Primary Active Transport Research Articles

Differential Modulation of the Human Liver Conjugate Transporters MRP2 and MRP3 by Bile Acids and Organic Anions

The multidrug resistance proteins MRP2 (ABCC2) and MRP3 (ABCC3) are key primary active transporters involved in anionic conjugate and drug extrusion from the human liver. The major physiological role of MRP2 is to transport conjugated metabolites into the bile canaliculus, whereas MRP3 is localized in the basolateral membrane of the hepatocytes and transports similar metabolites back to the bloodstream. Both proteins were shown to interact with a large variety of transported substrates, and earlier studies suggested that MRPs may work as co-transporters for different molecules. In the present study we expressed the human MRP2 and MRP3 proteins in insect cells and examined their transport and ATPase characteristics in isolated, inside-out membrane vesicles. We found that the primary active transport of estradiol-17-beta-d-glucuronide (E217betaG), a major product of human steroid metabolism, was differently modulated by bile acids and organic anions in the case of human MRP2 and MRP3. Active E217betaG transport by MRP2 was significantly stimulated by the organic anions indomethacin, furosemide, and probenecid and by several conjugated bile acids. In contrast, all of these agents inhibited E217betaG transport by MRP3. We found that in the case of MRP2, ATP-dependent vesicular bile acid transport was increased by E217betaG, and the results indicated an allosteric cross-stimulation, probably a co-transport of bile acids and glucuronate conjugates through this protein. There was no such stimulation of bile acid transport by MRP3. In conclusion, the different transport modulation of MRPs by bile acids and anionic drugs could play a major role in regulating physiological and pathological metabolite fluxes in the human liver.

Chloride ATPase pumps in nature: do they exist?

Five widely documented mechanisms for chloride transport across biological membranes are known: anion-coupled antiport, Na+ and H(+)-coupled symport, Cl- channels and an electrochemical coupling process. These transport processes for chloride are either secondarily active or are driven by the electrochemical gradient for chloride. Until recently, the evidence in favour of a primary active transport mechanism for chloride has been inconclusive despite numerous reports of cellular Cl(-)-stimulated ATPases coexisting, in the same tissue, with uphill ATP-dependent chloride transport. Cl(-)-stimulated ATPase activity is a ubiquitous property of practically all cells with the major location being of mitochondrial origin. It also appears that plasma membranes are sites of Cl(-)-stimulated ATPase pump activity. Recent studies of Cl(-) -stimulated ATPase activity and ATP-dependent chloride transport in the same plasma membrane system, including liposomes, strongly suggest a mediation by the ATPase in the net movement of chloride up its electrochemical gradient across the plasma membrane structure. Contemporary evidence points to the existence of Cl(-)-ATPase pumps; however, these primary active transporters exist as either P-, F- or V-type ATPase pumps depending upon the tissue under study.

Cholestasis

In contrast with urine formation, bile flow is not dependent on hydrostatic forces, but driven by osmotic pressure of solutes secreted across the apical membrane of hepatocytes and bile duct...

Oxidative damage to mouse lens in culture. Protective effect of pyruvate.

Studies have been conducted to examine the feasibility of preventing oxyradical-dependent oxidative stress to mouse lens in culture, using pyruvate as an antioxidant. The extent of oxidative damage to the tissue was assessed by measurement of the status of Na(+)-K(+) ATPase dependent active transport of rubidium 86Rb(+). The tissue levels of adenosine triphosphate (ATP), glutathione (GSH), malonaldehyde (MDA) and catalase were also determined. While the measurement of 86Rb(+) uptake provides an assessment of the integrity of the primary active transport system, measurement of the other components reflects the status of intracellular oxidative stress. ATP measurement also reflected on the overall status of metabolic integrity. Incubation of the lens with xanthine (XA)/xanthine oxidase (XO) system had an adverse effect on all these parameters. Incorporation of pyruvate was strikingly protective. The protective effect of pyruvate is apparently due to its ability to scavenge ROS generated in the medium with the possibility of its action on tissue metabolism as well. The findings are hence considered useful for further studies on the prevention of oxidative stress to tissues by exogenous supplementation with pyruvate, specially the human lens where the biochemistry of its antioxidant mechanisms is similar to the mouse lens, contrary to the rat lens.

Structural requirements for functional interaction of glutathione tripeptide analogs with the human multidrug resistance protein 1 (MRP1).

The human multidrug resistance protein 1 (MRP1) is a primary active transporter of reduced (GSH) and oxidized glutathione, as well as GSH-, glucuronate-, and sulfate-conjugated organic anions. In addition, the transport of certain MRP1 substrates is stimulated by the presence of GSH. To evaluate the structural features of GSH required for interaction with the protein, we investigated the ability of a series of GSH analogs to enhance GSH stimulatable transport of [(3)H]estrone 3-sulfate (E(1)SO(4)). We found that substitution of the gamma-Glu residue with Gly, beta-Asp, and alpha-Glu resulted in complete loss of transport stimulation. In contrast, substitution of Gly with Glu or beta-Ala resulted in only a partial loss of stimulatory activity. E(1)SO(4) transport activity surpassed GSH-stimulated levels in the presence of tripeptides in which Cys was substituted with the hydrophobic amino acids Leu, Phe, and homo-Phe. Moreover, polar substitutions of Cys did not enhance transport to the same extent as nonpolar substitutions of comparable size. gamma-Glu-Leu-Gly was 1.6-fold more effective than GSH in stimulating E(1)SO(4) uptake, and kinetic analysis indicated this was due to an increased V(max). In addition, this tripeptide was shown to be a competitive inhibitor of apigenin-stimulated GSH transport (K(i) value of 14 microM), confirming that it either interacts with the same site on MRP1 as GSH or that the binding of the two tripeptides is mutually exclusive. These data provide insight into the architecture of the GSH binding domain of MRP1.

Mechanism of Active Secretion of Phenolsulfonphthalein in the Liver via Mrp2 (abcc2), an Organic Anion Transporter

Phenolsulfonphthalein (PSP) has been selected as a model drug that is eliminated from both the kidney and liver in rats. Although the renal PSP transport system has been studied, few details of the biliary excretion of PSP have been reported. We investigated the biliary excretion system for PSP in rats. It has been reported that the biliary excretion of many organic anions from hepatocytes into bile is mediated by a primary active transporter, referred to as multidrug resistance-associated protein 2 (Mrp2/abcc2). The biliary excretion of PSP in SD rats was significantly decreased in the presence of Mrp2 inhibitors. The biliary excretion of PSP in Eisai hyperbilirubinemic rats (EHBR), hereditarily Mrp2-defective rats, was significantly lower than that in SD rats. Moreover, an efflux experiment using Caco-2 cells was carried out to confirm Mrp2-mediated PSP transport. Mrp2 inhibitors significantly decreased PSP efflux from Caco-2 cells. These results suggest that Mrp2 contributes to the biliary excretion of PSP in SD rats.

On the mechanism of solute uptake in Pseudomonas.

Pseudomonas species have over 300 known and putative nutrient uptake systems enabling them to metabolize a large number of organic compounds, and thus inhabit many diverse ecological niches. The outer membrane of these organisms acts as a semi-permeable barrier, excluding many classes of potentially toxic molecules from the cell. Nutrients use specialized water-filled channels called porins to traverse this barrier. Entry into the cell is mediated by one of four classes of cytoplasmic membrane transporters: glycerol facilitators, phosphotransferase systems, primary active transporters, and secondary active transporters. The class of transporter used is dependent on the environmental conditions, as well as the type and concentration of solute. The recent advances in elucidating the structures and functional mechanisms of these uptake systems will be discussed in this review.

Identity and regulation of ion transport mechanisms in the corneal endothelium.

Corneal transparency is dependent on regulation of the hydration of the corneal stroma. Water is driven into the cornea across the epithelial and endothelial cell layers by the stromal swelling pressure. This fluid leak into the cornea is counterbalanced by the corneal fluid pump, which is predominantly attributed to the ion and fluid transport capacity of the endothelial cell layer. Primary and secondary active transport mechanisms are responsible for generating a net ion flux from the stromal to anterior chamber side of the endothelium; however, the identity and location of all the components of this transport system are not known. The endothelial fluid pump is dependent on the presence of Cl(-) and HCO(3)(-), and can be slowed by carbonic anhydrase inhibitors. A number of anion transport mechanisms have been identified and characterized in the endothelium, including basolateral Na(+)/2HCO(3)(-) cotransport, Na(+)/K(+)/2Cl(-) cotransport, Cl(-)/HCO(3)(-) exchange, and apical anion channels permeable to both Cl(-) and HCO(3)(-). Furthermore, there is evidence for a carbonic anhydrase mediated CO(2)-diffusive mode of apical HCO(3)(-) flux. These findings are incorporated into a new model of transendothelial anion transport, which suggests that there are a number of alternate pathways for anion transport. There have been few studies on activation of signal transduction pathways that could stimulate endothelial fluid transport. Interestingly, recent studies show that multiple autocrine signaling pathways are in place that could be upregulated during physical stimulation and may be responsible for maintaining basal levels of fluid secretion.

Brain efflux index method. Characterization of efflux transport across the blood-brain barrier.

Efflux transport systems act as one of the detoxification systems at the blood-brain barrier (BBB) by eliminating xenobiotics and endogenous wastes from the brain into the blood (, , , , , ). P-glycoprotein is a well-known example (,,,). It is a primary active transporter for hydrophobic neutral and cationic compounds and mediates unidirectional transport from the inside to the outside of cells. The distribution of its substrates in the brain after its intravenous administration is significantly increased, by twofold to approx 20-fold, depending on the substrates in P-glycoprotein knockout mouse (,,,). Although the molecular mechanism has not been fully elucidated, the presence of an efflux transport system for organic anions on the brain capillary endothelial cells has been suggested (, , , , ). It is necessary to reveal the efflux transport mechanisms at the BBB to understand the disposition of nutrients and neurotransmitters and their metabolites in the central nervous system, and to deliver central acting drugs to the central nervous system efficiently. A method to determine in vivo efflux clearance across the BBB is required for that purpose.

Potential Cholestatic Activity of Various Therapeutic Agents Assessed by Bile Canalicular Membrane Vesicles Isolated from Rats and Humans

The active transport of solutes mediated by the bile salt export pump (BSEP/ABCB11) and multidrug resistance associated protein-2 (MRP2/ABCC2) are thought to involve bile acid-dependent and -independent bile formation, respectively. To evaluate the potential of therapeutic agents as inhibitors of such transporters on bile canalicular membranes, we examined the inhibition of the primary active transport of typical substrates by 15 drugs, clinically known to cause cholestasis in canalicular membrane vesicles. The inhibition by most of the compounds in rat canalicular membrane vesicles (CMVs) was minimal or observed at much higher concentrations than obtained in clinical situations. However, cloxacillin, cyclosporin A and midecamycin inhibited BSEP, and cyclosporin A and midecamycin inhibited MRP2 with an inhibition constant close to the clinical concentration. By comparing the inhibition potential between rat and human CMVs, the inhibition of BSEP- and MRP2-mediated transport by midecamycin and cyclosporin A was relatively similar whereas the inhibitory effect on BSEP-mediated transport by cloxacillin and glibenclamide was more marked in humans than in rats. These results suggest that the majority of cholestasis-inducing drugs have a minimal inhibitory effect on rat BSEP and MRP2 although species differences in inhibitory potential should be considered, especially in the case of BSEP.

Multiple Membrane-associated Tryptophan Residues Contribute to the Transport Activity and Substrate Specificity of the Human Multidrug Resistance Protein, MRP1

The multidrug resistance protein, MRP1, is a clinically important ATP-binding cassette transporter in which the three membrane-spanning domains (MSDs), which contain up to 17 transmembrane (TM) helices, and two nucleotide binding domains (NBDs) are configured MSD1-MSD2-NBD1-MSD3-NBD2. In tumor cells, MRP1 confers resistance to a broad spectrum of drugs, but in normal cells, it functions as a primary active transporter of organic anions such as leukotriene C(4) and 17beta-estradiol 17beta-(D-glucuronide). We have previously shown that mutation of TM17-Trp(1246) eliminates 17beta-estradiol 17beta-(D-glucuronide) transport and drug resistance conferred by MRP1 while leaving leukotriene C(4) transport intact. By mutating the 11 remaining Trp residues that are in predicted TM segments of MRP1, we have now determined that five of them are also major determinants of MRP1 function. Ala substitution of three of these residues, Trp(445) (TM8), Trp(553) (TM10), and Trp(1198) (TM16), eliminated or substantially reduced transport levels of five organic anion substrates of MRP1. In contrast, Ala substitutions of Trp(361) (TM7) and Trp(459) (TM9) caused a more moderate and substrate-selective reduction in MRP1 function. More conservative substitutions (Tyr and Phe) of the Trp(445), Trp(553), and Trp(1198) mutants resulted in substrate selective retention of transport in some cases (Trp(445) and Trp(1198)) but not others (Trp(553)). Our findings suggest that the bulky polar aromatic indole side chain of each of these five Trp residues contributes significantly to the transport activity and substrate specificity of MRP1.

Formulation of a coupled mechanism between solute diffusion, phosphatase–kinase reactions and membrane potentials for the primary active transport of phosphorylated substrates through biological membranes

Coupled interrelations occurring between a phosphatase/kinase reaction sequence acting in unstirred layers and on both sides of a charged biomembrane pore structure are presented as a plausible kinetic model for the primary active transport of phosphorylated molecules. Simulations conducted at the cell level and with credible numerical values demonstrate that the enzymes positions strongly regulate the membrane permeability for the transported substrate. Depending on both the enzymes positions (more or less far from the membrane) and the membrane charges, the membrane may appear either impervious, either permeable or able to actively transport a phosphorylated substrate. Globally all happens as if, in function of the enzymes positions, a permanent pore may be regulated, changing from a more closed to a more open conformation.

ATP dependant artificial enzymatic pumps for the primary active transport of small molecules

To be able to concentrate or selectively separate small molecules using chemical energy alone would be of considerable interest. In nature, biomembranes are capable of doing just this. The new concept of artificial enzymatic pumps has allowed the development of artificial biomimetic membranes capable of transporting specific small hydrophylic molecules against their concentration gradients at constant temperature and pressure. This primary active transport, which uses ATP as its energy source, already permits small molecules such as glycerol-3-phosphate to be concentrated 17-fold.

Loss of ATP-dependent Transport Activity in Pseudoxanthoma Elasticum-associated Mutants of Human ABCC6 (MRP6)

Mutations in the ABCC6 (MRP6) gene cause pseudoxanthoma elasticum (PXE), a rare heritable disorder resulting in the calcification of elastic fibers. In the present study a cDNA encoding a full-length normal variant of ABCC6 was amplified from a human kidney cDNA library, and the protein was expressed in Sf9 insect cells. In isolated membranes ATP binding as well as ATP-dependent active transport by ABCC6 was demonstrated. We found that glutathione conjugates, including leukotriene C(4) and N-ethylmaleimide S-glutathione (NEM-GS), were actively transported by human ABCC6. Organic anions (probenecid, benzbromarone, indomethacin), known to interfere with glutathione conjugate transport of human ABCC1 and ABCC2, inhibited the ABCC6-mediated NEM-GS transport in a specific manner, indicating that ABCC6 has a unique substrate specificity. We have also expressed three missense mutant forms of ABCC6, which have recently been shown to cause PXE. MgATP binding was normal in these proteins; ATP-dependent NEM-GS or leukotriene C(4) transport, however, was abolished. Our data indicate that human ABCC6 is a primary active transporter for organic anions. In the three ABCC6 mutant forms examined, the loss of transport activity suggests that these mutations result in a PXE phenotype through a direct influence on the transport activity of this ABC transporter.

Biliary excretion of tauroursodeoxycholate-3-sulfate in the rat.

Biliary organic anion excretion is mediated by an ATP-dependent primary active transporter, multidrug resistance protein 2. On the other hand, a multiplicity of canalicular organic anion transport has been suggested. Ursodeoxycholic acid, the 7beta-epimer of chenodeoxycholic acid, is clinically used for various hepatobiliary diseases. In our previous study, the contribution of multidrug resistance protein 2 for biliary excretion of taurine-conjugated bile acid sulfates depended on the numbers of hydroxyl residue. Therefore, to further examine the effect of hydrophobicity on the substrate specificity of multidrug resistance protein 2, we examined the effect of bile acid conjugates and organic anions on biliary excretion of tauroursodeoxycholate-3-sulfate, taurine and sulfonate-conjugated ursodeoxycholic acid, in rats. Biliary tauroursodeoxycholate-3-sulfate excretions was markedly delayed in Eisai hyperbilirubinemic rats. Taurolithocholate-3-sulfate inhibited but ursodeoxycholate-3,7-disulfate did not affect biliary tauroursodeoxycholate-3-sulfate excretion. Biliary tauroursodeoxycholate-3-sulfate excretion was inhibited by sulfobromophthalein, but was not inhibited by dibromosulfophthalein and cefpiramide. These findings indicate that tauroursodeoxycholate-3-sulfate is very specific for multidrug resistance protein 2.

Zur funktionellen Morphologie der Dickdarm‐Mukosa von Rind (Bos primigenius f. taurus), Schaf (Ovis ammon f. aries) und Ziege (Capra aegagrus f. hircus)*

The mucous membrane of the large intestine of the ox, sheep and goat was studied using light and electron microscopy. The surface epithelium possesses the well-known complement of organelles with a conspicuous number of mitochondria as a source of energy for absorptive activities. Occasionally, brush cells are found in the epithelium; the functional significance of this type of cell is still under discussion. In the present material, electron-lucid spheroids (diameter: 45 x 35 nm), which tend to fuse, appear in the supranuclearly located osmiophilic granules. The structure and appearance of the epithelium cells in the crypts of these species correspond to previous descriptions in other mammals. In the epithelial cells of the deep glandular region, dense accumulations of mainly rounded granules (diameter up to 400 nm) are found apically in the cytoplasm. Their functional significance is still under discussion. Acid phosphatase activity, as a marker enzyme for lysosomes, points to degenerative processes in connection with the physiological death of enterocytes. In contrast, the negative alkaline phosphatase reaction indicates that, according to our findings, the epithelial transport of substances in the large intestine of ruminants--in principle contrary to the situation in the small intestinal enterocytes--occurs without the aid of this enzyme. The demonstrated ATPase activity on the other hand, is related to a primary active ion transport linked with membrane exchange of non-electrolytes in connection with transepithelial events of absorption and secretion in the large intestine.

Cl(-)-ATPases: Novel primary active transporters in biology.

Five widely documented mechanisms of chloride transport across plasma membranes are anion-coupled antiport, sodium and hydrogen-coupled symport, Cl(-)channels, and an electrochemical coupling process. No genetic evidence has yet been provided for primary active chloride transport despite numerous reports of cellular Cl(-)-stimulated ATPases co-existing, in the same tissue, with uphill chloride transport that could not be accounted for by the five common chloride transport processes. Cl(-)-stimulated ATPase activity is a common property of practically all biological cells with the major location being of mitochondrial origin. It also appears that plasma membranes are sites of Cl(-)-stimulated ATPase activity. Recent studies of Cl(-)-stimulated ATPase activity and active chloride transport in the same membrane system, including liposomes, suggest a medication by the ATPase in net movement of chloride up its electrochemical gradient across plasma membranes. Further studies, especially from a molecular biological perspective, are required to confirm a direct transport role to plasma membrane-localized Cl(-)-stimulated ATPases. J. Exp. Zool. 289:215-223, 2001.

Physicochemical parameters responsible for the affinity of methotrexate analogs for rat canalicular multispecific organic anion transporter (cMOAT/MRP2).

Canalicular multispecific organic anion transporter (cMOAT/MRP2) is known to exhibit a broad substrate specificity toward amphiphatic organic anions, including methotrexate (MTX). The present study aims to identify the physicochemical properties of MTX derivatives that correlate with recognition specificity by cMOAT/MRP2. We examined the inhibitory effect of MTX and 24 analogs on the transport of [3H]-S-(2,4-dinitrophenyl)glutathione by cMOAT/MRP2. The affinity constants of these compounds were compared with their physicochemical parameters. The primary active transport of several compounds was also confirmed. The affinity constants closely correlated with the octanol/water partition coefficient (clogP), and a linear combination of polar and nonpolar surface areas. The affinity for cMOAT/MRP2 also closely correlated with the molecular weight, which also showed a significant correlation with nonpolar surface area and clogP. Recognition by cMOAT/MRP2 depends on a balance of dynamic surface properties between the polar and nonpolar regions of MTX analogs. The so-called "molecular weight threshold" for the cMOAT/MRP2 affinity of these compounds can be explained by their physicochemical parameters, especially their nonpolar surface areas.

Clinical Pharmacokinetics of Pravastatin

Pravastatin, one of the 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors (statins) widely used in the management of hypercholesterolaemia, has unique pharmacokinetic characteristics among the members of this class. Many in vivo and in vitro human and animal studies suggest that active transport mechanisms are involved in the pharmacokinetics of pravastatin. The oral bioavailability of pravastatin is low because of incomplete absorption and a first-pass effect. The drug is rapidly absorbed from the upper part of the small intestine, probably via proton-coupled carrier-mediated transport, and then taken up by the liver by a sodium-independent bile acid transporter. About half of the pravastatin that reaches the liver via the portal vein is extracted by the liver, and this hepatic extraction is mainly attributed to biliary excretion which is performed by a primary active transport mechanism. The major metabolites are produced by chemical degradation in the stomach rather than by cytochrome P450-dependent metabolism in the liver. The intact drug and its metabolites are cleared through both hepatic and renal routes, and tubular secretion is a predominant mechanism in renal excretion. The dual routes of pravastatin elimination reduce the need for dosage adjustment if the function of either the liver or kidney is impaired, and also reduce the possibility of drug interactions compared with other statins. which are largely eliminated by metabolism. The lower protein binding than other statins weakens the tendency for displacement of highly protein-bound drugs. Although all statins show a hepatoselective disposition, the mechanism for pravastatin is different from that of the others. There is high uptake of pravastatin by the liver via an active transport mechanism, but not by other tissues because of its hydrophilicity, whereas the disposition characteristics of other statins result from high hepatic extraction because of high lipophilicity. These pharmacokinetic properties of pravastatin may be the result of the drug being given in the pharmacologically active open hydroxy acid form and the fact that its hydrophilicity is markedly higher than that of other statins. The nature of the pravastatin transporters, particularly in humans, remains unknown at present. Further mechanistic studies are required to establish the pharmacokinetic-pharmacodynamic relationships of pravastatin and to provide the optimal therapeutic efficacy for various types of patients with hypercholesterolaemia.

The human multidrug resistance-associated protein (MRP) gene family: from biological function to drug molecular design.

The ATP-binding cassette transmembrane proteins play an important role in transport of drugs as well as of biologically active endogenous substances. The human multidrug resistance-associated protein (MRP) subfamily consists of at least six members, exhibiting a wide spectrum of biological functions. MRP1 operates as an ATP-dependent primary active transporter for substrates conjugated with glucuronide, sulfate or glutathione. Leukotriene C4 is an important endogenous substrate for MRP1. Glutathione serves as a cofactor in MRP1-mediated drug transport as well. Genes encoding both MRP1 and the catalytic subunit of gamma-glutamylcysteine synthetase (gamma-GCS) are coordinately regulated in cultured cancer cell lines as well as colorectal cancer tissues from colon cancer patients. The induction of MRP1 and gamma-GCS expression by oxidative stress varies among different cell lines, and p53 mutations are associated with elevated levels of induction. To modulate the transport function of MRP1, we have synthesized novel glutathione derivatives as photoreactive biochemical probes targeting the transporter protein. GIF-0019 restored the cellular sensitivity of MRP1-overexpressing drug-resistant cancer cells to anticancer prostaglandins in vitro, which was characterized by enhanced mRNA levels of the cyclin-dependent kinase inhibitor p21, suppressed c-myc expression and G1 arrest.