A transfected cell model for the renal toxin transporter, rOCT2.
A cDNA for the organic cation transporter (rOCT2) of the rat kidney was inserted into the retroviral plasmid pLXSN. This plasmid was used to stably transfect NIH3T3 cells. The transfected cell line exhibited an enhanced rate of tetraethylammonium (TEA) uptake and efflux compared to wild-type NIH3T3 cells. Uptake of TEA by the transfected cells was markedly reduced upon incubation at 4 degrees C. When the extracellular pH was lowered from 8.1 to 5.9, uptake was also reduced, suggesting inhibition of rOCT2 by extracellular protons. The apparent K(m) for TEA in the transfected cells was 141 microM. The classical organic cation transport inhibitors, cyanine 863 and cimetidine, produced noncompetitive inhibition with apparent Ki values of 0.81 and 198 microM, respectively. Daunomycin, vinblastine, and the deoxyadenosine analogs, 2'-deoxytubercidin and 2-chlorodeoxyadenosine, did not appear to be substrates for rOCT2. However, the anticancer drug, cisplatin, competitively inhibited TEA uptake by rOCT2 with an apparent Ki value of 925 microM, suggesting that rOCT2 may play a role in its renal secretion. In summary, transfected NIH3T3 cells provide a facile system by which this and other organic ion transporters can be studied.
Pan
B F
BF
Department of Experimental Pediatrics, University of Texas M. D. Anderson Cancer Center, Houston 77030, USA.
Sweet
D H
DH
Pritchard
J B
JB
Chen
R
R
Nelson
J A
JA
D016475:3T3 Cells; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D002352:Carrier Proteins / Q000235:genetics*; D002945:Cisplatin / Q000494:pharmacology; D018076:DNA, Complementary / Q000302:isolation & purification*; D003630:Daunorubicin / Q000494:pharmacology; D003839:Deoxyadenosines / Q000494:pharmacology; D007668:Kidney / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D008957:Models, Genetic; D027701:Organic Cation Transport Proteins; D000075102:Organic Cation Transporter 2; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013757:Tetraethylammonium Compounds / Q000378:metabolism; D014162:Transfection; D014747:Vinblastine / Q000494:pharmacology
D000970:Antineoplastic Agents; D002352:Carrier Proteins; D018076:DNA, Complementary; D003839:Deoxyadenosines; D027701:Organic Cation Transport Proteins; D000075102:Organic Cation Transporter 2; C491012:Slc22a2 protein, mouse; C100590:Slc22a2 protein, rat; D013757:Tetraethylammonium Compounds; D014747:Vinblastine; D002945:Cisplatin; D003630:Daunorubicin