Characterisation of Organic Cation Transport across the Apical Membrane of Proximal Tubular Cells with the Fluorescent Dye 4-Di-1-ASP

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Abstract
Recently, we introduced a new method to monitor organic cation transport using the fluorescent dye 4-(4-dimethylaminostyryl)-N-methylpyridinium (ASP+). We now examined properties of ASP+ uptake across the apical membrane of proximal tubular cells (LLC-PK1 and IHKE1) with this new method and compared the results with characteristics of organic cation transport obtained in transport studies using radio-labeled substrates. Cimetidine and choline inhibited ASP+ accumulation in LLC-PK1 cells with maximal inhibitions of 51 ± 5% (n = 6, at 1 mM) and 45 ± 14% (n = 6, at 50 mM), respectively. TMA+, TEA+ and THA+ showed bell-shaped concentration-response curves in LLC-PK1 and IHKE1 cells. Maximal inhibition was achieved in LLC-PK1 cells with 10 mMTMA+ (53 ± 4%, n = 6), 5 mMTEA+ (64 ± 4%, n = 4) and 10 µMTHA+ (97 ± 5%, n = 6) and in IHKE1 cells with > 50 mMTMA+ (51 ± 8%, n = 6), 1 mMTEA+ (53 ± 12%, n = 6) and 5 µMTHA+(100 ± 1%, n = 6). Higher concentrations of these cations led to a reduction of the inhibitory effect. ASP+ accumulation in LLC-PK1 cells was reduced at pH 6.0 by 60 ± 5% (n = 7) and increased at pH 8.4 by 18 ± 6% (n = 6). Membrane depolarization induced by increases in the extracelluar K+ concentration to 50 or 145 mMled to a reduction of the ASP+ accumulation in LLC-PK1 cells by 27 ± 7% (n = 5) or 37 ± 6% (n = 5), respectively. The data of this study demonstrate that ASP+ accumulation across the apical membrane of LLC-PK1 and IHKE1 cells measured fluorimetrically has properties comparable to those reported for the H+/organic cation exchanger described for the luminal membrane of the proximal tubule.

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