Basolateral membrane chloride permeability of A6 cells: implication in cell volume regulation.
The permeability to Cl- of the basolateral membrane (blm) was investigated in renal (A6) epithelial cells, assessing their role in transepithelial ion transport under steady-state conditions (isoosmotic) and following a hypoosmotic shock (i.e. in a regulatory volume decrease, RVD). Three different complementary studies were made by measuring: (1) the Cl- transport rates (delta F/Fo s-1 (x10(-3))), where F is the fluorescence of N-(6-methoxyquinoyl) acetoethyl ester, MQAE, and Fo the maximal fluorescence (x10(-3)) of both membranes by following the intracellular Cl- activities (ai Cl-, measured with MQAE) after extracellular Cl- substitution (2) the blm 86Rb and 36Cl uptakes and (3) the cellular potential and Cl- current using the whole-cell patch-clamp technique to differentiate between the different Cl- transport mechanisms. The permeability of the blm to Cl- was found to be much greater than that of the apical membranes under resting conditions: aiCl- changes were 5.3 +/- 0.7 mM and 25.5 +/- 1.05 mM (n = 79) when Cl- was substituted by NO3(-) in the media bathing apical and basolateral membranes. The Cl- transport rate of the blm was blocked by bumetanide (100 microM) and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, 50 microM) but not by N-phenylanthranilic acid (DPC, 100 microM). 86Rb and 36Cl uptake experiments confirmed the presence of a bumetanide- and a NPPB-sensitive Cl- pathway, the latter being approximately three times more important than the former (Na/K/2Cl cotransporter). Appli-cation of a hypoosmotic medium to the serosal side of the cell increased delta F/Fo s-1 (x10(-3)) after extracellular Cl- substitution (1.03 +/- 0.10 and 2.45 +/- 0.17 arbitrary fluorescent units s-1 for isoosmotic and hypoosmotic conditions respectively, n = 11); this delta F/Fo s-1 (x10(-3)) increase was totally blocked by serosal NPPB application; on the other hand, cotransporter activity was decreased by the hypoosmotic shock. Cellular Ca2+ depletion had no effect on delta F/Fo s-1 (x10(-3)) under isoosmotic conditions, but blocked the delta F/Fo s-1 (x10(-3)) increase induced by a hypoosmotic stress. Under isotonic conditions the measured cellular potential at rest was -37.2 +/- 4.0 mV but reached a maximal and transient depolarization of -25.1 +/- 3.7 mV (n = 9) under hypoosmotic conditions. The cellular current at a patch-clamping cellular potential of -85 mV (close to the Nernst equilibrium potential for K+) was blocked by NPPB and transiently increased by hypoosmotic shock (&ap;50% maximum increase). This study demonstrates that the major component of Cl- transport through the blm of the A6 monolayer is a conductive pathway (NPPB-sensitive Cl- channels) and not a Na/K/2Cl cotransporter. These channels could play a role in transepithelial Cl- absorption and cell volume regulation. The increase in the blm Cl- conductance, inducing a depolarization of these membranes, is proposed as one of the early events responsible for the stimulation of the 86Rb efflux involved in cell volume regulation.
Brochiero
E
E
Department of Cellular and Molecular Biology, CEA-URA 1855 (CNRS), Laboratoire Jean Maetz, BP 68, F-06230 Villefranche/Mer, France.
Banderali
U
U
Lindenthal
S
S
Raschi
C
C
Ehrenfeld
J
J
D000818:Animals; D001834:Body Water / Q000378:metabolism; D002034:Bumetanide / Q000494:pharmacology; D002352:Carrier Proteins / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002463:Cell Membrane Permeability; D048429:Cell Size / Q000502:physiology; D018118:Chloride Channels / Q000187:drug effects / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism*; D004232:Diuretics / Q000494:pharmacology; D005456:Fluorescent Dyes; D007668:Kidney / Q000166:cytology; D009994:Osmolar Concentration; D018408:Patch-Clamp Techniques; D011868:Radioisotopes; D012414:Rubidium Radioisotopes; D028021:Sodium-Potassium-Chloride Symporters; D014982:Xenopus laevis
D002352:Carrier Proteins; D018118:Chloride Channels; D002712:Chlorides; D004232:Diuretics; D005456:Fluorescent Dyes; D011868:Radioisotopes; D012414:Rubidium Radioisotopes; D028021:Sodium-Potassium-Chloride Symporters; D002034:Bumetanide