| The cytoplasmic Ca2+ signal is transferred to the mitochondrial matrix and activates mitochondrial dehydrogenases. The requirement for supramicromolar [Ca2+]c in perimitochondrial microdomains in this response has been suggested. In the first part of our work, we studied the mitochondrial calcium homeostasis in digitonin-permeabilized rat glomerulosa, insulinoma (INS-1/EK3) and human osteosarcoma (143B) cells, preventing the formation of high [Ca2+]c microdomains. Changes in [Ca2+]m were monitored using either the fluorescent dye rhod-2 or the bioluminescent probe aequorin. To compare the results obtained by these two methods, we applied in situ calibration for the signals and found that mt-aequorin luminescence in these cells shows one---one and a half order of magnitude higher calcium sensitivity, compared to the calibration equations obtained in vitro and used extensively in the literature. Using our in situ calibration curve, the two methods yielded compatible results. In all three cell types, step-by-step elevations of [Ca2+]c in the submicromolar range were followed by higher and higher steady-state [Ca2+] m elevations. In rat glomerulosa cells, the biological significance of the observed [Ca2+]m elevations was indicated by the parallel increase in mitochondrial reduced pyridine nucleotide (NAD(P)H) autofluorescence.; In the second part of our work, we studied the effect of two calcium mobilizing agonist, PGF2alpha and ATP on rat luteal cells. Our inicial finding was that although these agonists led to similar global cytoplasmic calcium signal, PGF2alpha evoked significantly larger increase in mitochondrial NAD(P)H. Similar results were found in the presence of the respiratory chain inhibitor rotenon. Using confocal microscopy and a custom algorithm evaluating the mean response of all the mitochondria measured, we could not find difference in the propagation into the mitochondrial matrix of Ca2+ signals evoked by PGF 2alpha and ATP. To study the non-[Ca2+]m mediated effects of the agonist, we compared the mitochondrial NAD(P)H elevations in calcium-ionophore treated cells at identical, permissive [Ca2+] c elevations in the presence or absence of the agonists, and found that in addition to the calcium-dependent activation of mitochondrial dehydrogenases shared by both agonists, PGF2alpha potentiates the Ca2+-induced generation of NAD(P)H by a non-Ca2+ mediated mechanism. |
