Mechanism of alloxan-induced calcium release from rat liver mitochondria

The objective of the present work was to investigate the mechanism of alloxan-induced Ca2+ release from rat liver mitochondria. Transport of Ca2+, oxidation and hydrolysis of mitochondrial pyridine nucleotides, changes in the mitochondrial membrane potential, and oxygen consumption by mitochondria were investigated. Alloxan does not inhibit the uptake of Ca2+ but stimulates the release of Ca2+ from liver mitochondria, which is accompanied by oxidation and hydrolysis of pyridine nucleotides. Oxidation of mitochondrial pyridine nucleotides by alloxan is not mediated by glutathione peroxidase and glutathione reductase and may occur largely nonenzymatically. Measurements of the mitochondrial membrane potential in combination with inhibitors of Ca2+ reuptake indicate that Ca2+ release takes place from intact liver mitochondria via a distinct pathway. Limited redox cycling of alloxan by mitochondria is indicated by measurements of the membrane potential and O2 consumption in the presence of cyanide. It is concluded that alloxan can cause Ca2+ release from intact rat liver mitochondria. Redox cycling of alloxan is not significantly involved in the Ca2+ release mechanism. Oxidation and hydrolysis of pyridine nucleotides, possibly in conjunction with oxidation of critical sulfhydryl groups, seem to be key events in the alloxan-induced Ca2+ release. Disturbance of cellular Ca2+ homeostasis may partly explain alloxan toxicity.     

Regulated release of Ca2+ from respiring mitochondria by Ca2+/2H+ antiport.

Simultaneous measurements of oxygen consumption and transmembrane transport of Ca2+, H+, and phosphate show that the efflux of Ca2+ from respiring tightly coupled rat liver mitochondria takes place by an electroneutral Ca2+/2H+ antiport process that is ruthenium red-insensitive and that is regulated by the oxidation-reduction state of the mitochondrial pyridine nucleotides. When mitochondrial pyridine nucleotides are kept in a reduced steady state, the efflux of Ca2+ is inhibited; when they are in an oxidized state, Ca2+ efflux is activated. These processes were demonstrated by allowing phosphate-depleted mitochondria respiring on succinate in the presence of rotenone to take up Ca2+ from the medium. Upon subsequent addition of ruthenium red to block Ca2+ transport via the electrophoretic influx pathway, and acetoacetate, to bring mitochondrial pyridine nucleotides into the oxidized state, Ca2+ efflux and H+ influx ensued. The observed H+ influx/Ca2+ efflux ratio was close to the value 2.0 predicted for the operation of an electrically neutral Ca2+/2H+ antiport process.     

Inhibition of pro-oxidant-induced mitochondrial pyridine nucleotide hydrolysis and calcium release by 4-hydroxynonenal.

Intra- and extra-mitochondrial Ca2+ participates in vital cellular processes. This work investigates the influence of 4-hydroxynonenal (HNE) on pro-oxidant-induced Ca2+ release from rat liver mitochondria. Ca2+ movements across the mitochondrial inner membrane, the pyridine nucleotide redox state and pyridine (nicotinamide) nucleotide hydrolysis were analysed. HNE did not influence Ca2+ uptake by mitochondria, but inhibited in a concentration-dependent manner Ca2+ release induced by t-butylhydroperoxide (tbh). Total inhibition was achieved with about 50 microM-HNE. Ca2+ release induced by the pro-oxidant alloxan was also inhibited by HNE. Oxidation of pyridine nucleotides, induced by tbh through the concerted action of glutathione peroxidase, glutathione reductase and the energy-linked transhydrogenase, was not affected by up to 50 microM-HNE. In contrast, HNE inhibited pyridine nucleotide hydrolysis in a concentration-dependent manner. The data suggest that HNE toxicity may be in part attributed to an impaired intramitochondrial Ca2+ homeostasis.     

Ca2+ releasing effect of perezone on adrenal cortex mitochondria

Ca2+ energy-coupled transport was analized in adrenal cortex mitochondria using the sesquiterpenic drug perezone. Perezone promotes Ca2+ efflux by inducing collapse of the membrane potential and oxidation of pyridine nucleotides. The effect of perezone on mitochondrial Ca2+ release follows a dose-response relationship and is dependent of the reduction of the drug. These data suggest that perezone may produce a cytotoxic effect through an impairment in Ca2+ homeostasis.     

p-Bromophenacyl bromide prevents cumene hydroperoxide-induced mitochondrial permeability transition by inhibiting pyridine nucleotide oxidation

Mitochondrial permeability transition is commonly characterized as a Ca2+ -dependent non-specific increase in inner membrane permeability that results in swelling of mitochondria and their de-energization. In the present study, the effect of different inhibitors of phospholipase A2--p-bromophenacyl bromide, dibucaine, and aristolochic acid--on hydroperoxide-induced permeability transitions in rat liver mitochondria was tested. p-Bromophenacyl bromide completely prevented the hydroperoxide-induced mitochondrial permeability transition while the effects of dibucaine or aristolochic acid were negligible. Organic hydroperoxides added to mitochondria undergo reduction to corresponding alcohols by mitochondrial glutathione peroxidase. This reduction occurs at the expense of GSH which, in turn, can be reduced by glutathione reductase via oxidation of mitochondrial pyridine nucleotides. The latter is considered a prerequisite step for mitochondrial permeability transition. Among all the inhibitors tested, only p-bromophenacyl bromide completely prevented hydroperoxide-induced oxidation of mitochondrial pyridine nucleotides. Interestingly, p-bromophenacyl bromide had no affect on mitochondrial glutathione peroxidase, but reacted with mitochondrial glutathione that prevented pyridine nucleotides from being oxidized. Our data suggest that p-bromophenacyl bromide prevents hydroperoxide-induced deterioration of mitochondria via interaction with glutathione rather than through inhibition of phospholipase A2.     

Quantitative and mechanistic aspects of the hydroperoxide-induced release of Ca2+ from rat liver mitochondria

We have previously demonstrated in rat liver mitochondria a hydroperoxide-induced hydrolysis of pyridine nucleotides and release of Ca2+ [L?tscher, H. R., Winterhalter, K. H., Carafoli, E. & Richter, C. (1979) Proc. Natl Acad. Sci. USA 76, 4340-4344, and L?tscher, H. R., Winterhalter, K. H., Carafoli, E. & Richter, C. (1980) J. Biol. Chem. 255, 9325-9330]. Here we investigate pyridine nucleotide hydrolysis and Ca2+ release under conditions of minimized Ca2+ cycling and with smaller Ca2+ loads. The extent of pyridine nucleotide hydrolysis, measured by pyridine-nucleotide-derived nicotinamide release from intact mitochondria, and the Ca2+ release rate show a very similar sigmoidal dependence on the mitochondrial Ca2+ load. The hydrolysis of oxidized pyridine nucleotides is limited under non-cycling conditions. Whereas pyridine nucleotide hydrolysis as measured by nicotinamide release is extensive, net loss of mitochondrial pyridine nucleotides is observed only at relatively high Ca2+ loads. Our results indicate the ability of mitochondria to resynthesize pyridine nucleotides after hydrolysis. Neither a decrease of reduced, nor an increase of oxidized, mitochondrial glutathione favour Ca2+ release. From these and previous findings it is concluded that the hydroperoxide-induced Ca2+ release is triggered by a factor which is distal to the oxidation of mitochondrial pyridine nucleotides. Ca2+ release is stimulated when the movement of protons across the inner mitochondrial membrane is facilitated, giving evidence for the operation of the hydroperoxide-induced release pathway as a Ca2+/H+ antiport.     

Induction of 2H+/Me2+ exchange in rat-liver mitochondria

The time dependency of CA2+ efflux from Ca2+-loaded rat liver mitochondria has been investigated. The rate of ruthenium-red-insensitive Ca2+ efflux is continuously increased during the retention as a result of induction of an electroneutral H+ Ca2+ exchange system. The activation of the Ca2+ efflux pathway takes place under the constant value of the membrane potential and is accompanied by oxidation of mitochondrial pyridine nucleotides. It has also been found that the ruthenium-red-insensitive H+/Sr2+ exchange occurs in mitochondria during Sr2+-induced oscillation of ion fluxes. The rate of H+/Sr2+ exchange is variable and depends on the stage of the oscillatory cycle.     

t-Butylhydroperoxide-induced Ca2+ efflux from liver mitochondria in the presence of physiological concentrations of Mg2+ and ATP

Isolated rat liver mitochondria, energized either by succinate oxidation or by ATP hydrolysis, present a transient increase in the rate of Ca2+ efflux concomitant to NAD(P)H oxidation by hydroperoxides when suspended in a medium containing 3 mM ATP, 4 mM Mg2+ and acetate as permeant anion. This is paralleled by an increase in the steady-state concentration of extramitochondrial Ca2+, a small decrease in delta psi and an increase in the rate of respiration and mitochondrial swelling. With the exception of mitochondrial swelling all other events were found to be reversible. If Ca2+ cycling was prevented by ruthenium red, the changes in delta psi, the rate of respiration and the extent of mitochondrial swelling were significantly diminished. In addition, there was no significant decrease in the content of mitochondrial pyridine nucleotides. Mitochondrial coupling was preserved after a cycle of Ca2+ release and re-uptake under these experimental conditions. It is concluded that hydroperoxide-induced Ca2+ efflux from intact mitochondria is related to the redox state of pyridine nucleotides.     

Alterations in intracellular calcium compartmentation following inhibition of calcium efflux from isolated hepatocytes

Addition of ATP to the incubation medium of freshly isolated rat hepatocytes causes a marked inhibition of the efflux of Ca2+ from the cells, and its accumulation in intracellular compartments. After an initial rise in cytosolic free Ca2+ concentration, as indicated by the activation of phosphorylase, Ca2+ is preferentially sequestered in the mitochondria, without any apparent contribution by the endoplasmic reticulum. Impairment of mitochondrial Ca2+ homeostasis by pyridine nucleotide oxidation associated with tert-butyl hydroperoxide metabolism, prevents the ATP-dependent cellular Ca2+ accumulation and causes a release of Ca2+ from the hepatocytes into the medium. Conversely, maintenance of the mitochondrial pyridine nucleotides in a more reduced state, e. g. in presence of 3-hydroxybutyrate in the medium, prevents this hydroperoxide-induced release of intracellular Ca2+. Under conditions of impaired mitochondrial Ca2+ sequestration, there appears to be a redistribution of a minor fraction of the intracellular Ca2+ from the mitochondria to the endoplasmic reticulum. Our results provide additional evidence for the critical involvement of the plasma membrane Ca2+-extruding system in the physiological regulation of the cytosolic free Ca2+ concentration in hepatocytes, and suggest that the mitochondria play a more important role than the endoplasmic reticulum in the regulation of the cytosolic free Ca2+ level when the plasma membrane Ca2+ pump is inhibited.     

Tyramine and monoamine oxidase inhibitors as modulators of the mitochondrial membrane permeability transition

Incubation of rat liver mitochondria with 100-500 mM tyramine, a substrate for monoamine oxidases A and B (MAOs), in the presence of 30 mM Ca2+ induces matrix swelling, accompanied by collapse of membrane potential, efflux of endogenous Mg2+ and accumulated Ca2+ and oxidation of endogenous pyridine nucleotides. These effects are completely abolished in the presence of cyclosporin A, ADP, dithioerythritol and N-ethylmaleimide, thus confirming the induction of the mitochondrial membrane permeability transition (MPT). The observed partial protective effect exerted by catalase indicates the involvement of both MAO-derived hydrogen peroxide and aldehyde. Higher concentrations of tyramine (1-2 mM) are less effective or even completely ineffective. At these high concentrations tyramine has an inhibitory effect when the MPT is induced by 100 mM Ca2+. The MAO inhibitors clorgyline (50 mM) and pargyline (500 mM) completely protect against MPT induction by 100 mM tyramine but also inhibit the phenomenon, although with different efficacy, when it is induced by 100 mM Ca2+ in the absence of tyramine. Taken together, our data suggest that tyramine, clorgyline and pargyline act as modulators of the MPT either through a direct inducing/protective effect or by controlling hydrogen peroxide and aldehyde generation.     

Temperature dependence of the atractyloside-induced mitochondrial Ca2+ release

1. Mitochondrial Ca2+, accumulated by succinate oxidation was released by addition of 50 microM atractyloside. Beside this Ca2+ efflux, a large oxidation of pyridine nucleotides and sustained membrane depolarization occurs. An absolute requirement for acetate to support Ca2+ release is demonstrated. 2. Membrane de-energization, NAD(P)H oxidation, and Ca2+ efflux as induced by atractyloside were temperature-dependent, since it occurs when mitochondria are incubated at 22 degrees C and was abolished at 4 degrees C. 3. Taking into account this latter, the effects of atractyloside on mitochondrial Ca2+ release appears not to be a simple result of the binding of the inhibitor to adenine nucleotide translocase. 4. It is proposed that the mechanism involved in atractyloside-driven membrane permeability to Ca2+ must be related with the transference of the conformational change of the carrier, to another membrane structure responsible for the maintenance permeability to ions.     

The role of hydroperoxides as calcium release agents in rat brain mitochondria

Hydroperoxides have previously been shown to induce Ca2+ release from intact rat liver mitochondria via a specific release pathway. Here it is reported that, in rat brain mitochondria, a hydroperoxide-induced Ca2+ release is also operative but is of minor importance. Hydroperoxide stimulates Ca2+ release in the presence of ruthenium red about twofold at a Ca2+ load of 40 nmol/mg mitochondrial protein. After addition of hydroperoxide, Ca2+ release from brain mitochondria can still be evoked by Na+. In the presence of succinate and rotenone, hydroperoxide induces only a very limited oxidation of pyridine nucleotides, most probably due to the low level of glutathione peroxidase (EC 1.11.1.9) and glutathione reductase (EC 1.6.4.2) found in brain mitochondria. Similar to liver mitochondria, a NADase (EC 3.2.2.5) activity is found in brain mitochondria. Its localization and sensitivity toward ADP and ATP, however, is different from that of the liver mitochondrial enzyme.     

The role of glutathione in the retention of Ca2+ by liver mitochondria

Concentrations of rhein and nitrofurantoin in the micromolar range induce Ca2+ release and the development of increased inner membrane permeability in liver mitochondria. Both compounds inhibit the mitochondrial glutathione reductase causing a depletion of GSH and an accumulation of GSSG in energized mitochondria. Under these conditions, the compounds also alter the oxidation state of pyridine nucleotides, NADH becoming oxidized while NADPH remains reduced. Using rhein or nitrofurantoin, together with t-butyl-hydroperoxide and beta-hydroxybutyrate, it is possible to selectively alter the NAD/NADH, the NADP/NADPH, and the GSSG/GSH ratios and to determine the effect of these different states on the ability of Ca2+ to produce a permeable inner membrane. No correlation between pyridine nucleotide ratios and sensitivity to Ca2+ was observed. Mitochondria are stable to Ca2+ when the GSH content is high, but become permeable when Ca2+ is present and GSH is converted to GSSG. It is proposed that the GSSG/GSH ratio, by controlling the reduction state of critical sulfhydryl groups, regulates lysophospholipid acyltransferase activity and, therefore, the ability of mitochondria to remain impermeable upon activation of the intramitochondrial Ca2+ requiring phospholipase A2.     

Hydroperoxide-induced loss of pyridine nucleotides and release of calcium from rat liver mitochondria

It has been previously reported (L?tscher, H. R., Winterhalter, K. H., Carafoli, E., and Richter, C. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 4340-4344) that in Ca2+-loaded mitochondria hydroperoxides induce a release of Ca2+ from mitochondria and an irreversible oxidation of mitochondrial pyridine nucleotides. Here we show that in the presence of Ca2+ oxidized mitochondrial pyridine nucleotides are hydrolyzed inside mitochondria and that nicotinamide is released from mitochondria. The extent of the hydrolysis of NAD(P)+ is dependent on the amount of both hydroperoxide and Ca2+. The hydrolysis is reversible in the presence of added nicotinamide. The release of Ca2+ from mitochondria is electroneutral, and is directly or indirectly dependent on oxidized mitochondrial pyridine nucleotides. By contrast, the uptake of Ca2+ most probably does not require the present of reduced pyridine nucleotides. Control experiments show that even under the most drastic conditions employed in this study (100 nmol of Ca2+ and 85 nmol of t-butylhydroperoxide/mg of protein) mitochondria retain a considerable degree of functional integrity.     

Mobilization of mitochondrial Ca2+ by hydroperoxy-eicosatetraenoic acid

Hydroperoxy-eicosatetraenoic acids (HPETEs) and less effectively, also hydroxy-eicosatetraenoic acids (HETEs) stimulated Ca2+ release from rat liver mitochondria. Ca2+ release is accompanied by intramitochondrial pyridine nucleotide oxidation and hydrolysis. Both Ca2+ release and pyridine nucleotide oxidation are impeded when the flow of electrons between pyridine nucleotides and HPETE is impaired. Measurements of the mitochondrial membrane potential indicate that HPETE-stimulated Ca2+ release is not due to uncoupling of mitochondria. It is suggested that HPETEs and HETEs may act as mobilizers of mitochondrial Ca2+ during signal transduction related to proliferation and tumor promotion.     

Perturbation in liver mitochondrial Ca2+ homeostasis in experimental iron overload: a possible factor in cell injury

The functional state of isolated mitochondria and specifically the integrity of the inner membrane, were investigated in the liver of rats made siderotic by dietary supplementation with carbonyl iron. The concentration of iron in the hepatic tissue increased progressively up to nearly 40 days and reached a steady-state level. When the iron content reached a threshold value (higher than 90 nmol/mg protein) the occurrence of in vivo lipid peroxidation in the mitochondrial membrane was detected. This process did not result in gross alterations in the mitochondrial membrane, as indicated by electron microscopy, phosphorylative capability and membrane potential measurements. On the contrary, the induction of lipoperoxidative reaction appeared to be associated with the activation of Ca2+ release from mitochondria. This was shown to occur as a consequence of rather subtle modifications in the inner membrane structure via a specific efflux route, which appeared to be linked to the oxidation level of mitochondrial pyridine nucleotides. The induction of this Ca2+ release from iron-treated mitochondria resulted in enhancement of Ca2+ cycling, a process which dissipates energy to reaccumulate into mitochondria the released Ca2+. The perturbation in mitochondrial Ca2+ homeostasis reported here may be a factor in the onset of cell damage in this experimental model of hepatic iron overload.     

The redox state of endogenous pyridine nucleotides can determine both the degree of mitochondrial oxidative stress and the solute selectivity of the permeability transition pore

Acetoacetate, an NADH oxidant, stimulated the ruthenium red-insensitive rat liver mitochondrial Ca(2+) efflux without significant release of state-4 respiration, disruption of membrane potential (Deltapsi) or mitochondrial swelling. This process is compatible with the opening of the currently designated low conductance state of the permeability transition pore (PTP) and, under our experimental conditions, was associated with a partial oxidation of the mitochondrial pyridine nucleotides. In contrast, diamide, a thiol oxidant, induced a fast mitochondrial Ca(2+) efflux associated with a release of state-4 respiration, a disruption of Deltapsi and a large amplitude mitochondrial swelling. This is compatible with the opening of the high conductance state of the PTP and was associated with extensive oxidation of pyridine nucleotides. Interestingly, the addition of carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone to the acetoacetate experiment promoted a fast shift from the low to the high conductance state of the PTP. Both acetoacetate and diamide-induced mitochondrial permeabilization were inhibited by exogenous catalase. We propose that the shift from a low to a high conductance state of the PTP can be promoted by the oxidation of NADPH. This impairs the antioxidant function of the glutathione reductase/peroxidase system, strongly strengthening the state of mitochondrial oxidative stress.     

The effect of ferric iron complex on isolated rat liver mitochondria. II. Ion movements

It has been found that addition of iron(III)-gluconate complex to rat liver mitochondria disturbed the mitochondrial Ca2+ transport. Indirect evidence when the changes in the membrane potential during the transport of Ca2+ were followed, as well as direct evidence, when the fluxes of Ca2+ were monitored by a Ca2+-selective electrode, indicated that this iron complex induced an efflux of Ca2+ from liver mitochondria. The mechanisms by which iron induced Ca2+ release appeared to be linked to the induction of lipoperoxidation of mitochondrial membrane. The mitochondrial membrane, however, did not become irreversibly damaged under these conditions, as indicated by its complete repolarization. It was also shown that the induction by iron of lipoperoxidation brought about an efflux of K+ from mitochondria.     

N-acetyl-p-benzoquinone imine induces Ca2+ release from mitochondria by stimulating pyridine nucleotide hydrolysis.

The mechanism of N-acetyl-p-benzoquinone imine (NAPQI)-induced release of Ca2+ from rat liver mitochondria was investigated. The addition of NAPQI or 3,5-Me2-NAPQI (a dimethylated analogue of NAPQI with only oxidizing properties) to mitochondria resulted in the rapid and extensive oxidation of NADH and NADPH. High-performance liquid chromatographic analysis of mitochondrial pyridine nucleotides revealed that the formation of NAD+ and NADP+ was followed by a time-dependent net loss of total pyridine nucleotides as a result of their hydrolysis, with the formation of nicotinamide. Preincubation of the mitochondria with cyclosporin A completely prevented the quinone imine-stimulated release of sequestered Ca2+ from mitochondria. Cyclosporin A did not affect the ability of NAPQI or 3,5-Me2-NAPQI to oxidize NAD(P)H but prevented the quinone imine-induced hydrolysis of the pyridine nucleotides. Although there was no detectable change in total protein-bound ADP-ribose content during quinone imine-induced Ca2+ release from mitochondria, meta-iodobenzylguanidine, a competitive inhibitor of protein mono(ADP-ribosylation), prevented Ca2+ release by NAPQI and 3,5-Me2-NAPQI; meta-iodobenzylguanidine did not inhibit the quinone imine-induced NAD(P)H oxidation and only partially blocked hydrolysis of the oxidized pyridine nucleotides. It is concluded that NAPQI causes the oxidation of mitochondrial NADH and NADPH, and stimulates Ca2+ release as a result of the further hydrolysis of the oxidized pyridine nucleotides and protein mono(ADP-ribosylation).     

Bcl-2 prevents mitochondrial permeability transition and cytochrome c release via maintenance of reduced pyridine nucleotides

Digitonin-permeabilized PC12 and GT1-7 neural cells exhibited a cyclosporin A-sensitive decrease in mitochondrial membrane potential, increased volume, and release of the pro-apoptotic factor cytochrome c in the presence of Ca2+ and the mitochondrial permeability transition (MPT) inducers t-butyl hydroperoxide (t-bOOH) or phenylarsine oxide (PhAsO). Although the concentration of PhAsO required to induce the MPT was similar for Bcl-2 negative and Bcl-2 overexpressing transfected cells (Bcl-2(+)), the level of t-bOOH necessary for triggering the MPT was much higher for Bcl-2(+) cells. A higher concentration of t-bOOH was also necessary for promoting the oxidation of mitochondrial pyridine nucleotides in Bcl-2(+) cells. The sensitivity of Bcl-2(- ) cell mitochondria to t-bOOH but not PhAsO could be overcome by the use of conditions that protect the pyridine nucleotides against oxidation. We conclude that the increased ability of Bcl-2(+) cells to maintain mitochondrial pyridine nucleotides in a reduced redox state is a sufficient explanation for their resistance to MPT under conditions of oxidative stress induced by Ca2+ plus t-bOOH.