ATP-dependent Transport Research Articles

The linker region of breast cancer resistance protein ABCG2 is critical for coupling of ATP-dependent drug transport.

The ATP-binding cassette (ABC) transporters of class G display a different domain organisation than P-glycoprotein/ABCB1 and bacterial homologues with a nucleotide-binding domain preceding the transmembrane domain. The linker region connecting these domains is unique and its function and structure cannot be predicted. Sequence analysis revealed that the human ABCG2 linker contains a LSGGE sequence, homologous to the canonical C-motif/ABC signature present in all ABC nucleotide-binding domains. Predictions of disorder and of secondary structures indicated that this C2-sequence was highly mobile and located between an α-helix and a loop similarly to the C-motif. Point mutations of the two first residues of the C2-sequence fully abolished the transport-coupled ATPase activity, and led to the complete loss of cell resistance to mitoxantrone. The interaction with potent, selective and non-competitive, ABCG2 inhibitors was also significantly altered upon mutation. These results suggest an important mechanistic role for the C2-sequence of the ABCG2 linker region in ATP binding and/or hydrolysis coupled to drug efflux.

ATP11C Encodes a Major Flippase in Human Erythrocyte and Its Genetic Defect Causes Congenital Non-Spherocytic Hemolytic Anemia

Flippases are members of the P-IV ATPase family of proteins, and contribute to localization of phosphatidylserine (PS) in inner leaflet by its ATP-dependent active transport from outer to inner leaflet of lipid bilayer of erythrocyte membranes. It is critical that erythrocytes maintain PS in the inner monolayer to ensure their 120-day survival in circulation since externalization of PS will be recognized as an eat-me signal, resulting in phagocytosis by splenic macrophages. In the present study, we identified that ATP11C gene encodes a major flippase in human erythrocytes. A 13 years-old boy was referred to our hospital for consultation for work up of an undiagnosed congenital hemolytic anemia. Since extensive biochemical and molecular analysis failed to identify hemoglobin, erythrocyte membranes and enzyme abnormalities for the pathogenesis of hemolysis, we performed the whole exome analysis by massively parallel sequencing. We identified that the proband is hemizyogous, and the mother is heterozygous for a missense mutation of ATP11C, c.1253C>A, corresponding to a single amino acid substitution, p.Thr418Asn. Flipping activity as measured by PS internalization was decreased to 10% in the red cells of the proband compared to a normal control, clearly demonstrating that ATP11C encodes a major flippase in the human erythrocyte membranes. The PS-positive erythrocytes were not significantly increased in the whole blood but only in the most dense senescent cells, suggesting that PS exposure did not occur until very late stages of lifespan. We showed that PS exposure mediated by Ca2+-stimulated phospholipid scrambling was not different between red cells of the proband and control. Taken together, our findings imply that suppressed scrambling activity rather than flippase activity is the major contributor to maintainance of PS in inner leaflet of normal red cells during their 120-day lifespan, and that PS exposure to cell surface as an 'eat-me' signal depends primarily on scramblase activity at the end of lifespan. Importantly, our study has enables us to identify the major flippase of human erythrocyte membrane. DisclosuresNo relevant conflicts of interest to declare.

Development of an acidic/neutral antibody flow-through polishing step using salt-tolerant anion exchange chromatography

6-Chloro- and 6-bromopurines can cross the blood-brain barrier and in situ give rise to substrates of multidrug resistance-associated proteins (MRPs). The electrophilic purines form glutathione conjugates in reactions catalyzed by intracellular glutathione transferases (GSTs), and the conjugates are subsequently exported from the cells by ATPdependent membrane transporters. In rodent model systems it has been demonstrated that suitably radiolabeled 6-halogenopurines by this scheme are pro-probes useful in monitoring the functionality of MRPs in intact brains using positron emission tomography. Prior to applications in human subjects it is imperative to establish the purine pro-probes as effective substrates for human GSTs occurring in brain and other tissues. We have developed a spectrophotometric assay for the glutathione conjugation and determined specific activities with a range of human GSTs as well as with some rat GSTs for comparison. The ubiquitous GST P1-1 showed the highest activities with the 6-halogenopurines, which bodes well for the application of pro-probes for human investigations.

New light in flavin autofluorescence.

Our attention was captured by the interesting debate on the identification of lipofuscins, lipofuscin-like lipopigments, or flavins as the responsible for intracellular autofluorescence (AF) detected in epithelial cancer stem cells when exciting at 480-490 nm. Evidence was provided leading to ascribe AF emission to flavins accumulating in cytoplasmic structures, bounded to membranes and bearing ATP-dependent ABCG2 transporters. Flavins were then proposed as an intrinsic AF biomarker of cancer stem cells, with advantageous implications on cell invasiveness and chemoresistance investigations. It is however worth recalling the huge amount of literature on flavins and NAD(P)H as AF biomarkers of cell energetic metabolism and redox state, an aspect that should not be overlooked in the renewed course to extend the potential of flavins as AF biomarkers, entailing also a recent proposal of Flavin-based fluorescent proteins as substitutes of Green fluorescent proteins.

Inflammation Modulates RLIP76/RALBP1 Electrophile-Glutathione Conjugate Transporter and Housekeeping Genes in Human Blood-Brain Barrier Endothelial Cells.

Endothelial cells are often present at inflammation sites. This is the case of endothelial cells of the blood-brain barrier (BBB) of patients afflicted with neurodegenerative disorders such as Alzheimer's, Parkinson's, or multiple sclerosis, as well as in cases of bacterial meningitis, trauma, or tumor-associated ischemia. Inflammation is a known modulator of gene expression through the activation of transcription factors, mostly NF-κB. RLIP76 (a.k.a. RALBP1), an ATP-dependent transporter of electrophile-glutathione conjugates, modulates BBB permeability through the regulation of tight junction function, cell adhesion, and exocytosis. Genes and pathways regulated by RLIP76 are transcriptional targets of tumor necrosis factor alpha (TNF-α) pro-inflammatory molecule, suggesting that RLIP76 may also be an inflammation target. To assess the effects of TNF-α on RLIP76, we faced the problem of choosing reference genes impervious to TNF-α. Since such genes were not known in human BBB endothelial cells, we subjected these to TNF-α, and measured by quantitative RT-PCR the expression of housekeeping genes commonly used as reference genes. We find most to be modulated, and analysis of several inflammation datasets as well as a metaanalysis of more than 5000 human tissue samples encompassing more than 300 cell types and diseases show that no single housekeeping gene may be used as a reference gene. Using three different algorithms, however, we uncovered a reference geneset impervious to TNF-α, and show for the first time that RLIP76 expression is induced by TNF-α and follows the induction kinetics of inflammation markers, suggesting that inflammation can influence RLIP76 expression at the BBB. We also show that MRP1 (a.k.a. ABCC1), another electrophile-glutathione transporter, is not modulated in the same cells and conditions, indicating that RLIP76 regulation by TNF-α is not a general property of glutathione transporters. The reference geneset uncovered herein should aid in future gene expression studies in inflammatory conditions of the BBB.

Increased efficiency of testicular tumor chemotherapy by ultrasound microbubble-mediated targeted transfection of siMDR1.

The MDR1 gene encoding P-glycoprotein (P-gp) is an ATP-dependent drug efflux transporter and is related to drug resistance of yolk sac tumors. Drug resistence may be an important factor for the low efficiency of chemotherapy in the treatment of testicular tumors. P-gp, encoded by the MDR1 gene, is an ATP-binding cassette transporter. P-gp exhibits high expression in capillary endothelial cells of the testis and prevents the intracellular accumulation of chemotherapy agents in testicular tumor cells, resulting in drug resistance. In the present study, we aimed to use specific siRNA to silence the expression of the MDR1 gene and P-gp, leading to the reversal of multidrug resistance of testicular tumors and contributing a suitable condition for chemotherapy. Ultrasound microbubble-mediated delivery is a safe and effective tool for gene delivery. In the present study, we demonstrated that ultrasound microbubble-mediated delivery effectively improved the siMDR1 gene transfection in interstitial capillary endothelial cells of the testis, inhibited the expression of P-gp and increased daunorubicin accumulation. The testis tumor model was successfully constructed by injecting 1x10(7) yolk sac tumor cells in 3-week-old Sprague-Dawley rats. Ultrasound microbubble-mediated siMDR1 gene therapy improved the effect of chemotherapy on the testicular tumors. The testicular volume was reduced, the number of tumor cells within the testicular tissues decreased, and pathological changes were mostly recovered. Therefore, the present study indicated that ultrasound microbubble-mediated siMDR1 gene therapy in vivo reversed drug resistance by regulating P-gp expression, providing a promising method for the treatment of testicular tumors.

Multidrug Resistance-Associated Protein 2 (MRP2) Mediated Transport of Oxaliplatin-Derived Platinum in Membrane Vesicles.

The platinum-based anticancer drug oxaliplatin is important clinically in cancer treatment. However, the role of multidrug resistance-associated protein 2 (MRP2) in controlling oxaliplatin membrane transport, in vivo handling, toxicity and therapeutic responses is unclear. In the current study, preparations of MRP2-expressing and control membrane vesicles, containing inside-out orientated vesicles, were used to directly characterise the membrane transport of oxaliplatin-derived platinum measured by inductively coupled plasma mass spectrometry. Oxaliplatin inhibited the ATP-dependent accumulation of the model MRP2 fluorescent probe, 5(6)-carboxy-2,'7'-dichlorofluorescein, in MRP2-expressing membrane vesicles. MRP2-expressing membrane vesicles accumulated up to 19-fold more platinum during their incubation with oxaliplatin and ATP as compared to control membrane vesicles and in the absence of ATP. The rate of ATP-dependent MRP2-mediated active transport of oxaliplatin-derived platinum increased non-linearly with increasing oxaliplatin exposure concentration, approaching a plateau value (Vmax) of 2680 pmol Pt/mg protein/10 minutes (95%CI, 2010 to 3360 pmol Pt/mg protein/10 minutes), with the half-maximal platinum accumulation rate (Km) at an oxaliplatin exposure concentration of 301 μM (95% CI, 163 to 438 μM), in accordance with Michaelis-Menten kinetics (r2 = 0.954). MRP2 inhibitors (myricetin and MK571) reduced the ATP-dependent accumulation of oxaliplatin-derived platinum in MRP2-expressing membrane vesicles in a concentration-dependent manner. To identify whether oxaliplatin, or perhaps a degradation product, was the likely substrate for this active transport, HPLC studies were undertaken showing that oxaliplatin degraded slowly in membrane vesicle incubation buffer containing chloride ions and glutathione, with approximately 95% remaining intact after a 10 minute incubation time and a degradation half-life of 2.24 hours (95%CI, 2.08 to 2.43 hours). In conclusion, MRP2 mediates the ATP-dependent active membrane transport of oxaliplatin-derived platinum. Intact oxaliplatin and its anionic monochloro oxalate ring-opened intermediate appear likely candidates as substrates for MRP2-mediated transport.

Evaluating Potential P-gp Substrates: Main Aspects to Choose the Adequate Permeability Model for Assessing Gastrointestinal Drug Absorption.

The success of an oral drug route administration depends on many factors that interfere in its bioavailability, therapeutic efficacy and clinical safety. In human cells, ATP-dependent efflux transporter proteins, such as P-glycoprotein (P-gp), BCRP and MRP2, reduce the absorption of drugs. A tiered approach chosen to evaluate drugs as substrates or inhibitors of efflux pumps, particularly P-gp, should be carefully selected, since each study method has advantages and intrinsic limitations to their processes. Depending on the adopted study conditions, the results may not correspond to the real characteristics of the drug regarding to its modulation by specific efflux proteins. This mini-review aims at summarizing the role of P-gp in the drugs oral absorption and correlating some of the most used permeability methods to determine the drug condition as P-gp substrate. Studies about P-gp have shown that it is a dynamic protein, facilitating secretion of endogenous compounds, as aldosterone, and protecting cells against xenobiotics. Different efflux assays are employed to evaluate drugs as P-gp substrates. In an initial planning, MDCK-MDR1 tend to be the chosen method for efflux studies due its ability of express P-gp, followed by studies conducted in Caco-2 models. However, it is necessary to evaluate the advantages and disadvantages of each method to generate sound results and to set the correlation in vitro x in situ x in vivo.

Functional and Biochemical Characterization of Cucumber Genes Encoding Two Copper ATPases CsHMA5.1 and CsHMA5.2

Plant copper P1B-type ATPases appear to be crucial for maintaining copper homeostasis within plant cells, but until now they have been studied mostly in model plant systems. Here, we present the molecular and biochemical characterization of two cucumber copper ATPases, CsHMA5.1 and CsHMA5.2, indicating a different function for HMA5-like proteins in different plants. When expressed in yeast, CsHMA5.1 and CsHMA5.2 localize to the vacuolar membrane and are activated by monovalent copper or silver ions and cysteine, showing different affinities to Cu(+) (Km ∼1 or 0.5 μM, respectively) and similar affinity to Ag(+) (Km ∼2.5 μM). Both proteins restore the growth of yeast mutants sensitive to copper excess and silver through intracellular copper sequestration, indicating that they contribute to copper and silver detoxification. Immunoblotting with specific antibodies revealed the presence of CsHMA5.1 and CsHMA5.2 in the tonoplast of cucumber cells. Interestingly, the root-specific CsHMA5.1 was not affected by copper stress, whereas the widely expressed CsHMA5.2 was up-regulated or down-regulated in roots upon copper excess or deficiency, respectively. The copper-induced increase in tonoplast CsHMA5.2 is consistent with the increased activity of ATP-dependent copper transport into tonoplast vesicles isolated from roots of plants grown under copper excess. These data identify CsHMA5.1 and CsHMA5.2 as high affinity Cu(+) transporters and suggest that CsHMA5.2 is responsible for the increased sequestration of copper in vacuoles of cucumber root cells under copper excess.

Interference with distinct steps of sphingolipid synthesis and signaling attenuates proliferation of U87MG glioma cells

Glioblastoma is the most common malignant brain tumor, which, despite combined radio- and chemotherapy, recurs and is invariably fatal for affected patients. Members of the sphingolipid (SL) family are potent effectors of glioma cell proliferation. In particular sphingosine-1-phosphate (S1P) and the corresponding G protein-coupled S1P receptors transmit proliferative signals to glioma cells. To investigate the contribution to glioma cell proliferation we inhibited the first step of de novo SL synthesis in p53wt and p53mut glioma cells, and interfered with S1P signaling specifically in p53wt U87MG cells. Subunit silencing (RNAi) or pharmacological antagonism (using myriocin) of serine palmitoyltransferase (SPT; catalyzing the first committed step of SL biosynthesis) reduced proliferation of p53wt but not p53mut GBM cells. In U87MG cells these observations were accompanied by decreased ceramide, sphingomyelin, and S1P content. Inhibition of SPT upregulated p53 and p21 expression and induced an increase in early and late apoptotic U87MG cells. Exogenously added S1P (complexed to physiological carriers) increased U87MG proliferation. In line, silencing of individual members of the S1P receptor family decreased U87MG proliferation. Silencing and pharmacological inhibition of the ATP-dependent cassette transporter A1 (ABCA1) that facilitates S1P efflux in astrocytes attenuated U87MG growth. Glyburide-mediated inhibition of ABCA1 resulted in intracellular accumulation of S1P raising the possibility that ABCA1 promotes S1P efflux in U87MG glioma cells thereby contributing to inside-out signaling. Our findings indicate that de novo SL synthesis, S1P receptor-mediated signaling, and ABCA1-mediated S1P efflux could provide pharmacological targets to interfere with glioma cell proliferation.

Development of Fourth Generation ABC Inhibitors from Natural Products: A Novel Approach to Overcome Cancer Multidrug Resistance

Multidrug resistance (MDR) in cancer caused due to overexpression of ABC drug transporters is a major problem in modern chemotherapy. Molecular investigations on MDR have revealed that the resistance is due to various transport proteins of the ABC superfamily which include Phosphoglycoprotein (P-gp/MDR1/ ABCB1), multidrug resistance-associated protein-1 (MRP1), and the breast cancer resistance protein (BCRP). They have been characterized functionally and are considered as major players in the development of MDR in cancer cells. These ATP-dependent transporter proteins cause MDR either by decreased uptake of the drug or increased efflux of the drug from the target organelles. Several MDR-reversing agents are being developed and are in various stages of clinical trials. The first three generations of ABC modulators such as quinine, verapamil, cyclosporine-A, tariquitor, PSC 833, LY335979, and GF120918 required to be administered in high doses to reverse MDR and were associated with adverse effects. Additionally, these modulators non-selectively inhibit ABC and adversely accumulate chemotherapeutic drugs in brain and kidney. Currently, research has stepped up towards reversing MDR by using natural products which exhibitted potential as chemosensitizers. Globally, there is a rich biodiversity of natural products which can be sourced for developing drugs. These products may provide more lead compounds with superior activity, foremost to the development of more effective therapies for MDR cancer cells. Here, we briefly review the status of natural products for reversing MDR modulators, and discuss the long term goal of MDR strategies in current clinical settings.

Editorial: multidrug resistance in cancer: pharmacological strategies from basic research to clinical issues.

EDITORIAL article Front. Oncol., 11 May 2015Sec. Pharmacology of Anti-Cancer Drugs https://doi.org/10.3389/fonc.2015.00105

In vitro effects of standardized extract of Bacopa monniera and its five individual active constituents on human P-glycoprotein activity

1. For centuries Bacopa monniera (BM) has been used as an herbal drug for the treatment of various mental ailments. A chemically standardized alcoholic extract of BM is clinically available over the counter herbal remedy for memory enhancement in children and adults. Consumption of herbal preparations has been reported to alter the function of membrane transporters, especially P-glycoprotein (P-gp), ATP-dependent drug efflux transporter responsible for the development of herb–drug interactions.2. In the present study, we evaluated the in vitro effect of BM extract and its five individual active constituents (namely, bacopaside I, bacopaside II and bacopasaponin C, bacoside A and bacoside A3) on P-gp function using luminescent P-gp ATPase assay and Rh123 transport assay across human MDR1 gene transfected LLC-GA5-COL150 cell line.3. It was observed that BM extract and its five individual constituents inhibited both basal activity as well as verapamil-stimulated ATPase activity, suggesting their affinity towards P-gp. Further, BM and its five active constituents inhibited the rhodamine 123 (Rh123) transport across LLC-GA5-COL150 cell monolayer with bacopaside II being the most potent inhibitor of P-gp, which decreased P-gp efflux ratio of Rh123 by fourfold in comparison to control.4. Our finding may prove beneficial in predicting the potential herb–drug interactions of BM on concomitant medication with P-gp substrate drugs in clinical settings.

The fuss over lipo"fuss"cin: not all autofluorescence is the same.

Since the first description of cellular autofluorescence over a century ago, we have now come to appreciate that autofluorescence should not be discarded as a biological artifact but embraced as a biological phenomenon with potentially important cellular relevance. Indeed, cellular and tissue autofluorescence has been attributed to a spectrum of unrelated molecules such as porphyrins, vitamins (vitamin A, riboflavin, thiamine), structural proteins, lipofuscin and ceroid pigments. We have recently shown that freshly isolated epithelial cancer stem cells (CSCs) bear autofluorescent vesicles in the cytoplasm. Our studies definitively prove that riboflavin and not lipofuscin is the source of autofluorescence in CSCs as the inhibition of ATP and not autophagy eliminates CSC autofluorescence, that the ATP-dependent transporter ABCG2, for which riboflavin is a substrate, is overexpressed in autofluorescent CSCs and co-localizes with the membrane of intracellular autofluorescent vesicles, the ABCG2-specific inhibitor Fumitremorgin C reversibly eliminates CSC autofluorescence, riboflavin is a substrate for ABCG2, and only the addition of riboflavin to vitamin-deprived CSC cultures is capable of restoring autofluorescence. Thus, the sum of these data unequivocally supports the conclusion that the source of CSC autofluorescence is the vitamin riboflavin.

Molecular factors influencing the affinity of flavonoid compounds on P-glycoprotein efflux transporter.

The most common mechanism of the so-called multidrug resistance (MDR), is mainly associated with an over expression of P-glycoprotein (Pgp). It is an ATP-dependent transport protein that limits the intracellular accumulation of a variety of structurally unrelated compounds within various organs and normal tissues such as kidney, small intestine and the blood brain barrier. Thus, the expression of Pgp has a major impact on the pharmacokinetic profile of many therapeutic agents and therefore, overcoming Pgp-mediated efflux constitutes an attractive means of potentially enhancing their therapeutic efficacy. The flavonoids comprise a large group of polyphenolic compounds that occur in plants and vegetables, and they have been shown to display a wide variety of biological activities. For example, anti-inflammatory, antioxidant, antiallergic, hepatoprotective, antithrombotic, antiviral, and anticarcinogenic activities. The interactions between flavonoids and Pgp have also been extensively studied and some quantitative structure-activity relationships (QSAR) have been reported. In the present work, we have employed 2D-QSAR analysis to evaluate the interactions between Pgp and several flavonoid compounds with the aim of identifying the molecular factors responsible for the Pgp-binding affinity evidenced by these compounds. Thus, the reported data for dissociation constants (KD) between Pgp and 62 flavonoid compounds were modeled by means of multiple regression analysis (MLR), and structures of the compounds under study were characterized by means of calculated physicochemical properties and several topological and constitutional descriptors, as well as geometrical and quantum chemical indexes. The obtained results suggest that the hydrophobic and especially geometric factors are of prime importance for binding, whereas in the case of flavonoid derivatives with flavone (flavonols), flavanone and isoflavone nuclei, the electronic factors are also involved in electron donor/acceptor interactions. In addition, in the case of chalcones, the results suggest that the affinity toward P-gp of such compounds is mainly governed by intermolecular dispersive interactions at the binding site.

Polymethyoxy Flavonoids: Possible Candidates for Overcoming Drug Resistance

The success of chemotherapy in cancer treatment is frequently limited by intrinsic or acquired multidrug resistance due to increased expression of a plasma membrane P-glycoprotein . P-glycoprotein is an ATP-dependent transporter that effluxes many lipophilic anticancer agents out of cells, thereby reducing intracellular drug concentration and results in cancer cell survival. It has been reported that certain kinds of natural compounds and their derivatives inhibit P-glycoprotein function, and overcome the multidrug resistance phenotype.

Nickel recognition by bacterial importer proteins.

Nickel supports the growth of microbes from a variety of very different growth environments that affect nickel speciation. The mechanisms of nickel uptake and the molecular bases for the selectivity of this process are emerging. The recent surge of Ni-importer protein structures provides an understanding of Ni-recognition in the initial binding step of the import process. This review compares the structural basis for Ni-recognition in the complexes (ABC and ECF-type) that dominate primary (ATP-dependent) transport, with a focus on how the structures suggest mechanisms for Ni selectivity. The structures raise key questions about the mechanisms of nickel-transfer reactions involved in import. There is also a discussion of key experimental approaches necessary to help establish the physiological importance of these structures.

P2X receptors trigger intracellular alkalization in isolated perfused mouse medullary thick ascending limb.

AimsExtracellular ATP is an important regulator of renal tubular transport. Recently, we found that basolateral ATP markedly inhibits Na+ and Cl− absorption in mouse medullary thick ascending limb (mTAL) via a P2X receptor. The underlying mechanism that mediates this ATP-dependent transport inhibition in mTAL is, however, unclear. The renal outer medullary K+ channel (ROMK) is sensitive to intracellular pH where a reduction leads to closing of ROMK. We speculated that P2X receptor stimulation in the TAL could lead to changes in pHi, leading to a reduction in NaCl transport.MethodsTo test this hypothesis, we measured pHi in single perfused mouse mTALs using the fluorescent ratiometric dye 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethylester.ResultsInterestingly, basolateral ATP (100 μm) caused a prominent, reversible intracellular alkalization of mTAL, with an average pHi increase of 0.14 ± 0.02 (n = 14). This was completely abolished by the P2X receptor antagonist periodate-oxidized ATP (50 μm). The P2X receptor-mediated intracellular alkalization required the activity of the apical Na+/H+ exchanger (NHE3). Typically, Gq-coupled receptors cause a significant acidification of tubular epithelial cells, which was confirmed in this study, by P2Y2 and Ca2+ sensing receptor stimulation.ConclusionThis study reports that stimulation of basolateral P2X receptors causes a substantial intracellular alkalization in the isolated perfused mouse mTAL. This intracellular alkalization is mediated through an increased apical NHE3 activity, similar to what we previously observed when tubular transport is inhibited with furosemide. This increased NHE3 activity causes H+ secretion in the mTAL and provides further support that the TAL is a site of urinary acidification.

Modulation of high affinity ATP-dependent cyclic nucleotide transporters by specific and non-specific cyclic nucleotide phosphodiesterase inhibitors

Intracellular cyclic nucleotides are eliminated by phosphodiesterases (PDEs) and by ATP Binding cassette transporters such as ABCC4 and ABCC5. PDE5 and ABCC5 have similar affinity for cGMP whereas ABCC5 has much higher affinity for cGMP compared with cAMP. Since the substrate (cGMP) is identical for these two eliminatory processes it is conceivable that various PDE inhibitors also modulate ABCC5-transport. Cyclic GMP is also transported by ABBC4 but the affinity is much lower with a Km 50–100 times higher than for that of ABBCC5. The present study aimed to determine Ki-values for specific or relative specific PDE5 inhibitors (vardenafil, tadalafil, zaprinast and dipyridamole) and the non-specific PDE inhibitors (IBMX, caffeine and theophylline) for ABCC5 andABCC4 transport. The transport of [3H]-cGMP (2µM) was concentration-dependently inhibited with the following Ki-values: vardenafil (0.62µM), tadalafil (14.1µM), zaprinast (0.68µM) and dipyridamole (1.2µM), IBMX (10µM), caffeine (48µM) and theophylline (69µM). The Ki-values for the inhibition of the [3H]-cAMP (2µM) transport were: vardenafil (3.4µM), tadalafil (194µM), zaprinast (2.8µM), dipyridamole (5.5µM), IBMX (16µM), caffeine (41µM) and theophylline (85µM). The specificity for ABCC5 we defined as ratio between Ki-values for inhibition of [3H]-cGMP and [3H]-cAMP transport. Tadalafil showed the highest specificity (Ki-ratio: 0.073) and caffeine the lowest (Ki-ratio: 1.2).

Gene replacement therapy for genetic hepatocellular jaundice.

Jaundice results from the systemic accumulation of bilirubin, the final product of the catabolism of haem. Inherited liver disorders of bilirubin metabolism and transport can result in reduced hepatic uptake, conjugation or biliary secretion of bilirubin. In patients with Rotor syndrome, bilirubin (re)uptake is impaired due to the deficiency of two basolateral/sinusoidal hepatocellular membrane proteins, organic anion-transporting polypeptide 1B1 (OATP1B1) and OATP1B3. Dubin-Johnson syndrome is caused by a defect in the ATP-dependent canalicular transporter, multidrug resistance-associated protein 2 (MRP2), which mediates the export of conjugated bilirubin into bile. Both disorders are benign and not progressive and are characterised by elevated serum levels of mainly conjugated bilirubin. Uridine diphospho-glucuronosyl transferase 1A1 (UGT1A1) is responsible for the glucuronidation of bilirubin; deficiency of this enzyme results in unconjugated hyperbilirubinaemia. Gilbert syndrome is the mild and benign form of inherited unconjugated hyperbilirubinaemia and is mostly caused by reduced promoter activity of the UGT1A1 gene. Crigler-Najjar syndrome is the severe inherited form of unconjugated hyperbilirubinaemia due to mutations in the UGT1A1 gene, which can cause kernicterus early in life and can be even lethal when left untreated. Due to major disadvantages of the current standard treatments for Crigler-Najjar syndrome, phototherapy and liver transplantation, new effective therapeutic strategies are under development. Here, we review the clinical features, pathophysiology and genetic background of these inherited disorders of bilirubin metabolism and transport. We also discuss the upcoming treatment option of viral gene therapy for genetic disorders such as Crigler-Najjar syndrome and the possible immunological consequences of this therapy.