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Am J Physiol Renal Physiol 251: F690-F701, 1986;
0363-6127/86 $5.00
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AJP - Renal Physiology, Vol 251, Issue 4 690-F701, Copyright © 1986 by American Physiological Society


ARTICLES

Regulation of cytosolic free calcium concentration in cultured renal epithelial cells

J. Y. Cheung, J. M. Constantine and J. V. Bonventre

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.


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