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Basolateral membrane chloride permeability of A6 cells: implication in cell volume regulation

  • Original Article
  • Transport Processes, Metabolism and Endocrinology; Kidney, Gastrointestinal Tract and Exorcrine Glands
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Abstract

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 (ΔF/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 μM) and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, 50 μM) but not by N-phenylanthranilic acid (DPC, 100 μM). 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 ΔF/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 ΔF/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 ΔF/F o·s−1 (×10−3) under isoosmotic conditions, but blocked the ΔF/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.

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Authors and Affiliations

  1. Department of Cellular and Molecular Biology, CEA-URA 1855 (CNRS), Laboratoire Jean Maetz, BP 68, F-06230, Villefranche/Mer, France

    E. Brochiero, U. Banderali, S. Lindenthal, C. Raschi & J. Ehrenfeld

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  1. E. Brochiero
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  2. U. Banderali
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  3. S. Lindenthal
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  4. C. Raschi
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  5. J. Ehrenfeld
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Brochiero, E., Banderali, U., Lindenthal, S. et al. Basolateral membrane chloride permeability of A6 cells: implication in cell volume regulation. Pflügers Arch. 431, 32–45 (1995). https://doi.org/10.1007/BF00374375

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  • DOI: https://doi.org/10.1007/BF00374375

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