Inhibition of 2,4-dinitrophenol-induced potassium efflux by adenine nucleotides in mitochondria

The influence of nucleotides on 2,4-dinitrophenol (DNP)-induced K+ efflux from intact rat liver mitochondria has been studied. ATP and ADP at micromolar concentrations were found to inhibit mitochondrial potassium transport, whereas GTP, GDP, CTP, and UTP did not show tha same effect. The values of half-maximal inhibition (IC50) were approximately 20 microM for ATP and approximately 60 microM for ADP. It is suggested that adenine nucleotides exert their inhibitory action at the matrix side of the inner mitochondrial membrane since the inhibitor of adenine nucleotide translocase atractyloside at concentration of 1 microM completely removed the inhibitory effect of ATP and ADP. The mitochondrial ATPase inhibitor oligomycin (2 microg/ml) was found to reduce slightly the rate of DNP-induced K+ efflux and had no effect on inhibition by adenine nucleotides; the latter was insensitive to Mg2+ and the changes in pH. It seems likely that the regulation of potassium transport is not due to phosphorylation of the channel-forming protein but to binding of the nucleotides in specific regulatory sites. The possibility of potassium efflux from mitochondria in the presence of uncoupler via the ATP-dependent potassium channel is discussed.     

Calcium stimulates ATP-Mg/Pi carrier activity in rat liver mitochondria

Adenine nucleotide transport over the carboxyatractyloside-insensitive ATP-Mg/Pi carrier was assayed in isolated rat liver mitochondria with the aim of investigating a possible regulatory role for Ca2+ on carrier activity. Net changes in the matrix adenine nucleotide content (ATP + ADP + AMP) occur when ATP-Mg exchanges for Pi over this carrier. The rates of net accumulation and net loss of adenine nucleotides were inhibited when free Ca2+ was chelated with EGTA and stimulated when buffered [Ca2+]free was increased from 1.0 to 4.0 microM. The unidirectional components of net change were similarly dependent on Ca2+; ATP influx and efflux were inhibited by EGTA in a concentration-dependent manner and stimulated by buffered free Ca2+ in the range 0.6-2.0 microM. For ATP influx, increasing the medium [Ca2+]free from 1.0 to 2.0 microM lowered the apparent Km for ATP from 4.44 to 2.44 mM with no effect on the apparent Vmax (3.55 and 3.76 nmol/min/mg with 1.0 and 2.0 microM [Ca2+]free, respectively). Stimulation of influx and efflux by [Ca2+]free was unaffected by either ruthenium red or the Ca2+ ionophore A23187. Calmodulin antagonists inhibited transport activity. In isolated hepatocytes, glucagon or vasopressin promoted an increased mitochondrial adenine nucleotide content. The effect of both hormones was blocked by EGTA, and for vasopressin, the effect was blocked also by neomycin. The results suggest that the increase in mitochondrial adenine nucleotide content that follows hormonal stimulation of hepatocytes is mediated by an increase in cytosolic [Ca2+]free that activates the ATP-Mg/Pi carrier.     

Control of the energizing of liver mitochondria at the adenine nucleotide carrier level under hypotonic conditions

The effects of ADP, carboxyatractyloside (CAT) and the local anaesthetic nupercaine on the energy-dependent Ca2+ uptake by rat liver mitochondria oxidizing succinate in the presence of oligomycin were compared, using incubation media of 320 mosM and 120 mosM tonicities. In hypotonic media the mitochondrial Ca2+ capacity was increased by 50%, and the mitochondria were more stable to the damaging effects of Ca + Pi. In the presence of ADP the Ca2+ capacities of mitochondria increased both in normotonic and hypotonic media; however, the absolute amounts of calcium consumed were levelled off. CAT abolished the effect of ADP on the mitochondrial Ca2+ uptake and equalized the Ca2+ capacities of rat liver mitochondria in the both media. The local anaesthetic nupercaine also increased the Ca2+ capacity of mitochondria. The effects of nupercaine and ADP were additive. CAT abolished the effect of ADP but not that of nupercaine. Measurements of the intramitochondrial contents of adenine nucleotides showed that in 120 mosM media there was a significant increase in the intramitochondrial content of ATP and the total pool of adenine nucleotides. It was concluded that in hypotonic media the mitochondrial adenine nucleotide carrier exists predominantly in the m-conformation thus facilitating the energization of mitochondria.     

Regulation of NAD+-linked isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase by Ca2+ ions within toluene-permeabilized rat heart mitochondria. Interactions with regulation by adenine nucleotides and NADH/NAD+ ratios.

1. Toluene-permeabilized rat heart mitochondria have been used to study the regulation of NAD+-linked isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase by Ca2+, adenine and nicotinamide nucleotides, and to compare the properties of the enzymes in situ, with those in mitochondrial extracts. 2. Although K0.5 values (concn. giving half-maximal effect) for Ca2+ of 2-oxoglutarate dehydrogenase were around 1 microM under all conditions, corresponding values for NAD+-linked isocitrate dehydrogenase were in the range 5-43 microM. 3. For both enzymes, K0.5 values for Ca2+ observed in the presence of ATP were 3-10-fold higher than those in the presence of ADP, with values increasing over the ADP/ATP range 0.0-1.0. 4. 2-Oxoglutarate dehydrogenase was less sensitive to inhibition by NADH when assayed in permeabilized mitochondria than in mitochondrial extracts. Similarly, the Km of NAD+-linked isocitrate dehydrogenase for threo-Ds-isocitrate was lower in permeabilized mitochondria than in extracts under all the conditions investigated. 5. It is concluded that in the intact heart Ca2+ activation of NAD+-linked isocitrate dehydrogenase may not necessarily occur in parallel with that of the other mitochondrial Ca2+-sensitive enzymes, 2-oxoglutarate dehydrogenase and the pyruvate dehydrogenase system.     

Control of mitochondrial content of adenine nucleotides by submicromolar calcium concentrations and its relationship to hormonal effects

Rat liver mitochondria were incubated at 30 degrees C with 4 mM ATP in a medium similar in electrolyte composition to that of hepatic cytosol. Under these conditions, a net increase in mitochondrial adenine nucleotides was observed that was dependent on the concentration of free Ca2+ [( Ca2+]) in the incubation medium. At 0.2 microM [Ca2+] or less, there was no demonstrable uptake of adenine nucleotides; at 0.4 microM [Ca2+], or greater, net uptake occurred. The calcium-dependent accumulation of nucleotides by mitochondria required Mg2+ in the incubation medium and was insensitive to carboxyatractyloside. The uptake of adenine nucleotides was enhanced by the addition of antimycin A or antimycin A together with oligomycin. Accumulation of nucleotides appeared to be associated with a small increase in mean mitochondrial volume, but the membrane potential was not affected. No uptake or loss of NAD-NADH by mitochondria was detected. Ruthenium red failed to inhibit the calcium-dependent uptake of adenine nucleotides by the mitochondria, indicating that stimulation of this process by Ca2+ does not involve transport of the cation into mitochondria by the Ca2+ uniporter. Because glucagon acts to elevate cytosolic [Ca2+] from approximately 0.2 microM to 0.6 microM, the same range affecting nucleotide uptake, it is proposed that the increase in mitochondrial adenine nucleotides that follows treatment with glucagon is mediated by the rise in cytosolic [Ca2+] produced by the hormone. This hypothesis was supported by the observation that epinephrine and A23187, agents that raise cytosolic [Ca2+], increased the content of mitochondrial adenine nucleotides in isolated hepatocytes. Furthermore, cells, incubated under calcium-depleting conditions, had a diminished response to glucagon.     

Free pyrimidine nucleotide pool of Ehrlich ascites-tumour cells. Characteristics related to quantitative studies of RNA metabolism

The atractyloside-insensitive accumulation of adenine nucleotides by rat liver mitochondria (as opposed to the exchange-diffusion catalysed by the adenine nucleotide translocase) has been measured by using the luciferin/luciferase assay as well as by measuring [14C]ATP uptake. In foetal rat liver mitochondria ATP is accumulated more rapidly than ADP, whereas AMP is not taken up. The uptake of ATP occurs against a concentration gradient, and the rate of ATP uptake is greater in foetal than in adult rat liver mitochondria. The accumulated [14C]ATP is shown to be present within the mitochondrial matrix space and is freely available to the adenine nucleotide translocase for exchange with ATP present in the external medium. The uptake is specific for ATP and ADP and is not inhibited by adenosine 5'-[beta gamma-imido] triphosphate, GTP, CTP, cyclic AMP or Pi, whereas dATP and AMP do inhibit ATP accumulation. The ATP accumulation is also inhibited by carbonyl cyanide m-chlorophenylhydrazone, KCN and mersalyl but is insensitive to atractyloside. The ATP uptake is concentration-dependent and exhibits Michaelis-Menten kinetics. The divalent cations Mg2+ and Ca2+ greatly enhance ATP accumulation, and the presence of hexokinase inhibits the uptake of ATP by foetal rat liver mitochondria. These latter effects provide an explanation for the low adenine nucleotide content of foetal rat liver mitochondria and the rapid increase that occurs in the mitochondrial adenine nucleotide concentration in vivo immediately after birth.     

Inhibitory Effects of Adenine Nucleotides on Brain Mitochondrial Permeability Transition

The adenine nucleotides ADP and ATP are probably the most important endogenous inhibitors of the mitochondrial permeability transition (MPT). We studied the inhibitory effects of adenine nucleotides on brain MPT by measuring mitochondrial swelling and Ca²⁺ and cytochrome c release. We observed that in the presence of either ADP or ATP, at 250 μM, brain mitochondria accumulated more than 1 μmol Ca²⁺ × mg protein⁻¹. ADP or ATP also prevented Ca²⁺-induced mitochondrial swelling and cytochrome c release. Interestingly, ATP lost most of its inhibitory effects on MPT when the experiments were carried out in the presence of ATP-regenerating systems. These results indicate that MPT inhibition observed in the presence of added ATP could be mainly due to hydrolysis of ATP to ADP. From mitochondrial swelling measurements, half-maximal inhibitory values (K _i) of 4.5 and 98 μM were obtained for ADP and ATP, respectively. In addition, a delayed mitochondrial swelling sensitive to higher ADP concentrations was observed. Mitochondrial anoxia/reoxygenation did not interfere with the inhibitory effect of ADP on Ca²⁺-induced MPT, but oxidative phosphorylation markedly decreased this effect. We conclude that ADP is a potent inhibitor of brain MPT whereas ATP is a weaker inhibitor of this phenomenon. Our results suggest that ADP can have an important protective role against MPT-mediated tissue damage under conditions of brain ischemia and hypoglycemia.     

Effects of extramitochondrial ADP on permeability transition of mouse liver mitochondria

Carboxyatractylate (CAT) and atractylate inhibit the mitochondrial adenine nucleotide translocator (ANT) and stimulate the opening of permeability transition pore (PTP). Following pretreatment of mouse liver mitochondria with 5 microM CAT and 75 microM Ca2+, the activity of PTP increased, but addition of 2 mM ADP inhibited the swelling of mitochondria. Extramitochondrial Ca2+ concentration measured with Calcium-Green 5N evidenced that 2 mM ADP did not remarkably decrease the free Ca2+ but the release of Ca2+ from loaded mitochondria was stopped effectively after addition of 2 mM ADP. CAT caused a remarkable decrease of the maximum amount of calcium ions, which can be accumulated by mitochondria. Addition of 2 mM ADP after 5 microM CAT did not change the respiration, but increased the mitochondrial capacity for Ca2+ at more than five times. Bongkrekic acid (BA) had a biphasic effect on PT. In the first minutes 5 microM BA increased the stability of mitochondrial membrane followed by a pronounced opening of PTP too. BA abolished the action about of 1 mM ADP, but was not able to induce swelling of mitochondria in the presence of 2 mM ADP. We conclude that the outer side of inner mitochondrial membrane has a low affinity sensor for ADP, modifying the activity of PTP. The pathophysiological importance of this process could be an endogenous prevention of PT at conditions of energetic depression.     

Arachidonic acid enhances intracellular [Ca2+]i increase and mitochondrial depolarization induced by glutamate in cerebellar granule cells

Maturation of primary neuronal cultures is accompanied by an increase in the proportion of cells that exhibit biphasic increase in free cytoplasmic Ca2+ ([Ca2+]i) followed by synchronic decrease in electrical potential difference across the inner mitochondrial membrane (DeltaPsim) in response to stimulation of glutamate receptors. In the present study we have examined whether the appearance of the second phase of [Ca2+]i change can be attributed to arachidonic acid (AA) release in response to the effect of glutamate (Glu) on neurons. Using primary culture of rat cerebellar granule cells we have investigated the effect of AA (1-20 microM) on [Ca2+]i, DeltaPsim, and [ATP] and changes in these parameters induced by neurotoxic concentrations of Glu (100 microM, 10-40 min). At =10 microM, AA caused insignificant decrease in DeltaPsim without any influence on [Ca2+]i. The mitochondrial ATPase inhibitor oligomycin enhanced AA-induced decrease in DeltaPsim; this suggests that AA may inhibit mitochondrial respiration. Addition of AA during the treatment with Glu resulted in more pronounced augmentation of [Ca2+]i and the decrease in DeltaPsim than the changes in these parameters observed during independent action of AA; removal of Glu did not abolish these changes. An inhibitor of the cyclooxygenase and lipoxygenase pathways of AA metabolism, 5,8,11,14-eicosatetraynoic acid, increased the proportion of neurons characterized by Glu-induced biphasic increase in [Ca2+]i and the decrease in DeltaPsim. Palmitic acid (30 microM) did not increase the percentage of neurons exhibiting biphasic response to Glu. Co-administration of AA and Glu caused 2-3 times more pronounced decrease in ATP concentrations than that observed during the independent effect of AA and Glu. The data suggest that AA may influence the functional state of mitochondria, and these changes may promote biphasic [Ca2+]i and DeltaPsim responses of neurons to the neurotoxic effect of Glu.     

Cyclic AMP-induced Mg2+ release from rat liver hepatocytes, permeabilized hepatocytes, and isolated mitochondria.

The addition of norepinephrine, epinephrine, or forskolin to collagenase-dispersed rat liver hepatocytes increase cAMP and result in a 15% loss in total cell Mg2+ within 5 min. Conversely, carbachol and vasopressin induce a 10-15% increase of total cell Mg2+. Permeabilized hepatocytes also mobilize a large pool of Mg2+ when stimulated by ADP or cAMP. This stimulation is completely inhibited by atractyloside and bongkrekic acid, two different specific inhibitors of the mitochondrial adenine nucleotide translocase. cAMP directly mobilizes Mg2+ efflux from isolated rat liver mitochondria. 50 nM cAMP or 250 microM ADP induces in 5 min a mitochondrial loss of about 6 nmol of Mg2+/mg of protein and a stimulation of ATP efflux. The effect of cAMP is specific, is not reproduced by other cyclic or noncyclic nucleotides, and is inhibited by inhibitors of the adenine nucleotide translocase. These data indicate that cAMP is a messenger for a major mobilization of Mg2+ in hepatocytes. A major target for the effect of cAMP are mitochondria, which lose up to 20-25% of their total Mg2+ in 5 min, both within the cell and after isolation. Evidence is presented suggesting that the adenine nucleotide translocase is the target of the cAMP-dependent Mg2+ efflux and that cAMP may change the operation of the translocase. This, in turn, could change within the matrix the substrate of choice of the translocase from ATP to ATP.Mg.     

Regulation of the specific and nonspecific ion conductivity of mitochondria by adenine nucleotide carriers]

The functioning of the adenine nucleotide carrier as a regulated pore which, depending on incubation conditions, operates as either a specific conductivity channel for H+ and K+ ions, or a nonspecific channel for low molecular weight metabolites, is reviewed. Both specific and nonspecific conductivities over this specific channel are controlled by the conformational state of the carrier protein molecule. The conformational states of the ATP/ADP-carrier alter by the specific effect of adenine nucleotides, ATP/ADP transport inhibitors, Ca2+ ions, medium tonicity, and energization of mitochondria. The transition of the ATP/ADP-carrier to the conformational state characterized by nonspecific conductivity is inhibited by cyclosporin A. The roles of the adenine nucleotide carrier in the realization of effects of Ca(2+)-mobilizing hormones and glucagon during mitochondrial function are discussed.     

Heterogeneity of the calcium-induced permeability transition in isolated non-synaptic brain mitochondria

Calcium overload of neural cell mitochondria plays a key role in excitotoxic and ischemic brain injury. This study tested the hypothesis that brain mitochondria consist of subpopulations with differential sensitivity to calcium-induced inner membrane permeability transition, and that this sensitivity is greatly reduced by physiological levels of adenine nucleotides. Isolated non-synaptosomal rat brain mitochondria were incubated in a potassium-based medium in the absence or presence of ATP or ADP. Measurements were made of medium and intramitochondrial free calcium, light scattering, mitochondrial ultrastructure, and the elemental composition of electron-opaque deposits within mitochondria treated with calcium. In the absence of adenine nucleotides, calcium induced a partial decrease in light scattering, accompanied by three distinct ultrastructural morphologies, including large-amplitude swelling, matrix vacuolization and a normal appearance. In the presence of ATP or ADP the mitochondrial calcium uptake capacity was greatly enhanced and calcium induced an increase rather than a decrease in mitochondrial light scattering. Approximately 10% of the mitochondria appeared damaged and the rest contained electron-dense precipitates that contained calcium, as determined by electron-energy loss spectroscopy. These results indicate that brain mitochondria are heterogeneous in their response to calcium. In the absence of adenine nucleotides, approximately 20% of the mitochondrial population exhibit morphological alterations consistent with activation of the permeability transition, but less than 10% exhibit evidence of osmotic swelling and membrane disruption in the presence of ATP or ADP.     

Net uptake of adenine nucleotides by newborn rat liver mitochondria

In newborn rat liver, the adenine nucleotide content (ATP + ADP + AMP) of mitochondria increases severalfold within 2 to 3 h of birth. The net increase in mitochondrial adenines suggests a novel mechanism by which mitochondria are able to accumulate adenine nucleotides from the cytosol (J. R. Aprille and G. K. Asimakis, 1980, Arch. Biochem. Biophys.201, 564.). This was investigated further in vitro. Isolated newborn liver mitochondria incubated with 1 mM ATP for 10 min at 30 °C doubled their adenine nucleotide content with effects on respiratory functions similar to those observed in vivo: State 3 respiration and adenine translocase activity increased, but uncoupled respiration was unchanged. The mechanism for net uptake of adenine nucleotides was found to be specific for ATP or ADP, but not AMP. Uptake was concentration dependent and saturable. The apparent K_m′s for ATP and ADP were 0.85 ± 0.27 mM and 0.41 ± 0.20 mM, respectively, measured by net uptake of [¹⁴C]ATP or [¹⁴C]ADP. The specific activities of net ATP and ADP uptake averaged 0.332 ± 0.062 and 0.103 ± 0.002 nmol/min/mg protein, respectively. ADP was a competitive inhibitor of net ATP uptake. If P_i was omitted from the incubations, net uptake of ATP or ADP was reduced by 51%. Either mersalyl or N-ethylmaleimide severely inhibited the accumulation of adenine nucleotides. Net ATP uptake was stoichiometrically dependent on MgCl₂, suggesting that Mg²⁺ is accumulated along with ATP (or ADP). Uptake was energy dependent as indicated by the following results: Net AdN uptake (especially ADP uptake) was stimulated by the addition of an oxidizable substrate (glutamate) and inhibited by FCCP (an uncoupler). Antimycin A had no effect on net ATP uptake but inhibited net ADP uptake, suggesting that ATP was able to serve as an energy source for its own accumulation. If carboxyatractyloside was added to inhibit the exchange translocase, thereby preventing rapid access of exogenous ATP to the matrix, net ATP uptake was inhibited; carboxyatractyloside had no effect on ADP uptake. It was concluded that the net uptake of adenine nucleotides from the extramitochondrial space occurs by a specific transport process distinct from the classic adenine nucleotide exchange translocase. The accumulation of adenine nucleotides may regulate matrix reactions which are allosterically affected by adenines or which require adenines as a substrate.     

The role of ADP in the modulation of the calcium-efflux pathway in rat brain mitochondria.

The role of ADP in the regulation of Ca2+ efflux in rat brain mitochondria was investigated. ADP was shown to inhibit Ruthenium-Red-insensitive H+- and Na+-dependent Ca2+-efflux rates if Pi was present, but had no effect in the absence of Pi. The primary effect of ADP is an inhibition of Pi efflux, and therefore it allows the formation of a matrix Ca2+-Pi complex at concentrations above 0.2 mM-Pi and 25 nmol of Ca2+/mg of protein, which maintains a constant free matrix Ca2+ concentration. ADP inhibition of Pi and Ca2+ efflux is nucleotide-specific, since in the presence of oligomycin and an inhibitor of adenylate kinase ATP does not substitute for ADP, is dependent on the amount of ADP present, and requires ADP concentrations in excess of the concentrations of translocase binding sites. Brain mitochondria incubated with 0.2 mM-Pi and ADP showed Ca2+-efflux rates dependent on Ca2+ loads at Ca2+ concentrations below those required for the formation of a Pi-Ca2+ complex, and behaved as perfect cytosolic buffers exclusively at high Ca2+ loads. The possible role of brain mitochondrial Ca2+ in the regulation of the tricarboxylic acid-cycle enzymes and in buffering cytosolic Ca2+ is discussed.     

Cyclosporin A-insensitive permeability transition in brain mitochondria: inhibition by 2-aminoethoxydiphenyl borate

The mitochondrial permeability transition pore (PTP) may operate as a physiological Ca2+ release mechanism and also contribute to mitochondrial deenergization and release of proapoptotic proteins after pathological stress, e.g. ischemia/reperfusion. Brain mitochondria exhibit unique PTP characteristics, including relative resistance to inhibition by cyclosporin A. In this study, we report that 2-aminoethoxydiphenyl borate blocks Ca2+-induced Ca2+ release in isolated, non-synaptosomal rat brain mitochondria in the presence of physiological concentrations of ATP and Mg2+. Ca2+ release was not mediated by the mitochondrial Na+/Ca2+ exchanger or by reversal of the uniporter responsible for energy-dependent Ca2+ uptake. Loss of mitochondrial Ca2+ was accompanied by release of cytochrome c and pyridine nucleotides, indicating an increase in permeability of both the inner and outer mitochondrial membranes. Under these conditions, Ca2+-induced opening of the PTP was not blocked by cyclosporin A, antioxidants, or inhibitors of phospholipase A2 or nitric-oxide synthase but was abolished by pretreatment with bongkrekic acid. These findings indicate that in the presence of adenine nucleotides and Mg2+,Ca2+-induced PTP in non-synaptosomal brain mitochondria exhibits a unique pattern of sensitivity to inhibitors and is particularly responsive to 2-aminoethoxydiphenyl borate.     

Dynamic compartmentation of adenine nucleotides in the mitochondrial intermembrane space of rat-heart mitochondria

To investigate whether or not the mitochondrial intermembrane space together with the extramitochondrial space form a homogeneous pool for adenine nucleotides, rat-heart mitochondria were studied in reconstituted systems with pyruvate kinase and ADP-producing enzymes with varied localization. In the hexokinase system, ADP is produced extramitochondrially by added yeast hexokinase, whereas in the creatine kinase system mitochondrial creatine kinase is responsible for ADP regeneration in the intermembrane space. The dependence of mitochondrial respiration on the extramitochondrial [ATP]/[ADP] ratio in both systems was investigated experimentally and by means of computer simulation. Near the resting state, higher [ATP]/[ADP] ratios were found in the creatine kinase system than in the hexokinase system at the same rate of respiration. This and the maintaining of a substantial creatine kinase-stimulated respiration in the presence of pyruvate kinase in excess is explained by a two-compartment model considering diffusion limitations of adenine nucleotides. A diffusion rate constant of (8.7 +/- 4.7) 10(4) microliters X mg-1 X min-1 for ADP and ATP was estimated, resulting in rate-dependent concentration differences up to 13.7 microM AdN between the extramitochondrial space and the AdN-translocator at the maximum rate of oxidative phosphorylation of rat-heart mitochondria. The results support the assumption that ADP diffusion towards the AdN-translocator is limited if its extramitochondrial concentration is low, resulting in a dynamic compartmentation of adenine nucleotides in the mitochondrial intermembrane space.     

Possible Mechanism for Formation and Regulation of the Palmitate-Induced Cyclosporin A-Insensitive Mitochondrial Pore

The mechanism of the palmitate-induced opening of the mitochondrial Ca2+-dependent cyclosporin A (CsA)-insensitive pore was studied, as well as the influence on this process of well-known modulators of the CsA-sensitive Ca2+-dependent pore. Palmitic acid, which can bind Ca2+ with high affinity, induced the cyclosporin A-insensitive swelling of mitochondria, whereas palmitoleic and 2-bromopalmitic acids, which have no such affinity for Ca2+, failed to induce the pore opening. The palmitate-induced Ca2+-dependent swelling of mitochondria was not affected by a well-known inhibitor of the CsA-sensitive pore (ADP) and an activator of this pore (inorganic phosphate, P(i)). However, this swelling was inhibited by physiological concentrations of ATP ([I]50 = 1.3 mM), but 100 microM ATP increased by 30% the rate of mitochondria swelling if Ca2+ had been added earlier. The effects of ATP (inhibition and activation) manifested themselves from different sides of the inner mitochondrial membrane. Mg2+ inhibited the palmitate-induced Ca2+-dependent swelling of mitochondria with [I]50 = 0.8 mM. It is concluded that palmitic acid induces the opening of the CsA-insensitive pore due to its ability for complexing with Ca2+. A possible mechanism of the pore formation and the influence of some modulators on this process are discussed.     

The effects of Mg2+ and adenine nucleotides on the sensitivity of the heart mitochondrial Na+-Ca2+ carrier to extramitochondrial Ca2+. A study using arsenazo III-loaded mitochondria.

The technique of reversible Ca2+-induced permeabilization [Al Nasser & Crompton (1986) Biochem. J. 239, 19-29, 31-40] has been applied to the preparation of heart mitochondria loaded with the Ca2+ indicator arsenazo III (2 nmol of arsenazo III/mg of mitochondrial protein). The loaded mitochondria ('mitosomes') were used to study the control of the Na+-Ca2+ carrier by extramitochondrial Ca2+ mediated by putative regulatory sites. The Vmax. of the Na+-Ca2+ carrier and the degree of regulatory-site-mediated inhibition were similar to normal heart mitochondria. Ca2+ occupation of the sites in mitosomes yields partial inhibition, which is half-maximal with 0.8 microM external free Ca2+. The inhibition consists of a small decrease in Vmax. and a relatively large increase in apparent Km for internal Ca2+. Mg2+ also appears to interact with the sites, but this is largely abolished by ATP and ADP (but not AMP) under conditions in which the free [Mg2+] is maintained constant. The results indicate that the regulatory sites are effective in controlling the Na+-Ca2+ carrier at physiological concentrations of adenine nucleotides, Mg2+, intra- and extra-mitochondrial free Ca2+.     

Effects of fatty acids in isolated mitochondria: implications for ischemic injury and cardioprotection

Fatty acids accumulate during myocardial ischemia and are implicated in ischemia-reperfusion injury and mitochondrial dysfunction. Because functional recovery after ischemia-reperfusion ultimately depends on the ability of the mitochondria to recover membrane potential (DeltaPsim), we studied the effects of fatty acids on DeltaPsim regulation, cytochrome c release, and Ca2+ handling in isolated mitochondria under conditions that mimicked aspects of ischemia-reperfusion. Long-chain but not short-chain free fatty acids caused a progressive and reversible (with BSA) increase in inner membrane leakiness (proton leak), which limited mitochondrial ability to support DeltaPsim. In comparison, long-chain activated fatty acids promoted 1). a slower depolarization that was not reversible with BSA, 2). cytochrome c loss that was unrelated to permeability transition pore opening, and 3). inhibition of the adenine nucleotide translocator. Together, these results impaired both mitochondrial ATP production and Ca2+ handling. Diazoxide, a selective opener of mitochondrial ATP-dependent potassium (KATP) channels, partially protected against these effects. These findings indicate that long-chain fatty acid accumulation during ischemia-reperfusion may predispose mitochondria to cytochrome c loss and irreversible injury and identify a novel cardioprotective action of diazoxide.     

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.