Role of Phosphatidylinositol 3 kinase (PI3‐kinase) in Regulation of Apical Membrane Expression of the Type IIa Sodium‐Phosphate Cotransporter (Npt2a)

Renal proximal tubule phosphate transport is dependent on apical membrane expression of Npt2a. Previous studies have suggested a role for PI3 kinase in endocytosis and exocytosis. We tested the hypothesis that PI3 kinase is required for apical membrane insertion of Npt2a by examining the effect of PI3 kinase inhibition and activation on Npt2a expression and phosphate uptake in opossum kidney (OK) cells, a model of renal proximal tubule. Treatment with the PI3 kinase inhibitors LY 294002 (LY) or wortmannin produced a time and dose dependent inhibition of phosphate uptake, 40–50% max after 24 h. Western blot showed that LY decreased Npt2a expression by 20%. PTH, a potent downregulator of Npt2a, did not further decrease phosphate uptake after maximal inhibition with LY but did so after submaximal inhibition by LY. Pretreatment with LY blunted the increase in phosphate uptake in response to incubation in low phosphate medium but did not block recovery of Npt2a expression. To determine the effect of PI3 kinase activation, OK cells were treated with products of PI3‐kinase activation, phosphatidylinositol 2 phosphate (PIP2) and phosphatidylinositol 3 phosphate (PIP3). PIP3 but not PIP2 increased apical membrane expression of Npt2a as shown by confocal microscopy. Treatment of the cells with the Golgi poison Brefeldin A followed by treatment with PIP3 blocked PIP3‐stimulated apical membrane expression of Npt2a. We conclude 1) PI3 kinase is required for apical membrane insertion of Npt2a but not for PTH‐mediated endocytosis; 2) PI3 kinase may play a role in membrane retention of Npt2a; 3) PI3 kinase effect is distal to the Golgi.