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J Biol Chem, Vol. 273, Issue 52, 34961-34969, December 25, 1998
From the Physiologisches Institut der
Albert-Ludwigs-Universität Freiburg, Hermann-Herder-Strasse
7, D-79104 Freiburg, Germany
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 µmol/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.
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