ATP-dependent transport of bile salts by rat multidrug resistance-associated protein 3 (Mrp3)

We have previously shown that cloned rat multidrug resistance-associated protein 3 (Mrp3) has the ability to transport organic anions such as 17beta-estradiol 17-beta-D-glucuronide (E(2)17betaG) and has a different substrate specificity from MRP1 and MRP2 in that glutathione conjugates are poor substrates for Mrp3 (Hirohashi, T., Suzuki, H., and Sugiyama, Y. (1999) J. Biol. Chem. 274, 15181-15185). In the present study, the involvement of Mrp3 in the transport of endogenous bile salts was investigated using membrane vesicles from LLC-PK1 cells transfected with rat Mrp3 cDNA. The ATP-dependent uptake of [(3)H]taurocholate (TC), [(14)C]glycocholate (GC), [(3)H]taurochenodeoxycholate-3-sulfate (TCDC-S), and [(3)H]taurolithocholate-3-sulfate (TLC-S) was markedly stimulated by Mrp3 transfection in LLC-PK1 cells. The extent of Mrp3-mediated transport of bile salts was in the order, TLC-S > TCDC-S > TC > GC. The K(m) and V(max) values for the uptake of TC and TLC-S were K(m) = 15.9 +/- 4.9 microM and V(max) = 50.1 +/- 9.3 pmol/min/mg of protein and K(m) = 3.06 +/- 0.57 microM and V(max) = 161.9 +/- 21.7 pmol/min/mg of protein, respectively. At 55 nM [(3)H]E(2)17betaG and 1.2 microM [(3)H]TC, the apparent K(m) values for ATP were 1.36 and 0.66 mM, respectively. TC, GC, and TCDC-S inhibited the transport of [(3)H]E(2)17betaG and [(3)H]TC to the same extent with an apparent IC(50) of approximately 10 microM. TLC-S inhibited the uptake of [(3)H]E(2)17betaG and [(3)H]TC most potently (IC(50) of approximately 1 microM) among the bile salts examined, whereas cholate weakly inhibited the uptake (IC(50) approximately 75 microM). Although TC and GC are transported by bile salt export pump/sister of P-glycoprotein, but not by MRP2, and TCDC-S and TLC-S are transported by MRP2, but not by bile salt export pump/sister of P-glycoprotein, it was found that Mrp3 accepts all these bile salts as substrates. This information, together with the finding that MRP3 is extensively expressed on the basolateral membrane of human cholangiocytes, suggests that MRP3/Mrp3 plays a significant role in the cholehepatic circulation of bile salts.     

Characterization of bile acid transport mediated by multidrug resistance associated protein 2 and bile salt export pump

Biliary excretion of certain bile acids is mediated by multidrug resistance associated protein 2 (Mrp2) and the bile salt export pump (Bsep). In the present study, the transport properties of several bile acids were characterized in canalicular membrane vesicles (CMVs) isolated from Sprague--Dawley (SD) rats and Eisai hyperbilirubinemic rats (EHBR) whose Mrp2 function is hereditarily defective and in membrane vesicles isolated from Sf9 cells infected with recombinant baculovirus containing cDNAs encoding Mrp2 and Bsep. ATP-dependent uptake of [(3)H]taurochenodeoxycholate sulfate (TCDC-S) (K(m)=8.8 microM) and [(3)H]taurolithocholate sulfate (TLC-S) (K(m)=1.5 microM) was observed in CMVs from SD rats, but not from EHBR. In addition, ATP-dependent uptake of [(3)H]TLC-S (K(m)=3.9 microM) and [(3)H]taurocholate (TC) (K(m)=7.5 microM) was also observed in Mrp2- and Bsep-expressing Sf9 membrane vesicles, respectively. TCDC-S and TLC-S inhibited the ATP-dependent TC uptake into CMVs from SD rats with IC(50) values of 4.6 microM and 1.2 microM, respectively. In contrast, the corresponding values for Sf9 cells expressing Bsep were 59 and 62 microM, respectively, which were similar to those determined in CMVs from EHBR (68 and 33 microM, respectively). By co-expressing Mrp2 with Bsep in Sf9 cells, IC(50) values for membrane vesicles from these cells shifted to values comparable with those in CMVs from SD rats (4.6 and 1.2 microM). Moreover, in membrane vesicles where both Mrp2 and Bsep are co-expressed, preincubation with the sulfated bile acids potentiated their inhibitory effect on Bsep-mediated TC transport. These results can be accounted for by assuming that the sulfated bile acids trans-inhibit the Bsep-mediated transport of TC.     

Bile salt excretion in skate liver is mediated by a functional analog of Bsep/Spgp, the bile salt export pump

Biliary secretion of bile salts in mammals is mediated in part by the liver-specific ATP-dependent canalicular membrane protein Bsep/Spgp, a member of the ATP-binding cassette superfamily. We examined whether a similar transport activity exists in the liver of the evolutionarily primitive marine fish Raja erinacea, the little skate, which synthesizes mainly sulfated bile alcohols rather than bile salts. Western blot analysis of skate liver plasma membranes using antiserum raised against rat liver Bsep/Spgp demonstrated a dominant protein band with an apparent molecular mass of 210 kDa, a size larger than that in rat liver canalicular membranes, approximately 160 kDa. Immunofluorescent localization with anti-Bsep/Spgp in isolated, polarized skate hepatocyte clusters revealed positive staining of the bile canaliculi, consistent with its selective apical localization in mammalian liver. Functional characterization of putative ATP-dependent canalicular bile salt transport activity was assessed in skate liver plasma membrane vesicles, with [(3)H]taurocholate as the substrate. [(3)H]taurocholate uptake into the vesicles was mediated by ATP-dependent and -independent mechanisms. The ATP-dependent component was saturable, with a Michaelis-Menten constant (K(m)) for taurocholate of 40+/-7 microM and a K(m) for ATP of 0.6+/-0.1 mM, and was competitively inhibited by scymnol sulfate (inhibition constant of 23 microM), the major bile salt in skate bile. ATP-dependent uptake of taurocholate into vesicles was inhibited by known substrates and inhibitors of Bsep/Spgp, including other bile salts and bile salt derivatives, but not by inhibitors of the multidrug resistance protein-1 or the canalicular multidrug resistance-associated protein, indicating a distinct transport mechanism. These findings provide functional and structural evidence for a Bsep/Spgp-like protein in the canalicular membrane of the skate liver. This transporter is expressed early in vertebrate evolution and transports both bile salts and bile alcohols.     

The effect of taurine depletion with guanidinoethanesulfonate on bile acid metabolism in the rat

Administration of guanidinoethanesulfonate (GES) to male rats for 5 weeks resulted in a 90% decrease in the hepatic taurine concentration. This depletion of hepatic taurine was associated with a 570% increase in the concentration of glycine-conjugated bile acids, a 30% decrease in the concentration of taurine-conjugated bile acids, and an increase in the ratio of glycine- to taurine-conjugated bile acids from 0.046 to 0.45. The total concentration of bile salts in the bile and the turnover of cholic acid were not affected by administration of GES. The data indicate that the taurine-depleted rat conserves taurine to some extent by using glycine instead of taurine for bile salt synthesis but not by decreasing the daily fractional turnover of bile acids.     

Characterization of the transport properties of cloned rat multidrug resistance-associated protein 3 (MRP3).

We have previously cloned rat MRP3 as an inducible transporter in the liver (Hirohashi, T., Suzuki, H., Ito, K., Ogawa, K., Kume, K., Shimizu, T., and Sugiyama, Y. (1998) Mol. Pharmacol. 53, 1068-1075). In the present study, the function of rat MRP3 was investigated using membrane vesicles isolated from LLC-PK1 and HeLa cell population transfected with corresponding cDNA. The ATP-dependent uptake of both 17beta estradiol 17-beta-D-glucuronide ([3H]E217betaG) and glucuronide of [14C] 6-hydroxy-5, 7-dimethyl-2-methylamino-4-(3-pyridylmethyl) benzothiazole (E3040), but not that of [3H]leukotriene C4 and [3H]2, 4-dinitrophenyl-S-glutathione, was markedly stimulated by MRP3 transfection in both cell lines. The Km and Vmax values for the uptake of [3H]E217betaG were 67 +/- 14 microM and 415 +/- 73 pmol/min/mg of protein, respectively, for MRP3-expressing membrane vesicles and 3.0 +/- 0.7 microM and 3.4 +/- 0.4 pmol/min/mg of protein, respectively, for the endogenous transporter expressed on HeLa cells. [3H]E217betaG had also a similar Km value for MRP3 when LLC-PK1 cells were used as the host. All glucuronide conjugates examined (E3040 glucuronide, 4-methylumbelliferone glucuronide, and naphthyl glucuronide) and methotrexate inhibited MRP3-mediated [3H]E217betaG transport in LLC-PK1 cells. Moreover, [3H]methotrexate was transported via MRP3. The inhibitory effect of estrone sulfate, [3H]2,4-dinitrophenyl-S-glutathione, and [3H]leukotriene C4 was moderate or minimal, whereas N-acetyl-2,4-dinitrophenylcysteine had no effect on the uptake of [3H]E217betaG. The uptake of [3H]E217betaG was enhanced by E3040 sulfate and 4-methylumbelliferone sulfate. Thus we were able to demonstrate that several kinds of organic anions are transported via MRP3, although the substrate specificity of MRP3 differs from that of MRP1 and cMOAT/MRP2 in that glutathione conjugates are poor substrates for MRP3.     

Involvement of γ-aminobutyric acid transporter 2 in the hepatic uptake of taurine in rats

Taurine is essential for the hepatic synthesis of bile salts and, although taurine is synthesized mainly in pericentral hepatocytes, taurine and taurine-conjugated bile acids are abundant in periportal hepatocytes. One possible explanation for this discrepancy is that the active supply of taurine to hepatocytes from the blood stream is a key regulatory factor. The purpose of the present study is to investigate and identify the transporter responsible for taurine uptake by periportal hepatocytes. An in vivo bolus injection of [(3)H]taurine into the rat portal vein demonstrated that 25% of the injected [(3)H]taurine was taken up by the liver on a single pass. The in vivo uptake was significantly inhibited by GABA, taurine, β-alanine, and nipecotic acid, a GABA transporter (GAT) inhibitor, each at a concentration of 10 mM. The characteristics of Na(+)- and Cl(-)-dependent [(3)H]taurine uptake by freshly isolated rat hepatocytes were consistent with those of GAT2 (solute carrier SLC6A13). Indeed, the K(m) value of the saturable uptake (594 μM) was close to that of mouse SLC6A13-mediated taurine transport. Although GABA, taurine, and β-alanine inhibited the [(3)H]taurine uptake by > 50%, each at a concentration of 10 mM, GABA caused a marked inhibition with an IC(50) value of 95 μM. The [(3)H]taurine uptake exhibited a significant reduction when the GAT2 gene was silenced. Immunohistochemical analysis showed that GAT2 was localized on the sinusoidal membrane of the hepatocytes predominantly in the periportal region. These results suggest that GAT2 is responsible for taurine transport from the circulating blood to hepatocytes predominantly in the periportal region.     

Quantitative estimation of the hydrophilic-hydrophobic balance of mixed bile salt solutions

This paper describes the derivation of a bile salt monomeric hydrophobicity index that quantitatively defines the composite hydrophilic-hydrophobic balance of a mixture of bile salts. The index is based on the logarithms of bile salt capacity factors determined using reversed phase high performance liquid chromatography (HPLC) (stationary phase octadecyl silane; mobile phase methanol-water 70:30 w/w, ionic strength 0.15). It has been standardized arbitrarily to set indices of taurocholate and taurolithocholate to 0 and 1, respectively. Indices of tauroursodeoxycholate, taurohyodeoxycholate, taurochenodeoxycholate, and taurodeoxycholate were found to be -0.47, -0.35, +0.46, and +0.59, respectively. Whereas capacity factors and hydrophobicity indices of taurine-conjugated bile salts were constant for pH 2.8-9.0, the hydrophilic-hydrophobic balance of glycine-conjugated and unconjugated bile salts was strongly influenced by pH. At alkaline pH (greater than 8.5), hydrophobicity indices of fully ionized unconjugated (n = 4) and glycine-conjugated (n = 6) bile salts differed by only 0.14 +/- 0.02 and 0.05 +/- 0.01, respectively, from those of the corresponding taurine conjugates. At acid pH (less than 3.5) the hydrophobicity indices of four unconjugated bile acids (protonated form) exceeded those of the corresponding salts (ionized form) by 0.76 +/- 0.04; indices of six glycine-conjugated bile acids exceeded those of the corresponding salts by only 0.26 +/- 0.03. Capacity factors of the salt forms of cholate and its conjugates increased dramatically with increasing ionic strength of the mobile phase; retention of the protonated forms (cholic and glycocholic acids) was only minimally influenced by ionic strength. Thus the difference in hydrophilic-hydrophobic balance between a bile acid and its corresponding salt decreases with increasing ionic strength. Examples are given of calculation of hydrophobicity indices for biliary bile salts (fully ionized) from four species under conditions of intact enterohepatic circulation. Mean values, from least to most hydrophobic, were: rat (-0.31) less than dog (0.11) less than hamster (0.22) less than human (0.32). This study provides a rational basis for calculating the hydrophilic-hydrophobic balance of mixed bile salt solutions over a broad range of pH.     

Bile acid solubility and precipitation in vitro and in vivo: the role of conjugation, pH, and Ca2+ ions.

The principles governing the in vitro solubility of the common natural conjugated and unconjugated bile acids and salts in relation to pH, micelle formation, and Ca2+ concentration are considered from a theoretical standpoint and then correlated first with experimental observations on model systems and second with the formation of precipitates containing bile acids in health and disease. In vitro, taurine-conjugated bile acids are soluble at strongly acidic pH; glycine-conjugated bile acids are poorly soluble at moderately acidic pH; and many of the common, natural unconjugated bile acids are insoluble at neutral pH. For both glycine-conjugated and unconjugated bile acids, solubility rises exponentially, with increasing pH, until the concentration of the anion reaches the critical micellization concentration (CMC) when micelle formation occurs and solubility becomes practically unlimited. In vivo, in health, conjugated bile acids are present in micellar form in the biliary and intestinal tract. Unconjugated bile acids formed in the large intestine remain at low monomeric concentrations because of the acidic pH of the proximal colon, binding to bacteria, and absorption across the intestinal mucosa. In diseases in which proximal small intestinal content is abnormally acidic, precipitation of glycine-conjugated bile acids (in protonated form) occurs. Increased bacterial formation of unconjugated bile acids occurs with stasis in the biliary tract and small intestine; in the intestine, unconjugated bile acids precipitate in the protonated form. If the precipitates aggregate, an enterolith may be formed. In vitro, the calcium salts of taurine conjugates are highly water soluble, whereas the calcium salts of glycine conjugates and unconjugated bile acids possess limited aqueous solubility that is strongly influenced by bile acid structure. Precipitation occurs extremely slowly from supersaturated solutions of glycine-conjugated bile acids because of metastability, whereas super-saturated solutions of unconjugated bile acids rapidly form precipitates of the calcium salt. In systems containing Ca2+ ions and unconjugated bile acids, pH is important, since it is the key determinant of the anion concentration. For bile acids with relatively soluble calcium salts (or with a low CMC), the concentration of the anion will reach the CMC and micelles will form, thus precluding formation of the insoluble calcium salt. For bile acids, with relatively insoluble calcium salts (or with a high CMC), the effect of increasing pH is to cause the anion to reach the solubility product of the calcium salt before reaching the CMC so that precipitation of the calcium salt occurs instead of micelle formation.(ABSTRACT TRUNCATED AT 400 WORDS)     

ATP-dependent transport of taurocholate across the hepatocyte canalicular membrane mediated by a 110-kDa glycoprotein binding ATP and bile salt

Direct photoaffinity labeling of liver plasma membrane subfractions enriched in sinusoidal and canalicular membranes using [35S]adenosine 5'-O-(thiotriphosphate) ([35S]ATP gamma S) allows the identification of ATP-binding proteins in these domains. Comparative photoaffinity labeling with [35S]ATP gamma S and with the photolabile bile salt derivative (7,7-azo-3 alpha, 12 alpha-dihydroxy-5 beta-[3 beta-3H]-cholan-24-oyl-2'- aminoethanesulfonate followed by immunoprecipitation with a monoclonal antibody (Be 9.2) revealed the identity of the ATP-binding and the bile salt-binding canalicular membrane glycoprotein with the apparent Mr of 110,000 (gp110). The isoelectric point of this glycoprotein was 3.7. Transport of bile salt was studied in vesicles enriched in canalicular and sinusoidal liver membranes. Incubation of canalicular membrane vesicles with [3H] taurocholate in the presence of ATP resulted in an uptake of the bile salt into the vesicles which was sensitive to vanadate. ATP-dependent taurocholate transport was also observed in membrane vesicles from mutant rats deficient in the ATP-dependent transport of cysteinyl leukotrienes and related amphiphilic anions. Substrates of the P-glycoprotein (gp170), such as verapamil and doxorubicin, did not interfere with the ATP-dependent transport of taurocholate. Reconstitution of purified gp110 into liposomes resulted in an ATP-dependent uptake of [3H]taurocholate. These results demonstrate that gp110 functions as carrier in the ATP-dependent transport of bile salts from the hepatocyte into bile. This export carrier is distinct from hitherto characterized ATP-dependent transport systems.     

Vectorial transport of unconjugated and conjugated bile salts by monolayers of LLC-PK1 cells doubly transfected with human NTCP and BSEP or with rat Ntcp and Bsep

Na(+)-taurocholate-cotransporting peptide (NTCP)/SLC10A1 and bile salt export pump (BSEP)/ABCB11 synergistically play an important role in the transport of bile salts by the hepatocyte. In this study, we transfected human NTCP and BSEP or rat Ntcp and Bsep into LLC-PK1 cells, a cell line devoid of bile salts transporters. Transport by these cells was characterized with a focus on substrate specificity between rats and humans. The basal to apical flux of taurocholate across NTCP- and BSEP-expressing LLC-PK1 monolayers was 10 times higher than that in the opposite direction, whereas the flux across the monolayer of control and NTCP or BSEP single-expressing cells did not show any vectorial transport. The basal to apical flux of taurocholate was saturated with a K(m) value of 20 microM. Vectorial transcellular transport was also observed for cholate, chenodeoxycholate, ursodeoxycholate, their taurine and glycine conjugates, and taurodeoxycholate and glycodeoxycholate, whereas no transport of lithocholate was detected. To evaluate the respective functions of NTCP and BSEP and to compare them with those of rat Ntcp and Bsep, we calculated the clearance by each transporter in this system. A good correlation in the clearance of the examined bile salts (cholate, chenodeoxycholate, ursodeoxycholate, and their taurine or glycine conjugates) was observed between transport by human and that of rat transporters in terms of their rank order: for NTCP, taurine conjugates > glycine conjugates > unconjugated bile salts, and for BSEP, unconjugated bile salts and glycine conjugates > taurine conjugates. In conclusion, the substrate specificity of human and rat NTCP and BSEP appear to be very similar at least for monovalent bile salts under physiological conditions.     

Blood-brain barrier is involved in the efflux transport of a neuroactive steroid, dehydroepiandrosterone sulfate, via organic anion transporting polypeptide 2

We have investigated the transport characteristics of dehydroepiandrosterone sulfate (DHEAS), a neuroactive steroid, at the blood-brain barrier (BBB) in a series of functional in vivo and in vitro studies. The apparent BBB efflux rate constant of [(3)H]DHEAS evaluated by the brain efflux index method was 2.68 x 10(-2) min(-1). DHEAS efflux transport was a saturable process with a Michaelis constant (K:(m)) of 32.6 microM: Significant amounts of [(3)H]DHEAS were determined in the jugular venous plasma by HPLC, providing direct evidence that most of the DHEAS is transported in intact form from brain to the circulating blood across the BBB. This efflux transport of [(3)H]DHEAS was significantly inhibited by common rat organic anion-transporting polypeptide (oatp) substrates such as taurocholate, cholate, sulfobromophthalein, and estrone-3-sulfate. Moreover, the apparent efflux clearance of [(3)H]DHEAS across the BBB (118 microl/min-g of brain) was 10.4-fold greater than its influx clearance estimated by the in situ brain perfusion technique (11.4 microl/min-g of brain), suggesting that DHEAS is predominantly transported from the brain to blood across the BBB. In cellular uptake studies using a conditionally immortalized mouse brain capillary endothelial cell line (TM-BBB4), [(3)H]DHEAS uptake by TM-BBB4 cells exhibited a concentration dependence with a K:(m) of 34.4 microM: and was significantly inhibited by the oatp2-specific substrate digoxin. Conversely, [(3)H]digoxin uptake by TM-BBB4 cells was significantly inhibited by DHEAS. Moreover, the net uptake of [(3)H]DHEAS at 30 min was significantly increased under ATP-depleted conditions, suggesting that an energy-dependent efflux process may also be involved in TM-BBB4. RT-PCR and sequence analysis suggest that an oatp2 is expressed in TM-BBB4 cells. In conclusion, DHEAS efflux transport takes place across the BBB, and studies involving in vitro DHEAS uptake and RT-PCR suggest that there is oatp2-mediated DHEAS transport at the BBB.     

Development of a scintillation proximity assay for analysis of Na+/Cl- -dependent neurotransmitter transporter activity

Human placental choriocarcinoma (JAR) cells endogenously expressing glycine transporter type 1a (GlyT1a) have been cultured in 96-well scintillating microplates to develop a homogenous screening assay for the detection of GlyT1 antagonists. In these microplates uptake of [14C]glycine was time dependent and saturable with a Michaelis-Menten constant (Km) of 27+/-3 microM. The GlyT1 transport inhibitors sarcosine, ALX-5407, and Org-24598 were tested and shown to block [14C]glycine uptake with expected IC50 values of 37.5+/-4.6 microM, 2.8+/-0.6 nM, and 6.9+/-0.9 nM, respectively. The [14C]glycine uptake process was sensitive to membrane Na+ gradient as blockade of membrane Na+/K+-ATPase by ouabain or Na+ exchanger by benzamil-disrupted glycine accumulation in JAR cells. Glycine influx was not affected by concentration of dimethyl sulfoxide up to 2%. The versatility of this technological approach was further confirmed by the characterization of a saturable [14C]taurine uptake in JAR cells. Taurine transport was of high affinity with a Km of 10.2+/-1.7 microM and fully inhibited by ALX-5407 (IC50=522 +/-83 nM). The developed assay is homogenous, rapid, versatile and amenable to automation for the discovery of new neurotransmitter transporter inhibitors.     

A purine-selective nucleobase/nucleoside transporter in PK15NTD cells

Nucleoside and nucleobase transporters are important for salvage of purines and pyrimidines and for transport of their analog drugs into cells. However, the pathways for nucleobase translocation in mammalian cells are not well characterized. We identified an Na-independent purine-selective nucleobase/nucleoside transport system in the nucleoside transporter-deficient PK15NTD cells. This transport system has 1,000-fold higher affinity for nucleobases than nucleosides with K(m) values of 2.5 +/- 0.7 microM for [(3)H]adenine, 6.4 +/- 0.5 microM for [(3)H]guanine, 1.1 +/- 0.1 mM for [(3)H]guanosine, and 4.2 +/- 0.5 mM [(3)H]adenosine. The uptake of [(3)H]guanine (0.05 microM) was inhibited by other nucleobases and nucleobase analog drugs (at 0.5-1 mM in the order of potency): 6-mercaptopurine = thioguanine = guanine > adenine > thymine = fluorouracil = uracil. Cytosine and methylcytosine had no effect. Nucleoside analog drugs with modification at 2' and/or 5 positions (all at 1 mM) were more potent than adenosine in competing the uptake of [(3)H]guanine: 2-chloro-2'-deoxyadenosine > 2-chloroadenosine > 2'3'-dideoxyadenosine = 2'-deoxyadenosine > 5-deoxyadenosine > adenosine. 2-Chloro-2'-deoxyadenosine and 2-chloroadenosine inhibited [(3)H]guanine uptake with IC(50) values of 68 +/- 5 and 99 +/- 10 microM, respectively. The nucleobase/nucleoside transporter was resistant to nitrobenzylthioinosine {6-[(4-nitrobenzyl) thiol]-9-beta-D-ribofuranosylpurine}, dipyridamole, and dilazep, but was inhibited by papaverine, the organic cation transporter inhibitor decynium-22 (IC(50) of approximately 1 microM), and by acidic pH (pH = 5.5). In conclusion, we have identified a mammalian purine-selective nucleobase/nucleoside transporter with high affinity for purine nucleobases. This transporter is potentially important for transporting naturally occurring purines and purine analog drugs into cells.     

Identification of phalloidin uptake systems of rat and human liver

To determine whether the liver toxin phalloidin is transported into hepatocytes by one of the known bile salt transporters, we expressed the sodium-dependent Na+/taurocholate cotransporting polypeptide (Ntcp) and several sodium-independent bile salt transporters of the organic anion transporting polypeptide (OATP/SLCO) superfamily in Xenopus laevis oocytes and measured uptake of the radiolabeled phalloidin derivative [3H]demethylphalloin. We found that rat Oatp1b2 (previously called Oatp4 (Slc21a10)) as well as human OATP1B1 (previously called OATP-C (SLC21A6)) and OATP1B3 (previously called OATP8 (SLC21A8)) mediate uptake of [3H]demethylphalloin when expressed in X. laevis oocytes. Transport of increasing [3H]demethylphalloin concentrations was saturable with apparent Km values of 5.7 microM (Oatp1b2), 17 microM (OATP1B1) and 7.5 microM (OATP1B3). All other tested Oatps/OATPs as well as the rat liver Ntcp did not transport [3H]demethylphalloin. Therefore, we conclude that rat Oatp1b2 as well as human OATP1B1 and OATP1B3 are responsible for phalloidin uptake into rat and human hepatocytes.     

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.     

ATP-dependent proton transport by isolated brain clathrin-coated vesicles. Role of clathrin and other determinants of acidification

We have systematically investigated certain characteristics of the ATP-dependent proton transport mechanism of bovine brain clathrin-coated vesicles. H+ transport specific activity was shown by column chromatograpy to co-purify with coated vesicles, however, the clathrin coat is not required for vesicle acidification as H+ transport was not altered by prior removal of the clathrin coat. Acidification of the vesicle interior, measured by fluorescence quenching of acridine orange, displayed considerable anion selectively (Cl- greater than Br- much greater than NO3- much greater than gluconate, SO2-(4), HPO2-(4), mannitol; Km for Cl- congruent to 15 mM), but was relatively insensitive to cation replacement as long as Cl- was present. Acidification was unaffected by ouabain or vanadate but was inhibited by N-ethylmaleimide (IC50 less than 10 microM), dicyclohexylcarbodiimide (DCCD) (IC50 congruent to 10 microM), chlorpromazine (IC50 congruent to 15 microM), and oligomycin (IC50 congruent to 3 microM). In contrast to N-ethylmaleimide, chlorpromazine rapidly dissipated preformed pH gradients. Valinomycin stimulated H+ transport in the presence of potassium salts (gluconate much greater than NO3- greater than Cl-), and the membrane-potential-sensitive dye Oxonol V demonstrated an ATP-dependent interior-positive vesicle membrane potential which was greater in the absence of permeant anions (mannitol greater than potassium gluconate greater than KCl) and was abolished by N-ethylmaleimide, protonophores or detergent. Total vesicle-associated ouabain-insensitive ATPase activity was inhibited 64% by 1 mM N-ethylmaleimide, and correlated poorly with H+ transport, however N-ethylmaleimide-sensitive ATPase activity correlated well with proton transport (r = 0.95) in the presence of various Cl- salts and KNO3. Finally, vesicles prepared from bovine brain synaptic membranes exhibited H+ transport activity similar to that of the coated vesicles.(ABSTRACT TRUNCATED AT 400 WORDS)     

Kinetics of binding of [(3)H]glycine to transport proteins in channel catfish brain

[(3)H]Glycine was observed to bind to channel catfish brain particles in a manner displaying saturation kinetics. The dissociation constant was calculated to be 7.38 +/- 2.11 microM. Though some binding occurred in the absence of Na(+) ions, the presence of such ions stimulated binding in a concentration-dependent manner. Similarly, chloride ions had a stimulatory effect on [(3)H]glycine binding. Several inhibitors of binding were identified, the most effective being beta-alanine, pipecolic acid, 2,3-pyrazine dicarboxylic acid and 3,5-pyrazole dicarboxylic acid. Each is a structural analogue of glycine. Harmaline, a known inhibitor of Na(+) binding, also inhibited glycine binding. A previous study had shown the presence of a sodium-dependent, active uptake system for glycine in synaptosomes derived from catfish brain. The present results suggest that the binding of [(3)H]glycine was to a glycine transporter and that the amino acid functions as a neurotransmitter in this species.     

Purification and functional reconstitution of the rat organic cation transporter OCT1

The rat organic cation transporter rOCT1 with six histidine residues added to the C-terminus was expressed in Sf9 insect cells, and expression of organic cation transport was demonstrated. To purify rOCT1 protein, Sf9 cells were lysed with 1% (w/v) CHAPS [3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate], centrifuged, and subjected to sequential affinity chromatography using lentil-lectin Sepharose and nickel(II)-charged nitrilotriacetic acid-agarose. This procedure yielded approximately 70 microg of purified rOCT1 protein from 10 standard culture plates. Using a freeze-thaw procedure, purified rOCT1 was reconstituted into proteoliposomes formed from phosphatidylcholine, phosphatidylserine, and cholesterol. Proteoliposomes exhibited uptake of [3H]-1-Methyl-4-phenylpyridinium ([3H]MPP) that was inhibited by quinine and stimulated by an inside-negative membrane potential. MPP uptake was saturable with an apparent K(m) of 30 +/- 17 microM. MPP uptake (0.1 microM) was inhibited by tetraethylammonium, tetrabutylammonium, and tetrapentylammonium with IC50 values of 197 +/- 11, 19 +/- 1, and 1.8 +/- 0.03 microM, respectively. With membrane potential clamped to 0 mV using valinomycin in the presence of 100 mM potassium on both sides of the membrane, uptake of 0.1 microM MPP was trans stimulated 3-fold by 2.5 mM intracellular choline, and efflux of 0.1 microM MPP was trans stimulated 4-fold by 9.5 mM extracellular choline. The data show that rOCT1 is capable and sufficient to mediate transport of organic cations. The observed trans stimulation under voltage-clamp conditions shows that rOCT1 operates as a transporter rather than a channel. Purification and reconstitution of functional active rOCT1 protein is an important step toward the biophysical characterization and crystallization.     

Choline transporter as a novel target for molecular imaging of cancer

Abnormalities of choline processing in cancer cells have been used as a basis for imaging of cancer with positron emission tomography and magnetic resonance spectroscopy. In this study, the transport mechanism for choline was investigated in cultured PC-3 prostate cancer cells. Furthermore, tritiated hemicholinium 3 (HC-3), a well-known inhibitor of choline transport, was studied as a prototypic molecular imaging probe in PC-3 cells and 9L glioma-bearing rats. [(3)H]Choline uptake by PC-3 cells was found to have both facilitative and nonfacilitative components. Facilitative transport was characterized by partial sodium dependence and intermediate affinity (K(M) = 9.7 +/- 0.8 microM). HC-3 inhibited choline with a K(I) of 10.5+/- 2.2 microM. Ouabain (1 mM) caused a 94% reduction in choline uptake. At physiologic choline concentration, phosphocholine was the rapid and predominant metabolic fate. The binding of [(3)H]HC-3 to PC-3 cells was rapid and specific (competitively blocked with unlabeled HC-3). Biodistribution of [(3)H]HC-3 in 9L glioma-bearing rats showed the ranking of uptake to be kidney > lung > tumor > liver > skeletal muscle congruent with blood > brain. In comparison with [(14)C]choline, [(3)H]HC-3 showed over twofold higher tumor uptake and favorable uptake ratios of tumor to blood, tumor to muscle, tumor to lung, and tumor to liver. The data demonstrate the quantitative importance of an intermediate-affinity, partially sodium-dependent choline transport system on choline processing in PC-3 cancer cells. The biodistribution properties of [(3)H]HC-3 in tumor-bearing rats encourage the development of molecular imaging probes based on choline transporter binding ligands.     

N-methyl-D-aspartate receptors mediating hippocampal noradrenaline and striatal dopamine release display differential sensitivity to quinolinic acid, the HIV-1 envelope protein gp120, external pH and protein kinase C inhibition

NMDA receptors regulating hippocampal noradrenaline (NA) and striatal dopamine (DA) release have been compared using superfused synaptosomes prelabelled with the [(3)H]catecholamines. Both receptors mediated release augmentation when exposed to NMDA plus glycine. Quinolinic acid (100 microM or 1 mM) plus glycine (1 microM)-elicited [(3)H]NA, but not [(3)H]DA release. The NMDA (100 microM)-evoked release of [(3)H]NA and [(3)H]DA was similar and concentration-dependently enhanced by glycine or D-serine (0.1-1 microM); in contrast, the HIV-1 envelope protein gp120 potently (30-100 pM) enhanced the NMDA-evoked release of [(3)H]NA, but not that of [(3)H]DA. Gp120 also potentiated quinolinate-evoked [(3)H]NA release. Ifenprodil (0.1-0.5 microM) or CP-101,606 (0.1-10 microM) inhibited the NMDA plus glycine-evoked release of both [(3)H]catecholamines. Zinc (0.1-1 microM) was ineffective. Lowering external pH from 7.4 to 6.6 strongly inhibited the release of [(3)H]NA elicited by NMDA plus glycine, whereas the release of [(3)H]DA was unaffected. The protein kinase C inhibitors GF 109203X (0.1 microM) or H7 (10 microM) selectively prevented the effect of NMDA plus glycine on the release of [(3)H]NA. GF 109203X also blocked the release of [(3)H]NA induced by NMDA or quinolinate plus gp120. It is concluded that the hippocampal NMDA receptor and the striatal NMDA receptor are pharmacologically distinct native subtypes, possibly containing NR2B subunits but different splice variants of the NR1 subunit.