A novel method exploiting the differential affinity of ADP and ATP to Mg(2+) was developed to measure mitochondrial ADP-ATP exchange rate. The rate of ATP appearing in the medium after addition of ADP to energized mitochondria, is calculated from the measured rate of change in free extramitochondrial [Mg(2+)] reported by the membrane-impermeable 5K(+) salt of the Mg(2+)-sensitive fluorescent indicator, Magnesium Green, using standard binding equations. The assay is designed such that the adenine nucleotide translocase (ANT) is the sole mediator of changes in [Mg(2+)] in the extramitochondrial volume, as a result of ADP-ATP exchange. We also provide data on the dependence of ATP efflux rate within the 6.8-7.8 matrix pH range as a function of membrane potential. Finally, by comparing the ATP-ADP steady-state exchange rate to the amount of the ANT in rat brain synaptic, brain nonsynaptic, heart and liver mitochondria, we provide molecular turnover numbers for the known ANT isotypes.

In pathological conditions, F(0)F(1)-ATPase hydrolyzes ATP in an attempt to maintain mitochondrial membrane potential. Using thermodynamic assumptions and computer modeling, we established that mitochondrial membrane potential can be more negative than the reversal potential of the adenine nucleotide translocase (ANT) but more positive than that of the F(0)F(1)-ATPase. Experiments on isolated mitochondria demonstrated that, when the electron transport chain is compromised, the F(0)F(1)-ATPase reverses, and the membrane potential is maintained as long as matrix substrate-level phosphorylation is functional, without a concomitant reversal of the ANT. Consistently, no cytosolic ATP consumption was observed using plasmalemmal K(ATP) channels as cytosolic ATP biosensors in cultured neurons, in which their in situ mitochondria were compromised by respiratory chain inhibitors. This finding was further corroborated by quantitative measurements of mitochondrial membrane potential, oxygen consumption, and extracellular acidification rates, indicating nonreversal of ANT of compromised in situ neuronal and astrocytic mitochondria; and by bioluminescence ATP measurements in COS-7 cells transfected with cytosolic- or nuclear-targeted luciferases and treated with mitochondrial respiratory chain inhibitors in the presence of glycolytic plus mitochondrial vs. only mitochondrial substrates. Our findings imply the possibility of a rescue mechanism that is protecting against cytosolic/nuclear ATP depletion under pathological conditions involving impaired respiration. This mechanism comes into play when mitochondria respire on substrates that support matrix substrate-level phosphorylation.

NAADP (nicotinic acid-adenine dinucleotide phosphate) is fast emerging as a new intracellular Ca2+-mobilizing messenger. NAADP induces Ca2+ release by a mechanism that is distinct from IP3 (inositol 1,4,5-trisphosphate)- and cADPR (cADP-ribose)-induced Ca2+ release. In the present study, we demonstrated that micromolar concentrations of NAADP trigger Ca2+ release from rat hepatocyte microsomes. Cross-desensitization to IP3 and cADPR by NAADP did not occur in liver microsomes. We report that non-activating concentrations of NAADP can fully inactivate the NAADP-sensitive Ca2+-release mechanism in hepatocyte microsomes. The ability of thapsigargin to block the NAADP-sensitive Ca2+ release is not observed in sea-urchin eggs or in intact mammalian cells. In contrast with the Ca2+ release induced by IP3 and cADPR, the Ca2+ release induced by NAADP was completely independent of the free extravesicular Ca2+ concentration and pH (in the range 6.4-7.8). The NAADP-elicited Ca2+ release cannot be blocked by the inhibitors of the IP3 receptors and the ryanodine receptor. On the other hand, verapamil and diltiazem do inhibit the NAADP- (but not IP3- or cADPR-) induced Ca2+ release.

One of the major roles of mitochondria in cellular signal transduction is the sequestration of Ca 2+ and its controlled release to the cytosol. This is achieved by the concerted action of various influx and efflux pathways located at the inner mitochondrial membrane, such as the uniporter, the permeability transition pore (PTP), the Na + ⁄ Ca 2+ exchanger, diacylglycerol-sensitive cationic channel(s), and other, less well-character- 2+ loading, mitochondria were allowed to phosphorylate 0.5 mm ADP. Opening of the permeability transition pore was additionally hampered by cyclosporin A, and was monitored by changes in light scattering. Na + was excluded from the medium, preventing Na + ⁄ Ca 2+ exchange. At both pH o values, DpH was in the range 0.11-0.15. Complete depolarization by uncoupling with or without oligomycin resulted in an approximately pH 0.05 acidic shift, but there was none in the case of stigmatellin plus oligomycin. At pH o 6.8 and in the presence of oligomycin, the uncoupler-induced Ca 2+ release started in the )80 to )50 mV range, whereas in the absence of oligomycin, the release occurred at approximately )15 mV. Stigmatellin induced minor Ca 2+ release only in the presence of oligomycin, starting at approximately )4 mV. At pH o 7.8, the uncoupler-induced Ca 2+ release started at approximately )11 mV, irrespective of the presence or absence of oligomycin. Unexpectedly, at this alkaline pH and in the presence of oligomycin, stigmatellin induced substantial Ca 2+ release, starting at approximately )10 mV. From the above findings, we conclude that matrix acidification cannot be the sole explanation for uncoupler-induced Ca 2+ release from mitochondria.Abbreviations ANT, adenine nucleotide translocase; AP 5 A, diadenosine pentaphosphate; BCECF, 2¢,7¢-bis(carboxyethyl)-5,6-carboxyfluorescein; BCECF-AM, 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein-acetoxymethyl ester; CaGr-5N, Calcium Green 5N; nBM, n-butyl-malonate; pH in , mitochondrial matrix pH; pH o , extramitochondrial pH; PTP, permeability transition pore; SF 6847, tyrphostin 9, RG-50872, malonaben, 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile, 2,6-di-t-butyl-4-(2',2'-dicyanovinyl)phenol.