NaPO4 cotransport type III (PiT1) expression in human embryonic kidney cells and regulation by PTH

The aim of the present study was to characterize the type(s) of NaPO4 cotra nsporter expressed in the human renal cell line HEK-293 and its regulation by parathyroid hormone (PTH) in wild-type cells and in cells transfected by the PTH/PTH-related protein (PTHrP) receptor. The results showed that huma n embryonic kidney HEK-293 cells expressed NaPO4 cotransporter type III (Pi T1) mRNA and protein. In contrast, type I (NPT1) or II (NPT2) cotransporter mRNA were not expressed. Nai-dependent phosphate uptake followed a Michael is-Menten model (apparent maximal transport rate and affinity constant: 23. 32 +/- 0.69 nmol PO4.mg protein(-1).10 min(-1) and 0.147 +/- 0.014 mM KH2PO 4, respectively), was stimulated by phosphate deprivation (maximal increase 24.5 +/- 0.8%, P < 0.001, after 15 h of phosphate deprivation), and was in hibited by increasing pH (3.6 +/- 0.2-fold decrease at pH 8.5, P < 0.0001). It was inhibited in a time- and concentration-dependent fashion by PTH in HEK-293 cells stably transfected by PTH/PTHrP receptors but not in parental HEK-293 cells. Maximal inhibition of Na+-dependent phosphate transport was observed at 30 min after the addition of 72 nM PTH-(1-34) (31.5 +/- 2.4% i nhibition, P < 0.01). PTH inhibition of phosphate transport was maintained in phosphate-deprived cells and reversed by both GF109203X (10(-6) M) or st aurosporine (5.5 nM), two protein kinase C inhibitors. Na+-dependent phosph ate uptake was also significantly inhibited by phorbol 12-myristate 13-acet ate (20.9 +/- 3.9% inhibition, P < 0.001) but not by dibutyril-cAMP (10(-4) M) or forskolin (50 mu M). The physiological role played by type III NaPO4 cotransport expression in the overall renal regulation of phosphate homeos tasis remains to be established.