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/F-o . s(-1) (x 10(-3))), where F is the fluorescence of N-(6-methoxyquinoyl) acetoethyl ester, MQAE, and F-o the maximal fluorescence (x 10(-3)) of both membranes by following the intracellular Cl- activities (alpha(i)Cl(-), measured with MQAE) after extracellular Cl- substitution (2) the blm Rb-86 and Cl-36 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: alpha(i)Cl(-) changes were 5.3 +/- 0.7 mM and 25.5 +/- 1.05 mM (n = 79) when Cl- was substituted by NO?- in the media bathing apical and basolateral membranes. The Cl- transport rate of the blm was blocked by bumetanide (100 mu M) and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, 50 mu M) but not by, N-phenylanthranilic acid (DPC, 100 mu M). Rb-86 and Cl-36 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). Application of a hypoosmotic medium to the serosal side of the cell increased Delta F/F-o . s(-1) (x 10(-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/F-o . s(-1) (x 10(-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/F-o . s(-1) (x 10(-3)) under isoosmotic conditions, but blocked the Delta F/F-o . s(-1) (x 10(-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 (approximate to 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 Rb-86 efflux involved in cell volume regulation.