SPATIAL-DISTRIBUTION AND QUANTITATION OF FREE LUMINAL [CA] WITHIN THEINSP(3)-SENSITIVE INTERNAL STORE OF INDIVIDUAL BHK-21-CELLS - ION DEPENDENCE OF INSP(3)-INDUCED CA RELEASE AND RELOADING

Free [Ga] 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 uniforml y 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 mu M). Examination of the spatial distribution of InsP(3 )-sensitive Ca stores showed that InsP(3) released Ca throughout all r egions of the cell, although we often noted a perinuclear region (whic h we speculate may correspond to the Golgi apparatus) with reduced res ponsiveness to InsP(3). InsP(3)-induced changes of intraluminal Mg cou ld not be detected. Cyclic ADP-ribose, ryanodine, caffeine, mitochondr ial 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 iono phores revealed that the average free intraorganellar [Ca] was initial ly 188 +/- 21 mu M in the presence of 170 nM free Ca and 3 mM ATP, and was reduced to 25 +/- 5 mu M upon stimulation with 6 mu M InsP(3). Th e ionic dependence of the release and reloading process was also inves tigated. The presence of either K, Na, or Cl could consistently suppor t both InsP(3)-induced release and the refilling of stores with Ca, bu t physiological concentrations of HCO3 were effective in sustaining th e response in only 24% of cells examined.