Rolf K. H. Kinne

Phosphate transport by isolated renal brush border vesicles

SummaryA 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

The use of isolated membrane vesicles to study epithelial transport processes

(C_{Na_o } > {\text{ }}C_{Na_i } )

Glucose transport in isolated brush-border and lateral-basal plasma-membrane vesicles from intestinal epithelila cells

. The amount of phosphate 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 slightly (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.

Properties of brush border vesicles isolated from rat kidney cortex by calcium precipitation.

SummaryUptake studies ofd- andl-glucose were performed on vesicles derived from brush-border and basal-lateral membranes. The uptake of the sugars into the vesicles was osmotically sensitive and independent of glucose metabolism. In brush-border vesiclesd-glucose but notl-glucose transport was Na+-dependent, was inhibited by phlorizin, and showed a transitory vesicle/medium ratio >1, in the presence of an initial Na+ gradient. Basal-lateral membranes take upd-glucose faster thanl-glucose, but thed-glucose uptake is significantly less sensitive to sodium removal and only moderately inhibited by phlorizin as compared to the brush-border fraction.

Phenylalanine uptake in isolated renal brush border vesicles

SummaryEpithelia are multicompartment and multicomponent systems performing transcellular and paracellular transport in a very complex manner. One way to get a deeper understanding of the function of such a complex system is to dissect it into the single components and then, after having defined the components under well-controlled conditions, to try to describe the behavior of the whole system on the basis of the properties of the single components.This article deals with the analysis of isolated plasma membranes derived from the luminal and contraluminal face of epithelial cells, predominantly renal proximal tubular and small intestinal cells. It is aimed to give an overview of methods used to isolate and separate plasma membranes, to study their transport properties as membrane vesicles, and also to address the question of how information gained with the isolated membranes corresponds to observations made in the intact cell using other, notably electrophysiological, measurements. The review also critically evaluates the limitations of the approach and thereby tries to set the work on isolated membranes in the proper perspective within the field of transport physiology.

Ammonium transport in medullary thick ascending limb of rabbit kidney: involvement of the Na+,K+,Cl(-)-cotransporter.

SummaryBasal-lateral plasma membrane vesicles and brush border membrane vesicles were isolated from rat kidney cortex and the uptake of p-amino-hippuric acid (PAH) into these vesicles was studied by Millipore filtration techniques.Both membrane preparations take up PAH into an osmotically reactive intravesicular space. The transport across the brush border membrane seems to involve only simple diffusion whereas in the basal-lateral plasma membrane in addition a specific transport system exists which is inhibited competitively by probenecid. The apparent affinity of this transport system for PAH is 5.4×10−4 M and for probenecid 5.4×10−5 M.PAH uptake into basal-lateral plasma membrane vésicles is influenced by alteration of the membrane potential. Maneuvers which render the intravesicular space more positive-as for example replacement of chloride by sulfate in the presence of a sodium gradient directed into the vesicles and addition of valinomycin in the presence of a potassium gradient directed into the vesicles-stimulate the uptake of PAH. Replacement of a sodium chloride gradient by a sodium thiocyanate gradient reduces the uptake probably by reducing the inside positive membrane potential.In the absence of salt gradients anion replacement and replacement of sodium by potassium does not affect PAH transport by basal-lateral plasma membranes.These results suggest that in isolated basal-lateral membranes transfer of PAH across the membrane is accompanied by a transfer of negative charge. They furthermore provide no evidence for the existence of a sodium-PAH cotransport system in this membrane preparation.

The effects of potassium and membrane potential on sodium-dependent glutamic acid uptake

Abstract Free-flow electrophoresis was used to separate microvilli from the lateral basal plasma membrane of the epithelial cells from rat small intestine. The activities of the marker enzyme for the microvillus membrane, i.e. alkaline phosphatase (EC 3.1.31), was clearly separated from the marker for the lateral-basal plasma membrane, i.e. the (Na+, K+)-ATPase (EC 3.6.1.3). A microvillus membrane fraction was obtained with a high specific activity of alkaline phosphatase (an 8-fold enrichement over the starting homogenate). The lateral-basal plasma membrane fraction contained (Na+, K+)-ATPase (5-fold over homogenate) with some alkaline phosphatase (2-fold over homogenate). Glucose transport was studied in both membrane fractions. The uptake of d -glucose was much faster than that of l -glucose in either plasma membrane, d -Glucose uptake could be accounted for completely by its transport into an osmotically active space. Interestingly, the characteristics of the glucose transport of the microvillus membrane were different from those of the lateral-basal plasma membrane. In particular: Na+ stimulated the d -glucose transport by the microvillus membrane, but not by the lateral-basal plasma membrane. In addition, the glucose transport of the microvillus membrane was much more sensitive to phlorizin inhibition than that of the lateral-basal plasma membrane. These experiments thus provide evidence not only for an asymmetrical distribution of the enzymes, but also for differences in the transport properties with respect to glucose between the two types of plasma membrane of the intestinal epithelial cell.

Carbonic anhydrase activity of isolated brush border and basal-lateral membranes of renal tubular cells.

Brush border membrane vesicles were isolated from rat kidney cortex by differential centrifugation in the presence of 10 mM calcium. Their properties were compared to brush border vesicles isolated by free-flow electrophoresis. By the calcium precipitation method membrane vesicles were obtained in a shorter time with a similar enrichment of brush border marker enzymes (11- to 12-fold for alkaline phosphatase and maltase), with a similarly reduced activity of the marker enzyme for basal-lateral plasma membranes and an almost identical protein composition as revealed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The transport properties of the two membrane preparations for D-glucose, L-phenylalanine, and phosphate are essentially the same; there is some indication for a lower sodium permeability of the vesicles prepared by the calcium precipitation method. The latter vesicles were also shown to exhibit sodium gradient stimulated uptake of L-glutamate.