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

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• 作者：Izabel Patik^a ; Daniella Kovacsics^a ; Orsolya Né ; met^a ; Melinda Gera^a ; Gyö ; rgy Vá ; rady^b ; Bruno Stieger^c ; Bruno Hagenbuch^d ; Gergely Szaká ; cs^a ; Csilla Ö ; zvegy-Laczka^a ; ^{laczka.csilla@ttk.mta.hu" class="auth_mail" title="E-mail the corresponding author}
• 关键词：Sodium fluorescein (PubChem CID ; 16850) ; Fluorescein-methotrexate (PubChem CID ; 86761749) ; Imatinib (PubChem CID ; 5291) ; Estradiol 17-β-d-glucuronide (PubChem CID ; 66424) ; Ursolic acid (PubChem CID ; 64945) ; Cyclosporin A (PubChem CID ; 5284373) ; Prostaglandin E2 (PubChem CID ; 5280360) ; Glycocholate (PubChem CID ; 5460316) ; Taurocholate (PubChem CID ; 6675)
• 刊名：Biochemical Pharmacology
• 出版年：2015
• 出版时间：15 December 2015
• 年：2015
• 卷：98
• 期：4
• 页码：649-658
• 全文大小：1784 K

文摘

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 (t_{1/2(fluorescein-methotrexate)} 2.64 and 4.16 min, and t_{1/2(fluorescein)} 6.71 and 5.58 min for OATP1B1 and 1B3, respectively) and high-affinity transport (K_{m(fluorescein-methotrexate)} 0.23 and 0.53 μM, and K_{m(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-β-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.