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Chloride secretion by avian and mammalian proximal tubule cells in culture
Author(s) -
Laverty Gary,
Anttila Ashley
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.a1225-c
Subject(s) - forskolin , bumetanide , chemistry , cotransporter , apical membrane , medicine , endocrinology , channel blocker , secretion , chloride channel , ibmx , biophysics , amiloride , epithelial polarity , matrigel , ion transporter , biochemistry , biology , membrane , calcium , cell , in vitro , organic chemistry , sodium
Our previous work with primary culture monolayers of chick proximal tubules (PT) revealed a chloride secretory pathway that was activated by parathyroid hormone and by cAMP agonists. Our data suggest that this Cl − flux is mediated by a CFTR (cystic fibrosis transmembrane regulator)‐like Cl − channel on the apical membrane. The goal of the present study was to examine the basolateral uptake mechanism for Cl − , and to determine whether a similar Cl − secretion pathway is present in mammalian PT cells. Chick (5 day white Leghorn) or mouse kidneys were collagenase‐digested and separated on a Percoll density gradient to yield PT fragments that were then seeded onto Nunc tissue culture inserts (0.02 μM filter pores). Chick cells were grown on collagen coated filters, while mouse cells required a dilute Matrigel coating. Under voltage‐clamped conditions, short circuit currents (I SC ) were measured. A positive Cl − current was induced in chick monolayers with 0.1–10 μM forskolin. This Cl − current could be partially inhibited by basolateral application of bumetanide (10–100 μM), an inhibitor of Na + ‐K + ‐2Cl − (NKCC) cotransport, and by barium, a blocker of K + channels. Mouse cells were more resistant to forskolin, but positive currents could be induced with 3‐isobutyl‐1‐methylxanthine (IBMX), a phosphodiesterase inhibitor. Cl − currents in both mouse and chick monolayers could be inhibited by apical addition of 100 μM 5‐nitro‐2‐(3‐phenylpropylamino) benzoic acid (NPPB), a Cl − channel blocker. These studies support the conclusion of a conserved chloride secretion pathway in the vertebrate proximal tubule, mediated, at least in part, by basolateral NKCC uptake and apical CFTR exit steps. Supported by NSF IBN0343478