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Massive amounts of Ca2+ can accumulate in mitochondria, owing to its complexation with matrix phosphate. Under conditions in which the mitochondrial uniporter is the foremost pathway for Ca2+ efflux, the release of sequestered Ca2+ by protonophoric uncouplers is invariably demonstrated. This has been recently ascribed to matrix acidification, which results in the dissociation of the Ca2+–phosphate complex. In the present study, we compared the effect of stepwise depolarization on Ca2+ release induced by either the complex III inhibitor stigmatellin or an uncoupler in energized Ca2+-loaded rat liver mitochondria in the presence of phosphate, at extramitochondrial pH (pHo) 6.8 and pHo 7.8. Both poisons were examined in the presence and absence of oligomycin. Prior to Ca2+ 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+/Ca2+ exchange. At both pHo values, ΔpH 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 pHo 6.8 and in the presence of oligomycin, the uncoupler-induced Ca2+ 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 Ca2+ release only in the presence of oligomycin, starting at approximately −4 mV. At pHo 7.8, the uncoupler-induced Ca2+ 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 Ca2+ release, starting at approximately −10 mV. From the above findings, we conclude that matrix acidification cannot be the sole explanation for uncoupler-induced Ca2+ release from mitochondria. [ABSTRACT FROM AUTHOR]

Many physiological processes are controlled by a great diversity of Ca2 + signals that depend on Ca2 + entry into the cell and/or Ca2 + release from internal Ca2 + stores. Ca2 + mobilization from intracellular stores is gated by a family of messengers including inositol-1,4,5-trisphosphate (InsP3), cyclic ADP-ribose (cADPR), and nicotinic acid adenine dinucleotide phosphate (NAADP). There is increasing evidence for a novel intracellular Ca2 + release channel that may be targeted by NAADP and that displays properties distinctly different from the well-characterized InsP3 and ryanodine receptors. These channels appear to localize on a wider range of intracellular organelles, including the acidic Ca2 + stores. Activation of the NAADP-sensitive Ca2 + channels evokes complex changes in cytoplasmic Ca2 + levels by means of channel chatter with other intracellular Ca2 + channels. The recent demonstration of changes in intracellular NAADP levels in response to physiologically relevant extracellular stimuli highlights the significance of NAADP as an important regulator of intracellular Ca2 + signaling. [ABSTRACT FROM AUTHOR]

Cyclophilin d (cypD)-deficient mice exhibit resistance to focal cerebral ischemia and to necrotic but not apoptotic stimuli. To address this disparity, we investigated isolated brain and in situ neuronal and astrocytic mitochondria from cypD-deficient and wild-type mice. Isolated mitochondria were challenged by high Ca2+, and the effects of substrates and respiratory chain inhibitors were evaluated on permeability transition pore opening by light scatter. In situ neuronal and astrocytic mitochondria were visualized by mito-DsRed2 targeting and challenged by calcimycin, and the effects of glucose, NaCN, and an uncoupler were evaluated by measuring mitochondrial volume. In isolated mitochondria, Ca2+ caused a large cypD-dependent change in light scatter in the absence of substrates that was insensitive to Ruthenium red or Ru360. Uniporter inhibitors only partially affected the entry of free Ca2+ in the matrix. Inhibition of complex III/IV negated the effect of substrates, but inhibition of complex I was protective. Mitochondria within neurons and astrocytes exhibited cypD-independent swelling that was dramatically hastened when NaCN and 2-deoxyglucose were present in a glucose-free medium during calcimycin treatment. In the presence of an uncoupler, cypD-deficient astrocytic mitochondria performed better than wild-type mitochondria, whereas the opposite was observed in neurons. Neuronal mitochondria were examined further during glutamate-induced delayed Ca2+ deregulation. CypD-knock-out mitochondria exhibited an absence or a delay in the onset of mitochondrial swelling after glutamate application. Apparently, some conditions involving deenergization render cypD an important modulator of PTP in the brain. These findings could explain why absence of cypD protects against necrotic (deenergized mitochondria), but not apoptotic (energized mitochondria) stimuli. [ABSTRACT FROM AUTHOR]