engleski

Characterization of zebrafish (Danio rerio) organic anion transporters Oat1 and Oat3

OATS/Oats are transmembrane proteins that transport a variety of compounds including drugs, environmental toxins and endogenous metabolites into the cell. Human OAT1 and OAT3 are primarily expressed in the kidney where their physiological role is uptake of urate into the kidneys, thus representing the first step in urate excretion. Substrates of human OAT1 and OAT3 largely overlap and include substances like cAMP, prostaglandins, urate, acidic neurotransmitter metabolites, and various pharmaceuticals. Yet, despite their physiological importance and role in cellular detoxification in mammals, the knowledge about Oats in non-mammalian species is scarce. Zebrafish (Danio rerio) has seven OAT orthologs: Oat1, Oat2a-e and Oat3. As OAT1 and OAT3 orthologs have been poorly studied in non- mammalian species, the goal of our study was to determine phylogenetic relationships, tissue distribution and substrate specificity of zebrafish Oat1 and Oat3. Our phylogenetic analysis of OAT/Oat genes points to similarities among mammalian and zebrafish Oat transporters. Considering tissue specific expression, we found that Oat1 has low expression in zebrafish tissues, while Oat3 is highly expressed in kidney and moderately in testes. We developed transport activity assays with HEK293T cells overexpressing zebrafish Oats and model fluorescent substrates: lucifer yellow (LY, Km = 8 µM) for Oat1 and 6- carboxyfluorescein (6-CF, Km = 2 µM) for Oat3. Finally, the initial screening of various endo- and xenobiotics showed significant inhibition of Oat1 mediated uptake of LY by endogenous compounds (α-ketoglutarate, bilirubin, DHEAS, estrone-3-sulfate and deoxycholic acid) and exogenous compounds (furosemide, indomethacin, MK- 571 and methotrexate). Oat3 mediated uptake of 6- CF was inhibited by the same endogenous compounds as for Oat1, except bilirubin, and same xenobiotics plus p-aminohippurate.

zebrafish ; transporters ; Oat1 ; Oat3

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