Organic cation transporters OCT1, 2, and 3 mediate high-affinity transport of the mutagenic vital dye ethidium in the kidney proximal tubule.
The positively charged fluorescent dyes ethidium (Et(+)) and propidium (Pr(2+)) are widely used as DNA and necrosis markers. Et(+) is cytotoxic and mutagenic. The polyspecific organic cation transporters OCT1 (SLC22A1), OCT2 (SLC22A2), and OCT3 (SLC22A3) mediate electrogenic facilitated diffusion of small (< or =500 Da) organic cations with broad specificities. In humans, OCT2 mediates basolateral uptake by kidney proximal tubules (PT), whereas in rodents OCT1/2 are involved. In mouse kidney, perfused Et(+) accumulated predominantly in the S2/S3 segments of the PT, but not Pr(2+). In cells stably overexpressing human OCTs (hOCTs), Et(+) uptake was observed with K(m) values of 0.8 +/- 0.2 microM (hOCT1), 1.7 +/- 0.5 microM (hOCT2), and 2.0 +/- 0.5 microM (hOCT3), whereas Pr(2+) was not transported. Accumulation of Et(+) was inhibited by OCT substrates quinine, 3-methyl-4-phenylpyridinium (MPP(+)), cimetidine, and tetraethylammonium (TEA(+)). For hOCT1 and hOCT2, the IC(50) values for MPP(+), TEA(+), and cimetidine were higher than for inhibition of previously tested transported substrates. For hOCT2, the inhibition of Et(+) uptake by MPP(+) and cimetidine was shown to be competitive. Et(+) also inhibited transport of 0.1 microM [(3)H]MPP(+) by all hOCT isoforms with IC(50) values between 0.4 and 1.3 microM, and the inhibition of hOCT1-mediated uptake of MPP(+) by Et(+) was competitive. In Oct1/2(-/-) mice, Et(+) uptake in the PT was almost abolished. The data demonstrate that Et(+) is taken up avidly by the PT, which is mediated by OCT1 and/or OCT2. Considering the high affinity of OCTs for Et(+) and their strong expression in various organs, strict safety guidelines for Et(+) handling should be reinforced.
Department of Physiology and Pathophysiology, Univ. of Witten/Herdecke, Stockumer Strasse 12, D-58453 Witten, Germany.
Wing-Kee
WK
Lee
Markus
M
Reichold
Bayram
B
Edemir
Giuliano
G
Ciarimboli
Richard
R
Warth
Hermann
H
Koepsell
Frank
F
Thévenod
D000818:Animals; D001693:Biological Transport, Active / Q000502:physiology; D016466:CHO Cells; D004396:Coloring Agents / Q000493:pharmacokinetics*; D006224:Cricetinae; D003412:Cricetulus; D004996:Ethidium / Q000493:pharmacokinetics*; D005786:Gene Expression Regulation / Q000502:physiology; D006801:Humans; D020128:Inhibitory Concentration 50; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D027701:Organic Cation Transport Proteins / Q000235:genetics* / Q000378:metabolism*; D010269:Paraquat / Q000737:chemistry / Q000494:pharmacology; D011419:Propidium / Q000737:chemistry / Q000494:pharmacology; D020033:Protein Isoforms; D013379:Substrate Specificity
D004396:Coloring Agents; D027701:Organic Cation Transport Proteins; D020033:Protein Isoforms; D011419:Propidium; D004996:Ethidium; D010269:Paraquat