Functional expression of the 11 human Organic Anion Transporting Polypeptides in insect cells reveals that sodium fluorescein is a general OATP substrate.

Patik, Izabel; Kovacsics, Daniella; Német, Orsolya; Gera, Melinda; Várady, György; Stieger, Bruno; Hagenbuch, Bruno; Szakács, Gergely; Özvegy-Laczka, Csilla

Patik, Izabel; Kovacsics, Daniella; Német, Orsolya; Gera, Melinda; Várady, György; Stieger, Bruno; Hagenbuch, Bruno; Szakács, Gergely; Özvegy-Laczka, Csilla.

Afiliación

Patik I; Momentum Membrane Protein Research Group, Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Magyar tudósok krt. 2, H-1117 Budapest, Hungary.
Kovacsics D; Momentum Membrane Protein Research Group, Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Magyar tudósok krt. 2, H-1117 Budapest, Hungary.
Német O; Momentum Membrane Protein Research Group, Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Magyar tudósok krt. 2, H-1117 Budapest, Hungary.
Gera M; Momentum Membrane Protein Research Group, Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Magyar tudósok krt. 2, H-1117 Budapest, Hungary.
Várady G; Laboratory of Molecular Cell Biology, Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Magyar tudósok krt. 2, H-1117 Budapest, Hungary.
Stieger B; Department of Clinical Pharmacology and Toxicology, University Hospital, 8091 Zurich, Switzerland.
Hagenbuch B; Department of Pharmacology, Toxicology and Therapeutics, The University of Kansas Medical Center, Kansas City, KA 66160, USA.
Szakács G; Momentum Membrane Protein Research Group, Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Magyar tudósok krt. 2, H-1117 Budapest, Hungary.
Özvegy-Laczka C; Momentum Membrane Protein Research Group, Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Magyar tudósok krt. 2, H-1117 Budapest, Hungary. Electronic address: laczka.csilla@ttk.mta.hu.

Biochem Pharmacol ; 98(4): 649-58, 2015 Dec 15.

Article en En | MEDLINE | ID: mdl-26415544

RESUMEN

Organic Anion Transporting Polypeptides (OATPs), encoded by genes of the Solute Carrier Organic Anion (SLCO) family, are transmembrane proteins involved in the uptake of various compounds of endogenous or exogenous origin. In addition to their physiological roles, OATPs influence the pharmacokinetics and drug-drug interactions of several clinically relevant compounds. To examine the function and molecular interactions of human OATPs, including several poorly characterized family members, we expressed all 11 human OATPs at high levels in the baculovirus-Sf9 cell system. We measured the temperature- and inhibitor-sensitive cellular accumulation of sodium fluorescein and fluorescein-methotrexate, two fluorescent substrates of the OATPs, OATP1B1 and 1B3. OATP1B1 and 1B3 were functional in Sf9 cells, showing rapid uptake (t1/2(fluorescein-methotrexate) 2.64 and 4.16 min, and t1/2(fluorescein) 6.71 and 5.58 min for OATP1B1 and 1B3, respectively) and high-affinity transport (Km(fluorescein-methotrexate) 0.23 and 0.53 µM, and Km(fluorescein) 25.73 and 38.55 µM for OATP1B1 and 1B3, respectively) of both substrates. We found that sodium fluorescein is a general substrate of all human OATPs: 1A2, 1B1, 1B3, 1C1, 2A1, 2B1, 3A1, 4A1, 4C1, 5A1 and 6A1, while fluorescein-methotrexate is only transported by 1B1, 1B3, 1A2 and 2B1. Acidic extracellular pH greatly facilitated fluorescein uptake by all OATPs, and new molecular interactions were detected (between OATP2B1 and Imatinib, OATP3A1, 5A1 and 6A1 and estradiol 17-ß-d-glucuronide, and OATP1C1 and 4C1 and prostaglandin E2). These studies demonstrate, for the first time, that the insect cell system is suitable for the functional analysis of the entire human OATP family, and for drug-OATP interaction screening.

Asunto(s)

Fluoresceína/metabolismo; Transportadores de Anión Orgánico/biosíntesis; Transportadores de Anión Orgánico/genética; Animales; Línea Celular; Regulación de la Expresión Génica; Humanos; Insectos; Transportadores de Anión Orgánico Sodio-Independiente/biosíntesis; Transportadores de Anión Orgánico Sodio-Independiente/genética; Especificidad por Sustrato/fisiología

Palabras clave

Cyclosporin A (PubChem CID: 5284373); Drug screening; Estradiol 17-ß-d-glucuronide (PubChem CID: 66424); Fluorescein-methotrexate (PubChem CID: 86761749); Fluorescent assay; Glycocholate (PubChem CID: 5460316); Imatinib (PubChem CID: 5291); New substrates; Organic Anion Transporting Polypeptide; Prostaglandin E(2) (PubChem CID: 5280360); Sodium fluorescein (PubChem CID: 16850); Taurocholate (PubChem CID: 6675); Ursolic acid (PubChem CID: 64945)

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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Fluoresceína / Transportadores de Anión Orgánico Límite: Animals / Humans Idioma: En Revista: Biochem Pharmacol Año: 2015 Tipo del documento: Article País de afiliación: Hungria Pais de publicación: Reino Unido

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