Time resolved secretion of chloride from a monolayer of mucin-secreting epithelial cells |
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Authors: | Sumitha Nair Rohit Kashyap Christian L Laboisse Ulrich Hopfer Miklos Gratzl |
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Institution: | (1) Department of Biomedical Engineering, Case School of Engineering, Case Western Reserve University, Cleveland, OH 44106, USA;(2) Institut National de la Santé et de la Recherche Médicale 94-04, Université de Nantes, 44035 Nantes, France;(3) Department of Physiology and Biophysics, School of Medicine, Case Western Reserve University, Cleveland, OH 44106, USA |
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Abstract: | Short-circuit current (Isc) measurement is used to quantify transepithelial ion flux. This technique provides a direct measure
of net charge transport across a cell monolayer. Isc however, lacks chemical selectivity. Chemically resolved ion fluxes may
be much greater than Isc, and differ in different biological processes. This work describes a novel experimental approach
and deconvolution method to obtain temporally resolved ion fluxes at epithelial cell monolayers. HT29-Cl.16E cells, a sub
clone of the human colonic cancer cell line HT29 was used as a model cell line to validate this approach in the context of
epithelial transport studies. This cell line is known to secrete chloride in response to purinergic stimulation. Changes in
chloride concentration after stimulation with 1 mM ATP plus 50 nM phorbol-myristate acetate (PMA) are recorded with a chloride
ion-selective electrode (ISE) at a short distance (∼50 μm) from the monolayer. The recorded concentrations are transformed
to corresponding chloride flux across the monolayer using a deconvolution algorithm for extracellular mass transport based
on minimization of the shape error function (Nair and Gratzl in Anal Chem 77:2875–2888, 2005). Simultaneous voltage clamp yields the associated net electrical charge flux (Isc). The dynamics of Cl− flux did correlate with that of the electrical flux, but was found to be greater in amplitude. This suggests that Cl− may not be the only ion secreted. The method of simultaneously assessing ionic and electrical fluxes with a temporal resolution
of seconds provides unique information about the dynamics of solute fluxes across the apical membrane.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. |
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