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

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. Using a Ca²⁺ ionophore, ionomycin, to clamp the [Ca²⁺](m) from 0 to 100 μM 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 Ca²⁺ sensitive manner, thereby allowing measurements of [Ca²⁺](m) in single astrocytes. 4. Exposure of fura-2-loaded astrocytes to 100 μM histamine produced a rapid and transient increase in cytosolic Ca²⁺ concentration ([Ca²⁺](m)) 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 Ca²⁺-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.