EFFECT OF PHENYLISOTHIOCYANATE ON ORGANIC CATION-TRANSPORT IN OPOSSUMKIDNEY-CELLS

In this study tetraethylammonium (TEA) was used as a model compound to determine the effect of the lysine selective modifying reagent, pheny lisothiocyanate (PITC), on organic cation transport across the brush-b order membrane of opossum kidney (OK) cells. TEA uptake in OK cell mon olayers treated with PITC was reduced in a time- and concentration- de pendent manner (IC50 200 mu M). Two lines of evidence suggested that a mine rather than sulfhydryl residues were being modified by PITC. Firs t, treatment of the cells with PITC at pH 8.5 was significantly more e ffective in reducing TEA uptake than PITC treatment at pH 7.4. Secondl y, dithiothreitol (10 mM) could not reverse the effect of PITC on TEA uptake. Preincubation of OK cell monolayers with PITC (50 mu M) result ed in a significant increase in the K-m of TEA (control cells: 81 +/- 10 mu M; treated cells: 165 +/- 54 mu M), whereas the V-max was unaffe cted. Incubation of the cells with quinine or unlabeled TEA significan tly altered the inhibitory effects of PITC on TEA uptake. At low conce ntrations, quinine protected the transporter from the inhibitory effec ts of PITC, whereas at higher concentrations (>2.5 mu M) quinine enhan ced PITC inhibition of TEA uptake. Other organic cations that are subs trates for the transporter including cimetidine and N-1-methylnicotina mide did not alter the effects of PITC on TEA transport. Collectively, these data suggest that covalent modification of amine groups reduces the transport of TEA in OK cells by decreasing the affinity of TEA to the transporter. The prior binding of TEA and quinine may increase th e exposure of amine residues to modification by PITC, resulting in an enhanced inhibitory effect of PITC on TEA transport.