Organic Anion Carrier Research Articles

HYPERTROPHIC OSTEOARTHROPATHY IN A PATIENT WITH HETEROZYGOUS MUTATION IN THE SLCO2A1 GENE: A CASE REPORT

Hypertrophic osteoarthropathy (HOA) is a condition characterized by aberrant skin and osseous tissue proliferation in the distal extremities. Mutations in the 15-hydroxyprostaglandin dehydrogenase gene (HPGD) and the soluble carrier organic anion carrier family member 2A1 gene (SLCO2A1) were associated with primary HOA. Secondary HOA, which is also called as 'hypertrophic pulmonary osteoarthropathy' is responsible for 95-97% of cases. Herein, we present a 19-year-old female patient with primary HOA and heterozygous mutation in the SLCO2A1 gene.

Mitochondrial Glutathione in Diabetic Nephropathy.

Although there are many etiologies for diabetic nephropathy (DN), one common characteristic of all cases involves mitochondrial oxidative stress and consequent bioenergetic dysfunction. As the predominant low-molecular-weight, intramitochondrial thiol reductant, the mitochondrial glutathione (mtGSH) pool plays important roles in how this organelle adapts to the chronic hyperglycemia and redox imbalances associated with DN. This review will summarize information about the processes by which this important GSH pool is regulated and how manipulation of these processes can affect mitochondrial and cellular function in the renal proximal tubule. Mitochondria in renal proximal tubular (PT) cells do not appear to synthesize GSH de novo but obtain it by transport from the cytoplasm. Two inner membrane organic anion carriers, the dicarboxylate carrier (DIC; Slc25a10) and 2-oxoglutarate carrier (OGC; Slc25a11) are responsible for this transport. Genetic modulation of DIC or OGC expression in vitro in PT cells from diabetic rats can alter mitochondrial function and susceptibility of renal PT cells to oxidants, with overexpression leading to reversion of bioenergetic conditions to a non-diabetic state and protection of cells from injury. These findings support the mtGSH carriers as potential therapeutic targets to correct the underlying metabolic disturbance in DN.

Bilitranslocase is Involved in the Uptake of Bromosulfophthalein in Rat and Human Liver

Hepatic disposition of bromosulfophthalein (BSP), bilirubin and bile salts partially overlap, as these anions share both uptake and excretion mechanisms. Multiple organic anion transporters mediate hepatic BSP uptake, i.e. members of the SLCO and SLC22 gene families and bilitranslocase (TCDB #2.A.65.1.1). This study aimed at evaluating the relative contribution of bilitranslocase in BSP uptake in precision-cut human and rat liver slices. To this purpose, two different anti-sequence bilitranslocase antibodies were used as specific, functional inhibitors of bilitranslocase. The intact liver physiology was accurately reproduced in this BSP uptake assay, since uptake was strongly temperature-dependentand inhibited by hepatotropic organic anions, such as 50 nM bilirubin, 1 μM nicotinic acid, 2 μM digoxin, 5 μMindocyanine green and 100 μM taurocholate. The bilitranslocase antibodies inhibited BSP uptake both in rat and human liver slices. The combined use of bilitranslocase antibodies and taurocholate caused additive-type inhibition, confirming that bilitranslocase is not a bile salt transporter; by contrast, bilirubin caused no additive-type inhibition. In conclusion this data, indicate the role of the bilirubin transporter bilitranslocase as one of the transporters involved in the uptake of anions like BSP in parallel with other organic anion carriers. Moreover this data indicate the value of precision-cut liver slices for phenotypic drug uptake studies.

Structural Analysis of a Peptide Fragment of Transmembrane Transporter Protein Bilitranslocase

Using a combination of genomic and post-genomic approaches is rapidly altering the number of identified human influx carriers. A transmembrane protein bilitranslocase (TCDB 2.A.65) has long attracted attention because of its function as an organic anion carrier. It has also been identified as a potential membrane transporter for cellular uptake of several drugs and due to its implication in drug uptake, it is extremely important to advance the knowledge about its structure. However, at present, only the primary structure of bilitranslocase is known. In our work, transmembrane subunits of bilitranslocase were predicted by a previously developed chemometrics model and the stability of these polypeptide chains were studied by molecular dynamics (MD) simulation. Furthermore, sodium dodecyl sulfate (SDS) micelles were used as a model of cell membrane and herein we present a high-resolution 3D structure of an 18 amino acid residues long peptide corresponding to the third transmembrane part of bilitranslocase obtained by use of multidimensional NMR spectroscopy. It has been experimentally confirmed that one of the transmembrane segments of bilitranslocase has alpha helical structure with hydrophilic amino acid residues oriented towards one side, thus capable of forming a channel in the membrane.

Mitochondrial glutathione in toxicology and disease of the kidneys

AbstractThe tripeptide glutathione (GSH), comprised of the amino acids l-cysteine, glycine, and l-glutamate, is found in all cells of aerobic organisms and plays numerous, critical roles as an antioxidant and nucleophile in regulating cellular homeostasis and drug metabolism. GSH is synthesized exclusively in the cytoplasm of most cells by two ATP-dependent reactions. Despite this compartmentation, GSH is found in other subcellular compartments, including mitochondria. As the GSH molecule has a net negative charge at physiological pH, it cannot cross cellular membranes by diffusion. Rather, GSH is a substrate for a variety of anion and amino acid transporters. Two organic anion carriers in the inner membrane of renal mitochondria, the dicarboxylate carrier (DIC; Slc25a10) and the 2-oxoglutarate carrier (OGC; Slc25a11), are responsible for most of the transport of GSH from cytoplasm into mitochondrial matrix. Genetic manipulation of DIC and/or OGC expression in renal cell lines demonstrated the ability to produce sustained increases in mitochondrial GSH content, which then protected these cells from cytotoxicity due to several oxidants and mitochondrial toxicants. Several diseases and pathological states are associated with mitochondrial dysfunction and oxidative stress, suggesting that the mitochondrial GSH pool may be a therapeutic target. One such disease that is of particular concern for public health is diabetic nephropathy. Another chronic, pathological state that is associated with bioenergetic and redox changes is compensatory renal hypertrophy that results from reductions in functional renal mass. This review summarizes pathways of mitochondrial GSH transport and discusses studies on its manipulation in toxicological and pathological states.

Indolicidin action on membrane permeability: Carrier mechanism versus pore formation

Indolicidin, a 13-residue cationic peptide with extremely high tryptophan content, exhibits broad-spectrum antimicrobial as well as hemolytic activity. To gain insight into the mechanism of indolicidin action on membrane permeability, liposome leakage induced by this peptide was studied by using various probes with vesicles of different lipid compositions. In liposomes containing negatively charged lipids, indolicidin induced rather unselective permeabilization. By contrast, the peptide appeared to be selective in provoking leakage of neutral, egg phosphatidylcholine (PC) liposomes: it effectively induced the release of negatively charged fluorescent dyes, carboxyfluorescein (CF), calcein and sulforhodamine B, but was unable to induce the leakage of a neutral compound, glucose, and that of positively charged doxorubicin. Moreover, organic anions, such as fatty acids, were found to suppress the indolicidin-induced CF leakage of egg PC liposomes. Based on these results, we concluded that indolicidin facilitates the dye release from uncharged lipid vesicles not by formation of membrane pores as it is generally accepted for the majority of antimicrobial peptides but rather via translocation of dye molecules across the membrane in the form of dye–peptide complexes, i.e. indolicidin operates as an organic anion carrier. This conclusion was supported by observing the formation of complexes between indolicidin and pyrenebutyrate in solution. The indolicidin analog having only one arginine was ineffective in pyrenebutyrate binding and CF transport. The mode of action proposed here for indolicidin can be related to that previously postulated for oligoarginine derivatives which are able to carry organic anions across liposomal and bulk phase membranes [Sakai N. & Matile S. J. Am. Chem. Soc. 2003, 125:14348–14356]. The newly identified mechanism of peptide ionophoric activity in uncharged lipid membranes may be involved in hemolytic action of indolicidin via induction of plasma membrane permeability for important anionic metabolites which disturbs regulation of osmotic balance ultimately leading to erythrocyte membrane rupture.

Mitochondrial glutathione transport: Physiological, pathological and toxicological implications

Although most cellular glutathione (GSH) is in the cytoplasm, a distinctly regulated pool is present in mitochondria. Inasmuch as GSH synthesis is primarily restricted to the cytoplasm, the mitochondrial pool must derive from transport of cytoplasmic GSH across the mitochondrial inner membrane. Early studies in liver mitochondria primarily focused on the relationship between GSH status and membrane permeability and energetics. Because GSH is an anion at physiological pH, this suggested that some of the organic anion carriers present in the inner membrane could function in GSH transport. Indeed, studies by Lash and colleagues in isolated mitochondria from rat kidney showed that most of the transport (>80%) in that tissue could be accounted for by function of the dicarboxylate carrier (DIC, Slc25a10) and the oxoglutarate carrier (OGC, Slc25a11), which mediate electroneutral exchange of dicarboxylates for inorganic phosphate and 2-oxoglutarate for other dicarboxylates, respectively. The identity and function of specific carrier proteins in other tissues is less certain, although the OGC is expressed in heart, liver, and brain and the DIC is expressed in liver and kidney. An additional carrier that transports 2-oxoglutarate, the oxodicarboxylate or oxoadipate carrier (ODC; Slc25a21), has been described in rat and human liver and its expression has a wide tissue distribution, although its potential function in GSH transport has not been investigated. Overexpression of the cDNA for the DIC and OGC in a renal proximal tubule-derived cell line, NRK-52E cells, showed that enhanced carrier expression and activity protects against oxidative stress and chemically induced apoptosis. This has implications for development of novel therapeutic approaches for treatment of human diseases and pathological states. Several conditions, such as alcoholic liver disease, cirrhosis or other chronic biliary obstructive diseases, and diabetic nephropathy, are associated with depletion or oxidation of the mitochondrial GSH pool in liver or kidney.

Role of Organic Anion and Amino Acid Carriers in Transport of Inorganic Mercury in Rat Renal Basolateral Membrane Vesicles: Influence of Compensatory Renal Growth

Susceptibility to renal injury induced by inorganic mercury (Hg(2+)) increases significantly as a result of compensatory renal growth (following reductions of renal mass). We hypothesize that this phenomenon is related in part to increased basolateral uptake of Hg(2+) by proximal tubular cells. To determine the mechanistic roles of various transporters, we studied uptake of Hg(2+), in the form of biologically relevant Hg(2+)-thiol conjugates, using basolateral membrane (BLM) vesicles isolated from the kidney(s) of control and uninephrectomized (NPX) rats. Binding of Hg(2+) to membranes, accounted for 52-86% of total Hg(2+) associated with membrane vesicles exposed to HgCl(2), decreased with increasing concentrations of HgCl(2), and decreased slightly in the presence of sodium ions. Conjugation of Hg(2+) with thiols (glutathione, L-cysteine (Cys), N-acetyl-L-cysteine) reduced binding by more than 50%. Under all conditions, BLM vesicles from NPX rats exhibited a markedly lower proportion of binding. Of the Hg(2+)-thiol conjugates studied, transport of Hg-(Cys)(2) was fastest. Selective inhibition of BLM carriers implicated the involvement of organic anion transporter(s) (Oat1 and/or Oat3; Slc22a6 and Slc22a8), amino acid transporter system ASC (Slc7a10), the dibasic amino acid transporter (Slc3a1), and the sodium-dicarboxylate carrier (SDCT2 or NADC3; Slc13a3). Uptake of each mercuric conjugate, when factored by membrane protein content, was higher in BLM vesicles from uninephrectomized (NPX) rats, with specific increases in transport by the carriers noted above. These results support the hypothesis that compensatory renal growth is associated with increased uptake of Hg(2+) in proximal tubular cells and we have identified specific transporters involved in the process.

Interaction of ochratoxin A with human intestinal Caco-2 cells: possible implication of a multidrug resistance-associated protein (MRP2)

Ochratoxin A (OTA), a nephrotoxic mycotoxin, is absorbed from small intestine and, in plasma, binds to serum albumin. Prolonged half-live results from reabsorption by proximal tubules and enterohepatic circulation. The mechanism whereby OTA crosses intestine was investigated by means of a cell culture system consisting of Caco-2 cells, as in vitro model of human intestinal epithelium. Cytotoxicity assays on proliferating Caco-2 cells showed that 0.4 μM OTA inhibits MTT reduction by 50%. Transepithelial transport and intracellular accumulation of OTA were studied in Caco-2 cells, differentiated in bicameral inserts. At pH 7.4, OTA is transported preferentially in basolateral (BL) to apical (AP) direction, suggesting a net secretion. Conditions closer to in vivo situation in duodenum (AP pH 6.0, BL pH 7.4) increase intracellular accumulation and transepithelial transport. AP to BL transport becomes higher than BL to AP transport, suggesting OTA absorption. Addition of serum albumin in BL compartment further increases OTA absorption across Caco-2 cells and suggests that in vivo OTA transport from serosal to luminal side of enterocytes is prevented, due to its binding to plasma proteins. Competition experiments showed that carrier systems for large neutral amino acids, H +/dipeptides cotransporter, organic anion ( p-aminohippurate) carrier and organic anion transporter (oatp) are not implicated in OTA transport across Caco-2 cells, in contrast to what was reported in kidney and liver. AP and BL transport and intracellular accumulation of OTA are increased in the presence of non specific inhibitors of MRPs (indomethacin, genistein and probenecid) and of 1-chloro-2,4-dinitrobenzene (biotransformed into 2,4-dinitrophenyl-gluthatione, a specific inhibitor of MRPs), but are affected by verapamil, an inhibitor of P-gp. This suggests that the multidrug resistance-associated protein (MRP2) could be implicated in transepithelial transport. Therefore, absorption of OTA across the intestinal mucosa would be limited thanks to its excretion through MRP2 at the apical pole of enterocytes.

Membrane Transport of Folates

The chapter reviews the current understanding of the transport mechanisms for folates in mammalian cells--their molecular identities and organization, tissue expression, regulation, structures, and their kinetic and thermodynamic properties. This encompasses a variety of diverse processes. Best characterized is the reduced folate carrier, a member of the SLC19 family of facilitative carriers. But other facilitative organic anion carriers (SLC21), largely expressed in epithelial tissues, transport folates as well. In addition to these bi-directional carrier systems are the membrane-localized folate receptors alpha and beta, that mediate folate uptake unidirectionally into cells via an endocytotic process. There are also several transporters, typified by the family of multidrug resistance-associated proteins, that unidirectionally export folates from cells. There are transport activities for folates, that function optimally at low pH, related in part to the reduced folate carrier, with at least one activity that is independent of this carrier. The reduced folate carrier-associated low-pH route mediates intestinal folate transport. This review considers how these different transport processes contribute to the generation of transmembrane folate gradients and to vectorial flows of folates across epithelia. The role of folate transporters in mouse development, as assessed by homologous deletion of folate receptors and the reduced folate carrier, is described. Much of the focus is on antifolate cancer chemotherapeutic agents that are often model surrogates for natural folates in transport studies. In particular, antifolate transport mediated by the reduced folate carrier is a major determinant of the activity of, and resistance to, these agents. Finally, many of the key in vitro findings on the properties of antifolate transporters are now beginning to be extended to patient specimens, thus setting the stage for understanding response to these drugs in the clinical setting at the molecular level.

Organic anion transporter 3 (Slc22a8) is a dicarboxylate exchanger indirectly coupled to the Na+ gradient.

Basolateral uptake of organic anions in renal proximal tubule cells is indirectly coupled to the Na(+) gradient through Na(+)-dicarboxylate cotransport and organic anion/dicarboxylate exchange. One member of the organic anion transporter (OAT) family, Oat1, is expressed in the proximal tubule and is an organic anion/dicarboxylate exchanger. However, a second organic anion carrier, Oat3, is also highly expressed in the renal proximal tubule, but its mechanism is unclear. Thus we have assessed Oat3 function in Xenopus laevis oocytes and rat renal cortical slices. Probenecid-sensitive uptake of p-aminohippurate (PAH, an Oat1 and Oat3 substrate) and estrone sulfate (ES, an Oat3 substrate) in rat Oat3-expressing oocytes was significantly trans-stimulated by preloading the oocytes with the dicarboxylate glutarate (GA). GA stimulation of ES transport by oocytes coexpressing rabbit Na(+)-dicarboxylate cotransporter 1 and rat Oat3 was significantly inhibited when the preloading medium contained Li(+) or methylsuccinate (MS) or when Na(+) was absent. All these treatments inhibit the Na(+)-dicarboxylate cotransporter, but not rat Oat3. Li(+), MS, and Na(+) removal had no effect when applied during the ES uptake step, rather than during the GA preloading step. Concentrative ES uptake in rat renal cortical slices was also demonstrated to be probenecid and Na(+) sensitive. Accumulation of ES was stimulated by GA, and this stimulation was completely blocked by probenecid, Li(+), MS, taurocholate, and removal of Na(+). Thus Oat3 functions as an organic anion/dicarboxylate exchanger that couples organic anion uptake indirectly to the Na(+) gradient.

AZT distribution in the fetal and postnatal rat central nervous system

The distribution of 3′‐azido‐3′‐deoxythymidine (AZT, zidovudine), an antiviral drug used in the treatment of human immunodeficiency virus, was investigated in gestation day‐20 (G‐20) fetuses and in postnatal day‐20 (PND‐20) rats. At both ages, a single dose of 150 mg/kg (1.78 mmol/kg) AZT was administered orally along with tracer amounts of 14C‐AZT, and rats were randomly killed at 15, 30, 60, 120, or 240 min after dosing. The fetuses, brains, and spinal cords were processed for autoradiography. The peak concentrations of AZT in plasma of G‐20 and PND‐20 rats were 92.2 μg/mL (0.345 μmol/mL) and 56.6 μg/mL (0.21 μmol/mL) at 15 and 30 min after intubation, respectively. The peak concentration of fetal tissue occurred in the colon at 60 min and was 205.8 μg/g tissue. In the G‐20 rats, the brain showed higher levels of AZT than spinal cord only at the 30‐min sample time, whereas in the PND‐20 rats, greater radioactivity was found in the spinal cord up to the 240‐min sample time. This pattern of AZT distribution in the central nervous system may hypothetically be attributed to the postnatal development of an organic anion carrier system believed to be responsible for transporting AZT from the brain to the blood, resulting in relatively greater overall exposure of the spinal cord to AZT than observed in the brain. © 2001 Wiley‐Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 90:1964–1971, 2001

Transport of bile acids in hepatic and non-hepatic tissues.

Bile acids are steroidal amphipathic molecules derived from the catabolism of cholesterol. They modulate bile flow and lipid secretion, are essential for the absorption of dietary fats and vitamins, and have been implicated in the regulation of all the key enzymes involved in cholesterol homeostasis. Bile acids recirculate through the liver, bile ducts, small intestine and portal vein to form an enterohepatic circuit. They exist as anions at physiological pH and, consequently, require a carrier for transport across the membranes of the enterohepatic tissues. Individual bile acid carriers have now been cloned from several species. Na+-dependent transporters that mediate uptake into hepatocytes and reabsorption from the intestine and biliary epithelium and an ATP-dependent transporter that pumps bile acids into bile comprise the classes of transporter that are specific for bile acids. In addition, at least four human and five rat genes that code for Na+-independent organic anion carriers with broad multi-substrate specificities that include bile acids have been discovered. Studies concerning the regulation of these carriers have permitted identification of molecular signals that dictate eventual changes in the uptake or excretion of bile acids, which in turn have profound physiological implications. This overview summarizes and compares all known bile acid transporters and highlights findings that have identified diseases linked to molecular defects in these carriers. Recent advances that have fostered a more complete appreciation for the elaborate disposition of bile acids in humans are emphasized.

Inhibition of initial transport rate of basolateral organic anion carrier in renal PT by BK and phenylephrine.

The effect of ligands for phospholipase C-coupled receptors and of protein kinase C (PKC) stimulation with phorbol ester [phorbol 12-myristate 13-acetate (PMA)] or 1,2-dioctanoyl-sn-glycerol on the activity of the basolateral organic anion transporter (OAT) in S2 segments of single, nonperfused rabbit proximal tubules (PT) was measured with the use of fluorescein and epifluorescence microscopy. The initial uptake rate (25 s, OAT activity) was measured in real time by using conditions similar to those found in vivo. Stimulation of PKC with PMA or 1,2-dioctanoyl-sn-glycerol led to an inhibition of OAT activity, which could be prevented by 10(-7) mol/l of the PKC-specific inhibitor bisindolylmaleimide. The alpha(1)-receptor agonist phenylephrine as well as the peptide hormone bradykinin induced a reversible decrease of OAT activity, which was prevented by bisindolylmaleimide. The observed effect was not due to a decrease in the concentration of the counterion alpha-ketoglutarate or to impaired alpha-ketoglutarate recycling, because it was unchanged in the continuous presence of alpha-ketoglutarate or methyl succinate. We conclude that physiological stimuli can inhibit the activity of OAT in rabbit PT via PKC. The effect is not mediated by alterations in counterion availability but by a direct action on the OAT.

Renal cellular transport of exogenous glutathione: heterogeneity at physiological and pharmacological concentrations

Properties and kinetics of GSH transport into proximal tubular (PT) and distal tubular (DT) cells from rat kidney were determined to validate further the hypothesis that cellular differences in handling of GSH contribute to the greater susceptibility of DT cells to oxidant injury. PT and DT cells were incubated with a broad range of GSH concentrations, encompassing physiologically relevant (0.001 to 0.1 mM) to pharmacological (0.25 to 5 mM) levels of GSH. GSH uptake in PT cells was rapid, exhibiting an overshoot with a maximum at 1-min incubation. GSH uptake in DT cells reached maximal intracellular levels at 2- to 5-min incubations. GSH uptake in PT cells was resolved into two kinetically distinct processes, with K m values of 41.7 and 540 μM and V max values of 183 and 4885 pmol/min per 10 6 cells. In contrast, GSH uptake in DT cells was best described by one process, with K m and V max values of 1480 μM and 2094 pmol/min per 10 6 cells, respectively. Rates of GSH synthesis from 1 mM precursor amino acids were approximately 3-fold faster in PT cells, but rates of cysteine accumulation were 3.5-fold faster in DT cells. Accumulation of intracellular GSH in PT cells was 8-fold faster after incubation with 1 mM GSH than after incubation with 1 mM precursor amino acids. At both a physiological (10 μM) and a pharmacological (5 mM) GSH concentration, uptake exhibited marked Na + and energy dependence, sensitivity to substrates for the organic anion and dicarboxylate carriers, and sensitivity to various γ-glutamyl amino acids in PT cells only. Na +-dependent GSH uptake in PT cells was accounted for completely by activity of the organic anion and dicarboxylate carriers. These results indicate that DT cells possess limited capacity to transport GSH and suggest that exogenous GSH may not be effective in protecting other segments of the nephron besides the PT region from oxidants or other agents that alter GSH status.

Bilitranslocase can exist in two metastable forms with different affinities for the substrates--evidence from cysteine and arginine modification.

Bilitranslocase is an organic anion carrier involved in bilirubin and phthalein uptake by the liver. In rat liver plasma membranes, its function is assayed by recording the electrogenic sulfobromophthalein movement. This has been found to be inhibited by both cysteine-specific and arginine-specific reagents. Inhibition is both partial and it occurs to the same extent, i.e. approximately 50%. The effects are not additive. Here we describe the mechanism underlying the above observations. It is concluded that bilitranslocase occurs in two possible states, featured by high and low affinity for the substrates (for sulfobromophthalein, Km = 5 microM and 37 microM, respectively). Cysteine- or arginine-reactive reagents, by reacting selectively with the low-affinity form, entrap it and shift the equilibrium between the two forms, so that, at completion, only the low-affinity form is present. The substrate concentration in the standard transport assay is 39 microM, a value at which the modified low-affinity form operates in the range of half-maximal velocity. This explains both the apparent half-inhibition measured after the chemical treatments and the lack of additivity. In addition, the substrates are shown to enhance the rate of conversion from the low-affinity to the high-affinity form of the translocator, thus favouring its high-affinity form under physiological conditions.

Molecular structure influence in the recognition of phthaleins by the electrogenic organic anion carrier at the sinusoidal plasma membrane level in the liver.

The hepatocytic uptake of cholephilic organic anions occurs by carrier-mediated mechanisms. Electrogenic and electroneutral transport systems have been described. The aim of this study was to determine the dissociation constant (Kd) of the Electrogenic Carrier System(s) (ECS) for tetrabromosulfophthalein (BSP), dibromosulfophthalein (DBSP), tetrabromophthalein (TBP), tetrabromosulfonephthalein (TBS) and thymol blue (TB). Kd (uM) values for ECS-organic anion complexes were: ECS-BSP = 3.61 +/- 0.18; ECS-DBSP = 11.61 +/- 1.32; ECS-TBP = 0.51 +/- 0.08; ECS-TBS = 1.31 +/- 0.25; ECS-TB = 9.44 +/- 1.80. From these data, it is possible to conclude that molecular characteristics of the organic anions are important factors in determining the dissociation constant for the electrogenic hepatic carrier(s). In this sense, the addition of two sulphonic groups on the phenolic ring; the presence of a sulphonic on the benzenic ring and the absence of two or four bromines on the molecule confers a lower affinity for ECS.

Biliary excretion of estrone metabolites in the rat

We previously reported that sulfobromophthalein infusion inhibited the biliary excretion of estradiol glucuronides, whereas dibromosulfophthalein had no effect. In the present study, we examined the biliary excretion of estrone metabolites in rats. Biliary excretion of a tracer dose of intravenously injected [ 3H]estrone to control rats or EHBR and effects of the infusion of sulfobromophthalein and dibromosulfophthalein were studied. Biliary excretion of estrone metabolites was delayed in EHBR. Analysis of the biliary estrone metabolites revealed a marked decrease of the glucuronides in EHBR. Sulfobromophthalein and dibromosulfophthalein infusion (0.2 μmol/min/100 g b.wt.) inhibited the biliary excretion of estrone metabolites. However, the decrease in biliary excretion of the glucuronides was observed only with sulfobromophthalein that is excreted mainly as the glutathione conjugate. These findings indicate that glucuronides of estrone and its metabolites are partly excreted into bile by a canalicular organic anion carrier for sulfobromophthalein-glutathione, but not for dibromosulfophthalein.

Biliary excretion of estradiol-17 beta-glucuronide in the rat

Estradiol-17beta-glucuronide (E217G) is a cholestatic agent and is considered to be related to the pathogenesis of intrahepatic cholestasis of pregnancy. In the current study, we examined the mechanism of the biliary excretion of E217G and estradiol metabolites in rats. Biliary excretion of tracer doses of [3H]estradiol-17beta-glucuronide and [14C]estradiol or [3H]taurocholate and ]14C]vinblastine, a P-glycoprotein (P-GP) substrate, intravenously administered as a bolus to bile-drained control rats or EHBR was studied. Biliary excretion of E217G and estradiol metabolites EHBR was markedly delayed. Analyses of biliary metabolites after estradiol injection showed less polar conjugates in EHBR. In contrast, the excretion of taurocholate and vinblastine (VLB) was only slightly delayed in EHBR. Although phenothiazine treatment to induce the expression of P-GP increased biliary vinblastine excretion, it did not affect biliary excretion of a tracer dose of [3H]estradiol-17beta-glucuronide. However, phenothiazine treatment inhibited the cholestasis induced by E217G infused at the rate of 0.075 micromol/min/100g for 20 minutes and increased biliary E217G excretion. Sulfobromophthalein infusion (0.2 micromol/min/100 g body 0 weight) markedly inhibited the biliary excretion of E217G and estradiol metabolites, whereas dibromosulfophthalein (DBSP) at the same infusion rate had no effect. These findings indicate that EG17G is excreted into bile by a canalicular organic anion carrier for sulfobromopthalein (BSP), not for DBSP, under physiological conditions, and that P-GP influences E217G excretion only at a high dose. under physiological conditions, and that P-GP influence s E217G excretion only at a high dose. (Hepatology 1996 Mar;23(3):607-13)

Biliary excretion of estradiol-17β-glucuronide in the rat

Estradiol-17β-glucuronide (E217G) is a cholestatic agent and is considered to be related to the pathogenesis of intrahepatic cholestasis of pregnancy. In the current study, we examined the mechanism of the biliary excretion of E217G and estradiol metabolites in rats. Biliary excretion of tracer doses of [3H]estradiol-17β-glucuronide and [14C]estradiol or [3H]taurocholate and ]14C]vinblastine, a P-glycoprotein (P-GP) substrate, intravenously administered as a bolus to bile-drained control rats or EHBR was studied. Biliary excretion of E217G and estradiol metabolites EHBR was markedly delayed. Analyses of biliary metabolites after estradiol injection showed less polar conjugates in EHBR. In contrast, the excretion of taurocholate and vinblastine (VLB) was only slightly delayed in EHBR. Although phenothiazine treatment to induce the expression of P-GP increased biliary vinblastine excretion, it did not affect biliary excretion of a tracer dose of [3H]estradiol-17β-glucuronide. However, phenothiazine treatment inhibited the cholestasis induced by E217G infused at the rate of 0.075 μmol/min/100g for 20 minutes and increased biliary E217G excretion. Sulfobromophthalein infusion (0.2 μmol/min/100 g body 0 weight) markedly inhibited the biliary excretion of E217G and estradiol metabolites, whereas dibromosulfophthalein (DBSP) at the same infusion rate had no effect. These findings indicate that EG17G is excreted into bile by a canalicular organic anion carrier for sulfobromopthalein (BSP), not for DBSP, under physiological conditions, and that P-GP influences E217G excretion only at a high dose. under physiological conditions, and that P-GP influence s E217G excretion only at a high dose.