Mitochondrial Calcium Dynamics Research Articles

Impaired presynaptic cytosolic and mitochondrial calcium dynamics in aged compared to young adult hippocampal CA1 synapses ameliorated by calcium chelation

Impaired regulation of presynaptic intracellular calcium is thought to adversely affect synaptic plasticity and cognition in the aged brain. We studied presynaptic cytosolic and mitochondrial calcium (Ca) dynamics using axonally loaded Calcium Green-AM and Rhod-2 AM fluorescence respectively in young (2–3 months) and aged (23–26 months) CA3 to CA1 Schaffer collateral excitatory synapses in hippocampal brain slices from Fisher 344 rats. After a tetanus (100 Hz, 200 ms), the presynaptic cytosolic Ca peaked at ∼10 s in the young and ∼12 s in the aged synapses. Administration of the membrane permeant Ca chelator, bis ( O-aminophenoxy)-ethane-N,N,N,N-tetraacetic acid (BAPTA-AM), significantly attenuated the Ca response in the aged slices, but not in the young slices. The presynaptic mitochondrial Ca signal was much slower, peaking at ∼90 s in both young and aged synapses, returning to baseline by 300 s. BAPTA-AM significantly attenuated the mitochondrial calcium signal only in the young synapses. Uncoupling mitochondrial respiration by carbonyl cyanide m-chlorophenylhydrazone (CCCP) application evoked a massive intracellular cytosolic Ca increase and a significant drop of mitochondrial Ca, especially in aged slices wherein the cytosolic Ca signal disappeared after ∼150 s of washout and the mitochondrial Ca signal disappeared after 25 s of washout. These signals were preserved in aged slices by BAPTA-AM. Five minutes of oxygen glucose deprivation (OGD) was associated with a significant increase in cytosolic Ca in both young and aged synapses, which was irreversible in the aged synapses. These responses were significantly attenuated by BAPTA-AM in both the young and aged synapses. These results support the hypothesis that increasing intracellular calcium neuronal buffering in aged rats ameliorates age-related impaired presynaptic Ca regulation.

Carboxyamidotriazole-induced inhibition of mitochondrial calcium import blocks capacitative calcium entry and cell proliferation in HEK-293 cells

Blocking calcium entry may prevent normal and pathological cell proliferation. There is evidence suggesting that molecules such as carboxyamidotriazole, widely used in anti-cancer therapy based on its ability to block calcium entry in nonexcitable cells, also have antiproliferative properties. We found that carboxyamidotriazole and the capacitative calcium entry blocker 2-aminoethoxydiphenyl borate inhibited proliferation in HEK-293 cells with IC50 values of 1.6 and 50 microM, respectively. Capacitative calcium entry is activated as a result of intracellular calcium store depletion. However, non-capacitative calcium entry pathways exist that are independent of store depletion and are activated by arachidonic acid and diacylglycerol, generated subsequent to G protein coupled receptor stimulation. We found that carboxyamidotriazole completely inhibited the capacitative calcium entry and had no effect on the amplitude of arachidonic-acid-activated non-capacitative calcium entry. However, investigation of the effects of carboxyamidotriazole on mitochondrial calcium dynamics induced by carbachol, capacitative calcium entry and exogenously set calcium loads in intact and digitonin-permeabilized cells revealed that carboxyamidotriazole inhibited both calcium entry and mitochondrial calcium uptake in a time-dependent manner. Mitochondrial inner-membrane potential was altered by carboxyamidotriazole treatment, suggesting that carboxyamidotriazole antagonizes mitochondrial calcium import and thus local calcium clearance, which is crucial for the maintenance of capacitative calcium entry.

1PA076 カエルの神経筋接合部シナプス前末端における細胞質内とミトコンドリアのCa^動態

1PA076 カエルの神経筋接合部シナプス前末端における細胞質内とミトコンドリアのCa^<2+>動態

Evidence that angiotensin II decreases mitochondrial calcium in the glomerulosa cell

The present studies were performed using primary monolayer cultures of bovine glomerulosa cells to determine whether the elevation in cytosolic calcium concentration produced by angiotensin II was accompanied by an elevation in mitochondrial calcium. Exchangeable mitochondria calcium content was assessed indirectly by measuring the changes in cytosolic calcium concentration and calcium efflux produced by the mitochondrial uncoupler, carbonyl cyanide m-chlorophenylhydrazone (CCCP). Total mitochondrial calcium content was also assessed directly by atomic absorption spectroscopy. CCCP had a direct effect to promote calcium release from an oligomycin/antimycin-sensitive (mitochondrial) calcium pool in permeabilized cells. In intact cells, CCCP caused rapid reductions in cellular ATP content and the ratio of ATP to ADP. Still, its effects on calcium dynamics were exerted primarily at the mitochondrial level as evidenced by inhibition with ruthenium red, but not dantrolene. As expected, angiotensin II produced a rapid increase in calcium efflux and an equally rapid and sustained increase in cytosolic calcium concentration. Nonetheless, CCCP-stimulated elevations in cytosolic calcium concentration and calcium efflux were reduced by angiotensin II in a concentration-dependent manner. Total mitochondrial calcium content was also lower in angiotensin-treated than in control cells. These results indicate that angiotensin II causes a net decrease in mitochondrial calcium stores. On the basis of these data, it is proposed that alterations in calcium metabolism initiated by angiotensin II are exerted not only at the membrane and cytosolic levels but also at the level of the mitochondria. Changes in mitochondrial calcium dynamics may directly contribute to the regulation of mitochondrial steroidogenic enzymes by angiotensin II.