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

Abstract

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.

Baseline Cytosolic Ca2+ Oscillations Derived from a Non-endoplasmic Reticulum Ca2+Store

“…The reduced passive Ca 2ϩ leak of the Pmr1-containing Ca 2ϩ store in permeabilized COS-1 cells corresponds with observations in LLC-PK 1 epithelial cells, Swiss 3T3 fibroblasts, and L5 rat myoblasts, where the Golgi apparatus was substantially more resistant to Ca 2ϩ depletion than the other regions of the cells (51). The difference in EC 50 for IP 3 ] within organelles in permeabilized BHK-21 cells, which showed a lower responsiveness to IP 3 in the perinuclear Golgi region than in the other regions of the cell (29).…”

A Reassessment of the Effects of Luminal [Ca2+] on Inositol 1,4,5-Trisphosphate-induced Ca2+ Release from Internal Stores

“…The opposite happens when the chelator in the medium is washed out. Therefore, by grading the TPEN concentration in the bath, it is possible to cause graded and reversible decreases of the [Ca 2ϩ ] within the lumen of the store (44,51 (47,50). Fig.…”

Calcium-Containing Organelles Display Unique Reactivity to Chemical Stimulation in Cultured Hippocampal Neurons

“…Other stimuli used include drugs that interact with the known calcium stores (Blatter, 1995;Hofer et al, 1995). Differences between cell types can be expected, and an interaction among calciosomes, the novel calcium compartment seen here, and different calcium stores may be unique to the cell types studied.…”