Spatial distribution and quantitation of free luminal [Ca] within the InsP3-sensitive internal store of individual BHK-21 cells: ion dependence of InsP3-induced Ca release and reloading.
Free [Ca] within organelles of permeabilized 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 InsP3 (6 microM). Examination of the spatial distribution of InsP3-sensitive Ca stores showed that InsP3 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 InsP3. InsP3-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 microM in the presence of 170 nM free Ca and 3 mM ATP, and was reduced to 25 +/- 5 microM upon stimulation with 6 microM InsP3. The ionic dependence of the release and reloading process was also investigated. The presence of either K, Na, or Cl could consistently support both InsP3-induced release and the refilling of stores with Ca, but physiological concentrations of HCO3 were effective in sustaining the response in only 24% of cells examined.
Hofer
A M
AM
Department of Molecular and Cell Biology, University of California, Berkeley 94720, USA.
Schlue
W R
WR
Curci
S
S
Machen
T E
TE
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002451:Cell Compartmentation / Q000187:drug effects*; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D006224:Cricetinae; D005347:Fibroblasts / Q000187:drug effects; D005456:Fluorescent Dyes / Q000378:metabolism; D016257:Fura-2 / Q000031:analogs & derivatives / Q000378:metabolism; D007091:Image Processing, Computer-Assisted; D007295:Inositol Phosphates / Q000494:pharmacology*; D008856:Microscopy, Fluorescence / Q000379:methods*; D015388:Organelles / Q000378:metabolism; D013729:Terpenes / Q000494:pharmacology; D019284:Thapsigargin
D005456:Fluorescent Dyes; D007295:Inositol Phosphates; D013729:Terpenes; C058776:2-(2-(5-carboxy)oxazole)-5-hydroxy-6-aminobenzofuran-N,N,O-triacetic acid; D019284:Thapsigargin; D000255:Adenosine Triphosphate; D002118:Calcium; D016257:Fura-2