Naslov
Functional characterization of zebrafish (Danio rerio) organic anion transporter 2d (Oat2d)

Autori
Dragojević, Jelena ; Mihaljević, Ivan ; Smital, Tvrtko

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
Book of abstracts of 10th European Zebrafish Meeting / - , 2017, 572-572

Skup
10th European Zebrafish Meeting

Mjesto i datum
Budimpešta, Mađarska, 03.07.2017. - 07.07.2017

Vrsta sudjelovanja
Poster

Vrsta recenzije
Recenziran

Ključne riječi
membrane proteins ; SLC22 ; organic anion transporters, Oat2d, zebrafish, phylogeny ; tissue expression ; cell localization ; functional analyses

Sažetak
The organic anion transporters (OATs in humans, Oats in other animal species) are a family of transmembrane proteins that are able to transport a variety of compounds including drugs, environmental toxins and endogenous metabolites into the cell and play an essential role in their elimination from the body. They belong to the SLC22 (solute carrier 22) subfamily of the major facilitator superfamily (MFS). OATs are polyspecific uptake (influx) transporters expressed in almost all barrier epithelia of the body where they mediate entrance of compounds into the cell. Consequently, they are key determinants of toxicological response to various xenobiotics. Despite their physiological importance and role in cellular detoxification, the knowledge about uptake transporters in non-mammalian species is scarce. Zebrafish (Danio rerio) has seven OAT orthologs: Oat1, Oat2a-e and Oat3. Mammalian OAT2 was characterized in human, rat and mouse and plays an important role in uptake and distribution of physiological compounds, as well as anionic toxins and drugs. Typical substrates of mammalian OAT2 are salicylate, acetylsalicylate, prostaglandin E2 (PGE2), dicarboxylates, and PAH. As OAT2 orthologs have been poorly studied in non-mammalian species, the goal of our study was to determine phylogenetic relationships, tissue distribution and substrate specifity zebrafish Oat2d. Phylogenetic analysis of OAT/Oat genes points to similarities among mammalian and zebrafish Oat transporters. Zebrafish Oat2d is highly expressed in intestine and moderately expressed in testes and brain. Using HEK293T cells transiently transfected with zebrafish Oat2d, our Western blot analysis revealed protein band of about 60 kDa (estimated size of zebrafish Oat2d is 62.92 kDa), and immunocitochemistry showed its correct localization in the plasma membrane. Functional studieds using the HEK293 cells overxpressing zebrafish Oat2d revealed two model fluorescent substrates of Oat2d: lucifer yellow (Km = 56.36 µM) and 6- carboxyfluorescein (Km = 210.1 µM). The initial screen with various endo- and xenobiotics showed significant inhibition of Oat2d mediated uptake of lucifer yellow by endogenous compounds (α-ketoglutarate, bilirubin and deoxycholic acid) and exogenous compounds (p- aminohippurate, perfluorooctanesulfonic acid, perfluorooctanoic acid, furosemid, chlorpyrifos, tetracycline and diclofenac). Our next research steps will be determination of their IC50 values, and Michaelis-Menten kinetic assays to determine the type of interaction (substrates vs. noncompetitive inhibitors) between tested compounds and zebrafish Oat2d.

Izvorni jezik
Engleski

Znanstvena područja
Biologija

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