Mitochondria have a well-established capacity to detect cytoplasmic Ca2+ signals resulting from the discharge of ER Ca2+ stores. Conversely, both the buffering of released Ca2+ and ATP production by mitochondria are predicted to influence ER Ca2+ handling, but this complex exchange has been difficult to assess in situ using conventional measurement techniques. Here we have examined this interaction in single intact BHK-21 cells by monitoring intraluminal ER [Ca2+] directly using trapped fluorescent low-affinity Ca2+ indicators. Treatment with mitochondrial inhibitors (FCCP, antimycin A, oligomycin, and rotenone) dramatically prolonged the refilling of stores after release with bradykinin. This effect was largely due to inhibition of Ca2+ entry pathways at the plasma membrane, but a significant component appears to arise from reduction of SERCA-mediated Ca2+ uptake, possibly as a consequence of ATP depletions in a localized subcellular domain. The rate of bradykinin-induced Ca2+ release was reduced to 51% of control by FCCP. This effect was largely overcome by loading cells with BAPTA-AM, highlighting the importance of mitochondrial Ca2+ buffering in shaping the release kinetics. However, mitochondria-specific ATP production was also a significant determinant of the release dynamic. Our data emphasize the localized nature of the interaction between these organelles, and show that competent mitochondria are essential for generating explosive Ca2+ signals.

/pdf/ca2-homeostasis-in-the-agonist-sensitive-internal-store-386d6ulsyx.pdf

Ca2+ Homeostasis in the Agonist-sensitive Internal Store: Functional Interactions Between Mitochondria and the ER Measured In Situ in Intact Cells

Although we and others have previously shown that newborn central mammalian neurons are more tolerant to anoxia than their adult counterparts, we do not know whether neonatal nerve cells accumulate free cytosolic calcium (Ca2+i) less than adults in response to O2 deprivation. In order to determine whether anoxia increases Ca2+i in adult and neonatal neurons, we monitored calcium in CA1 hippocampal neurons using the calcium-sensitive probe fluo-3 and confocal microscopy. These neurons were studied in the dissociated state in order to study their inherent response to anoxia without the influence of modulatory factors such as synaptic input and neurotransmitters. Severe anoxia caused a rapid increase in Ca2+i in adult CA1 hippocampal neurons, followed by swelling and bleb formation. In neonatal neurons, the latency of this calcium rise was about five times longer than in the adult. Removal of extracellular calcium and addition of calcium channel blockers (Co2+) greatly attenuated the increase in Ca2+i in response to anoxia but did not prevent cell swelling and injury. The addition of glutamate antagonists MK-801 and 6-cyano-7-nitroquinoxaline-2,3-dione did not affect the increase in Ca2+i induced by anoxia. Replacing extracellular sodium with impermeant cations (N-methyl-D-glucamine) prevented anoxia-induced nerve injury. In addition, Ca2+i levels dropped, rather than increased, during the anoxic period in the absence of sodium; Ca2+i returned toward baseline levels upon reoxygenation. Glutamate was also studied, in the presence of O2, to compare with the effects of anoxia. Adult neurons responded with similar latencies to both anoxia and glutamate (10-500 microM). Neonatal neurons, however, exhibited a more rapid increase in Ca2+i in response to glutamate than they did when exposed to anoxia. We conclude (1) that neonatal neurons increase Ca2+i more slowly than adults in response to anoxia and that this response is due to an influx of Ca2+o into the cytosol, (2) that an increase in Ca2+i is not necessary to induce nerve injury when nerve cells are deprived of O2, (3) that the removal of Nao+ prevents cell swelling and blebbing and is associated with a decrease in Ca2+i during anoxia, and finally (4) that glutamate exposure may not be a completely valid model for the direct action of anoxia on neurons.

https://www.jneurosci.org/content/jneuro/13/1/63.full.pdf

Major differences in Ca2+i response to anoxia between neonatal and adult rat CA1 neurons: role of Ca2+o and Na+o

Ca2+ extrusion across plasma membrane and Ca2+ uptake by intracellular stores.

Cyclosporin A protects hepatocytes against prooxidant-induced cell killing. A study on the role of mitochondrial Ca2+ cycling in cytotoxicity.

1. The free Ca2+ concentration of mitochondria ([Ca2+]m) in cultured rat brain astrocytes was measured with a fluorescent Ca2+ indicator, rhod-2, and laser confocal microscopy. 2. Confocal images revealed a rhod-2 distribution that matched mitochondrial localization. 3. Using a Ca2+ ionophore, ionomycin, to clamp the [Ca2+]m from 0 to 100 microM in order to obtain the minimal and maximal fluorescence of rhod-2 in situ, a 3.5 +/- 0.4-fold increase in fluorescence intensity was observed, suggesting that the fluorescence of intramitochondrial rhod-2 was responding in a Ca(2+)-sensitive manner, thereby allowing measurements of [Ca2+]m in single astrocytes. 4. Exposure of fura-2-loaded astrocytes to 100 microM histamine produced a rapid and transient increase in cytosolic Ca2+ concentration ([Ca2+]c) that lasted for several tens of seconds. The spike in [Ca2+]c was frequently followed by variable numbers of repetitive oscillations of Ca2+, which appeared to dampen in amplitude with time. 5. This pattern of histamine-induced [Ca2+]c oscillations was also observed in rhod-2-loaded cells suggesting that [Ca2+]m fluctuated with a similar frequency. 6. The oscillations of [Ca2+]m, but not of [Ca2+]c, were abolished by a proton ionophore, carbonyl cyanide m-chlorophenyl-hydrazone (CCCP), and by Ruthenium Red, a mitochondrial Ca(2+)-uniporter inhibitor. 7. These results suggest that the mitochondrial Ca2+ transport systems in cultured rat brain astrocytes are able to relay receptor-mediated [Ca2+]m oscillations into mitochondria.

https://physoc.onlinelibrary.wiley.com/doi/pdf/10.1113/jphysiol.1996.sp021769

Histamine induces oscillations of mitochondrial free Ca2+ concentration in single cultured rat brain astrocytes.

Measurement of mitochondrial and non-mitochondrial Ca2+ in isolated intact hepatocytes: a critical re-evaluation of the use of mitochondrial inhibitors.

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Measurement of mitochondrial and non-mitochondrial Ca2+ in isolated intact hepatocytes: a critical re-evaluation of the use of mitochondrial inhibitors.