Optimization and Application of a Biotinylation Method for Quantification of Plasma Membrane Expression of Transporters in Cells.

Quantitative proteomics, using LC-MS/MS, is increasingly used to quantify drug transporters present in tissues and cells. Most of these investigations quantify total transporter expression in the cells by utilizing a total membrane fraction, not only the plasma membrane. Here, we report development and optimization of a biotinylation method to quantify protein expression of transporters in the plasma membrane of cells. The Pierce cell surface isolation protocol was optimized for plasma membrane isolation. Incubation of OATP1B1-expressing CHO cells with 0.78 mg/mL of membrane impermeable biotinylation reagent (sulfo-NHS-SS-biotin) at 37°C for 1 h resulted in optimum isolation of the plasma membrane. Subsequently, the expression of transporters in the plasma membrane as a percent of the total was determined by quantitative proteomics using LC-MS/MS. Mean (±SD) plasma membrane expression of OATP1B1 in plated OATP1B1-expressing CHO, MDCKII, and HEK293 cells was found to be 79.7% (±4.7%), 67.7% (±12.2%), and 65.3% (±6.8%) of total cell OATP1B1 expression. Mean (±SD) plasma membrane expression of OATP1B3 in plated OATP1B3-expressing HEK293 cells, OATP2B1 in plated OATP2B1-expressing MDCKII cells, and sodium/taurocholate co-transporting polypeptide (NTCP) in plated NTCP-expressing CHO cells was 63.2% (±1.6%), 37.1% (±15.7%), and 71.7% (±1.2%), respectively. This method of quantifying transporter protein expression in the plasma membrane will be useful in the future to predict transporter-mediated drug disposition.

Insights into the diagnosis of hepatocellular carcinomas with hepatobiliary MRI.

The incidence of hepatocellular carcinomas (HCCs) has increased worldwide in line with an improved screening by high-resolution imaging of cirrhotic livers. Besides abdominal ultrasonography and computerised tomography, magnetic resonance imaging (MRI) is an important tool to detect HCCs. With commercialisation of MR hepatobiliary contrast agents that cross membrane transporters in hepatocytes or tumour cells, MRI adds new information to detect and characterise HCCs. When tumour cells lose organic anion transporting polypeptides (OATP1B1/B3) in cell membranes facing sinusoidal blood, tumours appear hypointense (decreased contrast agent concentrations) in comparison to surrounding normal or cirrhotic liver that retains OATP1B1/B3 expression. However, expression, regulation, and prognostic significance of transporter evolution along carcinogenesis are not completely known. Moreover, understanding signal intensities in focal lesions also relies on transport functions of cellular efflux transporters. This manuscript reviews all the publications that associate liver imaging with hepatobiliary contrast agents and expression of transporters. The regulation of transporters along carcinogenesis to anticipate the prognosis of focal lesions is also included.

Novel No-Wash Luminogenic Probes for the Detection of Transporter Uptake Activity.

Luminogenic probes were designed and synthesized for the detection of uptake transporter activity in a lytic cell-based assay. These probes rely on a self-cleavable trimethyl lock quinone-cyanobenzothiazole (TMQ-CNBT) or trimethyl lock quinone-luciferin (TMQ-Luc) linked to the anion transporter substrate fluorescein. Upon cellular transport, the TMQ is reduced by viable cells, resulting in the facile intramolecular lactonization and rapid release of the bioluminescent reporter molecule. The uptake transporter activity can then be detected without removing and washing off the extracellular substrates. Six probes were tested with OATP1B1*1a and OATP1B3 overexpressing HEK293 cells, and all compounds showed up to 10.2-fold enhancement in uptake when compared to control cells. Uptake of TMQ-luciferin compounds 2, 4, and 6 increased linearly over time up to 30 min at a concentration ranging from 40 nM to 20 µM. The apparent Km values obtained at different time intervals up to 30 min were nearly identical for a given compound, which validates the 30 min window as appropriate for uptake transporter assays. The average apparent Km values ranged from 0.3 to 0.8 µM and 0.2 to 1.3 µM for OATP1B1*1a and OATP1B3, respectively, indicating good affinities to these anion transporters. Furthermore, uptake of compound 2 was inhibited by two inhibitors of OATP1B1*1a and OATP1B3: rifampicin and ritonavir. The preliminary results obtained from compound 2 exhibited a time-dependent, saturatable, and inhibitable nature of uptake, indicating the feasibility of using the probe for the detection of a transporter-mediated process. This add-and-read homogeneous assay may provide a convenient, rapid, and facile way to detect changes in transporter activity in a high-throughput format, and this assay design strategy could create a new platform for a general cell uptake assay for biomaterials in the future.

Membrane trafficking of the human organic anion-transporting polypeptide C (hOATPC).

INTRODUCTION: The human organic anion transporting polypeptide C (OATPC) is one of the major transport proteins involved in the enterohepatic circulation of bile salts and plays an important role in vectorial transport of organic anions and drugs across hepatocytes. MATERIALS AND METHODS: In this study, the effects of biological reagents on the membrane localization of OATPC were investigated by confocal microscopy and estrone-3-sulfate transport. RESULTS: Our results demonstrated that the functional membrane expression of fluorescent chimera OATPC-GFP was achieved in non-polarized (COS7 and HEK293) and polarized (MDCK) cells. Both brefeldin A (a Golgi complex disruptor) and bafilomycin A1 (an inhibitor of vacuolar H+-ATPase) treatment significantly decreased the polarized membrane trafficking and markedly reduced the uptake of estrone-3-sulfate ( approximately 40-90%) in OATPC-GFP transfected cells, suggesting that membrane sorting of hOATPC-GFP was mediated by Golgi complex and vacuolar H+-ATPase-related vesicle transport pathways. Treatment with 8-Br-cAMP (a cAMP analog) stimulated OATPC-GFP membrane localization and enhanced estrone-3-sulfate uptake by approximately 20%. The protein kinase A (PKA) inhibitors (H89 and KT5720), but not a PKG inhibitor, blocked the polarized membrane expression of OATPC-GFP and reduced estrone-3-sulfate transport activity. The simultaneous treatment of cells with PKA activator/inhibitor and bafilomycin A1 demonstrated that bafilomycin A1 did not change the effects of 8-Br-cAMP and H89 on the membrane localization of OATPC-GFP compared with the use of 8-Br-cAMP and H89 alone. DISCUSSION: These data suggest that a cAMP-PKA sensitive membrane sorting pathway for OATPC-GFP is independent of the vacuolar H+-ATPase associated (bafilomycin A1 sensitive) vesicle mediated membrane sorting pathway. In contrast, with combined treatment with brefeldin A, neither the PKA-activator (8-Br-cAMP) nor the inhibitor (H89) further altered the plasma membrane expression and transport activity of OATPC-GFP compared with brefeldin A treatment alone. These data suggest that the cAMP-PKA regulation of OATPC membrane expression involves the Golgi complex. When the Golgi apparatus was disrupted by brefeldin A treatment, the effects of cAMP-PKA on the Golgi-to-basolateral surface sorting process of OATPC was also diminished. In summary, the plasma membrane localization of human OATPC is mediated by Golgi complex and vacuolar H+-ATPase vesicle mediated membrane sorting pathways. cAMP-PKA regulates sorting process through the Golgi complex but not the vacuolar H+-ATPase associated vesicular pathway.

Localization of organic anion transporting polypeptides in the rat and human ciliary body epithelium.

PURPOSE: To identify and localize the expression of multispecific organic anion transporting polypeptides (Oatps/OATPs) in the ciliary body epithelium and to investigate their possible involvement in the transport of the antiglaucoma agent unoprostone. METHODS: Oatps/OATPs were detected by immunoblot analysis and by immunofluorescence microscopy in homogenized and fixed rat and human ciliary body samples using specific polyclonal antibodies. Transport of 3H-labelled unoprostone was measured in Oatp/OATP expressing Xenopus laevis oocytes. RESULTS: Immunoblots of ciliary body extracts were positive for rat Oatp1a4, Oatp1a5 and Oatp1b2 and for human OATP1A2, OATP1C1, OATP2B1, OATP3A1 and OATP4A1. Confocal immunofluorescence microscopy localized Oatp1a4 and Oatp1b2 as well as all immunoblot positive human OATPs at the basolateral plasma membrane of the non-pigmented rat and human ciliary body epithelium, respectively. However, for human OATPs additional regional differences in expression were found with OATP1A2 and OATP1C1 being expressed only in the pars plana of human ciliary body epithelium. Furthermore, OATP1C1, OATP3A1 and OATP4A1 were also expressed at the basolateral plasma membrane of the pars plana pigmented epithelium. And finally, deesterified unoprostone carboxylate was found to be transported by OATP1A2, OATP2B1 and OATP4A1 with approximate K(m)-values of 93, 91 and 132 microm, respectively. CONCLUSIONS: Several multispecific organic anion transporting polypeptides are expressed at the basolateral plasma membrane of the non-pigmented, and to a lesser extent also of the pigmented, epithelium in rat and human ciliary body. These Oatps/OATPs can account for the previously suggested 'liver-like' transport functions of mammalian ciliary body epithelium.

Detection of the human organic anion transporters SLC21A6 (OATP2) and SLC21A8 (OATP8) in liver and hepatocellular carcinoma.

Transport proteins mediating the selective uptake of organic anions into human hepatocytes include the organic anion transporters SLC21A6 (also termed OATP2, OATP-C, or LST-1) and SLC21A8 (OATP8). Both transporters are localized to the basolateral membrane of human hepatocytes. Because of the importance of these transporters for hepatobiliary elimination, including the removal of bilirubin and its conjugates from the blood circulation, we have generated monoclonal antibodies for studies on the expression and localization of these transport proteins. We describe two antibodies, designated monoclonal antibody MDQ (mMDQ) and monoclonal antibody ESL (mESL), directed against the amino terminus and the carboxyl terminus of human SLC21A6, respectively. Both antibodies have been characterized by immunoblot analysis, immunoprecipitation, and immunofluorescence microscopy. While mESL reacted specifically with SLC21A6, mMDQ detects both SLC21A6 and SLC21A8. Neither of the two antibodies reacted with other human, or with dog, rat, or mouse liver SLC21A family members. Antibody mMDQ may be used for the simultaneous detection of SLC21A6 and SLC21A8 in immunoblotting because of its immunoreactivity with both molecules and because of the different molecular masses of both glycosylated proteins in human hepatocytes. This is exemplified in hepatocellular carcinomas where SLC21A6 and SLC21A8 were differentially synthesized and showed an irregular staining pattern. Both transport proteins have not been detected in human hepatoma HepG2 cells. In routine paraffin sections, 10 of 12 hepatocellular carcinomas were focally positive with antibody mMDQ. In contrast, cholangiocarcinomas and liver metastases of colorectal and pancreatic adenocarcinoma were negative without exception. This suggests the usefulness of SLC21A6/SLC21A8 within a panel of tumor markers for hepatocellular carcinomas. Moreover, both antibodies should be useful in studies on the expression and localization of two important uptake transporters of human hepatocytes under physiologic and pathophysiologic conditions.

Functional characterization of human organic anion transporting polypeptide B (OATP-B) in comparison with liver-specific OATP-C.

PURPOSE: To assess the functional characteristics of human organic anion transporter B (OATP-B) in comparison with those of the known, liver-specific OATP-C. METHODS: OATP-B or -C was expressed in HEK293 cells or Xenopus oocytes, and uptakes of estradiol-17beta-glucuronide and estrone-3-sulfate were measured using radiolabeled compounds. RESULTS: OATP-C transported both estrone-3-sulfate and estradiol-17beta-glucuronide, whereas OATP-B transported only the former. OATP-C-mediated uptake of estrone-3-sulfate exhibited biphasic saturation kinetics, whereas transports of estradiol-17beta-glucuronide by OATP-C and estrone-3-sulfate by OATP-B followed single-saturation kinetics. Inhibition kinetics showed that only the high-affinity site for estrone-3-sulfate on OATP-C was shared with glucuronide conjugates. Uptake of [3H]estrone-3-sulfate by OATP-B was inhibited by sulfate conjugates but not by glucuronide conjugates, whereas its uptake by OATP-C was inhibited by both types of conjugates. CONCLUSIONS: OATP-B accepted sulfate conjugates of steroids but not glucuronide conjugates, whereas OATP-C transported both types of steroid conjugates. Transport of estrone-3-sulfate by OATP-B and -C followed single- and biphasic-saturation kinetics, respectively, and the high-affinity site on OATP-C was the same as that for estradiol-17beta-glucuronide. Other OATPs, OATP-A and OATP-8, reportedly exhibit different preferences for steroid conjugates, and the specific recognition of sulfate conjugates seems to be unique to OATP-B.