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Quantification of calcium signal transmission from sarco‐endoplasmic reticulum to the mitochondria
Author(s) -
Pacher Pál,
Csordás György,
Schneider Timothy G.,
Hajnóczky György
Publication year - 2000
Publication title -
the journal of physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.802
H-Index - 240
eISSN - 1469-7793
pISSN - 0022-3751
DOI - 10.1111/j.1469-7793.2000.00553.x
Subject(s) - endoplasmic reticulum , ryanodine receptor , thapsigargin , mitochondrion , cytosol , calcium , calcium signaling , mitochondrial matrix , microbiology and biotechnology , biophysics , inositol trisphosphate receptor , ruthenium red , intracellular , chemistry , biology , biochemistry , receptor , inositol , organic chemistry , enzyme
1 Recent studies have shown that ryanodine and IP 3 receptor (RyR/IP 3 R)‐mediated cytosolic Ca 2+ signals propagate to the mitochondria, initiating chains of events vital in the regulation of different cellular functions. However, the fraction of released Ca 2+ utilized by the mitochondria during these processes has not been quantified. 2 To measure the amount of Ca 2+ taken up by the mitochondria, we used a novel approach that involves simultaneous fluorescence imaging of mitochondrial and cytosolic [Ca 2+ ] in permeabilized H9c2 myotubes and RBL‐2H3 mast cells. Communication between sarco‐endoplasmic reticulum (SR/ER) and mitochondria is maintained in these permeabilized cells, as evidenced by the large RyR/IP 3 R‐driven mitochondrial matrix [Ca 2+ ] and NAD(P)H signals and also by preservation of the morphology of the SR/ER‐mitochondrial junctions. 3 Ca 2+ was released from the SR/ER by addition of saturating caffeine or IP 3 and subsequently thapsigargin (Tg), an inhibitor of SR/ER Ca 2+ pumps. The amount of Ca 2+ transmitted to the mitochondria was determined by measuring increases of global [Ca 2+ ] in the incubation medium (cytosolic [Ca 2+ ] ([Ca 2+ ] c )). Mitochondrial Ca 2+ uptake was calculated from the difference between [Ca 2+ ] c responses recorded in the absence and presence of uncoupler or from [Ca 2+ ] c elevations evoked by uncoupler or ionophore applied after complete Ca 2+ mobilization from the SR/ER. [Ca 2+ ] c increases were calibrated by adding Ca 2+ pulses to the permeabilized cells. 4 In H9c2 cells, caffeine induced partial mobilization of SR Ca 2+ and mitochondria accumulated 26% of the released Ca 2+ . Sequential application of caffeine and Tg elicited complete discharge of SR Ca 2+ without further increase in mitochondrial Ca 2+ uptake. In RBL‐2H3 mast cells, IP 3 by itself elicited complete discharge of the ER Ca 2+ store and the increase of the ionophore‐releasable mitochondrial Ca 2+ content reached 50% of the Ca 2+ amount mobilized by IP 3 + Tg. Thus, RyR/IP 3 R direct a substantial fraction of released Ca 2+ to the mitochondria.