Blockade of mitochondrial Ca2+ uptake by mitochondrial inhibitors amplifies the glutamate‐induced calcium response in cultured cerebellar granule cells

FEBS Letters

Khodorov, B.²  Yuravichus, A.¹  Khaspekhov, L.³  Pinelis, V.¹  Storozhevykh, T.¹ 

[1] Scientific Centre of Children Health, Russian Academy of Medical Sciences, Moscow, Russia;Institute of General Pathology and Pathophysiology, Russian Academy of Medical Sciences, Moscow, Russia;Institute of Brain Research, Russian Academy of Medical Sciences, Moscow, Russia

关键词: Glutamate;    Intracellular Ca2+;    Mitochondrial inhibitor;    Mitochondrial depolarisation;    Cultured neuron;    [Ca2+]i;    cytosolic Ca2+ concentration;    fura-2/AM;    acetoxymethyl ester of fura-2;    fura-2FF/AM;    acetoxymethyl ester of fura-2FF;    ARA-C;    cytosine arabinoside;    HEPES;    4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid;    Glu;    glutamate;    Mem;    memantine;    MEM;    minimum essential medium;    MI;    mitochondrial inhibitors;    FCCP;    carbonyl cyanide p-(tri-fluoromethoxy) phenyl-hydrazone;    Rot;    rotenone;    Oli;    oligomycin;    HBSS;    HEPES-buffered salt solution;    V mit;    mitochondrial potential;    MCU;    mitochondrial calcium uptake;    MD;    mitochondrial depolarisation;   

DOI  :  10.1016/S0014-5793(99)01130-8

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

The objective of this study was to evaluate the role of mitochondrial Ca²⁺ uptake (MCU) in modulation (shaping) of the glutamate (Glu)-induced changes in neuronal cytoplasmic Ca²⁺ ([Ca²⁺]_i). In order to block MCU, nerve cells were treated with mitochondrial inhibitors (MI) inducing collapse of the mitochondrial potential (ΔΨ _m). Measurements of changes in [Ca²⁺]_i were performed using either the low-affinity (fura-2FF) or high-affinity (fura-2) Ca²⁺ indicators. Loading of nerve cells with rhodamine 123 made it possible to monitor changes in ΔΨ _m. In the first series of experiments it was shown that blockade of MCU in fura-2FF-loaded cells with a cocktail of rotenone (2 μM)+oligomycin (2.5 μg/ml) greatly (2.53±0.4 times, n=61) increased the [Ca²⁺]_i response to a 1-min Glu (100 μM) pulse. In fura-2-loaded cells, this increase was small (less than 1.3 times) or absent. In the second series of experiments, cocktails of rotenone+oligomycin or FCCP (1 μM)+oligomycin were applied during a prolonged Glu application. This produced strong mitochondrial depolarisation and an additional [Ca²⁺]_i increase. In most cells the latter could be reversed or prevented by a removal of external Ca²⁺. The MI-induced additional [Ca²⁺]_i increase was especially pronounced in cells loaded with fura-2FF. In some neurones a removal of external Ca²⁺ did not produce a decrease in [Ca²⁺]_i during combined Glu+MI application, suggesting an impairment of [Ca²⁺]_i extrusion mechanisms of these cells. The conclusion is drawn that MCU makes a considerable contribution to regulation of [Ca²⁺]_i responses caused by Ca²⁺ influx via Glu-activated ionic channels. The reasons for a quantitative difference between [Ca²⁺]_i responses observed in fura-2- and fura-2FF-loaded neurones are discussed.

【 授权许可】

Unknown