AJP - Renal Physiology, Vol 259, Issue 4 727-F731, Copyright © 1990 by American Physiological Society
Thapsigargin demonstrates calcium-dependent regulation of phosphate uptake in HeLa cells
J. P. Middleton, F. J. Albers, V. W. Dennis and J. R. Raymond
Division of Nephrology, Duke University Medical Center, Durham, North Carolina.
We used thapsigargin, a sesquiterpene lactone that mobilizes intracellular
Ca without increases in inositol phosphates or major activation of protein
kinase C (PKC), to test the specific effects of increasing cytosolic Ca on
Na-dependent phosphate uptake in HeLa cells. Thapsigargin increased the
Vmax for phosphate uptake from 5.40 +/- 0.26 to 7.86 +/- 0.43 nmol.mg
protein-1.3 min-1 (n = 7, P less than 0.001) without change in the apparent
Km for phosphate, which averaged 0.15 +/- 0.02 mM. The effect of
thapsigargin was dependent on concentration and time. Inactivation of PKC
by overnight exposure to 16 microM phorbol 12,13-dibutyrate did not
eliminate the effect of thapsigargin, although it completely abolished the
effects of phorbol ester on phosphate uptake. Thus thapsigargin are not
dependent on PKC. As in other cell systems, thapsigargin increased
cytosolic Ca concentration. Removal of extracellular Ca diminished the
increase in cytosolic Ca and eliminated the effect of thapsigargin on
phosphate uptake. Collectively, our data indicate that Na-dependent
phosphate uptake in HeLa cells can be regulated by at least three specific
signaling pathways: protein kinase A, PKC, and increased cytosolic Ca.
