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Influence of the flavonoids apigenin; kaempferol and quercetin on the function of organic anion transporting polypeptides 1A2 and 2B1, Biochemical Pharmacology (2010), doi:10.1016/j.bcp.2010 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.Page 1 of 37 A c c e p t e d M a n u s c r i p t 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 A c c e p t e d M a n u s c r i p t 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 A c c e p t e d M a n u s c r i p t 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 4 modification of OATP1A2 and OATP2B1 transport activity by apigenin, kaempferol, and quercetin may be a mechanism for food-drug or drug-drug interactions in humans.Page 5 of 37A c c e p t e d M a n u s c r i p t 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 A c c e p t e d M a n u s c r i p t Generation of a HEK293 cell line stably expressing OATP1A2The SLCO1A2 coding sequence (NM_134431. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 10 subcloned into the retroviral vector pQCXIN (Takara Bio Europe/Clontech, SaintGermain-en-Laye, France). Human embryonic kidney cells (HEK293) were transfected with the plasmid pQCXIN-OATP1A2 using a retroviral gene transfer and expression kit (Takara Bio Europe/Clontech, Saint-Germain-en-Laye, France). After geneticin (G-418; 500 µg/ml) treatment, single colonies were selected and characterized for OATP1A2 mRNA and protein expression using ...

Background and purpose: Organic anion transporting polypeptide 1B3 (OATP1B3) (SLCO1B3) mediates the uptake of endogenous substrates (e.g. estrone-3-sulphate) and drugs (e.g. pravastatin) from blood into hepatocytes. Structure-based modelling of OATP1B3 suggested that a pore with a positive electrostatic potential contributes to the transport mechanism. Therefore, we investigated the role of conserved positively charged amino acids for OATP1B3-mediated uptake of sulphobromophthalein (BSP) and pravastatin. Experimental approach: Residues Lys28, Lys41 and Arg580 in OATP1B3 were substituted by alanine, arginine, glutamine, glycine or lysine. Using immunofluorescence, immunoblot analysis and cellular uptake assays, the effect of these mutations on protein expression and transport activity was investigated. Key results: Immunofluorescence revealed that all mutants were localized in the plasma membrane with partial intracellular retention of the Arg580>Ala and Arg580>Lys mutants. Lys41>Ala, Lys41>Gln, Lys41>Gly, Arg580>Gly and Arg580>Lys showed significantly reduced transport for BSP and pravastatin. Kinetic analyses of BSP transport revealed a significant reduction of Vmax normalized to cell surface protein expression for Lys41>Ala (wild type: 190 Ϯ 8, Lys41>Ala:16 Ϯ 4 pmol (mg protein) -1 min -1 , P < 0.001), whereas Vmax of Lys41>Arg and Arg580>Lys (103 Ϯ 8 and 123 Ϯ 14 pmol (mg protein) -1 min -1, P > 0.05) did not change significantly. This suggests that the positive charges at positions 41 and 580 are important for transport activity of BSP. Structural modelling indicated that the positively charged side chain of Lys41 is flexible within the pore. The orientation of Arg580 is defined by adjacent residues Glu74 and Asn77, which was confirmed by kinetic analysis of Glu74>Ala. Conclusions and implications:We demonstrated that the conserved positively charged amino acids Lys41 and Arg580 are pivotal to the transport activity of OATP1B3.

The human organic anion-transporting polypeptide 2A1 (OATP2A1) is a prostaglandin transporter expressed in several tissues and plays an important role for local distribution of prostaglandins, which contribute to the integrity of gastric mucosa. Blockade of prostaglandin pathways by cyclooxygenase (COX) inhibitors has been associated with serious side effects such as gastrointestinal ulceration and bleeding. However, little is known regarding OATP2A1 expression in the upper gastrointestinal tract and the potential impact of cyclooxygenase inhibitors on OATP2A1 function. We first investigated the expression of OATP2A1 mRNA and protein in human gastroduodenal mucosa using human biopsy specimens obtained from antrum, corpus, and duodenum. The results indicate that OATP2A1 is expressed in the neck region and deep pyloric glands of antrum and in parietal cells of gastric corpus. Second, we examined various COX inhibitors for their effects on OATP2A1 transporter activity. Using HEK293 cells expressing OATP2A1, we found that diclofenac and lumiracoxib are potent inhibitors of OATP2A1-mediated transport of prostaglandin (PG) E 2 with IC 50 values of 6.2 Ϯ 1.2 and 3.1 Ϯ 1.2 M. In contrast, indomethacin, ketoprofen, and naproxen led to significant stimulation of OATP2A1-mediated PGE 2 transport by 162.7 Ϯ 13.9, 77.2 Ϯ 3.6, and 32.3 Ϯ 4.9%, respectively. Taken together, our results suggest that various clinically used COX inhibitors have differential impact on the function of the prostaglandin transporter OATP2A1 in human stomach and that these effects may contribute to differences in the gastrointestinal side effects of COX inhibitors.

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