Regulation of cytosolic free calcium concentration in cultured renal epithelial cells.
The relative contribution by intracellular organelles to the regulation of cytosolic free Ca2+ concentration was investigated in isolated cultured renal LLC-PK1 cells. Exchangeable mitochondrial and nonmitochondrial Ca2+ represented 10 and 50% of total cell Ca2+ content, respectively. In the absence of Mg2+, Ca2+ added to cells made permeable with digitonin was rapidly sequestered by the mitochondria so that basal cytosolic free Ca2+ concentration (approximately 100 nM) was reestablished within minutes. Provision of Mg2+ and ATP resulted in biphasic Ca2+ buffering behavior. A slow, low-capacity system buffered exogenous Ca2+ to a steady-state level of 50-300 nM. Ca2+ buffering at these low Ca2+ concentrations was likely by the endoplasmic reticulum, since vanadate but not ruthenium red blocked the Ca2+ buffering ability. After the endoplasmic reticulum was saturated with Ca2+, exogenous Ca2+ was buffered by a fast, high-capacity organelle to a new steady-state level of 600-1,500 nM. The high-capacity system was identified as the mitochondria, since ruthenium red but not vanadate abolished Ca2+ buffering and resulted in release of previously sequestered Ca2+. Addition of inositol-1,4,5-trisphosphate (IP3) resulted in rapid release from and reuptake of Ca2+ by the endoplasmic reticulum in digitonin-treated cells. We conclude that, under physiological conditions, most of the cell Ca2+ is stored in endoplasmic reticulum which is the functionally important organelle in buffering small changes in Ca2+ at the resting cytosolic free Ca2+ level. The mitochondrial compartment represents a high-capacity Ca2+ buffering system that may serve important physiological functions when large loads of Ca2+ are presented to the cell. In addition, IP3 mobilizes Ca2+ stored in the endoplasmic reticulum and may serve as the intracellular messenger to raise cytosolic free Ca2+ concentration transiently in response to stimuli.
D002118:Calcium / Q000378:metabolism*; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism*; D004847:Epithelial Cells; D004848:Epithelium / Q000378:metabolism; D015544:Inositol 1,4,5-Trisphosphate; D007295:Inositol Phosphates / Q000494:pharmacology; D007668:Kidney / Q000166:cytology / Q000378:metabolism*; D009994:Osmolar Concentration; D014357:Trypsin / Q000494:pharmacology
D007295:Inositol Phosphates; D015544:Inositol 1,4,5-Trisphosphate; D014357:Trypsin; D002118:Calcium