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 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 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 mu M). 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 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). 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 HCO3 were effective in sustaining the response in only 24% of cells examined.