Introduction

Top Abstract Introduction Experimental Procedures Results Discussion References

Rat organic anion transporting polypeptide oatp1 (Slc21a1) and oatp2 (Slc21a5) belong to the multispecific organic solute carrier family, and mediate sodium-independent uptake of a variety of chemicals from the portal blood across the hepatocellular sinusoidal membranes into hepatocytes (Jacquemin et al., 1994; Noe et al., 1997; Abe et al., 1998). The substrate spectrum of the oatp family is diverse, including anionic, neutral, and cationic compounds (Jacquemin et al., 1994; Bossuyt et al., 1996; Eckhardt et al., 1996; Kontaxi et al., 1996; Friesema et al., 1999). This broad substrate specificity indicates that the oatp family likely plays an important role in hepatic uptake, and facilitates the subsequent biotransformation and excretion of endogenous and exogenous chemicals (xenobiotics) from the circulation. Although rat oatp1 and oatp2 have overlapping substrate spectra with human OATP-A/OATP (SLC21A3) and OATP-C/OATP-2/LST-1 (SLC21A6), human orthologs for rat oatp1 and oatp2 have not been identified.

Long-term regulation of oatp1 and oatp2 expression has been studied in rats treated with testosterone (Lu et al., 1996), during liver regeneration and with bile-duct ligation (Dumont et al., 1997; Gerloff et al., 1999; Vos et al., 1999). However, there have been no studies regarding the short-term regulation of oatp activity by protein kinase A (PKA) and protein kinase C (PKC).

PKA and PKC have been shown to play important roles in the regulation of transporter activity. PKA is a cAMP-dependent protein kinase, whose activation has been shown to stimulate the fusion of membrane vesicles containing hepatic canalicular transporters, such as the multidrug-resistant protein 2 (mrp2) and the chloride/bicarbonate (Cl/HCO₃) exchanger, to the canalicular membranes, which results in increased transporting activity (Benedetti et al., 1994; Boyer and Soroka, 1995). In addition, the sodium-dependent taurocholate cotransporting polypeptide (ntcp), which is localized to the sinusoidal membrane of hepatocytes, is also up-regulated by protein kinase A activators (Grune et al., 1993; Mukhopadhayay et al., 1997, 1998a,b). The effects of PKC activation and the mechanisms by which PKC regulate the transporters are different for the various transporters. For example, the activity of the dopamine transporter DAT is suppressed by PKC activation via direct phosphorylation (Huff et al., 1997), whereas the sodium/glucose cotransporter, SGLT1, is suppressed by PKC through increased removal processes from the cell membrane to the cytosol (Hirsch et al., 1996). The mouse organic anion transporter is also suppressed by PKC activation (You et al., 2000). In contrast, the -aminobutyric acid transporter GAT-1 is activated by PKC activation by increased insertion of the transporter from the cytosol into the cell membrane (Corey et al., 1994). The effect of PKC activation on hepatic canalicular transporters has also been investigated. Activation of PKC increases the transport of dinitrophenyl-glutathione across hepatic canalicular membranes in isolated hepatocytes from normal rats, but not from TR rats that lack functional mrp2, indicating that mrp2 activity is increased by protein kinase C activation (Roelofsen et al., 1991; Pikula et al., 1994). Similarly, P-glycoprotein, the transporter associated with multiple drug resistance phenotype, is also increased by PKC activation (Endicott and Ling, 1989; Gottesman and Pastan, 1993). Activation of PKC decreases the accumulation of antineoplastic drugs in tumor cell lines expressing human multidrug resistance, suggesting that activation of PKC increases the transport activity of P-glycoproteins (Chambers et al., 1990a,b; Aftab et al., 1994). Recently, Noe et al. (2001) reported that phosphorylation of mouse bile salt export pump is increased by coexpression of mouse PKC catalytic subunit and phorbol ester treatment in Sf9 insect cells.

Amino acid sequence analysis of oatp1 and oatp2 predicts four putative PKA phosphorylation sites in oatp1 and five in oatp2. Two sites are common for both oatp1 and oatp2. Similarly, there are four PKC phosphorylation sites in oatp1 and oatp2. One is located near the carboxy terminus, two are near the amino terminus, and one is between transmembrane domains 8 and 9 (Jacquemin et al., 1994; Noe et al., 1997; Abe et al., 1998). These conserved phosphorylation sites suggest that oatp1 and oatp2 may be subject to PKA and PKC regulation.

High concentrations of bile acids have been shown to activate PKC via 1,2-diacylglycerol accumulation in hepatocytes (Beuers et al., 1996; Rao et al., 1997) and to inhibit organic anion uptake into the liver, but the signaling pathway involved is unknown (Ishii and Wolkoff, 1994). Rat oatp1 and oatp2 are two major Na⁺-independent anion uptake transporters in the liver; therefore, the purpose of the present study was to determine whether activation or inhibition of PKA or PKC affects the uptake mediated by oatp1 and oatp2.

	Experimental Procedures

Top Abstract Introduction Experimental Procedures Results Discussion References

Materials. [³H]Digoxin (specific activity 19 Ci/mmol) and [³H]estrone-3-sulfate (specific activity 53 Ci/mmol) were obtained from PerkinElmer Life Science Products (Boston, MA). Phorbol-12-myristate-13-acetate (PMA), 4-phorbal-12,13-didecanoate (4PDD), adenosine 3',5-cyclic monophosphate, N⁶-benzoyl-, sodium salt (N-6-benz-cAMP, sodium), H7, and bisindolylmaleimide were purchased from Calbiochem (San Diego, CA). Other chemicals were from Sigma (St. Louis, MO) unless otherwise indicated.

Animals. Mature female Xenopus laevis were purchased from Nasco (Fort Atkinson, WI) and kept under standard conditions according to the Guidelines of Laboratory Animal Research at University of Kansas Medical Center, Kansas City, KS.

cRNA Preparation. The plasmid pSPORT-1-oatp1 and pBK-CMV-oatp2 were kindly provided by Dr. Peter Meier (Department of Medicine, University Hospital, Zurich, Switzerland) and were linearized by restriction enzymes NotI and XhoI (Promega, Madison, WI) to release the full-length oatp1 and oatp2 cDNA, respectively. Oatp1 and oatp2 cDNA were used as templates to generate capped cRNA by in vitro transcription with T7 RNA polymerase (Promega) for oatp1, and T3 RNA polymerase (Promega) for oatp2. Capped RNA was dissolved in nuclease-free water and stored at 80°C.

Expression of oatp1 and oatp2 cRNA in X. laevis Oocytes. Oocytes were prepared as described previously (Jacquemin et al., 1994). Briefly, oocytes were removed from the ovary by laparotomy and transferred into Ca²⁺-free medium ND 96 (96 mM NaCl, 2 mM KCl, 1 mM MgCl₂, 5 mM HEPES, pH 7.6), supplemented with 2 mg/ml collagenase type A (Roche Molecular Biochemicals, Indianapolis, IN). After 1- to 2-h incubation at room temperature, oocytes were washed exhaustively with modified Barth's solution (MBS) consisting of 88 mM NaCl, 2.4 mM NaHCO₃, 15 mM HEPES, 0.30 mM CaNO₃(4H₂O), 0.41 mM CaCl₂(6H₂O), 0.82 mM MgSO₄(7H₂O), and 50 units/liter penicillin and streptomycin. Mature stage 5 and 6 oocytes were selected. After overnight incubation at 18°C in MBS, healthy oocytes were selected for microinjection of oatp1 or oatp2 cRNA (25 ng). Water-injected oocytes served as controls. Subsequently, oocytes were cultured for 2 to 3 days at 18°C with a daily change of MBS.

Oocyte Uptake Studies. Oocytes were washed in Na⁺-free uptake media [100 mM choline chloride, 1 mM KCl, 1 mM CaCl₂(2H₂O), 1 mM MgCl₂(6H₂O), and 10 mM HEPES, pH 7.5]. The uptake experiments were started by incubating 9 to 15 oocytes at 25°C in 150 µl of Na⁺-free uptake media supplemented with 18.1 µM [³H]estrone-3-sulfate for oatp1-cRNA-injected oocytes, or 0.52 µM [³H]digoxin for oatp2-cRNA-injected oocytes. Uptake was stopped at the indicated time points by adding 2 ml of ice-cold Na⁺-free uptake medium with 5% bovine serum albumin to stop uptake and also reduce nonspecific binding. These oocytes were subsequently washed three times with 10 ml of ice-cold Na⁺-free uptake medium. Then single oocytes were lysed in 0.5 ml of 10% SDS in a 7-ml scintillation vial, to which 4.5 ml of scintillation fluid (Cocktail Ultragold; Fisher Scientific, Pittsburgh, PA) was added after complete cell lysis. The oocyte-associated radioactivity was determined in a liquid scintillation detector (model 2200CA TRI-CARB; Packard Instrument Co., Meriden, CT).

Protein Kinase Activator and Inhibitor Treatment. N-6-Benz-cAMP, PMA, 4PDD, H7, and bisindolylmaleimide were diluted from stock solutions to the desired concentration with Na⁺-free uptake medium. Oocytes (9-15) were incubated in these solutions for various time periods indicated, followed by [³H]estrone-3-sulfate uptake for oatp1 and [³H]digoxin uptake for oatp2. Control oocytes were incubated with vehicle alone followed by uptake assay.

Statistics. Data were expressed as mean ± S.E. and were analyzed either with one-way analysis of variance, followed by Duncan's multiple range test, or the Student's t test. Level of significance was set at  = 0.05.

	Results

Top Abstract Introduction Experimental Procedures Results Discussion References

Effect of PKA on oatp1- and oatp2-Mediated Uptake. Both oatp1 and oatp2 have several potential PKA phosphorylation sites. It has been shown that the activity of rat ntcp is increased after PKA activation (Mukhopadhayay et al., 1997). Oatp1 and oatp2 are also expressed in the hepatic sinusoidal membranes and mediate sodium-independent uptake of many compounds; therefore, it was of interest to determine whether alteration of PKA activation also affects the uptake mediated by oatp1 and oatp2. Oatp1- or oatp2-cRNA-injected oocytes were incubated with a cell-permeable PKA activator, N-6-benz-cAMP, from 1 µM to 1 mM for 60 min before the determination of the uptake activity. In contrast to ntcp, neither oatp1-mediated [³H]estrone-3-sulfate uptake nor oatp2-mediated [³H]digoxin uptake was affected by this PKA activator (Fig. 1, top and bottom, respectively). When oatp1- and oatp2-injected-oocytes were incubated with the PKA inhibitor H7 from 5 to 100 µM (IC₅₀ is 3 µM to inhibit PKA), oatp1- and oatp2-mediated uptake was not affected by H7 (Fig. 2). Studies by Mukhopadhayay et al. (1997) showed that protein levels of oatp1 in rat hepatocyte membranes did not change after PKA activation.

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Effects of PKA activator N-6-benz-cAMP on the uptake activity of oatp1 and oatp2. Oocytes were treated with indicated concentrations of N-6-benz-cAMP for 1 h, followed by 30 min of [3H]estrone-3-sulfate uptake by oatp1-cRNA-injected oocytes (top), or [3H]digoxin uptake by oatp2-cRNA-injected oocytes (bottom). The x-axis is the log concentration of N-6-benz-cAMP. The squares indicate either oatp1- or oatp2-cRNA-injected oocytes and the circles water-injected oocytes.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Effects of PKA inhibitor H7 on the uptake activity mediated by oatp1 and oatp2. Oocytes were incubated with the indicated concentrations of H7 for 1 h followed by 30 min of [3H]estrone-3-sulfate uptake by oatp1-cRNA-injected oocytes (top), or [3H]digoxin uptake by oatp2-cRNA-injected oocytes (bottom). The x-axis is the log concentration of H7. The squares indicate either oatp1- or oatp2-injected oocytes and the circles the water-injected oocytes.

Effect of PKC on oatp1- and oatp2-Mediated Uptake. To determine whether oatp1 is regulated by PKC, oatp1-cRNA-injected oocytes were incubated with a potent PKC activator, PMA, for 10 min at concentrations ranging from 0.001 to 10 µM before determining the oatp1-mediated [³H]estrone-3-sulfate uptake. PMA suppressed the uptake activity of oatp1 in a concentration-dependent manner (Fig. 3, top). Uptake of [³H]estrone-3-sulfate into water-injected oocytes was not affected by PMA treatment.

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Effects of the PKC activator PMA and its inactive analog 4PDD on oatp1-mediated uptake in oatp1-cRNA-injected X. laevis oocytes. Oocytes injected with either oatp1 cRNA or water were incubated with the indicated concentrations of PMA (top) or 4PDD (bottom) for 10 min, followed by [3H]estrone-3-sulfate uptake for 30 min. The x-axis is the log concentration of PMA (top) or 4PDD (bottom). The squares are oatp1-injected oocytes and the circles represent water-injected oocytes. The * indicates statistical difference between untreated and PMA (top) or 4PDD (bottom)-treated oatp1-cRNA-injected oocytes.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Effects of the PKC activator PMA and its inactive analog 4PDD on oatp2-mediated uptake in oatp2-cRNA-injected X. laevis oocytes. Oocytes injected with oatp2 cRNA or water were incubated with indicated concentrations of PMA (top) or 4PDD (bottom) for 10 min, followed by [3H]digoxin uptake for 30 min. The x-axis is the log concentration of PMA (top) or 4PDD (bottom). The squares are oatp2-injected oocytes and the circles are water-injected oocytes. The * indicates statistical difference between untreated and PMA (top) or 4PDD (bottom)-treated oatp2-cRNA-injected oocytes.

Time Course of PMA Suppression of oatp1- and oatp2-Mediated Uptake. Oatp1- and oatp2-cRNA-injected oocytes were incubated with 1 µM PMA for various time intervals (5-60 min for oatp1 and 5-40 min for oatp2) followed by the uptake assays (Fig. 5). Incubation of oocytes with 1 µM PMA suppressed both oatp1 and oatp2 activity in as little as 5 min. The maximal suppression of oatp1-mediated uptake was achieved by 20 min, and prolonged incubation did not further suppress oatp1-mediated uptake (Fig. 5, top). Maximal suppression of oatp2-mediated uptake was observed at 10 min of PMA incubation, and prolonged incubation did not further suppress oatp2-mediated uptake (Fig. 5, bottom).

View larger version (20K): [in this window] [in a new window]   Fig. 5.   Time course for suppression of oatp1- and oatp2-mediated uptake by PMA, a PKC activator. Oocytes were treated with 1 µM PMA for the indicated time intervals, followed by 30 min of [3H]estrone-3-sulfate uptake by oatp1-cRNA-injected oocytes (top) or [3H]digoxin uptake by oatp2-cRNA-injected oocytes (bottom). The squares indicate either oatp1- or oatp2-cRNA-injected oocytes and the circles the water-injected oocytes. The * indicates statistical difference between untreated and PMA-treated oatp1-cRNA- (top) or oatp2-cRNA-injected (bottom) oocytes.

Effects of PKC Inhibition on oatp1- and oatp2-Mediated Uptake. The above-mentioned studies (Figs. 3-5) illustrate that activation of PKC suppresses oatp1- and oatp2-mediated uptake; therefore, further studies were designed to determine the effect of PKC inhibition on the uptake mediated by oatp1 and oatp2. Oatp1- and oatp2-cRNA-injected oocytes were incubated with bisindolylmaleimide, a specific PKC inhibitor (IC₅₀ = 27 nM) for 60 min before the uptake assays. Bisindolylmaleimide (0.001-10 µM) did not affect the basal level of oatp1- or oatp2-mediated substrate uptake into oocytes (Fig. 6, top and bottom). However, preincubation of oocytes with bisindolylmaleimide (1 µM) for 60 min followed by 10-min incubation with 1 µM PMA significantly reversed the suppression of PMA on oatp1- and oatp2-mediated uptake (Fig. 7, top and bottom).

View larger version (20K): [in this window] [in a new window]   Fig. 6.   Effects of bisindolylmaleimide, a specific PKC inhibitor, on oatp1- and oatp2-mediated uptake. Oocytes injected with oatp1 (top) or oatp2-cRNA (bottom) were incubated with the indicated concentrations of bisindolylmaleimide (BIL) for 1 h, followed by [3H]estrone-3-sulfate uptake by oatp1-cRNA-injected oocytes or [3H]digoxin uptake by oatp2-cRNA-injected oocytes for 30 min. The x-axis is the log concentration of bisindolylmaleimide. The squares indicate either oatp1- or oatp2-cRNA-injected oocytes and the circles the water-injected oocytes.

View larger version (29K): [in this window] [in a new window]   Fig. 7.   Effect of incubation with the PKC inhibitor bisindolylmaleimide before PMA treatment, on the uptake mediated by oatp1 and oatp2. Oatp1-or oatp2-cRNA-injected oocytes, as well as the water-injected oocytes were treated with the following paradigm, respectively: vehicle (0.1% dimethyl sulfoxide) for 60 min, PMA (1 µM) for 10 min, bisindolylmaleimide (1 µM) for 60 min, or bisindolylmaleimide (1 µM) for 60 min followed by PMA (1 µM) for 10 min. These treatments were followed by the uptake assays to determine the uptake mediated by oatp1 by measuring [3H]estrone-3-sulfate uptake (top) or by oatp2 by determining [3H]digoxin uptake (bottom). The black bars represent water-injected oocytes and the gray bars oatp1- or oatp2-cRNA-injected oocytes. The * indicates statistical difference between untreated and PMA-treated oatp1-cRNA- or oatp2-cRNA-injected oocytes.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

Rat oatp1 and oatp2 are nonspecific transporting peptides expressed in the sinusoidal membrane of hepatocytes that mediate Na⁺-independent uptake of various compounds (Jacquemin et al., 1994; Noe et al., 1997). Expression of oatp1 is also detected in the S₃ segment of proximal tubules and in the choroid plexus (Bergwerk et al., 1996; Angeletti et al., 1997). In addition, oatp2 has been reported to be widely distributed in the brain (Abe et al., 1998). The substrate spectrum of rat oatp1 and oatp2 includes organic anions, bulky type II organic cations, and neutral organic compounds. Many of these substrates are important and comprise widely used drugs and endogenous hormones that are critical in maintaining and regulating physiological homeostasis. As a result of their distribution and substrate spectrum, oatp1 and oatp2 appear to be important in facilitating the uptake of chemicals into the liver for their subsequent metabolism and elimination from the body.

Oatp1 mRNA levels can be up-regulated in kidney by testosterone treatment (Lu et al., 1996) and down-regulated in liver by bile duct ligation (Dumont et al., 1997), whereas both oatp1 and oatp2 mRNA levels are down-regulated during liver regeneration (Vos et al., 1999). However, studies addressing the short-term regulation of oatp1 and oatp2 uptake activity by protein kinases, such as PKA and PKC, have not been reported.

Short-term regulation by PKA of the sodium-dependent sinusoidal transporter ntcp has been investigated, and the results show that the mRNA and protein levels of ntcp in hepatocytes are increased after PKA activation. The mechanism by which PKA activation increases ntcp appears to be due to increased translocation of ntcp from cytosol to the sinusoidal membrane (Grune et al., 1993; Mukhopadhayay et al., 1997, 1998a,b). Extracellular ATP down-regulates the hepatic uptake of sulfobromophthalein; however, this phenomenon is not observed in HeLa cells transfected with oatp1 (Glavy et al., 2000). In the present study, both rat oatp1 and oatp2, when expressed in X. laevis oocytes, are insensitive to the cell-permeable PKA activator N-6-benz-cAMP, even over a wide range of concentrations, indicating that oatp1 and oatp2 are not subject to short-term PKA regulation. This is consistent with a study that showed that hepatic protein levels of oatp1 are not increased after PKA activator treatment (Mukhopadhyay et al., 1998a). In addition, a PKA inhibitor, H7, at concentrations from 5 to 100 µM, did not affect oatp1- or oatp2-mediated uptake either. The differential regulation of the sinusoidal transporters, namely, ntcp, oatp1, and oatp2, by PKA may reflect the different functions carried out by these transporters.

PKC activation has been shown to have opposite effects on various transporters expressed in X. laevis oocytes. Many transporters, such as the -aminobutyric acid transporter, sodium-glucose cotransporter, dopamine transporter, Mrp2, Bsep, and P-glycoprotein, are regulated by PKC. Interestingly, the mechanism by which PKC regulates the transporter activity appears to be different. PKC exerts its effect by either directly phosphorylating the transporters (Conradt and Stoffel, 1997; Huff et al., 1997) or by alternating the abundance of the transporters in the plasma membrane (Hirsch et al., 1996; Ramamoorthy et al., 1998; Pajor and Sun, 1999). In the present study, the PKC activator PMA dramatically suppressed the uptake mediated by rat oatp1 and oatp2. In addition, this suppression was shown to be concentration- and time-dependent. However, the mechanism(s) by which PKC activation suppresses the uptake mediated by rat oatp1 and oatp2 is not clear, which is an interesting area to investigate in the future. As mentioned earlier, PKC could be activated by high concentrations of bile acids (Beuers et al., 1996; Rao et al., 1997) that have been shown to decrease hepatic anion uptake (Ishii and Wolkoff, 1994). Taken together, one could speculate that high concentrations of bile acids might inhibit the hepatic anion uptake that is partially mediated by oatp1 and oatp2 by activation of PKC. Nevertheless, due to the fact that human orthologs of rat oatp1 and oatp2 are not identified, and the substrate spectrum differences of oatps do exist between species, extrapolation of the results of this study to other species should be done with caution.

In conclusion, the present study showed that although rat oatp1- and oatp2-mediated uptake is insensitive to the short-term regulation by the PKA activator and inhibitor, they are subject to the short-term regulation of PKC activation. PKC activator suppresses the uptake mediated by rat oatp1 and oatp2 in a concentration- and time-dependent manner, when expressed in X. laevis oocytes. In addition, the PKC specific inhibitor bisindolylmaleimide partially reversed PMA's suppressive effect on rat oatp1-mediated uptake, and almost completely reversed PMA's suppressive effect on rat oatp2-mediated uptake. Taken together, these studies implicate a role for PKC in the short-term regulation of the rat hepatic sinusoidal transporters oatp1 and oatp2. Furthermore, treatments or disease states that activate liver PKC may affect oatp-mediated uptake transport into the liver; however, potential species differences have to be considered when comparing rat and human oatps.