Multiple intracellular Ca2+ pools exist in human foreskin fibroblast cells: the effect of BK on release and filling of the non-cytosolic Ca2+ pools.
Previously, we have used the classical approach to examine intracellular calcium stores in human foreskin fibroblasts (HSWP) cells. In this classical protocol cells are first permeabilized and then allowed to fill their Ca2+ reservoirs with 45Ca2+ in the presence of ATP. In this paper we present an alternative method to examine intracellular calcium pools. In this alternate protocol, whole cells are loaded to isotopic steady-state with 45Ca2+ and then permeabilized using digitonin. Comparison of the Ca2+ content of cells treated with these two methodologies reveals that cells treated with the alternate protocol have a Ca2+ content 3 orders of magnitude higher than those treated with the classical protocol. Using this alternative technique we demonstrate that there are 3 intracellular calcium pools in HSWP cells. These pools are: (i) an IP3-sensitive, thapsigargin-sensitive Ca2+ pool; (ii) an IP3-insensitive, thapsigargin-sensitive Ca2+ pool; and (iii) an ionomycin sensitive Ca2+ pool. The relationship between the Ca2+ pool mobilized by BK treatment and by IP3 treatment is also explored. Microinjection data shown here suggest that IP3 can mobilize all of the intracellular Ca2+ mobilized by BK. However, in the permeabilized system BK pretreatment followed by IP3 treatment can release more Ca2+ than can be release by IP3 treatment alone. We suggest one plausible explanation for this observation: when cells are treated using the alternative permeabilization protocol, communication occurs between an IP3-sensitive and an IP3-insensitive calcium pool. Thus BK pretreatment would empty the IP3-sensitive Ca2+ pool. This pool would subsequently be refilled with Ca2+ from a previously untapped, IP3-insensitive, Ca2+ reservoir and more Ca2+ would be available for subsequent release by IP3 treatment.
Baumgarten
L B
LB
Department of Physiology and Biophysics, University of Illinois at Chicago, USA.
Lee
H C
HC
Villereal
M L
ML
D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000494:pharmacology; D007424:Intracellular Fluid / Q000187:drug effects / Q000378:metabolism*; D017136:Ion Transport / Q000187:drug effects; D007609:Kallidin / Q000494:pharmacology; D008297:Male; D013729:Terpenes / Q000494:pharmacology; D019284:Thapsigargin
D004791:Enzyme Inhibitors; D013729:Terpenes; D007609:Kallidin; D019284:Thapsigargin; D015544:Inositol 1,4,5-Trisphosphate; D000252:Calcium-Transporting ATPases; D002118:Calcium