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Spatial distribution and quantitation of free luminal [Ca] within the InsP 3 ‐sensitive internal store of individual BHK‐21 cells: ion dependence of InsP 3 .induced Ca releaee and reloading
Author(s) -
Hofer Aldebaran M.,
Schlue WolfRüdiger,
Curci Silvana,
Machen Terry E.
Publication year - 1995
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.9.9.7601343
Subject(s) - chemistry , biophysics , thapsigargin , fura 2 , calcium , ryanodine receptor , endoplasmic reticulum , analytical chemistry (journal) , biochemistry , biology , chromatography , cytosol , enzyme , organic chemistry
Free [Ca] within organelles of perme‐abilized BHK‐21 cells was measured using ratio imaging of compartmentalized mag‐fura‐2. In BHK‐21 cells, this dye monitors free [Ca] in principally one type of ATP‐dependent Ca‐sequestering organelle in which intrastore Ca was released uniformly and entirely by 100 nM thapsigargin or removal of ATP or Ca from the bath, and was reduced by 85% upon treatment with a supramaximal dose of InsP 3 (6 μM). Examination of the spatial distribution of InsP 3 ‐sensitive Ca stores showed that InsP 3 released Ca throughout all regions of the cell, although we often noted a perinuclear region (which we speculate may correspond to the Golgi apparatus) with reduced responsiveness to InsP 3 . InsP 3 ‐induced changes of intraluminal Mg could not be detected. Cyclic ADP‐ribose, ryanodine, caffeine, mitochondrial inhibitors, and GTP, agents known to influence intraorganellar Ca sequestration in other cell types, were all without effect on the mag‐fura‐2 ratio. In situ calibration of the mag‐fura‐2 ratio with Ca ionophores revealed that the average free intraorganellar [Ca] was initially 188 ± 21 μM in the presence of 170 nM free Ca and 3 mM ATP, and was reduced to 25 ± 5 μM upon stimulation with 6 μM InsP 3 . The ionic dependence of the release and reloading process was also investigated. The presence of either K., Na, or Cl could consistently support both InsP 3 ‐induced release and the refilling of stores with Ca, but physiological concentrations of HCO 3 were effective in sustaining the response in only 24% of cells examined.—Hofer, A. M., Schlue, W.‐R., Curci, S., Machen, T. E. Spatial distribution and quantitation of free luminal [Ca] within the InsP 3 ‐eensitive internal store of individual BHK‐21 cells: ion dependence of InsP 3 ‐induced Ca release and reloading. FASEB J. 9, 788‐798 (1995)