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Characterization of Ca2+ transport in rat renal brush-border membranes and its modulation by phosphatidic acid.

ABSTRACT: The Ca2+ transport process by isolated renal brush-border membranes was characterized and the influence of the acidic phospholipid phosphatidic acid (PtdA) on this transport process was assessed. Ca2+ uptake by brush-border membranes exhibited saturation kinetics. It was inhibitable by a variety of multivalent cations, as well as by Ca2+-entry inhibitors, including verapamil, Ruthenium Red and gentamicin. It was selective for Ca2+ compared with Mg2+. This process was also electrophoretic since generation of K+ and anion-diffusion potentials, negative inside the vesicle, increased Ca2+ uptake. Elevations in PtdA content of brush-border membranes by either exogenous addition or endogenous generation of PtdA by incubating brush-border membranes with MgATP2- elevated the rate of Ca2+ uptake. This ATP effect could not be attributed to (Ca2+ + Mg2+)-dependent ATPase or contaminating membrane fragments. PtdA also increased the magnitude and rate of Ca2+ efflux from brush-border membranes preloaded with Ca2+. These modulations in uptake and efflux were not observed with phosphatidylcholine or phosphatidylinositol. In summary, these results are consistent with the presence of an electrophoretic uniport system for Ca2+ in renal brush-border membranes, and demonstrate that PtdA uniquely among phospholipids tested appears to facilitate transmembrane flux of Ca2+ across this membrane preparation.

SUBMITTER: Somermeyer MG

PROVIDER: S-EPMC1152207 | biostudies-other | 1983 Jul

REPOSITORIES: biostudies-other

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Project description:Phosphate uptake by rat renal brush-border membrane vesicles was studied under experimental conditions where transmembrane electrical potential (delta psi) could be manipulated. Experiments were performed under initial rate conditions to avoid complications associated with the dissipation of ion gradients. First, phosphate uptake was shown to be strongly affected by the nature of Na+ co-anions, the highest rates of uptake being observed with 100 mM-NaSCN (1.010 +/- 0.086 pmol/5 s per micrograms of protein) and the lowest with 50 mM-Na2SO4 (0.331 +/- 0.046 pmol/5 s per micrograms of protein). Anion substitution studies showed that potency of the effect of the co-anions was in the order thiocyanate greater than nitrate greater than chloride greater than isethionate greater than gluconate greater than sulphate, which correlates with the known permeability of the membrane to these anions and thus to the generation of transmembrane electrical potentials of decreasing magnitude (inside negative). The stimulation by ion-diffusion-induced potential was observed from pH 6.5 to 8.5, indicating that the transport of both monovalent and divalent phosphate was affected. In addition, inside-negative membrane potentials were generated by valinomycin-induced diffusion of K+ from K+-loaded vesicles and showed a 57% stimulation of phosphate uptake, at pH 7.5. Similar experiments with H+-loaded vesicles, in the presence of carbonyl cyanide m-chlorophenylhydrazone gave a 50% stimulation compared with controls. Inside-positive membrane potentials were also induced by reversal of the K+ gradient (outside greater than inside) in the presence of valinomycin and gave 58% inhibition of phosphate uptake. The membrane-potential dependency of phosphate uptake was finally analysed under thermodynamic equilibrium, and a stimulation by inside-negative potential was observed. The transport of phosphate was thus driven against a concentration gradient by a membrane potential, implicating the net transfer of a positive charge during the translocation process. These results indicate a major contribution of electrical potential to phosphate uptake in renal brush-border membranes.

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Characterization of Ca2+ transport in rat renal brush-border membranes and its modulation by phosphatidic acid.

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