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 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 Ca2+ activity ([Ca2+]c, [Ca2+]n, and [Ca2+]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 [Ca2+]m lagged behind that of [Ca2+]c and [Ca2+]n by 10-20 s, and after removing the agonist, [Ca2+]m also decreased with a delay. A strong increase of [Ca2+]m occurred only when a certain threshold of [Ca2+]c (around 1 micromol/liter) was exceeded. In a very similar way, ATP, neurotensin, and thapsigargin increased [Ca2+]c and [Ca2+]m. Carbonyl cyanide p-trifluoromethoxyphenylhyrdrazone reversibly reduced the increase of [Ca2+]m. The source of the mitochondrial Ca2+ increase had intra- and extracellular components, as revealed by experiments in low extracellular Ca2+. We conclude that agonist-induced Ca2+ signals are transduced into mitochondria. 1) Mitochondria could serve as a Ca2+ sink, 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.