Mode of mitochondrial Ca2+ clearance and its influence on secretory responses in stimulated chromaffin cells

To study the role of mitochondrial Ca2+ clearance in stimulated cells, changes in free Ca2+ concentration in the cytosol, [Ca2+]c and that in mitochondria, [Ca2+]m along with secretory responses were observed using chromaffin cells co-loaded with Fura-2 and Rhod-2 in the perfused rat adrenal medulla. When the cells were stimulated with 40 mM K+ in the perfusate, the duration of [Ca2+]m response markedly increased with prolongation of the stimulation period, exhibiting a mean half-decay time of 21 min with 30 s stimulation, whereas its amplitude was not altered with stimulations of 10–30 s. A computer simulation analysis showed that such a mode of [Ca2+]m response can be produced if excess Ca2+ taken up by mitochondria precipitates as calcium phosphate (Pi) salt. In the presence of 5 μM rotenone plus 10 μM oligomycin, a decrease in the duration of [Ca2+]m response and a slight but significant increase (24%) in the secretory response to 30 s stimulation with 40 mM K+ were observed. Simulation analyses suggested that this effect of rotenone may be due to reduction in mitochondrial Ca2+ uptake induced by rotenone-elicited partial depolarization of the mitochondrial membrane potential. In chromaffin cells transsynaptically stimulated through the splanchnic nerve, the intensity of NAD(P)H autofluorescence changed with time courses similar to those of [Ca2+]m responses. The temporal profiles of those two responses were prolonged in a similar manner by application of an inhibitor of mitochondrial Na+/Ca2+ exchanger, CGP37157. Thus, due to the unique Ca2+ buffering mechanism, [Ca2+]m responses associated with massive mitochondrial Ca2+ uptake may occur within a limited concentration range in which Ca2+-sensitive dehydrogenases are activated to control the mitochondrial redox state in stimulated chromaffin cells.