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1 Institut National de la Santé et de la Recherche Médicale, Faculté de Médecine Xavier Bichat, 75018 Paris, France; and 2 Laboratoire d'Oncologie Moleculaire, Membrane Transport Research Group, Université du Québec à Montréal-Hôpital Ste-Justine, Montreal, Quebec, Canada H3C 3P8
The aim of the present study was to characterize the type(s) of
NaPO4 cotransporter 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 human
embryonic kidney HEK-293 cells expressed
NaPO4 cotransporter type III
(PiT1) mRNA and protein. In contrast, type I (NPT1) or II (NPT2)
cotransporter mRNA were not expressed.
Na+-dependent phosphate uptake
followed a Michaelis-Menten model (apparent maximal transport rate and
affinity constant: 23.32 ± 0.69 nmol
PO4 · mg
protein
1 · 10 min1 and 0.147 ± 0.014 mM
KH2PO4,
respectively), was stimulated by phosphate deprivation (maximal
increase 24.5 ± 0.8%, P < 0.001, after 15 h of phosphate deprivation), and was inhibited 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% inhibition, P < 0.01). PTH inhibition of phosphate transport was maintained in
phosphate-deprived cells and reversed by both GF109203X
(106 M) or staurosporine
(5.5 nM), two protein kinase C inhibitors. Na+-dependent phosphate uptake was
also significantly inhibited by phorbol 12-myristate 13-acetate (20.9 ± 3.9% inhibition, P < 0.001) but not by dibutyril-cAMP
(104 M) or forskolin (50 µM). The physiological role played by type III
NaPO4 cotransport expression in
the overall renal regulation of phosphate homeostasis remains to be established.
human renal cell line; parathyroid hormone; human embryonic kidney HEK-293 cells
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