A TP is the primary utilizable source of high-energy phosphate bonds within the cell and acts as an allosteric effector of numerous cell processes. Most intracellular ATP is derived from cytosolic glycolysis and mitochondrial oxidative phosphorylation. The latter process couples the oxidation of reduced cofactors via the respiratory chain to ATP synthesis by mitochondrial ATP synthase. The supply of reduced cofactors (NADH, FADH 2 ) is ensured by mitochondrial oxidation of substrates derived from glucose, fatty acids, and amino acids via different metabolic pathways. Therefore, oxidative phosphorylation is a complex process regulated at different levels by the interactions of mitochondrial and cytosolic metabolism (1). In this crosstalk, mitochondrial Ca 2ϩ homeostasis, a process that has attracted a large interest in the past few years (2-11), appears to play a major role. Indeed, three dehydrogenases of the Krebs cycle (pyruvate, isocitrate, and ␣-ketoglutarate dehydrogenase) are modulated by [Ca 2ϩ ] in the micromolar range (12, 13). Recently, it has been shown that, despite the low affinity of the mitochondrial Ca 2ϩ uptake systems, large increases in matrix [Ca 2ϩ ] parallel the cytosolic Ca 2ϩ signals, thanks to the close contact between mitochondria and the intracellular Ca 2ϩ stores (14). The final outcome of this Ca 2ϩ transfer is expected to be the enhancement of mitochondrial ATP production to balance the increased ATP demand of a stimulated cell (15)(16)(17)(18).In this paper, we directly addressed these issues by investigating, in intact living cells, the effect of cytosolic and mitochondrial Ca 2ϩ signaling on intramitochondrial ATP concentration. For this purpose, we have utilized a targeted recombinant Ca 2ϩ probe (mitochondrial aequorin, mtAEQ) (2) and constructed a tool, a specifically targeted chimera of the ATP-sensitive photoprotein luciferase, with the aim of dynamically monitoring the ATP concentration in the mitochondrial matrix ([ATP] m ). The basis for this approach to the measurement of this key cellular parameter was the observation that the affinity of luciferase for ATP, which in vitro is in the micromolar range, is drastically lower in vivo, presumably because of the presence of competing proteins and anions (19). Indeed, in the cellular environment and in the presence of luciferin, luciferase light emission is a linear function of [ATP] in a concentration range between 10 Ϫ3 and 10 Ϫ2 M, i.e., in the physiological range (20,21). We thus constructed a chimeric cDNA, which allows the selective targeting of luciferase to the mitochondrial matrix. With this tool, we could not only directly demonstrate that agonist-dependent changes in mitochondrial Ca 2ϩ concentration correlate with an enhancement in mitochondrial ATP concentration, provided that oxidizable substrates are available, but also identify a phenomenon of long-term memory, allowing a prolonged metabolic ''priming'' that lasts longer than the mitochondrial [Ca 2ϩ ] increase. These observations may clarify how mitoch...

We report Ca2(+)-induced release of Ca2+ from mitochondria (mCICR) dependent on transitory opening of the permeability transition pore (PTP) operating in a low conductance mode. The Ca2+ fluxes taking place during mCICR are a direct consequence of the mitochondrial depolarization spike (mDPS) caused by PTP opening. Both mDPS and mCICR can propagate from one mitochondrion to another in vitro, generating traveling depolarization and Ca2+ waves. Mitochondria thus appear to be excitable organelles capable of generating and conveying electrical and Ca2+ signals. In living cells, mDPS/mCICR is triggered during IP3-induced Ca2+ mobilization and results in the amplification of the Ca2+ signals primarily emitted from the endoplasmic reticulum.

Increases in the concentration of free ATP within the islet ␤-cell may couple elevations in blood glucose to insulin release by closing ATP-sensitive K ؉ (K ATP ) channels and activating Ca 2؉ influx. Here, we use recombinant targeted luciferases and photon counting imaging to monitor changes in free [ATP] Increases in extracellular glucose concentration stimulate the exocytosis of insulin from islet ␤-cells. This is probably achieved by an increase in glycolysis and flux through the citrate cycle (1), leading to elevated intracellular levels of likely coupling factors (2), including ATP. Closure of ATP-sensitive K ϩ channels (3-5) then leads to plasma membrane depolarization and the influx of Ca 2ϩ through voltage gated Ca 2ϩ channels. Increases in the total intracellular concentration of ATP have been measured in isolated islets (6) and cell lines (7) exposed to increases in extracellular glucose concentration. However, the measured changes are generally small and difficult to interpret because of the large depot of intragranular ATP, and the presence of non-␤-cells (8). Furthermore, such measurements give no indication of the concentration of unbound ATP. Unfortunately, measurements of free [ATP] in living cells, for example by 31 P NMR (9), cannot easily be extended to the islet micro-organ and do not provide sufficient sensitivity to detect changes at the cellular or subcellular level. This is an important question because differences in [ATP] at different intracellular sites have been predicted. In particular, locally high ATP consumption by the plasma membrane Na ϩ -K ϩ and Ca 2ϩ -ATPase, may mean that [ATP] is lower in this domain than in the bulk of the cell cytosol (1). Similarly, the electrogenic nature of the mitochondrial ATP/ADP translocase (10) is predicted to create differences in ATP/ADP ratio across the inner mitochondrial membrane (cytosolic high).The role of changes in free Ca 2ϩ ion concentration ([Ca 2ϩ ]) in regulating ␤-cell metabolism and ATP concentration is controversial. Increases in [Ca 2ϩ ] following plasma membrane depolarization act both to stimulate ATP requiring processes (i.e. secretory granule movement and exocytosis) (11) and possibly to enhance mitochondrial oxidative metabolism (12, 13). Recent measurements of total ATP content of whole islets have suggested that the former may dominate and that Ca 2ϩ influx may diminish glucose-induced increases in ATP/ADP ratio (14).The use of firefly luciferase, targeted to discrete intracellular domains, should provide an extremely sensitive method of monitoring free [ATP] dynamically and at the subcellular level. In previous studies, we have shown that photon counting imaging of total luciferase activity in single cells provides a convenient means to measure changes in gene expression in single cells (15,16). Luciferase has previously been employed to measure intracellular ATP concentration in single cardiac myocytes (17) and hepatocytes (18), but only after microinjection of the purified protein. In recent reports, Maechler...

In Xenopus oocytes, as well as other cells, inositol-1,4,5-trisphosphate (Ins(1,4,5)P3)-induced Ca2+ release is an excitable process that generates propagating Ca2+ waves that annihilate upon collision. The fundamental property responsible for excitability appears to be the Ca2+ dependency of the Ins(1,4,5)P3 receptor. Here we report that Ins(1,4,5)P3-induced Ca2+ wave activity is strengthened by oxidizable substrates that energize mitochondria, increasing Ca2+ wave amplitude, velocity and interwave period. The effects of pyruvate/malate are blocked by ruthenium red at the Ca2+ uniporter, by rotenone at complex I, and by antimycin A at complex III, and are subsequently rescued at complex IV by ascorbate tetramethylphenylenediamine (TMPD). Our data reveal that potential-driven mitochondrial Ca2+ uptake is a major factor in the regulation of Ins(1,4,5)P3-induced Ca2+ release and clearly demonstrate a physiological role of mitochondria in intracellular Ca2+ signalling.