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 (F/F _o · s^–1 (× 10^–3)), where F is the fluorescence of N-(6-methoxyquinoyl) acetoethyl ester, MQAE, and F _o the maximal fluorescence (×10^–3) of both membranes by following the intracellular Cl^–3 activities (a _iCl^–, measured with MQAE) after extracellular Cl^– substitution (2) the blm ⁸⁶Rb and ³⁶Cl uptakes and (3) the cellular potential and Cl^– current using the wholecell 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: a _iCl^– 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 M) and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, 50 M) but not by N-phenylanthranilic acid (DPC, 100 M). ⁸⁶Rb and ³⁶C1 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 F/F _o · s^–1 (×10^–3) after extracellular Cl^–3 substitution (1.03±0.10 and 2.45±0.17 arbitrary fluorescent units·s^–1 for isoosmotic and hypoosmotic conditions respectively, n=11); this F/F _o·s^–1 (×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 F/F _o·s^–1 (×10^–3) under isoosmotic conditions, but blocked the F/F _o·s^–1 (×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 ( 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.