Thapsigargin demonstrates calcium-dependent regulation of phosphate uptake in HeLa cells.
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.
Middleton, JP; Albers, FJ; Dennis, VW; Raymond, JR
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