Organic Anion Carrier Research Articles

Estradiol-17β-glucuronide (E 217G) is physiologically excreted into bile by a canalicular organic anion carrier for BSP, not by P-Glycoprotein (P-GP) : [formula omitted], R. Yamazaki, N. Sano, Y. Takamori, S. Tomita, K. Kitaura, M. Yamanaka Department of Medicine, Teikyo University School of Medicine, Tokyo

Estradiol-17β-glucuronide (E 217G) is physiologically excreted into bile by a canalicular organic anion carrier for BSP, not by P-Glycoprotein (P-GP) : [formula omitted], R. Yamazaki, N. Sano, Y. Takamori, S. Tomita, K. Kitaura, M. Yamanaka Department of Medicine, Teikyo University School of Medicine, Tokyo

In vitro and in vivo transport of zidovudine (AZT) across the blood-brain barrier and the effect of transport inhibitors.

The transport of the antiviral nucleoside analogue zidovudine (3'-azido-3'-deoxythymidine; AZT) into the central nervous system (CNS) was characterized in vitro and in vivo. The in vitro model consisted of primary cultures of isolated bovine capillary endothelial cells. The transport rate of AZT across the monolayer, expressed as endothelial permeability P, was determined following luminal and abluminal administration. P did not differ between the two administration sites (luminal, 1.65 +/- 0.44 cm/min/10(3); abluminal, 1.63 +/- 0.28 cm/min/10(3)). The transport of AZT across the endothelial cell monolayer was found to be concentration independent in the range between 0.4 and 50 micrograms/mL. AZT transport was not affected by pretreatment of the cells with either metabolic inhibitors (DODG and DODG/NaN3) or probenecid. This suggests that AZT passes the monolayer mainly by passive diffusion. The in vivo transport of AZT across the blood-brain barrier and the blood-CSF barrier was studied in male Wistar rats after coadministration of potential inhibitors of active transport of AZT: probenecid (organic anion transport) and thymidine (nucleoside transport). Intracerebroventricular and intravenous coadministration of probenecid caused a significant (P < 0.001) increase in the CSF/plasma concentration ratio compared to the control phase, indicating that the organic anion carrier is involved in AZT transport from CSF to blood. Since there was no effect of probenecid on the transport of AZT in vitro, it is suggested that this carrier is located at the choroid plexus. Coadministration of thymidine did not affect the CSF/plasma concentration ratio, suggesting that a nucleoside carrier system is not involved in AZT transport into or out of the CNS.

Functional integrity of hepatocyte canalicular membrane transport of taurocholate and bilirubin diglucuronide in eisai hyperbilirubinuria rats

Eisai hyperbilirubinuria rats (EHBR) are characterized by conjugated hyperbilirubinemia, and impaired or defective excretion of bilirubin, reduced glutathione and other organic anions from hepatocytes. Hepatocyte canalicular membrane vesicles (CMV) from EHBR and normal SD rats were studied with regard to taurocholate (TC) transport driven by ATP or a membrane potential and bicarbonate-stimulated bilirubin diglucuronide (BDG) transport. ATP-dependent uptake or association of BDG with CMV was also studied in both strains of rats. No significant differences in the uptake of TC and BDG by CMV were observed. This indicates the functional integrity of the canalicular transporters for both organic anions in EHBR. Biliary excretion of taurolithocholic acid sulfate (TLCS) is defective in EHBR. However, TLCS inhibited ATP-dependent TC uptake by SD rat CMV competitively, which may be against the hypothesis that a common organic anion carrier is defective in canalicular membranes of jaundiced rats.

Characterization of the transport of a synthetic bile salt, iodinated cholyl-glycyl-tyrosine, in isolated cultured rat hepatocytes.

The uptake of tri-hydroxy conjugated bile salts by hepatocytes is principally by a sodium-dependent carrier. We examined the uptake kinetics of the high-specific-activity, hydroxylated, conjugated bile salt 125I-labeled cholyl-glycyl-tyrosine, to determine whether this synthetic bile salt was transported by the sodium-dependent bile salt system. 125I-labeled cholyl-glycyl-tyrosine was synthesized, and its transport kinetics were studied in freshly cultured rat hepatocytes. Uptake into hepatocytes was time and temperature dependent and was decreased by the inhibitors diisothiocyanodisulfonic acid stilbene, probenecid and carbonyl cyanide chlorophenyl hydrazone, demonstrating carrier mediation and energy dependence. At concentrations of iodinated cholyl-glycyl-tyrosine less than 10 mumol/L, uptake was 27% +/- 5% sodium dependent, whereas at concentrations from 10 mumol/L to 40 mumol/L uptake was 52% +/- 4% sodium dependent. The apparent affinity for uptake of 125I-labeled cholyl-glycyl-tyrosine was 8 +/- 2 mumol/L, and the maximal velocity was 50 +/- 20 pmol/micrograms DNA/min. Both taurocholate and indocyanine green inhibited uptake of 125I-labeled cholyl-glycyl-tyrosine. Indocyanine green inhibited the uptake of 125I-labeled cholyl-glycyl-tyrosine (Ki = 10 microns) more effectively than taurocholate (Ki = 20 microns). We conclude that 125I-labeled cholyl-glycyl-tyrosine is not a specific probe for either sodium-dependent bile salt or sodium-independent organic anion carriers, but appears to use both systems in a concentration-dependent manner in cultured rat hepatocytes.

Canalicular transport of reduced glutathione in normal and mutant Eisai hyperbilirubinemic rats.

We have characterized the transport of GSH and the mechanism for impaired GSH transport in mutant Eisai hyperbilirubinemic rats (EHBR) using isolated canalicular membrane-enriched vesicles (cLPM). In control animals, the transport of GSH is an electrogenic process and is trans-stimulated by preloading the vesicles with GSH and is not enhanced in the presence of ATP. GSH transport in cLPM is saturable with a single component having a Km of approximately 16 mM and a Vmax of 6.7 nmol/mg/15 s. EHBR is a Sprague-Dawley rat with hyperbilirubinemia due to impaired bile secretion of organic anions by the ATP-dependent organic anion/GSH-conjugate transporter. In cLPM from EHBR we confirmed the defective stimulation by ATP of the transport of LTC4 and GSSG. In the mutant cLPM, the characteristics and kinetics of GSH transport were the same as in the controls. 2,4-(dinitrophenyl)-glutathione (DNP-GSH), which is a substrate for the ATP-dependent canalicular organic anion carrier, in the absence of ATP, cis-inhibited the transport of GSH into cLPM vesicles; however, when the vesicles were preloaded with DNP-GSH, there was a dose-dependent trans-stimulation of GSH transport. In contrast, in the presence of ATP, DNP-GSH enhanced GSH transport in cLPM vesicles; at 0.25 mM DNP-GSH, a concentration which did not cis-inhibit GSH, addition of ATP resulted in accelerated GSH transport; at 1.0 mM DNP-GSH, cis-inhibition was completely reversed by the addition of ATP despite a negligible fall in the medium DNP-GSH. Interestingly, sulfobromophthalein-glutathione (BSP-GSH) neither cis-inhibited nor trans-stimulated GSH transport in cLPM. This contrasts with bLPM where BSP-GSH interacts with the GSH carrier. Therefore, GSH is transported into bile by a multispecific low affinity electrogenic carrier which is distinct from the multispecific high affinity ATP-driven organic anion transporter. Although both carriers have overlapping specificities, BSP-GSH and GSH are uniquely specific for only one of the carriers. The near absence of GSH in the bile of mutant rats can be best explained as a secondary defect due to cis-inhibition from retained substrates for the defective carrier and/or loss of trans-stimulation by these same substrates which normally are concentratively transported into the bile. Other possibilities such as change in GSH carrier activity upon isolation or loss of a negative protein regulator during membrane isolation, although theoretical alternatives are less easily reconciled with the defect in the ATP-driven organic anion transporter.

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

The specific binding of [ 3H]estradiol-17β-(β- d-glucuronide) ([ 3H]E 217G) 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: K d1 = 26nM, B max1 = 0.26 pmol/mg protein; K d2 = 2.6 μM, B max2 = 27 pmol/mg protein for bLPM; and K d1 = 81 nM, B max1 = 0.61 pmol/mg protein; K d2 = 6.7 μM, B max2 = 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 (E 1SO 4) and bromosulfophthalein (BSP) were able to compete with both binding sites, whereas estriol-17β-(β- d-glucuronide) (E 317G), estriol-16α-(β- d-glucuronide) (E 316G), testosterone glucuronide (TG), estradiol-3-(β- d-glucuronide) (E 23G), estriol-3-(β- d-glucuronide) (E 33G), 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 317G, E 316G and TG) and TC were able to compete with both sites, whereas the non-cholestatic steroid A-ring glucuronides (E 23G and E 33G), 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.

Decreased transport of p-aminohippurate in renal basolateral membranes isolated from rats with acute renal failure.

Transport of D-glucose, p-aminohippurate (organic anion), and tetraethylammonium (organic cation) was studied in the renal basolateral membrane vesicles isolated from rats with acute renal failure (ARF). ARF was induced by a single injection of uranyl nitrate. Carrier-mediated transport of p-aminohippurate, estimated under anion-anion exchange condition, was significantly decreased in basolateral membrane vesicles isolated from ARF rats. In contrast, there were no significant differences in D-glucose and tetraethylammonium uptake between normal and ARF rats. When normal basolateral membrane vesicles were incubated in vitro with uranyl nitrate, no significant inhibition in p-aminohippurate uptake was observed. These results suggest that organic anion transport is decreased in renal basolateral membranes from ARF rats, and this transport dysfunction cannot be explained by the direct interaction of uranyl nitrate with the organic anion carrier.

Changes in renal function in cadmium-intoxicated rats.

Changes in renal function, Na+-K+-ATPase activity and PAH transport system in kidney cortex were studied in rats treated with cadmium. Subcutaneous injections of CdCl2 (2 mg Cd/kg.day) for 16 days induced a marked polyuria and a hyposthenuria. These changes were accompanied by increase in urinary protein, glucose, urea, calcium, phosphate, chloride and potassium excretions. The change in urine flow was proportional to the change in total osmotic solute excretion. Creatinine excretion and TcH2O remained unchanged. Na+ excretion was not increased, but the Na+-K+-ATPase of renal cortex was significantly inhibited. PAH uptake by renal cortical slices was markedly attenuated in Cd-treated rats. The Vmax for active PAH influx was drastically reduced, but the Km was not changed. The passive influx and efflux of PAH across the basolateral membrane and the renal tissue oxygen consumption were not apparently altered in Cd-treated animals. These results indicate that 1) the nature of Cd-induced polyuria and hyposthenuria is an osmotic diuresis induced by proximal tubular rejection of various substances, and 2) the mechanism of impaired renal PAH excretion in Cd-treated animals is a loss of organic anion carriers in proximal tubular basolateral membranes.

The quinoid structure is the molecular requirement for recognition of phthaleins by the organic anion carrier at the sinusoidal plasma membrane level in the liver

Sulfobromophthalein electrogenic uptake into rat liver plasma membrane vesicles was shown to admit only the quinoid, trivalent anion. The minimum requirement for this electrogenic process has been investigated in rat liver plasma membrane vesicles by using Thymol blue, a pH-indicator phthalein occurring either as a neutral, phenolic molecule or as a quinoid, monovalent anion. It has been found that Thymol blue is taken up electrogenically, in accordance with Michaelis-Menten kinetics. Parallel inhibition experiments have shown that both sulfobromophthalein and Thymol blue electrogenic uptakes are performed by the same carrier. It is, therefore, concluded that the phthalein structure recognized for transport is the quinoid molecule, with the dissociated acidic function on the benzene ring. Moreover, inhibitions by rifamycin-SV and bilirubin suggest that there exists a common uptake system for bilirubin, phthaleins and other anions. Taurocholate, on the contrary, does not appear to be involved in the same process.

Design of anion-selective membranes for clinically relevant sensors

Requirements for the applicability of anion-selective electrodes in clinical analysis are discussed in terms of selectivity, response time, lifetime and stability. Basically, classical anion-exchangers, electrically charged carriers and electrically neutral carriers may be used as anion-selective components in solvent polymeric membranes. While classical ion-exchanger-based membrane electrodes are only of limited practical relevance, sensors containing ion carriers seem to bear a broad potential with regard to the feasibility of realizing the required properties. The control of the selectivity determining properties of electrically neutral tin organic anion carriers probably allows the development of sufficiently selective membranes for the assay of clinically relevant anions. Furthermore, the incorporation of membrane technology into the design of such carrier membranes may lead to fully optimized sensors.

Mechanisms of taurocholate transport in canalicular and basolateral rat liver plasma membrane vesicles. Evidence for an electrogenic canalicular organic anion carrier.

The driving forces for taurocholate transport were determined in highly purified canalicular (cLPM) and basolateral rat liver plasma membrane (LPM) vesicles. Alanine transport was also examined for comparison. Inwardly directed Na+ but not K+ gradients transiently stimulated [3H]taurocholate (1 microM) and [3H]alanine (0.2 mM) uptake into basolateral LPM 3-4- fold above their respective equilibrium values (overshoots). Na+ also stimulated [3H]taurocholate countertransport and tracer exchange in basolateral LPM whereas valinomycin-induced inside negative K+ diffusion potentials stimulated alanine uptake but had no effect on taurocholate uptake. In contrast, in the "right-side out" oriented cLPM vesicles, [3H]taurocholate countertransport and tracer exchange were not dependent on Na+. Efflux of [3H]taurocholate from cLPM was also independent of Na+ and could be trans-stimulated by extra-vesicular taurocholate. Furthermore, an inside negative valinomycin-mediated K+ diffusion potential inhibited taurocholate uptake into and stimulated taurocholate efflux from the cLPM vesicles. These studies provide direct evidence for a "carrier mediated" and potential-sensitive conductive pathway for the canalicular excretion of taurocholate. In addition, they confirm the presence of a possibly electroneutral Na+-taurocholate cotransport system in basolateral membranes of the hepatocyte.

Postnatal development of carrier-mediated absorption of a foreign amino acid from the rat lung.

To investigate the postnatal development of the carrier-mediated transport process by which certain amino acids are absorbed from the air space of the lung into the blood-stream, a solution of the 14C-labeled nonmetabolized amino acid cycloleucine was administered into the lungs of anesthetized rats of various ages and the rate of absorption of the compound measured. Carrier transport was barely detectable in animals 3-6 days of age; it increased markedly by th age of 12 days, and it reached nearly mature levels by 18 days of age. The rate of development of this carrier process was considerably slower than that previously reported for the organic anion carrier system of the rat lung.

Identification, purification, and partial characterization of an organic anion binding protein from rat liver cell plasma membrane.

Uptake of bilirubin, sulfobromophthalein (BSP), and other organic anions by the liver is a process with kinetics consistent with carrier mediation. The molecular basis of this transport mechanism is unknown. In the search for the putative organic anion carrier or receptor, the interaction of BSP with rat liver cell plasma membrane (LPM) has been studied. Specific binding of [(35)S]BSP to LPM was determined using a filtration assay. Results revealed high affinity (K(a) = 0.27 muM(-1)), saturable (6.3 nmol/mg protein) binding, which was eliminated after preincubation with trypsin. Although [(35)S]BSP was strongly bound to LPM, the binding was rapidly reversible, preventing direct identification and study of a specific binding site(s). To avoid this problem, a photoaffinity probe was devised, in which [(35)S]BSP is covalently bound to LPM after exposure to ultraviolet light. Subsequent sodium dodecyl sulfate gel electrophoresis and fluorography revealed radioactivity predominantly associated with a single 55,000-mol wt protein. A protein with identical electrophoretic mobility was purified from deoxycholate solubilized LPM after affinity chromatography on glutathione-BSP-agarose gel. This protein migrated as a single band on sodium dodecyl sulfate gel electrophoresis and on urea gel isoelectric focusing. It contained 1-2 residues of sialic acid per 55,000-dalton protein, and was immunologically distinct from rat albumin and ligandin. It bound bilirubin with a K(d) of 20 muM, as determined by tryptophan fluorescence quenching. Although the high affinity of this LPM protein for organic anions suggests that it may function as a hepatocellular organic anion receptor, its role in transport of these compounds is unknown.

Molecular orbital study on a chlorine anion cryptate.

Closed shell LCAO-SCF-MO calculations were carried out for the chloride anion cryptate, which is of interest in connection with organic anion receptors and carriers. In the Cl- cryptate of [(C9H18)3(NH)2Cl]+Cl-, Cl- is encapsulated in the host molecule. Since the positions of the two protons bonded to the two nitrogens of the host molecule were not determined in the reported X-ray diffraction analyses, the positions of the two protons in the inclusion complex were decided by calculation using the ab initio molecular orbital method. A structure N+-H…Cl-…H-N+ in which Cl- is in between the two nitrogens was most stable at values of γ(NN) less than 7.0 A. The structure N+-H…Cl-…H-N+ was most stable at γ(NN)=6.023 A. In the most stable structure, the two protons are covalently bonded to the two nitrogens. The structure obtained by geometry optimization for the two ammonium ions and Cl- was similar to that determined by X-ray diffraction analysis.

The transport and oxidation of succinate by Ehrlich ascites-tumour cells.

The transport and oxidation of succinate by functionally intact Ehrlich ascites-tumour cells was investigated. On the basis of pH dependence and inhibitor sensitivity it was concluded that succinate may be transported across the cell membrane by the organic anion carrier system. Thus the ability of isolated Ehrlich cells to oxidize succinate is real, and is not necessarily a result of damage to cell integrity.

Translocation of Nickel in Xylem Exudate of Plants

Topped plants of tomato (Lycopersicon esculentum), cucumber (Cucumis sativus), corn (Zea mays), carrot (Daucus carota), and peanut (Arachis hypogaea) were treated with 0.5 to 50 micromolar Ni (containing (63)Ni) in nutrient solutions. Xylem exudate was collected for 10 hours or, in the case of corn, for 20 hours at 5-hour intervals. Electrophoresis of nutrient solution distributed all Ni cathodically as inorganic Ni(2+). Low concentrations of Ni in tomato exudate migrated anodically, presumably bound to organic anion (carrier). However, this carrier became saturated at about 2 micromolar Ni in exudate, and excess Ni ran cathodically. Most of the Ni in cucumber, corn, carrot, and peanut exudate ran anodically, and its migration rate was identical for all exudates. Peanut root sap contained 14 to 735 micromolar Ni. The anodic Ni carriers in root sap and exudate appear identical. The carrier in root sap became saturated near 100 micromolar Ni, as shown by cathodic streaking of Ni exceeding that concentration. It appears that all five species translocate low concentrations of Ni in the same anionic form.