Emerging transporters of clinical importance: an update from the International Transporter Consortium.

The International Transporter Consortium (ITC) has recently described seven transporters of particular relevance to drug development. Based on the second ITC transporter workshop in 2012, we have identified additional transporters of emerging importance in pharmacokinetics, interference of drugs with transport of endogenous compounds, and drug-drug interactions (DDIs) in humans. The multidrug and toxin extrusion proteins (MATEs, gene symbol SLC47A) mediate excretion of organic cations into bile and urine. MATEs are important in renal DDIs. Multidrug resistance proteins (MRPs or ABCCs) are drug and conjugate efflux pumps, and impaired activity of MRP2 results in conjugated hyperbilirubinemia. The bile salt export pump (BSEP or ABCB11) prevents accumulation of toxic bile salt concentrations in hepatocytes, and BSEP inhibition or deficiency may cause cholestasis and liver injury. In addition, examples are presented on the roles of nucleoside and peptide transporters in drug targeting and disposition.

In vitro methods to support transporter evaluation in drug discovery and development.

This white paper addresses current approaches and knowledge gaps concerning methods to assess the role of transport proteins in drug/metabolite disposition in humans. The discussion focuses on in vitro tools to address key questions in drug development, including vesicle- and cell-based systems. How these methods can be used to assess the liability of compounds for transporter-based drug-drug interactions (DDIs) in vivo is also explored. Existing challenges and approaches to examine the involvement of transporters in drug disposition are discussed.

Cholestasis and the role of basolateral efflux pumps.

ATP-dependent transport of biliary constituents, such as bile acids, reduced glutathione, and bilirubin glucuronosides across the hepatocyte canalicular membrane into bile represents the decisive driving force for the formation of biliary fluid. Functional characterization, cloning, and localization of hepatocellular transporter proteins has provided a molecular understanding of the mechanisms underlying bile flow and intrahepatic cholestasis. Genetic variants in humans and genetic knockout in rodents, or transporter inhibition have indicated that both the conjugate export pump MRP2 (multidrug resistance protein 2; ABCC2) and the bile salt export pump BSEP (ABCB11) are major contributors to bile acid-independent and bile acid-dependent bile flow, respectively. In humans, genetic variants of BSEP, leading to an impaired transport activity or localization of the protein in the canalicular membrane, are associated with severe intrahepatic cholestasis. Efflux pumps of the basolateral hepatocyte membrane, particularly MRP3 (multidrug resistance protein 3; ABCC3) and MRP4 (multidrug resistance protein 4; ABCC4) pump substances from hepatocytes into sinusoidal blood. These efflux pumps have been recognized in recent years to play an important compensatory role in cholestasis and to contribute to the balance between uptake and efflux of substances during the vectorial transport from sinusoidal blood into bile. This sinusoidal efflux not only enables subsequent renal elimination, but also re-uptake of substances into neighboring and more centrally located hepatocytes in the sinusoid.

Human multidrug resistance protein 8 (MRP8/ABCC11), an apical efflux pump for steroid sulfates, is an axonal protein of the CNS and peripheral nervous system.

Dehydroepiandrosterone 3-sulfate and other neurosteroids are synthesized in the CNS and peripheral nervous system where they may modulate neuronal excitability by interacting with ligand-gated ion channels. For this modulatory activity, neurosteroids have to be locally released from either neurons or glial cells. We here identify the integral membrane protein ABCC11 (multidrug resistance protein 8) as an ATP-dependent efflux pump for steroid sulfates, including dehydroepiandrosterone 3-sulfate, and localize it to axons of the human CNS and peripheral nervous system. ABCC11 mRNA was detected in human brain by real-time polymerase chain reaction. Antibodies raised against ABCC11 served to detect the protein in brain by immunoblotting and immunofluorescence microscopy. ABCC11 was preferentially found in the white matter of the brain and co-localized with neurofilaments indicating that it is an axonal protein. Additionally, ABCC11 was localized to axons of the peripheral nervous system. For functional studies, ABCC11 was expressed in polarized Madin-Darby canine kidney cells where it was sorted to the apical membrane. This apical sorting is in accordance with the localization of ABCC11 to the axonal membrane of neurons. Inside-out plasma membrane vesicles containing recombinant ABCC11 mediated ATP-dependent transport of dehydroepiandrosterone 3-sulfate with a Km value of 21 microM. This transport function together with the localization of the ABCC11 protein in vicinity to GABAA receptors is consistent with a role of ABCC11 in dehydroepiandrosterone 3-sulfate release from neurons to sites of dehydroepiandrosterone 3-sulfate-mediated receptor modulation. Our findings may provide a basis for the characterization of mutations in the human ABCC11 gene and their linkage with neurological disorders.

Expression and immunolocalization of the multidrug resistance proteins, MRP1-MRP6 (ABCC1-ABCC6), in human brain.

Multidrug resistance proteins (MRPs, symbol ABCC) are membrane glycoproteins that mediate the ATP-dependent export of organic anions, including cytotoxic and antiviral drugs, from cells. To identify MRP family members possibly involved in the intrinsic resistance of human brain to cytotoxic and antiviral drugs, we analyzed the expression and localization of MRP1-MRP6 in rapidly frozen perilesional samples of several regions of adult human brain obtained during neurosurgery. Quantitative polymerase chain reaction analysis showed expression of MRP1, MRP2, MRP3, MRP4, and MRP5 mRNA, whereas MRP6 mRNA was below detectability. However, immunofluorescence microscopy of cryosections from human brain showed no reactivity for the MRP2 or MRP3 proteins. The proteins MRP1, MRP4, and MRP5 were clearly localized by confocal laser scanning microscopy to the luminal side of brain capillary endothelial cells. The MRP4 and MRP5 proteins were also detected in astrocytes of the subcortical white matter. Notably, MRP5 protein was present in pyramidal neurons. MRP proteins may, thus, contribute to the cellular efflux of endogenous anionic glutathione or glucuronate conjugates (substrates for MRP1), cyclic nucleotides (substrates for MRP4 and MRP5), or glutathione (co-substrate for MRP1 and MRP4); in addition, they may play an important role in the resistance of the brain to several cytotoxic and antiviral drugs.

Expression of the multidrug resistance proteins MRP2 and MRP3 in human hepatocellular carcinoma.

Treatment of hepatocellular carcinoma (HCC) by chemotherapy is often impeded by the intrinsic multidrug resistance (MDR) of this frequent primary cancer of the liver. The MDR phenotype can be caused by ATP-dependent export of chemotherapeutic drugs across the plasma membrane being mediated by transporters of the MDR P-glycoprotein family or of the multidrug resistance protein (MRP) family. To elucidate the role of MRP family members in HCC, we analyzed the expression and subcellular localization of MRP1 (ABCC1), MRP2 (ABCC2) and MRP3 (ABCC3); all 3 isoforms have been shown to confer resistance to chemotherapeutic drugs. Semiquantitative RT-PCR demonstrated that MRP2 and MRP3 mRNA expression in HCC was at least 10-fold higher than MRP1 mRNA expression. MRP2 immunostaining was observed in 87% (33/38) of HCC samples. MRP2 was localized in the plasma membrane in a polarized fashion, either in trabecular structures resembling the canalicular membrane or in the luminal membrane when cells had a pseudoglandular arrangement. MRP3 was detected in all samples examined (9/9) by RT-PCR and by immunofluorescence microscopy. MRP3 was localized to the basolateral membrane of carcinoma cells. Double-label immunofluorescence microscopy with antibodies specific for MRP2 or MRP3 indicated that carcinoma cells expressed both MRP isoforms simultaneously. When MRP1 was detected by immunofluorescence microscopy, it was localized on the intracellular membranes of carcinoma cells. Thus, plasma membrane expression of MRP2 and MRP3, but not of MRP1, can contribute to the MDR phenotype of HCC.

Vectorial transport by double-transfected cells expressing the human uptake transporter SLC21A8 and the apical export pump ABCC2.

Vectorial transport of endogenous substances, drugs, and toxins is an important function of polarized cells. We have constructed a double-transfected Madin-Darby canine kidney (MDCK) cell line permanently expressing a recombinant uptake transporter for organic anions in the basolateral membrane and an ATP-dependent export pump for anionic conjugates in the apical membrane. Basolateral uptake was mediated by the human organic anion transporter 8 (OATP8; symbol SLC21A8) and subsequent apical export by the multidrug resistance protein 2 (MRP2; symbol ABCC2). Under physiological conditions, both transport proteins are strongly expressed in hepatocytes and contribute to the hepatobiliary elimination of organic anions. Expression and localization of OATP8 and MRP2 in MDCK cells growing on Transwell membrane inserts was demonstrated by immunoblotting and confocal laser scanning microscopy. (3)H-Labeled sulfobromophthalein (BSP) was a substrate for both transport proteins and was transferred from the basolateral to the apical compartment at a rate at least six times faster by double-transfected MDCK-MRP2/OATP8 cells than by single-transfected MDCK-OATP8 or MDCK-MRP2 cells. Vectorial transport at a much higher rate by double-transfected than by single-transfected cells was also observed for the (3)H-labeled substrates leukotriene C(4), 17 beta-glucuronosyl estradiol, and dehydroepiandrosterone sulfate, for the fluorescent anionic substrate fluo-3, and for the antibiotic rifampicin. Inhibition studies indicated that intracellular formation of S-(2,4-dinitrophenyl)-glutathione from 2,4-chlorodinitrobenzene selectively inhibits the transcellular transport of [(3)H]BSP at the site of MRP2-mediated export. The double-transfected cells provide a useful system for the identification of transport substrates and transport inhibitors including drug candidates.

Expression and localization of the multidrug resistance proteins MRP2 and MRP3 in human gallbladder epithelia.

BACKGROUND & AIMS: The multidrug resistance protein (MRP) isoforms MRP2 (ABCC2) and MRP3 (ABCC3) play a decisive role in the hepatic secretion of endogenous and xenobiotic conjugates and are differentially expressed in hepatocytes and cholangiocytes. The epithelium of the gallbladder considerably modifies the composition of primary hepatic bile by absorption and secretion; however, the underlying transport mechanisms were largely unknown. Localization of MRP2 and MRP3 may provide an explanation of how the products of phase II conjugation are effluxed from gallbladder epithelia. METHODS: Expression and localization of MRP2 and MRP3 were analyzed by reverse-transcription polymerase chain reaction (RT-PCR) and immunofluorescence microscopy of human gallbladder tissue. RESULTS: Expression of MRP2 and MRP3 was identified in all gallbladders by RT-PCR followed by sequencing of the amplified fragments. Double immunofluorescence microscopy using 2 specific antibodies for the respective MRP isoform showed the simultaneous expression of MRP2 in the apical membrane and MRP3 in the basolateral membrane of gallbladder epithelia. MRP1 protein expression was not detectable. CONCLUSIONS: Our findings show the expression of MRP2 and MRP3 in distinct plasma membrane domains of gallbladder epithelia and provide evidence for the capacity of the gallbladder to secrete xenobiotic and endogenous anionic conjugates into blood via MRP3 and into bile via MRP2.

Up-regulation of basolateral multidrug resistance protein 3 (Mrp3) in cholestatic rat liver.

Cholestasis induces down-regulation of multidrug resistance protein 2 (Mrp2, symbol Abcc2), which is localized to the canalicular membrane. Given the overlapping substrate specificities of Mrp2 and multidrug resistance protein 3 (Mrp3, symbol Abcc3), we examined the hypothesis of a different subcellular and lobular localization of these members of the Mrp family in rat liver after bile duct ligation. We raised a polyclonal antibody against rat Mrp3 and detected this protein in the basolateral plasma membrane of hepatocytes surrounding the central veins and of cholangiocytes. The Mrp3 protein level was less than 2% of the expression observed after 72 hours of obstructive cholestasis. After 48 hours of bile duct ligation, the Mrp3 protein was increased and was further enhanced after 72 hours. In 72-hour-cholestatic rat liver Mrp3 was expressed, in addition, in periportal hepatocytes. However, there was a preponderance of Mrp3 in the pericentral area of the liver lobule. In Mrp2-deficient mutant rat liver, the Mrp3 protein expression was most enhanced and its zonation was lost. The Mrp3 immunostaining of cholangiocytes was preserved in cholestatic and in Mrp2-deficient mutant liver. Canalicular Mrp2 decreased and amounted to 34% of normal after bile duct ligation for 72 hours. We conclude that the hepatocellular up-regulation of Mrp3 in cholestasis together with cholangiocellular Mrp3 may compensate for the biliary obstruction and impaired canalicular Mrp2 function by clearing cholephilic anionic substances into the blood.

Tauroursodeoxycholic acid inserts the apical conjugate export pump, Mrp2, into canalicular membranes and stimulates organic anion secretion by protein kinase C-dependent mechanisms in cholestatic rat liver.

Ursodeoxycholic acid (UDCA) exerts anticholestatic effects by undefined mechanisms. Previous work suggested that UDCA stimulates biliary exocytosis via Ca(++)- and protein kinase C (PKC)-dependent mechanisms. Therefore, the effect of taurine-conjugated UDCA (TUDCA) was studied in the experimental model of taurolithocholic acid (TLCA)-induced cholestasis on bile flow, hepatobiliary exocytosis, distribution of PKC isoforms, and density of the apical conjugate export pump, Mrp2, in canalicular membranes. Isolated perfused rat livers were preloaded with horseradish peroxidase (HRP), a marker of vesicular exocytosis, and were perfused with bile acids or dimethylsulfoxide (control) only. PKC isoform distribution and membrane density of Mrp2 were studied using immunoblotting and immunoelectron-microscopic techniques. Biliary secretion of the Mrp2 substrate, 2,4-dinitrophenyl-S-glutathione (GS-DNP), was studied in the presence or absence of the PKC inhibitor, bisindolylmaleimide I (BIM-I; 1 micromol/L). TLCA (10 micromol/L) impaired bile flow by 51%; biliary secretion of HRP and GS-DNP by 46% and 95%, respectively; membrane binding of the Ca(++)-sensitive alpha-isoform of PKC by 32%; and density of Mrp2 in the canalicular membrane by 79%. TUDCA (25 micromol/L) reversed the effects of TLCA on bile flow, secretion of HRP and GS-DNP, and distribution of alpha-PKC. TUDCA reduced membrane binding of epsilon-PKC and increased Mrp2 density 4-fold in canalicular membranes of cholestatic hepatocytes. BIM-I inhibited the effect of TUDCA on GS-DNP secretion in cholestatic livers by 49% without affecting secretion in controls. In conclusion, TUDCA may enhance the secretory capacity of cholestatic hepatocytes by stimulation of exocytosis and insertion of transport proteins into apical membranes via PKC-dependent mechanisms.

The multidrug resistance protein MRP1 mediates the release of glutathione disulfide from rat astrocytes during oxidative stress.

The release of glutathione disulfide has been considered an important process for the maintenance of a reduced thiol redox potential in cells during oxidative stress. In cultured rat astrocytes, permanent hydrogen peroxide-induced oxidative stress caused a rapid increase in intracellular glutathione disulfide, which was followed by the appearance of glutathione disulfide in the medium. Under these conditions, the viability of the cells was not compromised. In the presence of cyclosporin A and the quinoline-derivative MK571, inhibitors of multidrug resistance proteins (MRP1 and MRP2), glutathione disulfide accumulated in cells and the release of glutathione disulfide from astrocytes during H2O2 stress was potently inhibited, suggesting a contribution of MRP1 or MRP2 in the release of glutathione disulfide from astrocytes. Using RT-PCR we amplified a cDNA from astroglial RNA with a high degree of homology to MRP1 from humans and mouse. In contrast, no fragment was amplified by using primers specific for rat MRP2. In addition, the presence of MRP1 protein in astrocytes was demonstrated by its immunolocalization in cells expressing the astroglial marker protein glial fibrillary acidic protein. Our data identify rat astrocytes as a MRP1-expressin, brain cell type and demonstrate that this transporter participates in the release of glutathione disulfide from astrocytes during oxidative stress.

Hepatic uptake of bilirubin and its conjugates by the human organic anion transporter SLC21A6.

Bilirubin, the end product of heme catabolism, is taken up from the blood circulation into the liver. This work identifies a high-affinity transport protein mediating the uptake of bilirubin and its conjugates into human hepatocytes. Human embryonic kidney cells (HEK293) permanently expressing the recombinant organic anion-transporting polypeptide 2 (human OATP2, also known as LST-1 or OATP-C; symbol SLC21A6) showed uptake of [(3)H]monoglucuronosyl bilirubin, [(3)H]bisglucuronosyl bilirubin, and [(3)H]sulfobromophthalein with K(m) values of 0.10, 0.28, and 0.14 microm, respectively. High-affinity uptake of unconjugated [(3)H]bilirubin by OATP2 occurred in the presence of albumin and was not mediated by another basolateral hepatic uptake transporter, human OATP8 (symbol SLC21A8). OATP2 and OATP8 differed by their capacity to extract substrates from albumin before transport. In comparison to the high-affinity transport by OATP2, OATP8 transported [(3)H]sulfobromophthalein and [(3)H]monoglucuronosyl bilirubin with lower affinity, with K(m) values of 3.3 and 0.5 microm, respectively. The organic anion indocyanine green potently inhibited transport mediated by OATP2, with a K(i) value of 112 nm, but did not inhibit transport mediated by OATP8. Human OATP2 may play a key role in the prevention of hyperbilirubinemia by facilitating the selective entry of unconjugated bilirubin and its glucuronate conjugates into human hepatocytes.

Increased expression of mature cathepsin B in aging rat liver.

Senescence has been proposed as an important safeguard against neoplasia. One of the hallmarks of cellular senescence in vitro as well as human aging in vivo is a reduced intracellular protein catabolism. The pathways affected and the mechanisms responsible for the decrease in overall protein turnover in aging cells are not well understood. Our aim was to determine whether or not expression of one of the major hepatic lysosomal cysteine peptidases, cathepsin B, changes during aging of Sprague-Dawley rats. Cathepsin B activity was assessed in whole rat liver homogenates, and was found to be increased fourfold (P< or =0.001) in aged livers compared with younger counterparts. This was paralleled by an at least a twofold increase in mature cathepsin B protein. Nonetheless, Northern blot analysis of total liver RNA revealed no change in steady-state levels of cathepsin B mRNAs. These findings seem to contradict the present dogma according to which aging tissues have a reduced intracellular capacity to catabolise proteins. We propose that our earlier observation of the accumulation of T-kininogen, a potent but reversible cysteine peptidase inhibitor, in aging rat liver may provide a plausible explanation for this discrepancy.

Hepatic secretion of conjugated drugs and endogenous substances.

Conjugate export pumps of the multidrug resistance protein (MRP) family mediate the ATP-dependent secretion of anionic conjugates across the canalicular and the basolateral hepatocyte membrane into bile and sinusoidal blood, respectively. Xenobiotic and endogenous lipophilic substances may be conjugated with glutathione, glucuronate, sulfate, or other negatively charged groups and thus become substrates for export pumps of the MRP family. The apical isoform, MRP2 (gene symbol ABCC2), has been localized to the apical membrane of several polarized epithelia and particularly to the canalicular membrane of hepatocytes. Absence of functionally active MRP2 glycoprotein from this membrane domain prevents the secretion of many anionic conjugates into bile. Prototypic endogenous substrates of high affinity for recombinant human MRP2 include bisglucuronosyl bilirubin, monoglucuronosyl bilirubin, and the glutathione S-conjugate leukotriene C4. Several mutations in the human MRP2 gene have been identified that lead to the absence of MRP2 from the canalicular membrane and to the conjugated hyperbilirubinemia of Dubin-Johnson syndrome. MRP2-mediated conjugate export represents a decisive final step in the detoxification of drugs, toxins, and endogenous substances. The basolateral isoform, MRP3 (gene symbol ABCC3), is upregulated in MRP2 deficiency and in extrahepatic cholestasis. MRP3 mediates the ATP-dependent transport of anionic conjugates, particularly of glucuronides and sulfoconjugates, across the basolateral hepatocyte membrane into sinusoidal blood. The inverse regulation of MRP3 and MRP2 expression under many conditions is consistent with their distinct localization and with a compensatory role of MRP3 in the hepatic secretion of anionic conjugates during impaired transport into bile.

Impaired protein maturation of the conjugate export pump multidrug resistance protein 2 as a consequence of a deletion mutation in Dubin-Johnson syndrome.

The Dubin-Johnson syndrome is an inherited disorder characterized by conjugated hyperbilirubinemia. The deficient hepatobiliary transport of anionic conjugates is caused by the absence of a functional multidrug-resistance protein 2 (MRP2, symbol ABCC2) from the apical (canalicular) membrane of hepatocytes. Mechanisms underlying this deficiency may include rapid degradation of mutated MRP2 messenger RNA (mRNA) or impaired MRP2 protein maturation and trafficking. We investigated the consequences of the mutation MRP2Delta(R,M), which leads to the loss of 2 amino acids from the second ATP-binding domain of MRP2. The MRP2Delta(R,M) mutation is associated with the absence of the MRP2 glycoprotein from the apical membrane of hepatocytes. Transfection of mutated MRP2 complementary DNA (cDNA) led to an MRP2Delta(R,M) protein that was only core glycosylated, sensitive to endoglycosidase H digestion, and located in the endoplasmic reticulum (ER) of transfected HEK293 and HepG2 cells. This indicated that deletion of Arg1392 and Met1393 leads to impaired maturation and trafficking of the protein from the ER to the Golgi complex. Inhibition of proteasome function resulted in a paranuclear accumulation of the MRP2Delta(R,M) protein, suggesting that proteasomes are involved in the degradation of the mutant protein. This is the first mutation in Dubin-Johnson syndrome shown to cause deficient MRP2 maturation and impaired sorting of this glycoprotein to the apical membrane.

The multidrug resistance protein 5 functions as an ATP-dependent export pump for cyclic nucleotides.

Cellular export of cyclic nucleotides has been observed in various tissues and may represent an elimination pathway for these signaling molecules, in addition to degradation by phosphodiesterases. In the present study we provide evidence that this export is mediated by the multidrug resistance protein isoform MRP5 (gene symbol ABCC5). The transport function of MRP5 was studied in V79 hamster lung fibroblasts transfected with a human MRP5 cDNA. An MRP5-specific antibody detected an overexpression of the glycoprotein of 185 +/- 15 kDa in membranes from MRP5-transfected cells and a low basal expression of hamster Mrp5 in control membranes. ATP-dependent transport of 3',5'-cyclic GMP at a substrate concentration of 1 micrometer was 4-fold higher in membrane vesicles from MRP5-transfected cells than in control membranes. This transport was saturable with a K(m) value of 2.1 micrometer. MRP5-mediated transport was also detected for 3',5'-cyclic AMP at a lower affinity, with a K(m) value of 379 micrometer. A potent inhibition of MRP5-mediated transport was observed by several compounds, known as phosphodiesterase modulators, including trequinsin, with a K(i) of 240 nm, and sildenafil, with a K(i) value of 267 nm. Thus, cyclic nucleotides are physiological substrates for MRP5; moreover, MRP5 may represent a novel pharmacological target for the enhancement of tissue levels of cGMP.

Enhanced urinary excretion of cysteinyl leukotrienes in patients with acute alcohol intoxication.

BACKGROUND & AIMS: Leukotrienes are proinflammatory mediators. Ethanol inhibits the catabolism of both cysteinyl leukotrienes (leukotriene E(4) [LTE(4)] and N-acetyl-LTE(4)) and leukotriene B(4) (LTB(4)) in hepatocytes. We examined the metabolic derangement of leukotriene inactivation by ethanol in humans in vivo. METHODS: LTE(4), N-acetyl-LTE(4), LTB(4), and 20-hydroxy-LTB(4) were quantified in urine samples from 16 patients with acute alcohol intoxication (mean blood ethanol, 75 mmol/L). In 9 healthy volunteers, urinary LTE(4) was determined before and after ethanol consumption (mean blood ethanol, 14 mmol/L). RESULTS: The excretion of LTE(4) during alcohol intoxication was 286 compared with 36 nmol/mol creatinine in healthy subjects (P < 0.01); the corresponding values for N-acetyl-LTE(4) were 101 and 11 nmol/mol creatinine, respectively (P < 0.001). This excretion of cysteinyl leukotrienes decreased when the blood ethanol concentration returned to normal. LTB(4) and 20-hydroxy-LTB(4) were detectable only in patients with excessive blood ethanol concentrations (mean, 95 mmol/L). In healthy volunteers, LTE(4) excretion increased 3-5 hours after ethanol consumption (mean peak concentration of 1.5 nmol/L compared with 0.5 nmol/L for basal values; P < 0.005). CONCLUSIONS: Ethanol at high concentration induces increased leukotriene excretion into urine. These changes are consistent with inhibition of leukotriene catabolism and inactivation induced by ethanol, as well as with a higher leukotriene formation caused by ethanol-induced endotoxemia.

Fluorescent microplate assay for cancer cell-associated cathepsin B.

Cathepsin B and in particular cell-surface and secreted cathepsin B has been implicated in the invasive and metastatic phenotype of numerous types of cancer. We describe here a method to easily survey cancer cell lines for cathepsin B activity using the highly selective substrate Z-Arg-Arg-AMC. Intact human U87 glioma cells hydrolyze Z-Arg-Arg-AMC with a Km of 460 microM at pH 7.0 and 37 degrees C. This is nearly the same as the Km of 430 microM obtained with purified cathepsin B assayed under the same conditions. The pericellular (i.e. both cell-surface and released) cathepsin B activity was inhibited by the cysteine protease inhibitors E-64, leupeptin, Mu-Np2-HphVS-2Np, Mu-Leu-HpHVSPh and the cathepsin B selective inhibitor Mu-Tyr(3,5 I2)-HphVSPh with IC50 values similar to those observed for the inhibition of purified human liver cathepsin B. Other human cancer cell lines with measurable pericellular cathepsin B activity included HT-1080 fibrosarcoma, MiaPaCa pancreatic, PC-3 prostate and HCT-116 colon. Cathepsin B activity correlated with protein levels of cathepsin B as determined by immunoblot analysis. Pericellular cathepsin B activity was also detected in the rat cell lines MatLyLu prostate and Mat B III adenocarcinoma and in the murine lines B16a melanoma and Lewis lung carcinoma. The ability to determine pericellular cathepsin B activity will be useful in selecting appropriate cell lines for use in vivo when analyzing the effects of inhibiting cathepsin B activity on tumor growth and metastasis.

ATP-dependent para-aminohippurate transport by apical multidrug resistance protein MRP2.

BACKGROUND: Para-aminohippurate (PAH), a widely used model substrate for organic anion transport in proximal tubule epithelia, was investigated as a substrate for the apical multidrug resistance protein MRP2 (symbol ABCC2). This ATP-dependent export pump for anionic conjugates and additional amphiphilic anions was cloned recently and localized to the apical membrane of proximal tubules in human and rat kidney. METHODS: Membrane vesicles from HEK-MRP2 cells containing recombinant human MRP2 and from control vector-transfected HEK-Co cells were incubated with various concentrations of [3H]PAH, and the net ATP-dependent transport into inside-out vesicles was determined. Comparative studies were performed with membrane vesicles containing recombinant human MRP1. RESULTS: Transport rates at 10 micromol/L PAH were 21.9 +/- 1.9 and 1.6 +/- 0.4 pmol x mg protein-1 x min-1 (means +/- SEM, N = 10) with membrane vesicles from HEK-MRP2 and HEK-Co cells, respectively. The Km value for PAH was 880 micromol/L. The high-affinity substrate leukotriene C4 and the inhibitor of MRP-mediated transport, MK571, inhibited MRP2-mediated transport of PAH (100 nmol/L) with IC50 values of 3.3 and 4.0 micromol/L, respectively. The nephrotoxic mycotoxin ochratoxin A inhibited MRP2-mediated PAH transport with an IC50 value of 58 micromol/L. Ochratoxin A was itself a substrate for MRP2. CONCLUSIONS: PAH is a good substrate for the ATP-dependent export pump MRP2. The localization and function of MRP2 indicate that this unidirectional transport protein contributes to the secretion of PAH and other amphiphilic anions into the lumen of kidney proximal tubules.