title: An effect of Ca2+ on the Intrinsic Cl(-)-conductance of rat kidney cortex brush border membrane vesicles.
abstract: 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).
authors.affiliation: Department of Physiological Sciences, Medical School, University of Newcastle-upon-Tyne, U.K.
authors.forename: S
authors.initials: S
authors.lastname: Vayro
authors.forename: N L
authors.initials: NL
authors.lastname: Simmons
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002118:Calcium / Q000494:pharmacology*; D002712:Chlorides / Q000378:metabolism*; D004789:Enzyme Activation; D005947:Glucose / Q000378:metabolism; D007672:Kidney Cortex / Q000187:drug effects / Q000378:metabolism*; D008274:Magnesium / Q000494:pharmacology; D008297:Male; D008871:Microvilli / Q000378:metabolism; D008969:Molecular Sequence Data; D009355:Neomycin / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D011392:Proline / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D011722:Pyrethrins / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D012964:Sodium / Q000494:pharmacology; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors
chemical_list: D002712:Chlorides; D011722:Pyrethrins; D011392:Proline; D012964:Sodium; D011493:Protein Kinase C; D010749:Phosphoprotein Phosphatases; D010738:Type C Phospholipases; D009355:Neomycin; D008274:Magnesium; D005947:Glucose; D002118:Calcium