Sodium-dependent transport of Pi by an established intestinal epithelial cell line (CaCo-2)

Abstract

The uptake of inorganic phosphate (Pi) was analyzed in monolayers and in apical membrane vesicles (AMV) of the established intestinal cell line CaCo-2. AMV, prepared by a MgCl2 precipitation technique, were enriched approximately 10-fold in alkaline phosphatase activity. Pi uptake into intact cells as well as into AMV was specifically dependent on the presence of sodium. In the presence of high sodium concentrations, the apparent Km for Pi was 214 +/- 17 mumol/l in monolayers and 300 +/- 19.7 mumol/l in AMV. Increasing the sodium concentration increased the apparent affinity of the transport system for Pi but hardly affected the maximal velocity (Vmax). At 0.1 mmol/l Pi and pH 7.4, the apparent Km for sodium was approximately 70 mmol/l in intact cells as well as in AMV. The results obtained in both systems suggested the involvement of two sodium ions and one phosphate ion in the transport process. Advancing confluence--independently of the age of the monolayers--reduced sodium-dependent uptake of Pi significantly by a decrease in Vmax, whereas the apparent Km for Pi remained unchanged. It is concluded that the apical membrane of CaCo-2 cells contains a sodium-dependent transport system for Pi.