Cyclosporin A increases mitochondrial calcium uptake capacity in cortical astrocytes but not cerebellar granule neurons

Abstract

Isolated brain mitochondria are a heterogeneous mixture from different cell types and these subsets may have differing sensitivities to Ca2+-induced membrane permeability transition (MPT) and to inhibition of the MPT by cyclosporin A (CsA). This study tested the hypothesis that mitochondria within primary cultures of astrocytes and neurons exhibit different energy-dependent Ca2+ uptake capacities and different degrees to which CsA increases their uptake capacity. Astrocytes and neurons were suspended in a cytosol-like medium containing respiratory substrates, ATP, and Mg2+ in the presence of digitonin to selectively permeabilize the plasma membrane. Uptake of added Ca2+ by mitochondria within the cells was measured by Calcium Green 5N fluorescent monitoring of the medium [Ca2+]. Permeabilized astrocytes had a fourfold higher Ca2+ uptake capacity, relative to neurons and a twofold higher content based on relative contents of mitochondria assessed by measurements of mitochondrial DNA and cytochrome oxidase subunit 1 protein. In astrocytes the Ca2+ uptake capacity was increased twofold by preincubation with 2-5 microM CsA, while in neurons CsA had no effect. Similar results were obtained using measurements of the effects of added Ca2+ on mitochondrial membrane potential. FK506, a drug similar to CsA but without MPT inhibitory activity, had no effect on either cell type. These results are consistent with the presence of a calcium-induced MPT in astrocytes, even in the presence of ATP, and indicate that the MPT in cerebellar granule neurons is resistant to CsA inhibition. Some of the protective effects of CsA in vivo may therefore be mediated by preservation of mitochondrial functional integrity within astrocytes.

Fig. 1

Calcium uptake in permeabilized astrocytes and neurons in the absence (top traces) or presence (bottom traces) of 2 μM CsA. Extramitochondrial calcium was measured by Calcium Green fluorescence. Arrows indicate additions of CaCl₂. Calcium Green fluorescence increased with each addition of calcium and decreased as the calcium was sequestered by the mitochondria. 10⁷ cells/experiment, 75 nmoles of calcium/addition. Astrocytes took up more calcium/cell than neurons and the uptake capacity of astrocytes, but not neurons, was increased by CsA

Fig. 2

Maximal calcium uptake in astrocytes and neurons in the absence or presence of CsA (2 μM). Bars are Mean ± SEM for 7–9 experiments for astrocytes and 4–5 experiments for neurons. *Significantly different from 0 CsA by t-test, p < 0.01