Simultaneous measurements of cytosolic and mitochondrial Ca2+ transients in HT29 cells

Loading of HT29 cells with the Ca2+ dye fura-2/AM resulted in an nonhomogen eous intracellular distribution of the dye. Cellular compartments with high fura-2 concentrations were identified by correlation with mitochondrial ma rkers, cellular autofluorescence induced by UV, and dynamic measurement of autofluorescence after inhibition of oxidative phosphorylation. Stimulation with carbachol (10(-4) mol/liter) increased cytosolic, nuclear, and mitoch ondrial Ca2+ activity ([Ca2+](c), [Ca2+](n), and [Ca2+](m), respectively) m easured by UV confocal and conventional imaging. Similar results were obtai ned with a prototype two-photon microscope (Zeiss, Jena, Germany) allowing for fura-2 excitation. The increase of [Ca2+](m) lagged behind that of [Ca2 +](c) and [Ca2+](n) by 10-20 s, and after removing the agonist, [Ca2+](m) a lso decreased with a delay. A strong increase of [Ca2+](m) occurred only wh en a certain threshold of [Ca2+](c) (around 1 mu mol/liter) was exceeded. I n a very similar way, ATP, neurotensin, and thapsigargin increased [Ca2+](c ) and [Ca2+](m). Carbonyl cyanide p-trifluoromethoxyphenylhyrdrazone revers ibly reduced the increase of [Ca2+](m). The source of the mitochondrial Ca2 + increase had intra- and extracellular components, as revealed by experime nts in low extracellular Ca2+. We conclude that agonist-induced Ca2+ signal s are transduced into mitochondria, 1) Mitochondria could serve as a Ca2+ s ink, 2) mitochondria could allow the modulation of [Ca2+](c) and [Ca2+](n) signals, and 3) [Ca2+](m) may serve as a stimulatory metabolic signal when a cell is highly stimulated.