Effect of phenylisothiocyanate on organic cation transport in opossum kidney cells.
In this study tetraethylammonium (TEA) was used as a model compound to determine the effect of the lysine selective modifying reagent, phenylisothiocyanate (PITC), on organic cation transport across the brush-border membrane of opossum kidney (OK) cells. TEA uptake in OK cell monolayers treated with PITC was reduced in a time- and concentration- dependent manner (IC50, 200 microM). Two lines of evidence suggested that amine rather than sulfhydryl residues were being modified by PITC. First, treatment of the cells with PITC at pH 8.5 was significantly more effective in reducing TEA uptake than PITC treatment at pH 7.4. Secondly, dithiothreitol (10 mM) could not reverse the effect of PITC on TEA uptake. Preincubation of OK cell monolayers with PITC (50 microM) resulted in a significant increase in the Km of TEA (control cells: 81 +/- 10 microM; treated cells: 165 +/- 54 microM), whereas the Vmax was unaffected. Incubation of the cells with quinine or unlabeled TEA significantly altered the inhibitory effects of PITC on TEA uptake. At low concentrations, quinine protected the transporter from the inhibitory effects of PITC, whereas at higher concentrations (> 2.5 microM) quinine enhanced PITC inhibition of TEA uptake. Other organic cations that are substrates for the transporter including cimetidine and N1-methylnicotinamide 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Ott
R J
RJ
School of Pharmacy, Department of Pharmacy, University of California, San Francisco.
Chan
J K
JK
Ramanathan
V K
VK
Hui
A C
AC
Giacomini
K M
KM
D000818:Animals; D001693:Biological Transport, Active; D002478:Cells, Cultured; D017879:Isothiocyanates; D007668:Kidney / Q000187:drug effects* / Q000378:metabolism* / Q000648:ultrastructure; D007700:Kinetics; D008239:Lysine / Q000378:metabolism; D008871:Microvilli / Q000187:drug effects / Q000378:metabolism; D009893:Opossums; D026902:Potassium Channel Blockers; D011803:Quinine / Q000494:pharmacology; D019789:Tetraethylammonium; D013757:Tetraethylammonium Compounds / Q000493:pharmacokinetics*; D013861:Thiocyanates / Q000494:pharmacology*
D017879:Isothiocyanates; D026902:Potassium Channel Blockers; D013757:Tetraethylammonium Compounds; D013861:Thiocyanates; C005441:phenylisothiocyanate; D019789:Tetraethylammonium; D011803:Quinine; D008239:Lysine