Pyridine Nucleotide Hydrolysis Research Articles

Regulation of cyclosporin A sensitive mitochondrial permeability transition by the redox state of pyridine nucleotides7

The mechanisms involved in the induction of cyclosporine A sensitive mitochondrial swelling by oxidative stress were investigated in isolated guinea pig liver mitochondria. The aim of our study was to investigate, if swelling is inevitably associated with the oxidation of pyridine nucleotides, and if the oxidized pyridine nucleotides have to be hydrolysed for the induction of mitochondrial swelling. Quantitative measurement of oxidized pyridine nucleotides was performed with HPLC. Mitochondrial swelling was recorded by monitoring the decrease in light scattering of the mitochondrial suspension. Reduction and oxidation of pyridine nucleotides were followed by monitoring the changes of the autofluorescence signal of reduced pyridine nucleotides. Qualitative measurement of mitochondrial membrane potential was performed with the fluorescence indicator rhodamine 123. Neither t-butyl hydroperoxide nor the dissipation of the mitochondrial inner membrane potential with FCCP (carbonyl cyanide- p-trifluoromethoxyphenyl hydrazone) induced the opening of the membrane permeability transition pore, unless an extensive oxidation of mitochondrial pyridine nucleotides took place. Mitochondrial swelling induced by our experimental conditions was always sensitive to cyclosporine A and accompanied by a cyclosporine A sensitive release of inner mitochondrial pyridine nucleotides without pyridine nucleotide hydrolysis. Not the cycling of calcium across the mitochondrial inner membrane but the accumulation of calcium inside the mitochondria was a prerequisite for mitochondrial swelling. The mitochondrial membrane permeability transition is neither caused nor accompanied by the hydrolysis of mitochondrial pyridine nucleotides.

Control of the pyridine nucleotide-linked Ca 2+ release from mitochondria by respiratory substrates

Oxidation of mitochondrial pyridine nucleotides followed by their hydrolysis promotes Ca 2+ release from intact liver mitochondria. In most of the previous studies oxidation was achieved with pro-oxidants which were added to mitochondria respiring on succinate in the presence of rotenone, a site I-specific inhibitor of the respiratory chain. Here we investigate pro-oxidant dependent and independent Ca 2+ release from mitochondria when respiration is supported either by the NAD +-linked substrate β-hydroxybutyrate, or by succinate. In the presence, as well as in the absence, of the pro-oxidant t-butylhydroperoxide mitochondria retain Ca 2+ much better with succinate than with β-hydroxybutyrate, as respiratory substrate. When Ca 2+ release is induced by t-butylhydroperoxide succinate-supported Ca 2+ retention is impeded by rotenone. Ca 2+ release (pro-oxidant dependent or independent) is paralleled by oxidation and hydrolysis of intramitochondrial pyridine nucleotides, and Ca 2+ retention is paralleled by reduction of pyridine nucleotides. It is concluded that the pyridine nucleotide-linked Ca 2+ release from mitochondria can be controlled by respiratory substrates which regulate the intramitochondrial hydrolysis of oxidized pyridine nucleotides.

Peroxynitrite stimulates the pyridine nucleotide-linked Ca2+ release from intact rat liver mitochondria.

Rat liver mitochondria contain a specific Ca2+ release pathway which operates when oxidized mitochondrial pyridine nucleotides are hydrolyzed in a Ca2+-dependent manner to ADP-ribose and nicotinamide. We have previously shown that NAD+ hydrolysis is inhibited by cyclosporin A and is possible only when some vicinal thiols are cross-linked. Here we report that the thiol oxidant peroxynitrite (ONOO-), which can form from nitric oxide (nitrogen monoxide, NO.) and superoxide anion (O2-), at low concentrations stimulates the specific Ca2+ release pathway. Both peroxynitrite-induced pyridine nucleotide hydrolysis and Ca2+ release are inhibited by cyclosporin A, and peroxynitrite is ineffective when pyridine nucleotides are kept reduced. Ca2+ release induced by peroxynitrite occurs with maintenance of the mitochondrial membrane potential and is not accompanied by entry of sucrose into mitochondria. The results suggest that peroxynitrite stimulates the specific Ca2+ release from intact mitochondria by modifying critical mitochondrial thiols other than glutathione in such a way that hydrolysis of oxidized pyridine nucleotides is achieved. These findings provide further insight into the regulation of Ca2+ release from mitochondria by nitric oxide and its congeners.

Pyridine nucleotide hydrolysis and interconversion in rat hepatocytes during oxidative stress

A characteristic feature of many types of chemically induced oxidative stress is a depletion of the pyridine nucleotide NAD +. This has been attributed to either its hydrolysis to nicotinamide and ADP-ribose or to its phosphorylation (interconversion) to NADP +. In this study the exposure of rat hepatocytes to either tert-butyl hydroperoxide (250–750 μM) or 2,3-dimethoxy-1,4-naphthoquinone (50 μM) resulted in a rapid depletion of NAD + with no change in the level of NAD. The depletion of NAD + was accompanied by an increase in nicotinamide. The rate of NAD + deletion induced by tert-butyl hydroperoxide (500 μM) and 2,3-dimethoxy-1,4-naphthoquinone (50 μM) was reduced by preincubating the hepatocytes for 1 hr with either 3-aminobenzamide (20 mM), nicotinamide (10 mM) or theophylline (7.5 mM), potent inhibitors of poly(ADP-ribose)polymerase. In cells exposed to 2,3-dimethoxy-1, 4-naphthoquinone (50 μM) extensive oxidation of NADPH to NADP + was observed; this was followed by an increase in the level of NADP + + NADPH (NADP(H)). However, no change in the total pyridine nucleotide ( NAD( H) + NADP( H)) pool was detected. Exposure to tert-butyl hydroperoxide resulted in the oxidation of NADPH to NADP + and a decrease in total pyridine nucleotide pool. These results suggest that during oxidative stress, NAD + is hydrolysed to nicotinamide, possibly by the activation of poly(ADP-ribose)polymerase and that the depletion of NAD + is independent of the increase in NADP +. Furthermore, no evidence of an interconversion of NAD + to NADP + was found.

Gliotoxin stimulates Ca2+ release from intact rat liver mitochondria.

Gliotoxin is an epidithiodioxopiperazine compound which can both react with sulfhydryl groups and form hydrogen peroxide. Rat liver mitochondria contain a prooxidant-regulated specific Ca2+ release pathway. Here we report that gliotoxin at low concentrations stimulates Ca2+ release via this pathway in isolated mitochondria. Ca2+ release is not promoted by gliotoxin exposed to disulfide-reducing reagents prior to addition to mitochondria or when its disulfide moiety is dimethylated. Gliotoxin is equally effective in glutathione-depleted and glutathione-adequate mitochondria. This and the unchanged mitochondrial oxygen consumption in the presence of gliotoxin suggest that the compound stimulates Ca2+ release by reacting with critical mitochondrial thiol compounds and not by increasing hydrogen peroxide formation in mitochondria. The gliotoxin-induced Ca2+ release is paralleled by hydrolysis of mitochondrial pyridine nucleotides, and both pyridine nucleotide hydrolysis and Ca2+ release are inhibited by cyclosporin A. These findings provide further insight into the regulation of Ca2+ release from intact mitochondria.

Phenylarsine oxide stimulates pyridine nucleotide-linked Ca 2+ release from rat liver mitochondria

Rat liver mitochondria contain a specific Ca 2+ release pathway which operates when oxidized mitochondrial pyridine nucleotides are hydrolysed to ADPribose and nicotinamide. Here we report that the hydrophobic bifunctional thiol reagent phenylarsine oxide (PhAsO) at low concentrations stimulates this pathway by promoting a Ca 2+-dependent hydrolysis of oxidized mitochondrial pyridine nucleotides. Ca 2+ release is inhibited by cyclosporine A or m-iodobenzylguanidine, compounds known to prevent intramitochondrial pyridine nucleotide hydrolysis or protein mono(ADPribosyl)ation, respectively. At higher concentrations, PhAsO causes non-specific leakiness of mitochondria.

Cyclosporine A protects mitochondria in an in vitro model of hypoxia/reperfusion injury

Hypoxia/reperfusion injury is a major clinical problem. One of its hallmarks is an increased cytosolic Ca 2+ content and an increased generation of reactive oxygen species in the cytosol and in mitochondria. In the present study of an in vitro model of hypoxia/reperfusion injury, mitochondria are exposed to Ca 2+ in combination with extra- and intramitochondrially acting prooxidants. In this model mitochondria are damaged in a Ca 2+-dependent manner. The extent and the site(s) of damage depend on both the kind of respiratory substrate and prooxidant used. The major damage occurs specifically at site I of the respiratory chain, and is due to hydrolysis of oxidized pyridine nucleotides and Ca 2+ release followed by Ca 2+ re-uptake (Ca 2+ ‘cycling’). Cyclosporine A completely protects against this damage. The protection is due to inhibition of pyridine nucleotide hydrolysis, an obligatory step in the sequence of events that links prooxidants to Ca 2+ release from intact mitochondria.

Sensitivity of mitochondrial peptidyl-prolyl cis-trans isomerase, pyridine nucleotide hydrolysis and Ca 2+ release to cyclosporine a and related compounds

Prooxidants activate a specific Ca 2+ release pathway from mitochondria. Here we investigate the inhibitory potency of cyclosporine A and six related compounds with respect to peptidyl-prolyl cis-trans isomerase (PPIase), pyridine nucleotide hydrolysis and Ca 2+ release. Whereas the absolute inhibitory potency of the compounds varies by about three orders of magnitude, a given compound is always most effective on PPIase, followed by pyridine nucleotide hydrolysis, and least effective in Ca 2+ release inhibition. The data show that pyridine nucleotide hydrolysis is a prerequisite but not a consequence of Ca 2+ release. They also strongly suggest that PPIase participates in the Ca 2+ release mechanism from intact mitochondria by regulating the intramitochondrial NAD + glycohydrolase, and thereby ascribe a physiological function to the protein. Furthermore, a complete lack of correlation between the inhibitory potencies described here and the reported immunosuppressive activities of the drugs is evident.

Submitochondrial localization of the NAD+ glycohydrolase. Implications for the role of pyridine nucleotide hydrolysis in mitochondrial calcium fluxes.

The submitochondrial location of the NAD+ glycohydrolase (NADase) and its role in mitochondrial ADP-ribosyl transfer reactions were investigated in isolated rat liver mitochondria. The NADase catalyzes the hydrolysis of NAD+ to ADP-ribose and nicotinamide. Hydrolysis of intramitochondrial NAD+ has been suggested to be the first step in a nonenzymatic mono(ADP-ribosylation) by free ADP-ribose of an inner membrane-associated acceptor peptide and that this protein is involved in regulating calcium efflux from mitochondria during exposure to oxidants. The results of the present study indicate that mitochondrial NADase activity lies outside the matrix space, however. This was determined by assessing the rates of hydrolysis of externally added NAD+ by intact mitochondria and comparing these rates with those obtained when the mitochondria were permeabilized with detergent. No significant difference was observed in the rate of NAD+ hydrolysis when detergent was added indicating that NAD+ hydrolysis by mitochondria does not necessitate its access to the matrix space. The submitochondrial location of the NADase was investigated further by digitonin titration of isolated mitochondria. Digitonin titration of the mitochondria released NADase activity with the outer membrane marker, monoamine oxidase. The digitonin titration data also suggest that the outer membrane is the exclusive location of the NADase. The incorporation of radioactive label derived from [3H]NAD+ into submitochondrial particles proceeds at comparable rates in the absence or presence of NADase activity, indicating that the production of free ADP-ribose is not necessary for intramitochondrial ADP-ribosyl transfer reactions. Thus the conclusions of this study suggest that due to the submitochondrial location of the NADase, it does not participate in intramitochondrial pyridine nucleotide hydrolysis or nonenzymatic mono(ADP-ribosylation) during oxidative stress in mitochondria.

Events that precede and that follow S-(1,2-dichlorovinyl)- l-cysteine-induced release of mitochondrial Ca 2+ and their association with cytotoxicity to renal cells

Previous studies showed that S-(1,2-dichlorovinyl)- l-cysteine perturbs intracellular Ca 2+ homeostasis [Vamvakas et al., Mol Pharmacol 38: 455–461, 1990]. The objective of the present study was to investigate the cellular events that precede and that follow S-(1,2-dichlorovinyl)- l-cysteine-induced mitochondrial Ca 2+ release. In incubations with isolated kidney mitochondria, S-(1,2-dichlorovinyl)- l-cysteine-induced Ca 2+ efflux is preceded by increased oxidation of mitochondrial pyridine nucleotides and is prevented by ATP, an inhibitor of the hydrolysis of pyridine nucleotides, and by meto-iodobenzylguanidine, an acceptor of ADP-ribose moieties. In LLC-PK 1 cells, elevation in the cytosolic Ca 2+ concentration is followed by a several-fold increase in DNA double-strand breaks which is attributed to the activation of Ca 2+- and Mg 2+-dependent endonucleases. The formation of DNA double-strand breaks is followed by increased poly(ADP-ribosylation) of nuclear proteins. S-(1,2-Dichlorovinyl)- l-cysteine-induced cytotoxicity in LLC-PKin1 cells is blocked by chelation of cytosolic Ca 2+ with Quin-2, by inhibition of DNA fragmentation with aurintricarboxylic acid and by inhibition of increased poly(ADP-ribosyl)transferase activity by 3-aminobenzamide. These findings indicate that 5-(1,2-dichlorovinyl)- l-cysteine bioactivation in renal cells may initiate the following cascade of events: increased oxidation and hydrolysis of mitochondrial pyridine nucleotides resulting in the modification of mitochondrial membrane proteins by pyridine nucleotide-derived ADP-ribose moieties, followed by Ca 2+ release. Elevated Ca 2+ concentrations may activate Ca 2+-dependent endonucleases, which leads to DNA fragmentation followed by increased poly(ADP-ribosylation) of nuclear proteins and, finally, cytotoxicity.

Butylated hydroxytoluene prevents cumene hydroperoxide-induced Ca 2+ release from liver mitochondria by inhibiting pyridine nucleotide hydrolysis

The mechanism by which the free radical scavenger butylated hydroxytoluene (BHT) prevents cumene hydroperoxide-induced Ca 2+ release from rat liver mitochondria was studied. In Ca 2+-loaded mitochondria cumene hydroperoxide induced a rapid oxidation and subsequent hydrolysis of the pyridine nucleotides. In the presence of BHT, pyridine nucleotide oxidation by cumene hydroperoxide occurred but was reversible as hydrolysis was prevented by BHT. However, the addition of BHT directly to rat liver submitochondrial particles did not inhibit NAD + hydrolysis or the formation of ADP-ribose from NAD +. Thus, whilst BHT prevented NAD + hydrolysis in isolated mitochondria, this appeared not to be due to a direct effect of BHT on the NADase. It is concluded that the mechanism of action of BHT on cumene hydroperoxide-induced Ca 2+ release from mitochondria involves the inhibition of pyridine nucleotide hydrolysis by an indirect mechanism rather than the radical scavenging properties of BHT.

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).

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.

Cyclosporine a inhibits mitochondrial pyridine nucleotide hydrolysis and calcium release

Mitochondria participate in the maintenance of cellular Ca 2+ homeostasis. Here we show that the immunosuppressive drug cyclosporine A at low concentrations inhibits release but not uptake of Ca 2+ by mitochondria. Prevention of Ca 2+ release is due to inhibition of intramitochondrial enzymatic hydrolysis of NAD to ADP-ribose and nicotinamide. These findings suggest a mechanism by which cyclosporine A interferes with cellular Ca 2+ homeostasis, and may be related to the immunosuppressive and cytotoxic properties of the drug.

The frooxidant-induced and spontaneous mitochondrial calcium release: Inhibition by meta-iodo-benzylguanidine (mibg), a substrate for mono (adpribosylation)

The norepinephrine analogue meta-iodo-benzylguanidine (MIBG), a substrate for mono(ADP-ribosylation) and inhibitor of eukaryotic ADP-ribosyltransferases, inhibits the prooxidant-induced and spontaneous calcium release from intact rat liver mitochondria without affecting pyridine nucleotide oxidation and hydrolysis. This finding strongly suggests regulation of calcium release by ADP-ribosylation in mitochondria, and may be relevant for the cellular and pharmacological effects of MIBG.

Kinetic analysis of the transglycosidation reaction catalyzed by rabbit spleen pyridine nucleotide glycohydrolase.

Properties of the transglycosidation reaction catalyzed by rabbit spleen pyridine nucleotide glycohydrolase were characterized using a modified cyanide addition method by which initial velocities of the transglycosidation (vT) and hydrolysis (vH) of pyridine nucleotides could be monitored simultaneously. (1) The vT was routinely determined with NMN and nicotinic acid used as substrates and was observed to be maximal at pH 6. Arrhenius plots of vT and vH indicated that the activation energies for transglycosidation and hydrolysis were 8.7 and 10.7 kcal/mol, respectively. (2) The enzyme showed a broad spectrum of substrate specificity with respect to both pyridine nucleotides and bases. Of the compounds tested, NMN and nicotinic acid were shown to be the best substrates when compared on the basis of Vmax/Km values. Kinetic constants for the enzyme-catalyzed transglycosidation reaction were as follows; Km(NMN) = 0.53 mM, Km(nicotinic acid), as acid form = 15 mM, apparent Vmax = 7.8 mumol/min/mg protein, in the presence of 0.2 M nicotinic acid. (3) The ratio of vT/vH was shown to be dependent on both pH and nicotinic acid concentration. However, transglycosidation versus hydrolysis partition at a fixed pH was constant regardless of the nicotinic acid concentration employed and approximated to be 1.2 x 10(4) at the maximal pH. (4) Nicotinamide, one of the most potent inhibitors for the enzyme-catalyzed hydrolysis, was shown to function as an antagonist for the transglycosidation reaction with NMN and nicotinic acid used as substrates. The inhibition mechanism with nicotinamide was purely noncompetitive with respect to nicotinic acid; on the other hand, the double reciprocal plot of the transglycosidation velocity against NMN concentration at a fixed concentration of nicotinamide was concave downwards. (5) The equilibrium constant of the reaction, NMN + 3-acetylpyridine----3-acetylpyridine mononucleotide + nicotinamide, was 0.61, whereas the conversion of NMN with nicotinic acid to nicotinic acid mononucleotide was essentially irreversible. These enzymatic properties of rabbit spleen pyridine nucleotide glycohydrolase suggested that the enzyme should not function as a glycohydrolase but as a transglycosidase and could serve in an important mechanism for an alternative biosynthetic pathway of nicotinic acid mononucleotide, one of the precursors for NAD synthesis, when nicotinic acid is supplied.

Mobilization of mitochondrial Ca 2+ by hydroperoxy-eicosatetraenoic acid

Hydroperoxy-eicosatetraenoic acids (HPETEs) and less effectively, also hydroxy-eicosatetraenoic acids (HETEs) stimulated Ca 2+ release from rat liver mitochondria. Ca 2+ release is accompanied by intramitochondrial pyridine nucleotide oxidation and hydrolysis. Both Ca 2+ 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 Ca 2+ release is not due to uncoupling of mitochondria. It is suggested that HPETEs and HETEs may act as mobilizers of mitochondria. Ca 2+ during signal transduction related to proliferation and tumor promotion.

N-Methyl-4-phenylpyridine (MMP + together with 6-hydroxydopamine or dopamine stimulates Ca 2+ release from mitochondria

The nigrostriatal neurotoxin N-methyl-1,2,3,6-tetrahydropyridine (MPTP) causes Parkinsonism in humans and laboratory animals. MPTP neurotoxicity is dependent on its oxidation to N-methyl-4-phenylpyridine (MPP +). The mechanism by which MPP + causes destruction of dopamine-containing nigrostriatal cells is unknown. Here we show that MPP + but not MPTP is taken up by energized mitochondria. MPP + in the presence of dopamine and particularly of 6-hydroxydopamine stimulates Ca 2+ release from mitochondria. Ca 2+ release is accompanied by hydrolysis of intramitochondrial pyridine nucleotides. Our findings suggest that the MPTP-induced model of Parkinson's disease may be due to a disturbed Ca 2+ homeostasis in dopamine neurons.

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.

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+ [Lötscher, H. R., Winterhalter, K. H., Carafoli, E. & Richter, C. (1979) Proc. Natl Acad. Sci. USA 76, 4340-4344, and Lötscher, 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.