Halide fluxes in epithelial cells measured with an automated cell plate reader

Abstract

A method is introduced to measure chloride permeability in cultured epithelial cells using 6-methoxy- N-(3-sulfopropyl)quinolinium (SPQ) and 6-methoxy- N-ethylquinolinium iodide quinolinium (MEQ) as fluorescent chloride-sensitive probes. The method involves growing cells in multiwell plates, incubating cells with SPQ or MEQ, and then exchanging intracellular or extracellular halide ions with nitrate. The resulting time course of SPQ or MEQ fluorescence is followed by repetitive readings with a multiwell fluorescence plate reader. Exchange times are extracted by fitting the time course with a single exponential function of time. The method was validated by measuring the effect of chloride channel activators and blockers in A6 and MDCK cells. The baseline iodide/nitrate exchange time was 200–300 s. Isoproterenol (a modulator of cAMP-activated chloride channels) increased the exchange rate by a factor of 1.4±0.1; A23187 (a modulator of calcium-activated chloride channels) increased the rate by 3.4±0.4; bradykinin (also a modulator of calcium-activated chloride channels) increased the rate by 2.0±0.4; forskolin (a direct stimulator of adenylate cyclase) increased the rate by 2.7±0.3. Diphenylamine-2-carboxylate (a chloride channel blocker) decreased the rate by 0.12±0.03. These results indicate that our method is a valid indicator of halide–nitrate exchange in cultured epithelial cells.