AM Hofer, WR Schlue, S Curci, and TE Machen (1995). Spatial distribution and quantitation of free luminal [Ca] within the IP3-sensitive internal store of individual BHK-21 cells: ion dependence of IP3-induced Ca release and reloading. FASEB Journal. 9, 788-798.
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.