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. 3. Using a Ca2+ ionophore, ionomycin, to clamp the [Ca2+](m) from 0 to 100 mu M 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 mu M histamine produced a rapid and transient increase in cytosolic Ca2+ concentration ([Ca2+](c)) that lasted for several tens of seconds. The spike in [Ca2+](m) 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.