Mechanisms of phosphate uptake into brush-border membrane vesicles from goat jejunum.

Abstract

This study concerns the uptake of inorganic phosphate into brush-border membrane vesicles prepared from jejunal tissues of either control or Ca- and/or P-depleted goats. The brush-border membrane vesicles showed a time-dependent accumulation of inorganic phosphate with a typical overshoot phenomenon in the presence of an inwardly direct Na+ gradient. The Na+-dependent inorganic phosphate uptake was completely inhibited by application of 5 mmol.1(-1) sodium arsenate. Half-maximal stimulation of inorganic phosphate uptake into brush-border membrane vesicles was found with Na+ concentrations in the order of 5 mmol.1(-1). Inorganic phosphate accumulation was not affected by a K+ diffusion potential (inside negative), suggesting an electroneutral transport process. Stoichiometry suggested an interaction of two or more Na ions with one inorganic phosphate ion at pH 7.4 Na+-dependent inorganic phosphate uptake into jejunal brush-border membrane vesicles from normal goats as a function of inorganic phosphate concentration showed typical Michaelis-Menten kinetic with Vmax = 0.42 +/- 0.08 nmol.mg-1 protein per 15S-1 and Km = 0.03 +/- 0.01 mmol.1(-1) (n = 4, mean +/- SEM). Long-term P depletion had no effect on these kinetic parameters. Increased plasma calcitriol concentrations in Ca-depleted goats, however, were associated with significant increases of Vmax by 35-80%, irrespective of the level of P intake. In the presence of an inwardly directed Na+ gradient inorganic phosphate uptake was significantly stimulated by almost 60% when the external pH was decreased to 5.4 (pHout/pHin = 5.4/7.4). The proton gradient had no effect on inorganic phosphate uptake in absence of Na+. In summary, in goats Na+ and calcitriol-dependent mechanisms are involved in inorganic phosphate transport into jejunal brush-border membrane vesicles which can be stimulated by protons.