Abstract
The goal of this study was to determine the mechanisms involved in the transport of the organic cation, tetraethylammonium (TEA), across the apical membrane of OK cells. [14C]TEA accumulated in OK cell monolayers reaching equilibrium in 2 h. The uptake of [14C]TEA at equilibrium was dependent upon temperature and was inhibited by sodium azide and by various organic cations, including N1-methylnicotinamide (NMN), mepiperphenidol, and cimetidine but not by the organic anion, p-aminohippuric acid. The initial uptake of [14C]TEA was characterized by a saturable process. The mean +/- S.D. Km was 27.8 +/- 2.6 microM and the Vmax was 414 +/- 26.5 pmol/mg protein/min. Both an accelerated efflux and influx of [14C]TEA in the presence of a trans-gradient of unlabeled TEA and NMN was observed, whereas a deaccelerated influx and efflux was observed in the presence of a trans-gradient of mepiperphenidol. The mechanism of interaction between NMN and TEA was examined. NMN significantly increased the apparent Km (mean +/- S.D.) of TEA to 82.8 +/- 16.4 microM (p less than 0.001), whereas the Vmax (mean +/- S.D.) was only slightly affected (478 +/- 72 pmol/mg protein/min) suggesting a competitive inhibition. The stimulatory effect of trans-gradients of NMN on TEA transport was due to an increase in the Vmax of TEA suggesting that NMN trans-stimulates TEA transport by increasing the turnover rate of the exchanger. In the presence of an inwardly directed proton gradient, the efflux at 30 s of [14C]TEA from the OK cell monolayers was significantly accelerated (p less than 0.05). Studies with the pH-sensitive fluorescent probe, 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein, suggested that TEA could drive the countertransport of protons. In apical membrane vesicles prepared from OK cells, the uptake of [3H]NMN exhibited an apparent "overshoot phenomenon" in the presence of an initial outwardly directed proton gradient. Protons competitively inhibited TEA uptake suggesting that the proton/organic cation and the organic cation/organic cation self exchange mechanism are the same mechanism. This is the first report describing both TEA self-exchange and proton/TEA exchange in the apical membrane of a continuous cell line. OK cells are an excellent model for the study of organic cation transport across the apical membrane.
Highlights
(mean f S.D.) was only slightly affected (478 f 72 pmol/mg proteinlmin) suggesting a competitive inhibition.Thestimulatoryeffect of trans-gradients of that organic cations temporarily accumulate againasctoncentration gradient in the presenceof a n outwardly directed proton gradient
TOdetermine the time at which organic cation transport is expressed in OK cell monolayers grown in culture, we examined the uptake of [14C]TEAin cells that had been subcultured for various days
We observed that the organic cation, TEA, accumulated in OK cell monolayers across the apical membrane
Summary
P4C]TEA U ~ ~ U ~ ~ - - [ ' ~ Ca]cTcuEmAulated with time in the OK cellmonolayers reaching equilibrium after approximately 150min (Fig. 1).The cell water space, determined as described under "Experimental Procedures", was 9.5f 1.7pl/mgprotein cause it was the earliest timethat reproducible data could be obtained. A saturable process appears to be involved in the transport of TEA. The kinetics of TEA transporwt ere described by the followingrelationship:. This value is similar to that reported where Vmaxand K,,, are the Michaelis-Menten constants and by others (Bosch et al, 1989). From the mean intracellular C is the media TEA concentration. The weighted mean data water space and the equilibrium uptake of [14C]TEAat a
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