Iodide efflux measurements with an iodide-selective electrode: A non-radioactive procedure for monitoring cellular chloride transport

A nonradioactive procedure using an I^–-selective electrode has been developed for assaying cellular Cl^– transport. NIH 3T3 fibroblasts stably transfected with the Cystic Fibrosis Trans-membrane Conductance Regulator (CFTR) Cl^– channel were grown in standard 35 mm culture dishes and used to test this assay system. For efflux measurements, the fibroblasts were first incubated in an I^–-loading buffer and then exposed to an I^–-free buffer. Efflux was monitored using the I^–-selective electrode. Application of either forskolin (5 µM) or 8-chlorphenylthio cAMP (500 µ M), to activate the CFTR Cl^– channels, resulted in over a 5-fold increase in I^– efflux as compared with control, untreated fibroblasts. No increase over basal efflux levels was observed in nontransfected NIH 3T3 fibroblasts treated with forskolin. The Cl^– channel blockers diphenylamine-2-carboxylate (DPC) (1 mM) and 5-nitro-2-(3- phenylpropylamino)-benzoic acid (NPPB) (25 µM) reduced forskolin- stimulated efflux by 50% and 23%, respectively. In addition to forskolin, both the tyrosine kinase inhibitor genistein and the protein kinase C activator phorbol 12,13-dibutyrate, were capable of stimulating I^– efflux. Thus, use of the I^–-selective electrode provides a fast and convenient method for studying Cl^– channels. The I^– efflux assay should be useful for monitoring drug and hormone-activated Cl^– transport pathways in a wide variety of cell types.