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Organic anion transporter 2 transcript variant 1 shows broad ligand selectivity when expressed in multiple cell lines.

Hotchkiss AG, Berrigan L, Pelis RM - Front Pharmacol (2015)

Bottom Line: We cloned OAT2-tv1 and OAT2-tv2, but were unsuccessful at amplifying mRNA for OAT2-tv3 from human kidney.OAT2-tv1 trafficked to the plasma membrane of all three cell types, but OAT2-tv2 did not.Not surprising given its lack of plasma membrane expression, OAT2-tv2 failed to transport any of the organic solutes examined, including penciclovir.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Dalhousie University Halifax, NS, Canada.

ABSTRACT
Organic anion transporter 2 (OAT2) is likely important for renal and hepatic drug elimination. Three variants of the OAT2 peptide sequence have been described - OAT2 transcript variant 1 (OAT2-tv1), OAT2 transcript variant 2 (OAT2-tv2), and OAT2 transcript variant 3 (OAT2-tv3). Early studies helping to define the ligand selectivity of OAT2 failed to identify the variant used, and the studies used several heterologous expression systems. In preliminary studies using OAT2-tv1, we failed to observe transport of several previously identified substrates, leading us to speculate that ligand selectivity of OAT2 differs with variant and/or heterologous expression system. The purpose was to further investigate the ligand selectivity of the OAT2 variants expressed in multiple cell types. We cloned OAT2-tv1 and OAT2-tv2, but were unsuccessful at amplifying mRNA for OAT2-tv3 from human kidney. OAT2-tv1 and OAT2-tv2 were individually expressed in human embryonic kidney (HEK), Madin-Darby canine kidney (MDCK), or Chinese hamster ovary (CHO) cells. mRNA for OAT2-tv1 and OAT2-tv2 was demonstrated in each cell type transfected with the respective construct, indicating their expression. OAT2-tv1 trafficked to the plasma membrane of all three cell types, but OAT2-tv2 did not. OAT2-tv1 transported penciclovir in all three cell types, but failed to transport para-aminohippurate, succinate, glutarate, estrone-3-sulfate, paclitaxel or dehydroepiandrosterone sulfate - previously identified substrates of OAT2-tv2. Not surprising given its lack of plasma membrane expression, OAT2-tv2 failed to transport any of the organic solutes examined, including penciclovir. Penciclovir transport by OAT2-tv1 was sensitive to large (e.g., cyclosporine A) and small (e.g., allopurinol) organic compounds, as well as organic anions, cations and neutral compounds, highlighting the multiselectivity of OAT2-tv1. The potencies with which indomethacin, furosemide, cyclosporine A and cimetidine inhibited OAT2-tv1 are in good agreement with previous studies using this variant, but inconsistent with studies using OAT2 with an unidentified sequence. This study shows that organic molecules with diverse physicochemical properties interact with OAT2-tv1, making it a likely site of drug interactions. Many previously identified substrates of OAT2 are not transported by OAT2-tv1, suggesting that variant and/or expression system may contribute. Future work should establish the expression pattern and ligand selectivity of OAT2-tv3.

No MeSH data available.


Cellular accumulation of [14C]succinate (18.5 μM) by CHO parental cells, CHO-OAT2-tv1 cells or CHO cells stably expressing the Na-dicarboxylate cotransporter 3 (NaDC3). Uptake was conducted for 5 min. Values are the mean ± standard error of the mean of four experiments for CHO parental and CHO-OAT2-tv1 cells. A single uptake experiment was performed with CHO-NaDC3 cells. There was no significant difference in [14C]succinate uptake by CHO-OAT2-tv1 cells compared to parental CHO cells (P > 0.05, two-tailed unpaired student’s t-test.
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Figure 4: Cellular accumulation of [14C]succinate (18.5 μM) by CHO parental cells, CHO-OAT2-tv1 cells or CHO cells stably expressing the Na-dicarboxylate cotransporter 3 (NaDC3). Uptake was conducted for 5 min. Values are the mean ± standard error of the mean of four experiments for CHO parental and CHO-OAT2-tv1 cells. A single uptake experiment was performed with CHO-NaDC3 cells. There was no significant difference in [14C]succinate uptake by CHO-OAT2-tv1 cells compared to parental CHO cells (P > 0.05, two-tailed unpaired student’s t-test.

Mentions: It was previously reported that succinate and fumarate are capable of trans-stimulating OAT2-mediated substrate uptake (Kobayashi et al., 2005a), suggesting that OAT2 may be an organic anion/dicarboxylate exchanger. Given these previous results we hypothesized that if dicarboxylates interact as substrates of OAT2 that they may also inhibit the transport protein. Thus, we examined the ability of a variety of Kreb’s cycle intermediates (succinate, fumarate, α-ketoglutarate, oxaloacetate, citrate, cis-aconitate, and D-malate) to cis-inhibit penciclovir uptake into CHO-OAT2-tv1 cells. Even at a high concentration (1 mM), none of the Kreb’s cycle intermediates tested, including succinate and fumarate, cis-inhibited penciclovir uptake (Figure 3). Conversely, the OAT2 inhibitors glutamate (1 mM) and furosemide (200 μM) reduced penciclovir uptake by 70–85% compared to control, confirming that OAT2-tv1 was functional (Figure 3). We also examined the uptake of [14C]-succinate by CHO-OAT2-tv1 cells to determine if it is in fact a substrate. The uptake of [14C]succinate by CHO-OAT2-tv1 cells was not different than uptake into the parental CHO cells (Figure 4). In a single experiment (n = 1), the uptake of [14C]succinate by CHO cells stably expressing the Na-dicarboxylate cotransporter 3 (NaDC3) was ∼30-fold higher than control.


Organic anion transporter 2 transcript variant 1 shows broad ligand selectivity when expressed in multiple cell lines.

Hotchkiss AG, Berrigan L, Pelis RM - Front Pharmacol (2015)

Cellular accumulation of [14C]succinate (18.5 μM) by CHO parental cells, CHO-OAT2-tv1 cells or CHO cells stably expressing the Na-dicarboxylate cotransporter 3 (NaDC3). Uptake was conducted for 5 min. Values are the mean ± standard error of the mean of four experiments for CHO parental and CHO-OAT2-tv1 cells. A single uptake experiment was performed with CHO-NaDC3 cells. There was no significant difference in [14C]succinate uptake by CHO-OAT2-tv1 cells compared to parental CHO cells (P > 0.05, two-tailed unpaired student’s t-test.
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Figure 4: Cellular accumulation of [14C]succinate (18.5 μM) by CHO parental cells, CHO-OAT2-tv1 cells or CHO cells stably expressing the Na-dicarboxylate cotransporter 3 (NaDC3). Uptake was conducted for 5 min. Values are the mean ± standard error of the mean of four experiments for CHO parental and CHO-OAT2-tv1 cells. A single uptake experiment was performed with CHO-NaDC3 cells. There was no significant difference in [14C]succinate uptake by CHO-OAT2-tv1 cells compared to parental CHO cells (P > 0.05, two-tailed unpaired student’s t-test.
Mentions: It was previously reported that succinate and fumarate are capable of trans-stimulating OAT2-mediated substrate uptake (Kobayashi et al., 2005a), suggesting that OAT2 may be an organic anion/dicarboxylate exchanger. Given these previous results we hypothesized that if dicarboxylates interact as substrates of OAT2 that they may also inhibit the transport protein. Thus, we examined the ability of a variety of Kreb’s cycle intermediates (succinate, fumarate, α-ketoglutarate, oxaloacetate, citrate, cis-aconitate, and D-malate) to cis-inhibit penciclovir uptake into CHO-OAT2-tv1 cells. Even at a high concentration (1 mM), none of the Kreb’s cycle intermediates tested, including succinate and fumarate, cis-inhibited penciclovir uptake (Figure 3). Conversely, the OAT2 inhibitors glutamate (1 mM) and furosemide (200 μM) reduced penciclovir uptake by 70–85% compared to control, confirming that OAT2-tv1 was functional (Figure 3). We also examined the uptake of [14C]-succinate by CHO-OAT2-tv1 cells to determine if it is in fact a substrate. The uptake of [14C]succinate by CHO-OAT2-tv1 cells was not different than uptake into the parental CHO cells (Figure 4). In a single experiment (n = 1), the uptake of [14C]succinate by CHO cells stably expressing the Na-dicarboxylate cotransporter 3 (NaDC3) was ∼30-fold higher than control.

Bottom Line: We cloned OAT2-tv1 and OAT2-tv2, but were unsuccessful at amplifying mRNA for OAT2-tv3 from human kidney.OAT2-tv1 trafficked to the plasma membrane of all three cell types, but OAT2-tv2 did not.Not surprising given its lack of plasma membrane expression, OAT2-tv2 failed to transport any of the organic solutes examined, including penciclovir.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Dalhousie University Halifax, NS, Canada.

ABSTRACT
Organic anion transporter 2 (OAT2) is likely important for renal and hepatic drug elimination. Three variants of the OAT2 peptide sequence have been described - OAT2 transcript variant 1 (OAT2-tv1), OAT2 transcript variant 2 (OAT2-tv2), and OAT2 transcript variant 3 (OAT2-tv3). Early studies helping to define the ligand selectivity of OAT2 failed to identify the variant used, and the studies used several heterologous expression systems. In preliminary studies using OAT2-tv1, we failed to observe transport of several previously identified substrates, leading us to speculate that ligand selectivity of OAT2 differs with variant and/or heterologous expression system. The purpose was to further investigate the ligand selectivity of the OAT2 variants expressed in multiple cell types. We cloned OAT2-tv1 and OAT2-tv2, but were unsuccessful at amplifying mRNA for OAT2-tv3 from human kidney. OAT2-tv1 and OAT2-tv2 were individually expressed in human embryonic kidney (HEK), Madin-Darby canine kidney (MDCK), or Chinese hamster ovary (CHO) cells. mRNA for OAT2-tv1 and OAT2-tv2 was demonstrated in each cell type transfected with the respective construct, indicating their expression. OAT2-tv1 trafficked to the plasma membrane of all three cell types, but OAT2-tv2 did not. OAT2-tv1 transported penciclovir in all three cell types, but failed to transport para-aminohippurate, succinate, glutarate, estrone-3-sulfate, paclitaxel or dehydroepiandrosterone sulfate - previously identified substrates of OAT2-tv2. Not surprising given its lack of plasma membrane expression, OAT2-tv2 failed to transport any of the organic solutes examined, including penciclovir. Penciclovir transport by OAT2-tv1 was sensitive to large (e.g., cyclosporine A) and small (e.g., allopurinol) organic compounds, as well as organic anions, cations and neutral compounds, highlighting the multiselectivity of OAT2-tv1. The potencies with which indomethacin, furosemide, cyclosporine A and cimetidine inhibited OAT2-tv1 are in good agreement with previous studies using this variant, but inconsistent with studies using OAT2 with an unidentified sequence. This study shows that organic molecules with diverse physicochemical properties interact with OAT2-tv1, making it a likely site of drug interactions. Many previously identified substrates of OAT2 are not transported by OAT2-tv1, suggesting that variant and/or expression system may contribute. Future work should establish the expression pattern and ligand selectivity of OAT2-tv3.

No MeSH data available.