Interaction of sulfonylureas with the transport of bile acids into hepatocytes.

The sulfonylurea compounds glisoxepide and glibenclamide inhibit the uptake of bile acids into isolated rat hepatocytes. The Ki values for the inhibition of cholate uptake was 9 microM with glibenclamide and 200 microM with glisoxepide. The inhibition of cholate uptake by both sulfonylureas was noncompetitive. Uptake of the conjugated bile acid taurocholate was inhibited by glibenclamide, Ki = 75 microM. Again the inhibition was noncompetitive. Glisoxepide inhibited taurocholate uptake only in the absence of sodium ions. Under sodium-free conditions glisoxepide also strongly inhibited cholate uptake. The inhibition was competitive, Ki = 42 microM. Both bile acids interfered with the hepatocellular uptake of [3H]glisoxepide, with IC50 values of 375 and 467 microM for cholate and taurocholate, respectively. The uptake of [3H]glibenclamide was inhibited by cholate, IC50 = 328 microM, but not by taurocholate. Glisoxepide uptake was further inhibited by blockers of the hepatocellular monocarboxylate transporter, by the loop diuretic bumetanide, by 4,4'-diisothiocyano-2,2'-stilbenedisulfonate (DIDS) and by sulfate. Glibenclamide uptake was weakly inhibited by DIDS and by anthracene-9-carboxylic acid (A-9-C) but not by bumetanide and sulfate. Neither bromosulfophthalein nor the fatty acid oleate inhibited glisoxepide or glibenclamide uptake. These results are consistent with the transport of glisoxepide via the transport system for the unconjugated bile acid cholate. Glibenclamide uptake is mediated by a still unknown hepatocellular transport system.     

The effect of pregnancy and treatment with 17 beta-estradiol on the transport of organic anions into isolated rat hepatocytes

The uptake of the bile acid taurocholate (TC), and the organic anions, estradiol-17 beta (beta-D-glucuronide) (E217G), estradiol-3-(beta-D-glucuronide) (E23G), estriol-16 alpha (beta-D-glucuronide) (E316G), and morphine glucuronide (MG) were evaluated in hepatocytes isolated from nonpregnant female, pregnant (19-21 days of gestation) and E2-treated (1 mg/kg/day sc for 14 days) rats. Pregnancy significantly decreased the uptake of TC, E217G, E23G, and MG whereas E2 treatment decreased only the uptake of E217G. The Vmax (nmol/min/mg protein) for E217G uptake was significantly decreased from 1.45 +/- 0.2 (mean +/- SE) in hepatocytes from nonpregnant female rats to 0.70 +/- 0.11 and 0.64 +/- 0.13 in cells from pregnant and E2-treated rats, respectively. The Vmax for uptake of TC was decreased, but not significantly, from 0.56 +/- 0.16 in hepatocytes from nonpregnant female rats to 0.34 +/- 0.08 in cells from pregnant rats.     

Mirex exposure inhibits the uptake of estradiol-17 beta(beta-D-glucuronide), taurocholate, and L-alanine into isolated rat hepatocytes.

The insecticides mirex and chlordecone have previously been found to suppress the biliary excretion of a wide variety of compounds. In the present studies, the effects of mirex, chlordecone, and phenobarbital on the uptake of two endogenous organic anions, estradiol-17 beta(beta-D-glucuronide) (E217G), an estrogen metabolite, taurocholate (TC), a common bile acid, and an essential amino acid, L-alanine (L-Ala) (0.5 mM), into isolated rat hepatocytes was investigated. Female Sprague-Dawley rats were orally dosed with mirex (12.5, 25, and 50 mg/kg) or chlordecone (6.25, 12.5, and 18.75 mg/kg) dissolved in corn oil for 3 days and isolated rat hepatocytes were prepared 2 days later. Rats were also dosed orally with phenobarbital (50 mg/kg on the first day and 80 mg/kg for the next 4 days) dissolved in distilled deionized water, and isolated hepatocytes were prepared on the sixth day. Mirex significantly reduced the uptake of both organic anions (0.5, 10, and 50 microM E2 17G; 10 microM TC) into hepatocytes by 40-70%, whereas chlordecone had no effect on their uptake. Mirex at 50 mg/kg significantly reduced the Vmax for the low- and high-affinity E217G uptake sites by 70% and decreased the Km for the low affinity uptake site by 60%. Mirex also significantly decreased the Vmax for TC uptake from 1.11 to 0.82 nmol/min/mg protein but had no effect on its Km (23.2 vs 22.9 microM). Mirex at 50 mg/kg was also found to reduce the uptake of 0.5 mM L-Ala by nearly 40%. Phenobarbital had no effect on the uptake of E217G (0.5 microM), TC (10 microM), or L-Ala (0.5 mM). Mirex treatment had no effect on hepatic plasma membrane Na+,K(+)- or Mg2(+)-ATPase activity. Neither mirex nor chlordecone at 50-100 microM had any effect on the uptake of 10 microM TC when added directly to hepatocytes from naive rats. These results indicate that mirex decreases the transport of organic anions and L-Ala across the basolateral domain of the hepatocyte in addition to its inhibitory effects on biliary excretion.     

Functional analysis of mouse and monkey multidrug resistance-associated protein 2 (Mrp2).

We investigated the intrinsic transport activity of mouse and monkey Mrp2 and compared it with that of rat and dog Mrp2 reported previously. Mrp2 cDNAs were isolated from BALB/c and Macaca fascicularis liver, respectively, and vesicle transport studies were performed using recombinant Mrp2s expressed in insect Sf9 cells. ATP-dependent transport of [3H]leukotriene C4 (LTC4), [3H]17beta-estradiol 17-(beta-D-glucuronide) (E217betaG), [3H]bromosulfophthalein (BSP), and [3H]cholecystokinin octapeptide (CCK-8) were readily detected for all Mrp2s. A species difference in the intrinsic transport activity was apparent for LTC4 (monkey > mouse, dog > rat) and BSP (rat, dog, monkey > mouse). In addition to the difference in the transport activity, complex kinetic profiles were also evident in CCK-8, where a cooperative transport site was observed. Moreover, the transport of [3H]E217betaG by mouse and monkey Mrp2 was quite different from that of rat and dog Mrp2 in that 1) there was practically only nonsaturable uptake for [3H]E217betaG and 2) 4-methylumbelliferon glucuronide (Mrp2 modulator) showed a concentration-dependent stimulatory effect on the transport of [3H]E217betaG in mouse and monkey Mrp2, whereas rat and dog transport activity was inhibited by the modulator. In conclusion, although the substrate specificity is similar, the intrinsic transport activity differs from one species to another. This is due not only to the difference in the Km and Vmax values, but also the qualitatively different mode of substrate and modulator recognition exhibited by different species.     

Characterization of [3H]estradiol-17 beta-(beta-D-glucuronide) binding sites in basolateral and canalicular liver plasma membranes

The specific binding of [3H]estradiol-17 beta-(beta-D-glucuronide) ([3H]E217G) was examined in isolated basolateral (bLPM) and canalicular (cLPM) liver plasma membranes. Two distinct binding sites were identified in each membrane fraction by competition and saturation experiments. Binding parameters obtained from competition studies were: Kd1 = 26 nM, Bmax1 = 0.26 pmol/mg protein; Kd2 = 2.6 microM, Bmax2 = 27 pmol/mg protein for bLPM; and Kd1 = 81 nM, Bmax1 = 0.61 pmol/mg protein; Kd2 = 6.7 microM, Bmax2 = 79 pmol/mg protein for cLPM. Binding parameters obtained from saturation experiments were not significantly different. There was no Na+ requirement for binding. Kinetic dissociation experiments showed that binding was reversible and revealed two components. The dissociation rate constants did not vary with the method of dilution of radioligand, i.e. by "infinite" volume, or excess unlabeled ligand, thus ruling out the possibility of cooperativity. The ability of a series of compounds to inhibit the binding of [3H]E217G was also examined. In bLPM, taurocholate (TC), estrone sulfate (E1SO4) and bromosulfophthalein (BSP) were able to compete with both binding sites, whereas estriol-17 beta-(beta-D-glucuronide) (E317G), estriol-16 alpha-(beta-D-glucuronide) (E316G), testosterone glucuronide (TG), estradiol-3-(beta-D-glucuronide) (E23G), estriol-3-(beta-D-glucuronide) (E(3)3G), cholate and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) were able to inhibit binding to only the low-affinity site. In cLPM, only the cholestatic steroid D-ring glucuronides (E(3)17G, E(3)16G and TG) and TC were able to compete with both sites, whereas the non-cholestatic steroid A-ring glucuronides (E(2)3G and E(3)3G), BSP and DIDS competed for only the low-affinity site. Based on the observed substrate specificities, the low-affinity sites in bLPM and cLPM are postulated to represent multispecific organic anion carriers. The high-affinity site in cLPM may play a role in mediating steroid D-ring glucuronide-induced cholestasis.     

Antagonistic actions of renal dopamine and 5-hydroxytryptamine: effects of amine precursors on the cell inward transfer and decarboxylation.

1. The present work was designed to examine the interference of L-3,4-dihydroxyphenylalanine (L-DOPA) on the cell inward transport of L-5-hydroxytryptophan (L-5-HTP) and on its decarboxylation by aromatic L-amino acid decarboxylase (AAAD) in rat isolated renal tubules. 2. The accumulation of both L-5-HTP and L-DOPA in renal tubules was found to occur through non-saturable and saturable mechanisms. The kinetics of the saturable component L-5-HTP and L-DOPA uptake in renal tubules were as follows: L-5-HTP, Vmax = 24.9 +/- 4.5 nmol mg-1 protein h-1 and Km = 121 (95% confidence limits: 75, 193) microM (n = 5); L-DOPA, Vmax = 58.0 +/- 4.3 nmol mg-1 protein h-1 and Km = 135 (97, 188) microM (n = 5). When the saturation curve of L-5-HTP tubular uptake was performed in the presence of L-DOPA (250 microM), the maximal rate of accumulation of L-5-HTP in renal tubules was found to be markedly (P < 0.01) reduced (Vmax = 10.5 +/- 1.7 nmol mg-1 protein h-1, n = 4); this was accompanied by a significant (P < 0.05) increase in Km values (325 [199, 531] microM, n = 4). 3. L-DOPA (50 to 2000 microM) was found to produce a concentration-dependent decrease (38% to 91% reduction) in the tubular uptake of 5-HTP; the Ki value (in microM) of L-DOPA for inhibition of L-5-HTP uptake was found to be 29.1 (13.8, 61.5) (n = 6). 4. At the highest concentration tested the organic anion inhibitor, probenecid (10 microM) produced no significant (P = 0.09) changes in L-5-HTP and L-DOPA uptake (18% and 22% reduction, respectively). The organic cation inhibitor, cyanine 863 (1-ethyl-2-[1,4-dimethyl-2-phenyl-6-pyrimidinylidene)methyl]-quino linium) produced a potent inhibitory effect on the tubular uptake of L-5-HTP (Ki = 212 [35, 1289] nM, n = 8), being slightly less effective against L-DOPA uptake (Ki = 903 [584, 1396] nM, n = 5). The cyanine derivatives 1,1-diethyl-2,4-cyanine (decynium 24) and 1,1-diethyl-2,2-cyanine (decynium 22) potently inhibited the tubular uptake of both L-5-HTP (Ki = 100 [49, 204] and 120 [26, 561] nM, n = 4-6, respectively) and L-DOPA (Ki = 100 [40, 290] and 415 [157, 1094] nM, n = 5, respectively). 5. The Vmax and Km values for AAAD using L-DOPA as the substrate (Vmax = 479.9 +/- 74.0 nmol mg-1 protein h-1; Km = 2380 [1630, 3476] microM; n = 4) were both found to be significantly (P < 0.01) higher than those observed when using L-5-HTP (Vmax = 81.4 +/- 5.2 nmol mg-1 protein h-1, Km = 97 [87, 107] microM, n = 10). The addition of 5 mM L-DOPA to the incubation medium reduced by 30% (P < 0.02) the maximal rate of decarboxylation of L-5-HTP (Vmax = 56.7 +/- 3.1 nmol mg-1 protein h-1, n = 10) and resulted in a significant (P < 0.05) increase in Km values (249 [228, 270] microM, n = 10). 6. The results presented suggest that L-5-HTP and L-DOPA are using the same transporter (most probably, the organic cation transporter) in order to be taken up into renal tubular cells; L-DOPA exerts a competitive type of inhibition upon the tubular uptake and decarboxylation of L-5-HTP. The decrease in the formation of 5-HT as induced by L-DOPA may also depend on a decrease in the rate of its decarboxylation by AAAD.     

Comparison of the uptake of [3H]-gabapentin with the uptake of L-[3H]-leucine into rat brain synaptosomes.

1. Gabapentin is a novel anticonvulsant with an unknown mechanism of action. Homogenate binding studies described elsewhere have suggested that [3H]-gabapentin binds to a site in brain similar to the large neutral amino acid (LNAA) uptake site, termed system-L. 2. This study describes an investigation into the uptake of [3H]-gabapentin into a crude synaptosomal preparation from cerebral cortex of rat brain. Characterization studies showed that [3H]-gabapentin is taken up into synaptosomes by a system that is similar to that responsible for the uptake of L-[3H]-leucine. This system is sodium-independent, temperature-sensitive and requires ATP for function. 3. Kinetic studies of [3H]-gabapentin uptake produced a Michaelis constant (KM = 160 microM) similar to that observed for L-[3H]-leucine (KM = 110.3 microM). Vmax values were 837.1 pmol mg-1 protein min-1 and 2.192 nmol mg-1 protein min-1 respectively. 4. Gabapentin and L-leucine mutually inhibit their uptake. Lineweaver-Burke plots of these data demonstrate that inhibition occurs by a competitive mechanism. Further to this the Dixon transformation of the data illustrates that these two substrates share a common uptake site by the similarity between their calculated Ki and KM values (gabapentin inhibition of L-[3H]-leucine uptake: Ki = 160 microM; L-leucine inhibition of [3H]-gabapentin uptake: Ki = 262 microM). 5. Studies into the effect of gabapentin, the system-L-specific ligand 2-(-)-endoamino-bicycloheptane-2-carboxylic acid (BCH), and the system-A-specific ligand alpha-(methyl-amino)-isobutyric acid (MeAIB), on the initial rate of uptake of [3H]-glycine, L-[3H]-glutamate, L-[3H]-glutamine, and L-[3H]-leucine were performed. At 100 microM, gabapentin significantly inhibited initial rate of uptake of [3H]-glycine (29%), L-[3H]-glutamate (22%) and L-[3H]-leucine (40%). 6. Gabapentin is taken up into synaptosomes by a system similar to system-L, responsible for the uptake of large neutral amino acids. Gabapentin will also inhibit the uptake of certain excitatory amino acids in this synaptosomal preparation. The implications of these findings for the mechanism of action for gabapentin are unclear. The data presented here may suggest an intracellular site for mechanism of action for this compound. Similarly changes in levels of amino acid pools may be involved in the mechanism of gabapentin's anticonvulsant action.     

Characterization of the bile acid sensitive methotrexate carrier of rat liver cells

The chemotherapeutic agent methotrexate is widely used in tumor therapy for different forms of leukemia and for the therapy of arthritis. Methotrexate is eliminated from systemic blood circulation by the liver and its transport into hepatocytes is therefore described in detail in this paper. Methotrexate uptake is energy- and sodium-dependent. The K _m and the V _max are 23 μM and 36 pmol/mg protein min, respectively. The apparent activation energy (E _app) of methotrexate uptake (5 μM [³H]methotrexate) is 53.73 kJ/mol, which indicates an energy-dependent carrier-mediated process. Although methotrexate is a folate derivative, folate itself does not inhibit methotrexate uptake, whereas the reduced folates, dihydrofolate and tetrahydro-folate are weak uncompetitive inhibitors. In contrast, the bile acids taurocholate and cholate are effective competitive inhibitors of methotrexate uptake into hepatocytes. Further strong inhibitors are the loop diuretic bumetanide, the mycotoxin ochratoxin A and bromosulfophthalein. Because tumor patients develop drug resistance during methotrexate therapy, the uptake of methotrexate was tested in different hepatoma cell lines. In HepG2-cells and Reuber hepatoma Fao-cells the transport was non-existent or very small. However, the hepatocytoma fusion cell line HPCT-1E3, a hybrid cell line between primary rat hepatocytes and rat Reuber Fao-cells, shows an intermediate transport activity with a threefold increase of the methotrexate uptake. These results indicate the presence of a bile acid sensitive methotrexate carrier in hepatocytes which is absent in dedifferentiated hepatoma cells. The carrier differs from previously described transporters for the uptake of organic anions. Received: 26 October 1998 / Accepted: 25 January 1999     

Evidence for a saturable, energy-dependent and carrier-mediated uptake of oral antidiabetics into rat hepatocytes.

The hepatic uptake of two sulfonylureas, glisoxepide and glibenclamide, was investigated in isolated rat hepatocytes. Two transport processes were defined: passive physical diffusion and saturable carrier transport. For diffusion at pH 7.4 the permeability coefficients were 3.3 x 10(-6) cm/s for glisoxepide and 10.6 x 10(-6) cm/s for glibenclamide. Saturable uptake differed among the sulfonylureas. Glibenclamide uptake was neither energy- nor sodium-dependent and temperature dependence was linear. The apparent activation energy for saturable glibenclamide uptake was 15.2 kJ/mol and Q10 values for uptake between 7 and 37 degrees C were 1.17 +/- 0.12. Saturable glibenclamide uptake exhibited a Km = 3.1 microM and a Vmax = 416 pmol/mg cell protein per min. Thus glibenclamide uptake was defined kinetically as a facilitated diffusion process. Glisoxepide uptake revealed two Km values: Km1 = 2-3 microM and Vmax1 = 200 pmol/mg protein per min, and Km2 = 110 microM and Vmax2 = 1600 pmol/mg protein per min. Uptake at low and high substrate concentration was energy-dependent, sodium-dependent and was inhibited by ouabain. Temperature dependence increased markedly beyond 22 degrees C and the apparent activation energy was 59.7 kJ/mol at low Km1 glisoxepide concentrations and 60.3 kJ/mol at high Km2 concentrations. Whereas glisoxepide was slowly taken up into AS-30D hepatoma cells, glibenclamide was not. The hepatic uptake of glibenclamide was not inhibited by glisoxepide but glibenclamide inhibited glisoxepide uptake. The inhibition by glibenclamide was noncompetitive. Isolated hepatocytes accumulated the sulfonylureas markedly and metabolized both. The metabolized radioligands were slowly released into the incubation buffer. The results indicate that the hepatic uptake of the two sulfonylureas is by carrier-mediated transport. The uptake processes are, however, strikingly different, indicating heterogeneity of sulfonylurea transporters.     

Metabolism of harmol and transport of harmol conjugates in isolated rat hepatocytes

The conjugation of harmol and the disposition of harmol metabolites by isolated rat hepatocytes were investigated. Harmol sulfation was saturated at very low concentrations and exhibited a maximum velocity of 1.2 +/- 0.2 nmol/min/10(6) hepatocytes. Glucuronidation of harmol proceeded with a Km of 17 +/- 5.7 microM and a maximal velocity of 2.1 +/- 0.3 nmol/min/10(6) hepatocytes. After synthesis, harmol glucuronide and harmol sulfate were distributed between cells and incubation media, and at equilibrium, a large concentration gradient between cells and media existed for both conjugates. Experiments with preformed metabolites indicated that hepatocytes removed harmol glucuronide from the incubation media by single Michaelis-Menten process with a Km of 384 +/- 63 microM and Vmax of 1.8 +/- 0.3 nmol/min/10(6) hepatocytes. Harmol sulfate was taken up by two Michaelis-Menten processes, a high affinity process with a Km of 33 +/- 8 microM and a Vmax of 0.97 +/- 0.2 nmol/min/10(6) cells and a low affinity process with a Km of 452 +/- 71 microM and Vmax of 25.2 +/- 4.1/min/10(6) hepatocytes. Harmol sulfate was released from hepatocytes by a process which did not exhibit saturation. Hepatocytes converted preformed harmol glucuronide to harmol sulfate.     

Deuterium isotope effect on the metabolism of N-nitrosodimethylamine and related compounds by cytochrome P4502E1.

1. Deuteration of N-nitrosodimethylamine (NDMA) decreases its carcinogenicity, and produces an isotope effect on its metabolism in vivo. Consistent with these results are the observations that deuteration caused a 5-fold increase in the apparent Km, but not the Vmax for the demethylation and denitrozation of NDMA in acetone-induced rat liver microsomes. These microsomes are a good source of cytochrome P4502E1. 2. For demethylation of Z-[2H3]NDMA and E-[2H3]NDMA, the Km values were indistinguishable, and were between the values for those of NDMA and [2H6]NDMA. Almost all the formaldehyde formed was derived from the non-deuterated methyl group, indicating a lack of stereoselectivity in the demethylation of NDMA. 3. NDMA and [2H6]NDMA displayed apparent Ki values of 59 and 441 microM, respectively, for N-nitrosodiethylamine deethylase, showing an apparent isotope effect of 0.13, and displayed an isotope effect of 0.21 in the Ki values for p-nitrophenol hydroxylase. 4. With acetone and deuterated acetone as inhibitors for p-nitrophenol hydroxylase, the isotope effect on the Ki was 0.11. Similar deuterium isotope effects were also observed with acetone and dimethylformamide as competitive inhibitors for NDMA demethylase. 5. In the microsomal oxidation of ethanol, a deuterium isotope effect of about five was observed in the Vmax/Km when carbon-1 was deuterated, but was not observed in the Vmax. 6. Results illustrate a unique deuterium isotope effect on the Km values of reactions catalysed by P4502E1.     

Hepatic clearance and biliary secretory rate maximum of taurocholate in the recirculating and single pass isolated perfused rat liver. Effects of the cholestatic agent, estradiol-17 beta-(beta-D-glucuronide)

The ability of the cholestatic steroid glucuronide, estradiol-17 beta-(beta-D-glucuronide) (E(2)17G), to inhibit the hepatic clearance (ClH) and biliary secretory rate maximum (SRm) of taurocholate was investigated in the recirculating and single pass isolated perfused male rat liver. In the recirculating perfused liver, E(2)17G (0, 2, 4, or 6 mumol) was added as a bolus dose to the reservoir at zero time while taurocholate was infused into the portal vein in increasing amounts (15, 30, 45, or 60 mumol/mL; 1 mL/hr for 15 min each). E(2)17G (4 mumol) caused a significant (P less than 0.05) inhibition of bile flow and bile acid secretion at 10-15 min during infusion of 15 mumol/hr taurocholate but did not inhibit the SRm which occurred at 42 min, indicating that E(2)17G had not caused an irreversible inhibition of taurocholate transport. E(2)17G (6 mumol) caused a profound and irreversible inhibition of bile flow attributable to retention of E(2)17G in the liver. The noncholestatic estradiol-3-(beta-D-glucuronide) (E(2)3G; 6 mumol) had no significant effect on bile flow or the SRm. In the single pass perfused liver (10 mL/min flow rate), E(2)17G (0, 1, 2, 5, or 10 nmol/mL) or E(2)3G (2 nmol/mL) was added to the perfusate resulting in a stable infusion to the liver. [3H]Taurocholate was infused into the portal vein in increasing amounts to give inflow concentrations (Cin) of 25, 50, 75 or 100 nmol/mL. In the absence of E(2)17G, taurocholate ClH decreased from 0.92 to 0.70 mL/min/g liver with increasing taurocholate concentrations. Neither E(2)17G nor E(2)3G altered the ClH of 25 nmol/mL taurocholate. E(2)17G (10 nmol/mL) inhibited bile flow and bile acid secretion first at 20-25 min, followed by inhibition of ClH of 75 and 100 nmol/mL taurocholate (35-60 min). In contrast, E(2)3G stimulated bile acid secretion and increased the SRm by 80%. Thus, at doses that did not block its own elimination, E(2)17G did not cause an irreversible inhibition of taurocholate transport into bile. E(2)17G did not directly inhibit the uptake of taurocholate into the liver but first inhibited the biliary excretion of taurocholate, resulting in its intrahepatic accumulation and decreased clearance from the perfusate.     

High and low affinity binding of [3H]cholate to rat liver plasma membranes

The transport of bile acids across sinusoidal and canalicular membranes of hepatocytes is characterized as carrier mediated. Such a carrier should specifically bind bile acids at physiological concentrations. We examined the binding of [3H]cholate to rat liver plasma membranes using a microcentrifugation technique and detected high (KD = 1.23 +/- 0.44 microM, Bmax = 21.8 +/- 3.3 pmol/mg protein) and low (KD = 1.97 +/- 1.33 mM, Bmax = 41.5 +/- 25.3 nmol/mg protein) affinity binding sites. Maximal binding was achieved within 15-45 sec and was stable for 2 min at 37 degrees. Binding to the high affinity site was reversible, was not Na+ dependent or attributable to vesicular uptake, and exhibited a broad pH optimum. Binding to this site was negligible or not detected in liver mitochondrial and microsomal fractions, was saturable, and was inhibited by other bile acids. The IC50 values for bile acids as inhibitors of [3H]cholate binding at the high affinity site were: taurocholate, 1.9 nM; glycodeoxycholate, 3.1 nM; chenodeoxycholate, 5.6 nM; taurochenodeoxycholate, 7.3 nM; glycochenodeoxycholate, 11 nM; lithocholate, 13 nM; taurodeoxycholate, 20 nM; glycocholate, 3.6 microM; and deoxycholate, 5.6 microM. [3H]Cholate specific binding was inhibited by 10(-5) M bromosulfophthalein, bilirubin and indocyanin green. These data support the hypothesis that the high affinity binding site represents a carrier which is shared by bile acids and nonbile acid organic anions.     

A micromethod for the determination of the activities of kininases in rat plasma. Kinetics and inhibitory characteristics.

Kininase II (EC 3.4.15.1) (KII) and kininase I (KI) (EC 3.4.12.7) activities of rat plasma were characterized by the hydrolysis of hippuryl-L-histidyl-L-leucine (HHL), hippuryl-L-arginine (HLA) [expressed as carboxypeptidase N1 (CN1) activity] and hippuryl-L-lysine (HLL) [expressed as carboxypeptidase N2 (CN2) activity]. Using a spectrophotometric assay, biochemical characteristics of the three enzymes were investigated. The Michaelis-Menten constants were as follows: KII: Km 2.55 +/- 0.22 mM, Vmax 0.357 +/- 0.017 mumol/min/mL; CN1: Km 6.93 +/- 0.32 mM, Vmax 0.748 +/- 0.019 mumol/min/mL; and CN2: Km 35.8 +/- 1.52 mM, Vmax 13.11 +/- 0.40 mumol/min/mL. EDTA and O-phenanthroline inhibited the three enzyme assays at the same Ki, whereas captopril and 2-mercaptomethyl-3-guanidinoethylthiopropanoic acid (MERGETPA), allowed for the demonstration of the specificity of each assay. Furthermore, Ki values of MERGETPA against both CN1 (4.75 microM) and CN2 (2.36 microM) activities do not support the hypothesis that KI activity may be accounted for by the presence of isoenzymes in rat plasma.     

Estradiol 3-glucuronide is transported by the multidrug resistance-associated protein 2 but does not activate the allosteric site bound by estradiol 17-glucuronide.

beta-estradiol 17-(beta-D-glucuronide) (E217G) is a well known cholestatic agent and substrate of multidrug resistance-associated protein 2 (Mrp2), whereas beta-estradiol 3-(beta-D-glucuronide) (E23G) is a noncholestatic regioisomer of E217G with unknown transport properties. The purpose of this study was to compare and contrast the Mrp2-mediated transport of E217G and E23G. The full coding region of rat Mrp2 was cloned into the baculovirus genome, the recombinant baculovirus used to infect Sf9 cells, and ATP-dependent transport of 3H-E23G and 3H-E217G in Sf9 cell membranes was characterized. Mrp2 transported E23G into an osmotically sensitive space, requiring ATP, with S50=55.7 microM, Vmax=326 pmol.mg(-1).min(-1), and a Hill coefficient of 0.88. ATP-dependent Mrp2-mediated E217G transport was markedly stimulated at high E217G concentrations, consistent with positive cooperativity (Hill coefficient 1.5). E217G (5-125 microM) increased S50 but not Vmax for E23G transport, consistent with competitive inhibition. E23G (0.4-400 microM) completely, potently (IC50=14.2 microM), and competitively inhibited E217G transport, but E217G (0.01-250 microM) inhibited only 53% of E23G transport (IC50=33.4 microM). Estriol 16alpha-(beta-D-glucuronide) potently and completely inhibited transport of E23G (IC50=2.23 microM), as did beta-estradiol 3-sulfate 17-(beta-D-glucuronide) (5-50 microM). In summary, E217G binds not only to an Mrp2 transport site, but also to an allosteric site that activates Mrp2 with positive cooperativity, thus activating its own transport and potentially that of other Mrp2 substrates, such as E23G. The noncholestatic E23G is an Mrp2 substrate and competes with E217G for transport, but does not activate the allosteric site.     

Donepezil, tacrine and α-phenyl-n-tert-butyl nitrone (PBN) inhibit choline transport by conditionally immortalized rat brain capillary endothelial cell lines (TR-BBB)

In the present study, we have characterized the choline transport system and examined the influence of various amine drugs on the choline transporter using a conditionally immortalized rat brain capillary endothelial cell line (TR-BBB) in vitro. The cell-to-medium (C/M) ratio of [3H]choline in TR-BBB cells increased time-dependently. The initial uptake rate of [3H]choline was concentration-dependent with a Michaelis-Menten value, Km, of 26.2 +/- 2.7 microM. The [3H]choline uptake into TR-BBB was Na+-independent, but was membrane potential-dependent. The [3H]choline uptake was susceptible to inhibition by hemicholinium-3, and tetraethylammonium (TEA), which are organic cation transporter substrates. Also, the uptake of [3H]choline was competitively inhibited with Ki values of 274 microM, 251 microM and 180 microM in the presence of donepezil hydrochloride, tacrine and alpha-phenyl-n-tert-butyl nitrone (PBN), respectively. These characteristics of choline transport are consistent with those of the organic cation transporter (OCT). OCT2 mRNA was expressed in TR-BBB cells, while the expression of OCT3 or choline transporter (CHT) was not detected. Accordingly, these results suggest that OCT2 is a candidate for choline transport at the BBB and may influence the BBB permeability of amine drugs.     

Inhibition of the intestinal absorption of bile acids using cationic derivatives: mechanism and repercussions

To pharmacologically interrupt bile acid enterohepatic circulation, two compounds named BAPA-3 and BAPA-6, with a steroid structure and 1 or 2 positive charges, were obtained by conjugation of N-(3-aminopropyl)-1,3-propanediamine with one or two moieties of glycocholic acid (GC). Both BAPA-3 and BAPA-6 inhibited Na+-dependent taurocholate (TC) uptake by Xenopus laevis oocytes expressing rat Asbt, with Ki values of 28 and 16 microM, respectively. BAPA-3 reduced Vmax without affecting Km. In contrast, BAPA-6 increased Km, with no effect on Vmax. Uptake of [14C]-GC by the last 10 cm of the rat ileum, perfused in situ over 60 min, was inhibited to a similar extent by unlabeled GC, BAPA-3 and BAPA-6. However, the intestinal absorption of these compounds was lower (BAPA-6) or much lower (BAPA-3) than that of GC. When administered orally to mice, both compounds (BAPA-3>BAPA-6) reduced the bile acid pool size, which was accompanied by up-regulation of hepatic Cyp7a1 and Hmgcr and intestinal Ostalpha/Ostbeta. A tendency towards a decreased expression of hepatic Ntcp and an enhanced expression of intestinal Asbt was also observed. Serum biochemical parameters were not affected by treatment with these compounds, except for a moderate increase in serum triglyceride concentrations. In sum, our results suggest that these compounds, in particular BAPA-3, are potentially useful tools for inhibiting the intestinal absorption of bile acids in a non-competitive manner.     

Inhibition of [3H]D-aspartate release by deramciclane.

The effects of the 5-HT2C receptor inverse agonist deramciclane on the gamma-aminobutyric acid (GABA) uptake and excitatory amino acid release processes were compared in rat cerebrocortical homogenates containing resealed plasmalemma fragments and nerve endings. Deramciclane non-competitively inhibited the uptake of [3H]GABA with a Ki value of 13.7 +/- 0.5 microM and partially displaced specifically bound [3H](R,S)-N-[4,4-bis(3-methyl-2-thienyl)-3-butenyl]nipecotic acid ([3H]NNC-328) with high affinity (IC50 = 2.0 +/- 0.7 nM). Depolarization by 4-aminopyridine or by 4-aminopyridine with (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate [(S)-AMPA] induced the release of [3H]D-aspartate. Deramciclane (10 microM) partially (approximately 50%) inhibited the release of [3H]D-aspartate without affecting [3H]D-aspartate uptake. These results suggest a role for presynaptic inhibition of excitatory amino acid release and GABA uptake in the anxiolytic properties of deramciclane.     

In rat alveolar macrophages lipopolysaccharides exert divergent effects on the transport of the cationic amino acids l-arginine and l-ornithine

In rat alveolar macrophages (AMphi) it was tested whether induction of iNOS by lipopolysaccharides (LPS) is accompanied by changes in L-arginine transport and whether L-ornithine, the product of arginase released from AMphi, could, via inhibition of L-arginine uptake, act as a paracrine inhibitor of NO synthesis. Rat AMphi (cultured for 20 h in the absence or presence of 1 microg/ml LPS) were incubated in Krebs-HEPES solution containing [3H]-L-arginine (0.1 microM for 2 min or 100 microM for 5 min) and the cellular radioactivity was determined as a measure of L-arginine uptake. In parallel, cells were incubated for 6 h in Krebs-HEPES solution containing 0-1 mM L-arginine and nitrite accumulation was determined. [3H]-L-arginine uptake (0.1 microM or 100 microM) occurred independently of sodium ions and was inhibited by L-ornithine (EC50: 117 and 562 microM, respectively) and with similar potencies by L-lysine. In LPS-treated AMphi the concentration inhibition curve of L-ornithine was shifted to the right by about a factor of 4, whereas that of L-lysine was only marginally shifted to the right. L-Leucine (0.1 and 1 mM) inhibited [3H]-L-arginine (0.1 microM) by 43 and 58%, respectively, and the effect of 0.1 mM L-leucine was partially sodium dependent. In LPS-treated AMphi, 0.1 mM L-leucine no longer inhibited [3H]-L-arginine and the effect of 1 mM L-leucine was attenuated. Kinetic analysis of the transport of [3H]-L-arginine and [14C]-L-ornithine revealed two components for each amino acid with Km values of 21 and 114 microM (L-arginine) and 39 and 1050 microM (L-ornithine), respectively. After LPS treatment Km2 of L-arginine transport was reduced to 63 microM and Vmax of both components was increased, whereas Km2 of L-ornithine transport was enhanced to 1392 microM and Vmax1 reduced. LPS-stimulated AMphi, incubated in amino acid-free Krebs-HEPES solution, produced about 4 nmol nitrite/10(6) cells per 6 h, and L-arginine enhanced nitrite accumulation maximally about threefold (EC50: 30 microM). L-ornithine, up to 3 mM, failed to affect significantly nitrite accumulation observed in the presence of 30 or 100 microM L-arginine. Rat AMphi express mRNA for two cationic amino acid transporters (CAT-1 and CAT-2B), and LPS markedly up-regulated mRNA for CAT-2B in parallel with mRNA for iNOS, but had no effect on that for CAT-1. In conclusion, in rat AMphi LPS up-regulates L-arginine transport and induces changes in the characteristics of the cationic amino acid transport resulting in preferential transport of L-arginine. These effects may be regarded as cellular measures to ensure a high L-arginine supply for iNOS.     

In-vivo and in-vitro evidence of a carrier-mediated efflux transport system for oestrone-3-sulphate across the blood-cerebrospinal fluid barrier

The efflux transport of oestrone-3-sulphate, a steroid hormone sulphate, across the blood-cerebrospinal fluid barrier has been examined following its intracerebroventricular administration. [3H]Oestrone-3-sulphate was eliminated from cerebrospinal fluid (CSF) with an apparent efflux clearance of 205 microL min(-1) per rat. There was 25% of unmetabolized [3H]oestrone-3-sulphate in the plasma 5 min after intracerebroventricular administration, indicating that at least a part of [3H]oestrone-3-sulphate is transported from CSF to the circulating blood across the blood-CSF barrier. This efflux transport was inhibited by co-administration of excess oestrone-3-sulphate (25 mM 10 microL = 0.25 micromol) into rat cerebral ventricle. To characterize the oestrone-3-sulphate transport process, an in-vitro uptake experiment was performed using isolated rat choroid plexus. Oestrone-3-sulphate uptake by isolated rat choroid plexus was found to be a saturable process with a Michaelis-Menten constant (Km) of 18.1 +/- 6.3 microM, and a maximum uptake rate (Vmax) of 48.0 +/- 15.1 pmol min(-1) microL(-1) of tissue. The oestrone-3-sulphate transport process was temperature dependent and was inhibited by metabolic inhibitors such as 2,4-dinitrophenol and rotenone, suggesting an energy dependence. This uptake process was also inhibited by steroid hormone sulphates (1 mM dehydroepiandrosterone sulphate and 1 mM oestrone sulphate), bile acids (1 mM taurocholic acid and 1 mM cholic acid) and organic anions (1 mM sulphobromophthalein and 1 mM phenolsulphonphthalein), whereas 1 mM p-aminohippuric acid, 1 mM p-nitrophenol sulphate, 0.1 mM methotrexate and the cardiac glycoside, 2.5 microM digoxin, had little effect. In conclusion, these results provide evidence that oestrone-3-sulphate is transported from CSF to the circulating blood across the blood-CSF barrier via a carrier-mediated efflux transport system.