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Vayro S, Simmons NL: An effect of Ca2+ on the Intrinsic Cl (-)-conductance of rat kidney cortex brush border membrane vesicles. J Membr Biol. 1996 Mar;150(2):163-73.
Brush-border membrane vesicles (BBMV) were prepared from superficial rat renal cortex by a divalent (2+)-precipitation technique using either CaCl2 or MgCl2. The dependence of the initial [14C]-D-glucose (or [3H]-L-proline) uptake rate and the extent of the overshoot of D-glucose or L-proline uphill accumulation from solutions containing 100 mM Na+ salt, was found to be dependent upon the precipitating divalent cation. With Mg2+ precipitation the initial uptake and overshoot accumulation of either D-glucose or L-proline were enhanced compared to BBMV prepared by Ca2+ precipitation. When the anion composition of the media was varied (uptake in Cl- media in comparison to gluconate (-)-containing media) it was found that the Cl (-)-dependent component of the initial uptake was markedly depressed with Ca (2+)-prepared BBMV (104.99 +/- 33.31 vs. 13.83 +/- 1.44 pmoles/sec/mg protein for Mg2+ and Ca2+ prepared vesicles respectively). When Ca2+ was loaded into Mg2+ prepared BBMV using a freeze-thaw technique, it was found that the magnitude and Cl- enhancement of D-glucose transport was reduced in a dose-dependent manner. Neomycin, an inhibitor of phospholipase C, had no effect on the reduction of D-glucose uptake by Ca2+ in Mg2+ prepared vesicles. In contrast, phosphatase inhibitors such as vanadate and fluoride were able to partially reverse the Ca2+ inhibition of D-glucose uptake and restore the enhancement due to Cl- media. In addition, inhibitors of protein phosphatase 2B, deltamethrin (50 nM) and trifluoperazine (10 microM), caused partial reversal of Ca2 (+)-dependent inhibition of D-glucose uptake. Direct measurement of changes in the bi-ionic (Cl-vs. gluconate-) transmembrane electrical potential differences using the cyanine dye, 3,3'-dipropylthiodicarbocyanine iodide DiSC3-(5) confirmed that Cl- conductance was reduced in Ca (2+)-prepared vesicles. We conclude that a Cl- conductance coexists with Na+ cotransport in rat renal BBMV and this may be subject to negative regulation by Ca2+ via stimulation of protein phosphatase (PP2B). |
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