Phosphate transport by isolated renal brush border vesicles

Abstract

A sodium dependent specific transport system for phosphate is present in the brush border microvilli but absent from the basal-lateral plasma membranes. The apparent affinity of this transport system for phosphate is 0.08 mM at 100 mM sodium and pH 7.4. It is inhibited competitively by arsenate with an apparent inhibitor constant of 1.1 mM (100 mM sodium, pH 7.4). Sodium dependent phosphate uptake is two times higher at pH 8 compared to the uptake observed at pH 6. The apparent affinity of the transport system for sodium is also pH-dependent, half-maximal stimulation of uptake is found at pH 6 with 129 mM sodium, at pH 7.4 with 60 mM sodium and at pH 8 with 50 mM sodium. Under all conditions a nonhyperbolic dependence of phosphate uptake on the sodium concentration is observed. The uptake of phosphate by brush border microvilli vesicles shows a typical overshoot phenomenon in the presence of sodium gradient across the membrane (CNao greater than CNai). The amount of pohsphate taken up after 2 min is about twice the equilibrium value reached after 2 h of incubation. At pH 7.4 the initial rate of uptake is increased only slighyly (12%) by inside negative membrane diffusion potentials and inhibited to the same extent by inside positive membrane diffusion potentials. These results indicate that the entry of phosphate across the brush border membrane into the epithelial cell of the proximal tubule is coupled to the entry of sodium. The transfer of phosphate is dependent on its concentration gradient and on the concentration difference of sodium. The data are best explained by the following hypothesis: Both the primary phosphate as well as the secondary phosphate are transported in cotransport with sodium. The divalent form however seems to be transported preferentially. Its transport occurs electroneutral with 2 sodium ions; the monovalent phosphate also enters the cell together with 2 sodium ions but as a positively charged complex. The exit of phosphate across the contraluminal cell border is sodium independent and is favoured by the high intracellular phosphate concentration and the inside negative membrane potential.

Phosphate Transport Kinetics and Structure-Function Relationships of SLC34 and SLC20 Proteins

“…Early steady-state kinetic studies using native tissue established that Na-coupled P i cotransport proceeds in an ordered manner with Na + binding first (e.g. (Hoffmann, Thees et al 1976)). Studies on NaPi-IIa expressed in Xenopus oocytes refined this model by proposing that 1 Na + ion interacts with the protein before P i binds (Forster, Hernando et al 1998;Forster 2007), consistent with the finding that the Na + -dependence of the uncoupled leak (see below) showed Michaelian kinetics (Forster, Hernando et al 1998).…”

“…Transport studies in the pre-cloning era using membrane vesicles from renal and intestinal epithelial tissue documented secondary-active, Na + -dependent P i transport ((Berner, Kinne et al 1976;Hoffmann, Thees et al 1976)). Further studies used both cloned transporters and native tissue to characterize the kinetics and regulation of this transport (for review see (Murer, Hernando et al 2000;Biber, Hernando et al 2009).…”

Renal Adaptation to a Low Phosphate Diet in Rats

“…Uptake of both 32Pi and D-[3H]glucose in the presence of NaCl increased rapidly and reached a peak in the case of 32Pi within 1-2 min, and in the case of D-glucose within 0.5-1.0 min, so-called "overshoot" (10,32,36,37), and then gradually declined to a much lower level of "equilibrium point" at 120 min. This time interval for equilibrium point was chosen, as in our previous study (10), at 120 min primarily because of slow equilibration of Pi across the BBM (32).…”

“…Uptake of 32P-phosphate and D-[3H]glucose by isolated BBM vesicles were measured by the Millipore filtration technique (Millipore Corp., Bedford, Mass.) described in detail by Beck and Sacktor (17) and used also by Hoffman et al (32), and in our previous study (10). The final composition of the incubation medium used for measurement of Pi uptake was 100 mM mannitol, 100 mM NaCl, 5 mM Hepes-Tris, pH 8.5, 0.1 mM K2H 32P04 (=3 x 105 cpm/tube).…”

Physiological Mechanisms of Intestinal Absorption of Protein Hydrolysates and Their Relevance to Bacterial Growth in the Small Intestine

“…For the proliferation of bacteria, the presence of utilizable nitrogen compounds, such as amino acids and oligopeptides, and inorganic phosphate is particularly important. It has been already well established that free amino acids and phosphate are efficiently absorbed by Na+-dependent active transport mechanisms (7,16) .…”