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Basolateral membrane chloride permeability of A6 cells: implication in cell volume regulation
- Source :
- Pflügers Archiv European Journal of Physiology; November 1995, Vol. 431 Issue: 1 p32-45, 14p
- Publication Year :
- 1995
-
Abstract
- The permeability to Cl<superscript>-</superscript> 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<superscript>-</superscript> transport rates (?F/F<subscript>o</subscript> · s<superscript>-1</superscript> (× 10<superscript>-3</superscript>)), where F is the fluorescence of N-(6-methoxyquinoyl) acetoethyl ester, MQAE, and F<subscript>o</subscript> the maximal fluorescence (×10<superscript>-3</superscript>) of both membranes by following the intracellular Cl<superscript>-3</superscript> activities (a<subscript>i</subscript>Cl<superscript>-</superscript>, measured with MQAE) after extracellular Cl<superscript>-</superscript> substitution (2) the blm <superscript>86</superscript>Rb and <superscript>36</superscript>Cl uptakes and (3) the cellular potential and Cl<superscript>-</superscript> current using the wholecell patch-clamp technique to differentiate between the different Cl<superscript>-</superscript> transport mechanisms. The permeability of the blm to Cl<superscript>-</superscript> was found to be much greater than that of the apical membranes under resting conditions: a<subscript>i</subscript>Cl<superscript>-</superscript> changes were 5.3±0.7 mM and 25.5±1.05 mM (n=79) when Cl<superscript>-</superscript> was substituted by NO<subscript>3</subscript><superscript>-</superscript> in the media bathing apical and basolateral membranes. The Cl<superscript>-</superscript> 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). <superscript>86</superscript>Rb and <superscript>36</superscript>C1 uptake experiments confirmed the presence of a bumetanide- and a NPPB-sensitive Cl<superscript>-</superscript> 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<subscript>o</subscript> · s<superscript>-1</superscript> (×10<superscript>-3</superscript>) after extracellular Cl<superscript>-3</superscript> substitution (1.03±0.10 and 2.45±0.17 arbitrary fluorescent units·s<superscript>-1</superscript> for isoosmotic and hypoosmotic conditions respectively, n=11); this ?F/F<subscript>o</subscript>·s<superscript>-1</superscript> (×10<superscript>-3</superscript>) increase was totally blocked by serosal NPPB application; on the other hand, cotransporter activity was decreased by the hypoosmotic shock. Cellular Ca<superscript>2+</superscript> depletion had no effect on ?F/F<subscript>o</subscript>·s<superscript>-1</superscript> (×10<superscript>-3</superscript>) under isoosmotic conditions, but blocked the ?F/F<subscript>o</subscript>·s<superscript>-1</superscript> (×10<superscript>-3</superscript>) 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<superscript>+</superscript>) was blocked by NPPB and transiently increased by hypoosmotic shock (˜ 50% maximum increase). This study demonstrates that the major component of Cl<superscript>-</superscript> transport through the blm of the A6 monolayer is a conductive pathway (NPPB-sensitive Cl<superscript>-</superscript> channels) and not a Na/K/2Cl cotransporter. These channels could play a role in transepithelial Cl<superscript>-</superscript> absorption and cell volume regulation. The increase in the blm Cl<superscript>-</superscript> conductance, inducing a depolarization of these membranes, is proposed as one of the early events responsible for the stimulation of the <superscript>86</superscript>Rb efflux involved in cell volume regulation.
Details
- Language :
- English
- ISSN :
- 00316768 and 14322013
- Volume :
- 431
- Issue :
- 1
- Database :
- Supplemental Index
- Journal :
- Pflügers Archiv European Journal of Physiology
- Publication Type :
- Periodical
- Accession number :
- ejs16302991
- Full Text :
- https://doi.org/10.1007/BF00374375