The activity of the Na'-dependent Pi transporter present in the brush-border membrane of the proximal tubule region of the nephron is important for the homoeostatic control of phosphate in mammals (Dennis et ul., 1979; Gmaj & Murer, 1086;Butterworth, 1987). The activity of the transporter is stimulated when the dietary intake of phosphate is low.Isolated chick proximal tubule cells (Grahn & Butterworth, 1986) and the kidney epithelial cell line LLC-PK, (Caverzasio et u/., 1985) respond to a direct stimulus of low ambient PI with an increase in Na+-dependent P, uptake, suggesting therefore that other humoral factors are not essential for the process whereby kidney reabsorption of PI is adjusted to meet the physiological demand for the anion.The experiments with cultured LLC-PK, cells (Caverzasio et ul., 1985) suggested that adaptation of transport to low P, occurs in two stages: a rapid increase that is independent of protein synthesis followed by a slower protein synthesisdependent component. It is possible that rapid adjustment requires the insertion into the brush-border membrane of extra molecules of transporter derived from intracellular stores, and/or an increase of the intrinsic activity of the existing complement of transporter. In the absence of methodology that permits quantification of the number of membrane PI transport sites, indirect methods are being used in order to investigate altered transport capacity subsequent to P, depletion.Proximal tubule cells were isolated from mice kidneys immediately after killing, by digestion at 37°C with collagenase and hyaluronidase dissolved in Krebs-Ringer-bicarbonate medium containing 10 mM-glucose and 10 mMpyruvate. The suspension was stirred continuously and gassed gently with 95%02/5%C02 and allowed to digest for 30 min. Proximal tubule cells were selectively prepared from the digest by several 30 s spins at 2000 rev./min on an MSE Microcentaur bench centrifuge. The final cell pellet was resuspended in warm isolation medium containing bovine serum albumin ( 1 mg/ml). The kidneys from four mice produced about 15 mg of cell protein.To prepare phosphate-depleted cells, the same isolation procedure was followed except that the medium was devoid of P, during the collagenase digestion and also the washing steps (a total period of 45 min). The cells were finally suspended in Pi-free isolation medium. Na+-dependent P, uptake by the cells was stimulated by a depletion episode. V,;,, was increased by 80n/0 and K , decreased by SOYO and so the efficiency of P, reabsorption from the luminal fluid would be increased by these changes in kinetic parameters. The temperature dependence of transport was different for the two cell types (Fig. 1). The energies of activation were 28 kJ/mol and 65 kJ/mol for normal and *r / 1 C -Fig. 1. Arrhenius plot of' Na + -dependent I', uptuke by mouse proximal tubule cells 0 , Normal phosphate-replete cells; m, phosphatc-depleted cells. The data were obtained from three separate experiments conducted with each of the cell types.phosphate-...
