Properties of chloride-conductive pathways in rat kidney cortical and outer-medulla brush-border membranes--inhibition by anti-(cystic fibrosis transmembrane regulator) mAbs.
The activity of the Cl(-)-conductive pathways, their regulation by protein kinase A (PKA) and their relationship to the cystic fibrosis transmembrane regulator (CFTR) protein were assessed in rat kidney cortical brush-border-membrane vesicles (cBBMV) and outer medullary vesicles (OMV) by measuring the rate of valinomycin-induced microsomal swelling by light scattering in the presence of an inward Cl- gradient. Valinomycin increased the rate of swelling of cBBMV and OMV, which is consistent with the presence of a Cl(-)-conductive pathway. PKA further increased these rates. This effect was blocked by the inhibitor of protein kinase A, suggesting that phosphorylation by PKA activates these pathways. Four anion-transport inhibitors were tested ¿N-phenylanthranilic acid (PhNHPhCOOH), 5-nitro-2-(3-phenylpropylamino)benzoic acid [N(PhPrNH2)BzOH], glybenclamide and 4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulfonic acid¿. Ph2COOH and 4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulfonic acid inhibited the basal Cl(-)-conductive pathways, while PKA-treated microsomes were sensitive also to N(PhPrNH2)BzOH and glybenclamide, suggesting that additional Cl- pathways were activated by phosphorylation. The pharmacological properties of these pathways were similar to those of the CFTR Cl- channel. Two anti-CFTR mAbs inhibited PKA-activated valinomycin-induced swelling in cBBMV and OMV, while immunoblot analysis of the corresponding proteins with the same antibodies indicated the presence of a 170-kDa protein. The results thus indicate the presence of a PKA-activated Cl(-)-conductive pathway in cBBMV and OMV, and suggest that CFTR protein is involved in PKA-activated Cl- fluxes in these vesicles.
Benharouga
M
M
INSERM U.467, CHU Necker, Paris, France.
Fritsch
J
J
Banting
G
G
Edelman
A
A
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D015153:Blotting, Western; D018118:Chloride Channels / Q000037:antagonists & inhibitors / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000276:immunology*; D007672:Kidney Cortex / Q000378:metabolism*; D007679:Kidney Medulla / Q000378:metabolism*; D008871:Microvilli / Q000378:metabolism; D051381:Rats
D000911:Antibodies, Monoclonal; D018118:Chloride Channels; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D017868:Cyclic AMP-Dependent Protein Kinases