinar cells (original) (raw)
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American Journal of Physiology-cell Physiology, 2008
and in particular the transition to an irreversible "Ca 2+ overload" response, has been implicated in various pathophysiological states. In some diseases, including pancreatitis, oxidative stress has been suggested to mediate this Ca 2+ overload and the associated cell injury. We have previously demonstrated that oxidative stress with hydrogen peroxide (H 2 O 2 ), evokes a Ca 2+ overload response and inhibition of plasma membrane Ca 2+ -ATPase (PMCA) in rat pancreatic acinar cells (Bruce JI and Elliott AC. Am J Physiol Cell Physiol 293: C938-950, 2007). The aim of the present study was to further examine this oxidant-impaired inhibition of the PMCA, focussing on the role of the mitochondria. Using a [Ca 2+ ] i clearance assay in which mitochondrial Ca 2+ uptake was blocked with Ru360, H 2 O 2 (50uM-1mM) markedly inhibited the PMCA activity. This H 2 O 2 -induced inhibition of the PMCA correlated with mitochondrial depolarisation (assessed using tetramethylrhodamine methylester fluorescence) but could occur without significant ATP depletion (assessed using magesium green fluorescence). The H 2 O 2 -induced PMCA inhibition was sensitive to the mitochondrial permeability transition pore (mPTP) inhibitors, cyclosporin-A and bongkrekic acid.
Journal of Membrane Biology, 2001
In the present study we have studied how [Ca 2+ ] i is influenced by H 2 O 2 in collagenase-dispersed mouse pancreatic acinar cells and the mechanism underlying this effect by using a digital microspectrofluorimetric system. In the presence of normal extracellular calcium concentration, perfusion of pancreatic acinar cells with 1 mM H 2 O 2 caused a slow sustained [Ca 2+ ] i increase, reaching a stable plateau after 10-15 min of perfusion. This increase induced by H 2 O 2 was also observed in a nominally calcium-free medium, reflecting the release of calcium from intracellular store(s). Application of 1 mM H 2 O 2 to acinar cells, in which nonmitochondrial agonist-releasable calcium pools had been previously depleted by a maximal concentration of CCK-8 (1 nM) or thapsigargin (0.5 M) was still able to induce calcium release. Similar results were observed when thapsigargin was substituted for the mitochondrial uncoupler FCCP (0.5 M). By contrast, simultaneous addition of thapsigargin and FCCP clearly abolished the H 2 O 2 -induced calcium increase. Interestingly, coincubation of intact pancreatic acinar cells with CCK-8 plus thapsigargin and FCCP in the presence of H 2 O 2 did not significantly affect the transient calcium spike induced by the depletion of nonmitochondrial and mitochondrial agonist-releasable calcium pools, but was followed by a sustained increase of [Ca 2+ ] i . In addition, H 2 O 2 was able to block calcium efflux evoked by CCK and thapsigargin. Finally, the transient increase in [Ca 2+ ] i induced by H 2 O 2 was abolished by an addition of 2 mM dithiothreitol (DTT), a sulfhydryl reducing agent. Our results show that H 2 O 2 releases calcium from CCK-8-and thapsigargin-sensitive intracellular stores and from mitochondria. The action of H 2 O 2 is likely medi-ated by oxidation of sulfhydryl groups of calcium-ATPases.
Cell Calcium, 1991
Isolated rat hepatocytes treated with mitochondrial inhibitors FCCP or antimycin A release discrete amounts of Ca2+ in a Ca2+-free extracellular medium as revealed by changes in the absorbance of the Ca2+ indicator arsenazo Ill. The process is completed in 2 min and the amount of Ca2' released is not affected by the type of the mitochondrial poison employed. The subsequent treatment with the cation ionophore A23187 causes a further release of Ca2' that does not appear related to the specificity of the previous treatment with FCCP or antimycin A. Both FCCP and antimycin A cause a progresslve loss of cellular ATP associated with a decrease in the ATP/ADP ratio from 6 to 2-1.6. However, this decrease does not significantly prevent 45Ca2t accumulation in isolated liver microsomes. Moreover, the decrease of the ATP/ADP ratio to I, does not promote a significant release of 45Ca2+ from %a2+-preloaded microsomes. Finally, experiments with Fura-P-loaded hepatocytes reveal that agents specifically releasing Ca2+ from non-mitochondrial stores (vasopressin and 2,5-di-fefibutyl-1-4-benzohydroquinone) are still able to increase the cytosolic Ca2+ concentration in FCCP-treated cells. Taken together, these findings demonstrate that, in freshly isolated hepatocytes, FCW s@eciflcally releases Ca2' from mitochondrial stores without significantly affecting active Ca + sequestration in other cellular pools. For these reasons, FCCP can be used to release and quantitate mitochondrial Ca2+ in liver cells. Abbreviations If'3: myo-inositol 1,4,%risphosphate; EGTA: ethylene The increasing recognition of the importance of glycol bis(@uninoethyl ether) N,N,N',N'-tetraacetic acid; Ca2' as a cell regulator has been accompanied by HEPES: 4-(2-hydroxyethyl)-l-piperazineethanesulfofonic the development of methods for measuring cellular acid; FCCF? carbonyl cyanide p-fluoromethoxyphenyl-levels and fluxes of Ca2'. To date levels of and hydrazone; Fura-2: l-[2-(Scarboxyoxazol-2-yl)-6-amino-fluctuations in, intracellular free 'Ca2+ can be benzo!kran-5-oxy]-2-(2'-amino-S-methylphenoxy)-ethane-selectively, sensitively and kinetically measured NN,N',N'-tetraacetic acid; Fur&YAM: Fura-2-acetoxy-with fluorescent Ca2' indicators [l-3]. However, methyl ester, MOPS: 3-(N-morpholino)propanesulfonic acid; tBuBHQ: 2,5-di-(ferf-butyl)-1,4-benzohydroquinone difplties still exist cuncfzrning the evaluation of Ca distribution among the various cellular 431
Mitochondria and Ca2+ in cell physiology and pathophysiology
Cell Calcium, 2000
There is now a consensus that mitochondria take up and accumulate Ca 2; during physiological [Ca 2; ] c signalling. This contribution will consider some of the functional consequences of mitochondrial Ca 2; uptake for cell physiology and pathophysiology. The ability to remove Ca 2; from local cytosol enables mitochondria to regulate the [Ca 2; ] in microdomains close to IP3-sensitive Ca 2; -release channels. The [Ca 2; ] sensitivity of these channels means that, by regulating local [Ca 2; ] c , mitochondrial Ca 2; uptake modulates the rate and extent of propagation of [Ca 2; ] c waves in a variety of cell types. The coincidence of mitochondrial Ca 2; uptake with oxidative stress may open the mitochondrial permeability transition pore (mPTP). This is a catastrophic event for the cell that will initiate pathways to cell death either by necrotic or apoptotic pathways. A model is presented in which illumination of an intramitochondrial fluorophore is used to generate oxygen radical species within mitochondria. This causes mitochondrial Ca 2; loading from SR and triggers mPTP opening. In cardiomyocytes, mPTP opening leads to ATP consumption by the mitochondrial ATPase and so results in ATP depletion, rigor and necrotic cell death. In central mammalian neurons exposed to glutamate, a cellular Ca 2; overload coincident with NO production also causes loss of mitochondrial potential and cell death, but mPTP involvement has proven more difficult to demonstrate unequivocally. A cartoon to illustrate the features of the mitochondrial permeability transition pore and its pharmacology. The mPTP seems to consist of an association between the adenine nucleotide translocase (ANT), the voltage dependent anion channel (VDAC) of the outer mitochondrial membrane and cyclophilin D (Cyp D). Opening of the pore is modulated primarily by [Ca 2; ] m , ROS, NO, many sulphydryl reagents and by bongrekate or atractyloside, amongst many agents and conditions (from Duchen, 2000, with permission).
1991
Isolated rat hepatocytes treated with mitochondrial inhibitors FCCP or antimycin A release discrete amounts of Ca2+ in a Ca2+-free extracellular medium as revealed by changes in the absorbance of the Ca2+ indicator arsenazo Ill. The process is completed in 2 min and the amount of Ca2' released is not affected by the type of the mitochondrial poison employed. The subsequent treatment with the cation ionophore A23187 causes a further release of Ca2' that does not appear related to the specificity of the previous treatment with FCCP or antimycin A. Both FCCP and antimycin A cause a progresslve loss of cellular ATP associated with a decrease in the ATP/ADP ratio from 6 to 2-1.6. However, this decrease does not significantly prevent 45Ca2t accumulation in isolated liver microsomes. Moreover, the decrease of the ATP/ADP ratio to I, does not promote a significant release of 45Ca2+ from %a2+-preloaded microsomes. Finally, experiments with Fura-P-loaded hepatocytes reveal that agents specifically releasing Ca2+ from non-mitochondrial stores (vasopressin and 2,5-di-fefibutyl-1-4-benzohydroquinone) are still able to increase the cytosolic Ca2+ concentration in FCCP-treated cells. Taken together, these findings demonstrate that, in freshly isolated hepatocytes, FCW s@eciflcally releases Ca2' from mitochondrial stores without significantly affecting active Ca + sequestration in other cellular pools. For these reasons, FCCP can be used to release and quantitate mitochondrial Ca2+ in liver cells. Abbreviations If'3: myo-inositol 1,4,%risphosphate; EGTA: ethylene The increasing recognition of the importance of glycol bis(@uninoethyl ether) N,N,N',N'-tetraacetic acid; Ca2' as a cell regulator has been accompanied by HEPES: 4-(2-hydroxyethyl)-l-piperazineethanesulfofonic the development of methods for measuring cellular acid; FCCF? carbonyl cyanide p-fluoromethoxyphenyl-levels and fluxes of Ca2'. To date levels of and hydrazone; Fura-2: l-[2-(Scarboxyoxazol-2-yl)-6-amino-fluctuations in, intracellular free 'Ca2+ can be benzo!kran-5-oxy]-2-(2'-amino-S-methylphenoxy)-ethane-selectively, sensitively and kinetically measured NN,N',N'-tetraacetic acid; Fur&YAM: Fura-2-acetoxy-with fluorescent Ca2' indicators [l-3]. However, methyl ester, MOPS: 3-(N-morpholino)propanesulfonic acid; tBuBHQ: 2,5-di-(ferf-butyl)-1,4-benzohydroquinone difplties still exist cuncfzrning the evaluation of Ca distribution among the various cellular 431
Diverse Effects of Hydrogen Peroxide on Cytosolic Ca2+ Homeostasis in Rat Pancreatic .BETA.-cells
Cell Structure and Function, 2000
Oxygen-free radicals are thought to be a major cause of b-cell dysfunction in diabetic animals induced by alloxan or streptozotocin. We evaluated the effect of H2O2 on cytosolic Ca 2+ concentration ([Ca 2+ ]i) and the activity of ATP-sensitive potassium (K + ATP) channels in isolated rat pancreatic b-cells using microfluorometry and patch clamp techniques. Exposure to 0.1 mM H2O2 in the presence of 2.8 mM glucose increased [Ca 2+ ]i from 114.3±15.4 nM to 531.1±71.9 nM (n=6) and also increased frequency of K + ATP channel openings. The intensity of NAD(P)H autofluorescence was conversely reduced, suggesting that H2O2 inhibited the cellular metabolism. These three types of cellular parameters were reversed to the control level on washout of H2O2, followed by a transient increase in [Ca 2+ ]i, the transient inhibition of K + ATP channels associated with action currents and increase of the NAD(P)H intensity with an overshoot. In the absence of external Ca 2+ , 0.1 mM H2O2 increased [Ca 2+ ]i from 88.8±7.2 nM to 134.6±8.3 nM. Magnitude of [Ca 2+ ]i increase induced by 0.1 mM H2O2 was decreased after treatment of cells with 0.5 mM thapsigargin, an inhibitor of endoplasmic reticulum Ca 2+ pump (45.8±4.9 nM vs 15.0±4.8 nM). Small increase in [Ca 2+ ]i in response to an increase of external Ca 2+ from zero to 2 mM was further facilitated by 0.1 mM H2O2 (330.5±122.7 nM). We concluded that H2O2 not only activates K + ATP channels in association with metabolic inhibition, but also increases partly the Ca 2+ permeability of the thapsigargin-sensitive intracellular stores and of the plasma membrane in pancreatic b-cells.
Ca2+-dependent and independent mitochondrial damage in hepatocellular injury
Cell Calcium, 1991
The alterations of mitochondrial membrane potential during the development of irreversible cell damage were investigated by measuring rhodamine-123 uptake and distribution in primary cultures as well as in suspensions of rat hepatocytes exposed to different toxic agents. Direct and indirect mechanisms of mitochondrial damage have been identified and a role for Ca2+ in the development of this type of injury by selected compounds was assessed by using extracellular as well as intracellular Ca2+ chelators. In addition, mitochondrial uncoupling by carbonylcyanide-m-chloro-phenylhydrazone (CCCP) resulted in a marked depletion of cellular ATP that was followed by an increase in cytosolic Ca2+ concentration, immediately preceding cell death. These results support the existence of a close relationship linking, in a sort of reverberating circuit, the occurrence of mitochondrial dysfunction and the alterations in cellular Ca2+ homeostasis during hepatocyte injury.
Simultaneous Measurements of Cytosolic and Mitochondrial Ca2+ Transients in HT29 Cells
Journal of Biological Chemistry, 1998
Loading of HT 29 cells with the Ca 2؉ dye fura-2/AM resulted in an nonhomogeneous intracellular distribution of the dye. Cellular compartments with high fura-2 concentrations were identified by correlation with mitochondrial markers, cellular autofluorescence induced by UV, and dynamic measurement of autofluorescence after inhibition of oxidative phosphorylation. Stimulation with carbachol (10 ؊4 mol/liter) increased cytosolic, nuclear, and mitochondrial Ca 2؉ activity ([Ca 2؉ ] c , [Ca 2؉ ] n , and [Ca 2؉ ] m , respectively) measured by UV confocal and conventional imaging. Similar results were obtained with a prototype two-photon microscope (Zeiss, Jena, Germany) allowing for fura-2 excitation. The increase of [Ca 2؉ ] m lagged behind that of [Ca 2؉ ] c and [Ca 2؉ ] n by 10-20 s, and after removing the agonist, [Ca 2؉ ] m also decreased with a delay. A strong increase of [Ca 2؉ ] m occurred only when a certain threshold of [Ca 2؉ ] c (around 1 mol/liter) was exceeded. In a very similar way, ATP, neurotensin, and thapsigargin increased [Ca 2؉ ] c and [Ca 2؉ ] m. Carbonyl cyanide p-trifluoromethoxyphenylhyrdrazone reversibly reduced the increase of [Ca 2؉ ] m. The source of the mitochondrial Ca 2؉ increase had intra-and extracellular components, as revealed by experiments in low extracellular Ca 2؉. We conclude that agonist-induced Ca 2؉ signals are transduced into mitochondria. 1) Mitochondria could serve as a Ca 2؉ sink, 2) mitochondria could allow the modulation of [Ca 2؉ ] c and [Ca 2؉ ] n signals, and 3) [Ca 2؉ ] m may serve as a stimulatory metabolic signal when a cell is highly stimulated.
Circulation Research, 2007
We sought to understand the effect of a transient exposure of cardiac myocytes to H 2 O 2 at a concentration that did not induce apoptosis. Myocytes were exposed to 30 mol/L H 2 O 2 for 5 minutes followed by 10 U/mL catalase for 5 minutes to degrade the H 2 O 2 . Cellular superoxide was measured using dihydroethidium. Transient exposure to H 2 O 2 caused a 66.4% increase in dihydroethidium signal compared with controls exposed to only catalase, without activation of caspase 3 or evidence of necrosis. The increase in dihydroethidium signal was attenuated by the mitochondrial inhibitors myxothiazol or carbonyl cyanide p-(trifluoromethoxy)phenyl-hydrazone and when calcium uptake by the mitochondria was inhibited with Ru360. We investigated the L-type Ca 2ϩ channel (I Ca-L ) as a source of calcium influx. Nisoldipine, an inhibitor of I Ca-L , attenuated the increase in superoxide. Basal channel activity increased from 5.4 to 8.9 pA/pF. Diastolic calcium was significantly increased in quiescent and contracting myocytes after H 2 O 2 . The response of I Ca-L to -adrenergic receptor stimulation was used as a functional reporter because decreasing intracellular H 2 O 2 alters the sensitivity of I Ca-L to isoproterenol. H 2 O 2 increased the K 0.5 required for activation of I Ca-L by isoproterenol from 5.8 to 27.8 nmol/L. This effect and the increase in basal current density persisted for several hours after H 2 O 2 . We propose that extracellular H 2 O 2 is associated with an increase in superoxide from the mitochondria caused by an increase in Ca 2ϩ influx from I Ca-L . The effect persists because a positive feedback exists among increased basal channel activity, elevated intracellular calcium, and superoxide production by the mitochondria.
Journal of Biological Chemistry, 1986
With a variety of forms of ischemic and toxic tissue lhjury, cellular accumulation of Ca2+ and generation of ortygen free radicals may have adverse effects upon cel ular and, in particular, mitochondrial membranes. Da i age to mitochondria, resulting in impaired ATP synthesis and diminished activity of cellular energydependent processes, could contribute to cell death. In order to model, in vitro, conditions present post-ischemia or during toxin exposure, the interactions between Ca2+ and oxygen free radicals on isolated renal mitochondria were characterized. The oxygen free radicals were generated by hypoxanthine and xanthine oxidase to simulate in vitro one of the sources of oxygen free radicals in the early post-ischemic period in vivo. With site I substrates, pyruvate and malate, Ca" pretreatment, followed by exposure to oxygen free radicals, resulted in an inhibition of electron transport chain function and complete uncoupling of oxidative phosphorylation. These effects were partially mitigated by dibucaine, a phospholipase Az inhibitor. With the site I1 substrate, succinate, the electron transport chain defect was not manifest and respiration remained partially coupled. The electron transport chain defect produced by Ca2+ and oxygen free radicals was localized to NADH CoQ reductase. Calcium and oxygen free radicals reduced mitochondrial ATPase activity by 55% and adenine nucleotide translocase activity by 66%. By contrast oxygen free radicals alone reduced ATPase activity by 32% and had no deleterious effects on translocase activity. Dibucaine partially prevented the Ca2+-dependent reduction in ATPase activity and totally prevented the Caz+dependent translocase damage observed in the presence of oxygen free radicals. These findings indicate that calcium potentiates oxygen free radical injury to mitochondria. The Ca2+induced potentiation of oxygen free radical injury likely is due in part to activation of phospholipase Az. This detrimental interaction associated with Ca2+ uptake by mitochondria and exposure of the mitochondria to oxygen free radicals may explain the enhanced cellular injury observed during post-ischemic reperfusion.