Simultaneous Measurements of Cytosolic and Mitochondrial Ca2+ Transients in HT29 Cells

Loading of HT 29 cells with the Ca 2+ dye fura-2/AM resulted in an nonhomogeneous intracellular distribution of the dye. Cellular compartments with high fura-2 concentrations were identified by correlation with mitochondrial markers, 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 mitochondrial Ca 2+ activity ([Ca 2+ ] c , [Ca 2+ ] n , and [Ca 2+ ] m , respectively) measured by UV confocal and conventional imaging. Similar results were obtained with a prototype two-photon microscope (Zeiss, Jena, Germany) allowing for fura-2 excitation. The increase of [Ca 2+ ] m lagged behind that of [Ca 2+ ] c and [Ca 2+ ] n by 10–20 s, and after removing the agonist, [Ca 2+ ] m also decreased with a delay. A strong increase of [Ca 2+ ] m occurred only when a certain threshold of [Ca 2+ ] c (around 1 μmol/liter) was exceeded. In a very similar way, ATP, neurotensin, and thapsigargin increased [Ca 2+ ] c and [Ca 2+ ] m . Carbonyl cyanide p -trifluoromethoxyphenylhyrdrazone reversibly reduced the increase of [Ca 2+ ] m . The source of the mitochondrial Ca 2+ increase had intra- and extracellular components, as revealed by experiments in low extracellular Ca 2+ . We conclude that agonist-induced Ca 2+ signals are transduced into mitochondria. 1) Mitochondria could serve as a Ca 2+ sink, 2) mitochondria could allow the modulation of [Ca 2+ ] c and [Ca 2+ ] n signals, and 3) [Ca 2+ ] m may serve as a stimulatory metabolic signal when a cell is highly stimulated.