ABSTRACT
Cleft palate surgery has traditionally presented numerous problems for cleft surgeons including ergonomics, limited visual fields restricting the opportunity for demonstration and teaching. Additionally, the move toward online teaching means the ability to record or livestream video is paramount. The following report of eight cleft palate repairs highlights the novel use of the Vitom 3D® microvideoscope as an innovative technique for cleft palate repair with our early experience demonstrating significant ergonomic and teaching benefits.
Subject(s)
Cleft Lip , Cleft Palate , Oral Surgical Procedures , Surgeons , Surgery, Oral , Cleft Lip/surgery , Cleft Palate/surgery , Ergonomics , HumansABSTRACT
Organic anion transporting polypeptides (OATPs) are polyspecific organic anion transporters, which are expressed in the blood-brain barrier, the choroid plexus, and other organs. The physiologic function of OATPs in extrahepatic tissues remains ambiguous. In rat retina, members of the OATP family are expressed. We therefore investigated the human retina for the expression of OATP1A2 and OATP2B1 and extended the study to human brain. Furthermore, we searched for peptide neurotransmitters as novel OATP substrates. OATP1A2 displayed a broad expression pattern in human retina as assessed by immunofluorescence localization. It is expressed in photoreceptor bodies and somas of amacrine cells. OATP1B2 expression is restricted to the inner nuclear layer and to the inner plexiform layer. Using paraffin sections from human cortex, cerebellum, and hippocampus, OATP1A2 was localized to neurons and neuronal processes, while OATP2B1 is expressed in endothelial cells of brain capillaries. Substance P and vasoactive intestinal peptide were identified as substrates for OATP1A2 and OATP2B1. Double-labeling immunofluorescence of human retina demonstrated the presence of substance P and of vasoactive intestinal peptides in neurons expressing OATP1A2 and OATP2B1, respectively. The expression of OATP1A2 and OATP2B1 in retinal neurons implies a role of these transporters in the reuptake of peptide neurotransmitters released from retinal neurons. The abundant expression of OATP1A2 in brain neurons points to the possibility that OATP1A2 could be involved in the homeostasis of neurosteroids. The high expression of OATP2B1 in brain capillaries supports an important function of OATPs in substance penetration across the blood-brain barrier.
Subject(s)
Brain/metabolism , Organic Anion Transporters/metabolism , Retina/metabolism , Substance P/metabolism , Vasoactive Intestinal Peptide/metabolism , Adult , Aged , Aged, 80 and over , Animals , Biological Transport, Active , Female , Humans , Male , Middle Aged , Organ Specificity , Organic Anion Transporters/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , XenopusABSTRACT
This article summarizes the impact of the pharmacogenetics of drug transporters expressed in the enterohepatic circulation on the pharmacokinetics and pharmacodynamics of drugs. The role of pharmacogenetics in the function of drug transporter proteins in vitro is now well established and evidence is rapidly accumulating from in vivo pharmacokinetic studies, which suggests that genetic variants of drug transporter proteins can translate into clinically relevant phenotypes. However, a large amount of conflicting information on the clinical relevance of drug transporter proteins has so far precluded the emergence of a clear picture regarding the role of drug transporter pharmacogenetics in medical practice. This is very well exemplified by the case of P-glycoprotein (MDR1, ABCB1). The challenge is now to develop pharmacogenetic models with sufficient predictive power to allow for translation into drug therapy. This will require a combination of pharmacogenetics of drug transporters, drug metabolism and pharmacodynamics of the respective drugs.
Subject(s)
Enterohepatic Circulation/physiology , Membrane Transport Proteins/genetics , Membrane Transport Proteins/metabolism , Pharmaceutical Preparations/metabolism , Pharmacogenetics/methods , ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Animals , Biological Transport/physiology , Humans , Polymorphism, Genetic/physiologyABSTRACT
Intrahepatic cholestasis of pregnancy and drug-induced cholestasis are two clinically important forms of acquired cholestatic liver disease. The understanding of the underlying mechanisms of acquired cholestasis has recently made considerable progress by the identification of canalicular ATP-binding cassette (ABC) transporters as likely targets for these forms of cholestasis. Cholestasis of pregnancy is linked to estrogen and progesterone metabolites. These metabolites have been shown to impair the bile salt export pump (BSEP) function by an indirect mechanism. In addition, genetic variants (as well as mutants) of the genes coding for the phosphatidylcholine translocator MDR3 and BSEP and for the farnesoid X receptor, which is critical in the transcriptional activation of MDR3 ( ABCB4) and BSEP ( ABCB11) have been associated with intrahepatic cholestasis of pregnancy. The pathogenesis of drug-induced liver injury encompasses a wide spectrum of mechanisms, some of which are still poorly understood. BSEP is now known to be subject to drug inhibition in susceptible patients. Information on genetic factors rendering individuals susceptible to inhibition of BSEP by drugs or their metabolites is still scarce. Besides rare mutations that have been linked to drug-induced cholestasis, the common p.V444A polymorphism of BSEP has been identified as a potential risk factor. In this review, the authors summarize key concepts of physiology of bile formation, diagnostic principles to indentify these forms of acquired cholestasis, as well as pathogenetic mechanisms leading to intrahepatic cholestasis of pregnancy or drug-induced cholestasis. In addition, they review the current knowledge on genetic susceptibility factors for these two forms of cholestasis.
Subject(s)
Cholestasis, Intrahepatic/genetics , Cholestasis/chemically induced , Cholestasis/genetics , Pregnancy Complications/genetics , ATP Binding Cassette Transporter, Subfamily B/physiology , ATP Binding Cassette Transporter, Subfamily B, Member 11 , ATP-Binding Cassette Transporters/physiology , Bile/metabolism , Bile Acids and Salts/metabolism , Cholestasis/physiopathology , Cholestasis, Intrahepatic/diagnosis , Cholestasis, Intrahepatic/physiopathology , Female , Genetic Predisposition to Disease , Hepatocytes/drug effects , Humans , Organic Anion Transporters/physiology , Pregnancy , Pregnancy Complications/diagnosis , Pregnancy Complications/physiopathologyABSTRACT
UNLABELLED: The canalicular plasma membrane is constantly exposed to bile acids acting as detergents. Bile acids are essential to mediate release of biliary lipids from the canalicular membrane. Membrane microdomains (previously called lipid rafts) are biochemically defined by their resistance to detergent solubilization at cold temperature. We aimed to investigate the canalicular plasma membrane for the presence of microdomains, which could protect this membrane against the detergent action of bile acids. Highly purified rat liver canalicular plasma membrane vesicles were extracted with 1% Triton X-100 or 1% Lubrol WX at 4 degrees C and subjected to flotation through sucrose step gradients. Both detergents yielded detergent-resistant membranes containing the microdomain markers alkaline phosphatase and sphingomyelin. However, cholesterol was resistant to Lubrol WX solubilization, whereas it was only marginally resistant to solubilization by Triton X-100. The microdomain marker caveolin-1 was localized to the canalicular plasma membrane domain and was resistant to Lubrol WX, but to a large extent solubilized by Triton X-100. The two additional microdomain markers, reggie-1 and reggie-2, were localized to the basolateral and canalicular plasma membrane and were partially resistant to Lubrol WX but resistant to Triton X-100. The canalicular transporters bile salt export pump, multidrug resistance protein 2, multidrug resistance-associated protein 2, and Abcg5 were largely resistant to Lubrol WX but were solubilized by Triton X-100. CONCLUSION: These results indicate the presence of two different types of microdomains in the canalicular plasma membrane: "Lubrol-microdomains" and "Triton-microdomains". "Lubrol-microdomains" contain the machinery for canalicular bile formation and may be the starting place for canalicular lipid secretion.
Subject(s)
ATP-Binding Cassette Transporters/metabolism , Bile Canaliculi/metabolism , Detergents/pharmacology , Hepatocytes/metabolism , Membrane Microdomains/metabolism , Animals , Caveolin 1/metabolism , Male , Membrane Microdomains/drug effects , Membrane Proteins/metabolism , Octoxynol/pharmacology , Polyethylene Glycols/pharmacology , Rats , Rats, Sprague-DawleyABSTRACT
Organic anion transporting polypeptides (humans OATPs, rodents Oatps) are expressed in most mammalian tissues and mediate cellular uptake of a wide variety of amphipathic organic compounds such as bile salts, steroid conjugates, oligopeptides, and a large list of drugs, probably by acting as anion exchangers. In the present study we aimed to investigate the role of the extracellular pH on the transport activity of nine human and four rat OATPs/Oatps. Furthermore, we aimed to test the concept that OATP/Oatp transport activity is accompanied by extrusion of bicarbonate. By using amphibian Xenopus laevis oocytes expressing OATPs/Oatps and mammalian cell lines stably transfected with OATPs/Oatps, we could demonstrate that in all OATPs/Oatps investigated, with the exception of OATP1C1, a low extracellular pH stimulated transport activity. This stimulation was accompanied by an increased substrate affinity as evidenced by lower apparent Michaelis-Menten constant values. OATP1C1 is lacking a highly conserved histidine in the third transmembrane domain, which was shown by site-directed mutagenesis to be critically involved in the pH dependency of OATPs/Oatps. Using online intracellular pH measurements in OATP/Oatp-transfected Chinese Hamster Ovary (CHO)-K1 cells, we could demonstrate the presence of a 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid-sensitive chloride/bicarbonate exchanger in CHO-K1 cells and that OATP/Oatp-mediated substrate transport is paralleled by bicarbonate efflux. We conclude that the pH dependency of OATPs/Oatps may lead to a stimulation of substrate transport in an acidic microenvironment and that the OATP/Oatp-mediated substrate transport into cells is generally compensated or accompanied by bicarbonate efflux.
Subject(s)
Organic Anion Transporters/metabolism , 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid/pharmacology , Acetazolamide/pharmacology , Amiloride/pharmacology , Animals , Bicarbonates/metabolism , Biological Transport , CHO Cells , Carbonic Anhydrase Inhibitors/pharmacology , Carbonic Anhydrases/metabolism , Chlorides/metabolism , Cricetinae , Cricetulus , Dogs , Female , Histidine , Humans , Hydrogen-Ion Concentration , Kinetics , Mutagenesis, Site-Directed , Mutation , Organic Anion Transporters/antagonists & inhibitors , Organic Anion Transporters/chemistry , Organic Anion Transporters/genetics , Protein Structure, Tertiary , Rats , Sodium-Hydrogen Exchangers/antagonists & inhibitors , Sodium-Hydrogen Exchangers/metabolism , Transfection , Xenopus laevisABSTRACT
AIM: To study the association of three common ABCB11 and ABCC2 polymorphisms (ABCB11: 1331T>C --> V444A; ABCC2: 3563T>A --> V1188E and 4544G>A --> C1515Y) with intrahepatic cholestasis of pregnancy (ICP) and contraceptive-induced cholestasis (CIC). METHODS: ABCB11 and ABCC2 genotyping data were available from four CIC patients and from 42 and 33 ICP patients, respectively. Allele-frequencies of the studied polymorphisms were compared with those in healthy pregnant controls and Caucasian individuals. Furthermore, serum bile acid levels were correlated with the presence or absence of the 1331 C allele. RESULTS: The ABCB11 1331T>C polymorphism was significantly more frequent in cholestatic patients than in pregnant controls: C allele 76.2% (CI, 58.0-94.4) vs 51.3% (CI 35.8-66.7), respectively (P = 0.0007); and CC allele 57.1% (CI 36.0-78.3) vs 20% (CI 7.6-32.4), respectively (P = 0.0065). All four CIC patients were homozygous carriers of the C allele. In contrast, none of the studied ABCC2 polymorphism was overrepresented in ICP or CIC patients. Higher serum bile acid levels were found in carriers of the 1331CC genotype compared to carriers of the TT genotype. CONCLUSION: Our data support a role for the ABCB11 1331T>C polymorphism as a susceptibility factor for the development of estrogen-induced cholestasis, whereas no such association was found for ABCC2. Serum bile acid and gamma-glutamyl transferase levels might help to distinguish ABCB4- and ABCB11-related forms of ICP and CIC.
Subject(s)
ATP-Binding Cassette Transporters/genetics , Cholestasis, Intrahepatic/genetics , Contraceptive Agents, Female/adverse effects , Pregnancy Complications/genetics , ATP Binding Cassette Transporter, Subfamily B, Member 11 , Adolescent , Adult , Cholestasis, Intrahepatic/chemically induced , Female , Genetic Predisposition to Disease , Heterozygote , Humans , Membrane Transport Proteins/genetics , Multidrug Resistance-Associated Protein 2 , Multidrug Resistance-Associated Proteins/genetics , Polymorphism, Genetic , Pregnancy , Pregnancy Complications/chemically inducedABSTRACT
The liver has a large regenerative capacity in response to injury. However, in severe cases of liver injury, its regenerative capacity may prove insufficent and the liver injury may progress to liver failure, and in such situations liver transplantation is the only treatment option. An alternative, less invasive approach may be transplantation of hepatocytes or hepatocyte precursor cells. In the adult liver two candidate progenitor cells have been identified: oval cells and small hepatocytes. The former are induced by liver injury under conditions preventing cell division of mature hepatocytes, while the latter are present in small numbers in normal liver. Both cell types have the capacity to expand and differentiate into hepatocytes. In recent years evidence has been presented that bone-marrow derived stem cells can also be expanded and differentiated into hepatocyte progenitor cells. Such cells may be a source for hepatocyte transplantation and hence have the potential to offer a novel therapy for liver failure.
ABSTRACT
OBJECTIVES: Increasing evidence suggests that a genetically determined functional impairment of the hepatocellular efflux transporters bile salt export pump (BSEP, ABCB11) and multidrug resistance protein 3 (MDR3, ABCB4) play a pathophysiological role in the development of drug-induced liver injury. The aim of this study was therefore to describe the extent of genetic variability in ABCB11 and ABCB4 in patients with drug-induced liver injury and to in vitro functionally characterize newly detected ABCB11 mutations and polymorphisms. METHODS: ABCB11 and ABCB4 were sequenced in 23 patients with drug-induced cholestasis and 13 patients with drug-induced hepatocellular injury. Ninety-five healthy Caucasians served as the control group. Reference and mutant BSEP were expressed in Sf9 cells and ATP-dependent transport of [H]-taurocholate was measured in a rapid filtration assay. RESULTS: Four highly conserved nonsynonymous mutations were specific for drug-induced liver injury [ABCB11: D676Y (drug-induced cholestasis) and G855R (drug-induced cholestasis); ABCB4: I764L (drug-induced cholestasis) and L1082Q (drug-induced hepatocellular injury)]. Furthermore, a polymorphism in exon 13 of ABCB11 (V444A), which is associated with decreased hepatic BSEP expression was significantly more frequent in drug-induced cholestasis patients than in drug-induced hepatocellular injury patients and healthy controls (76 versus 50 and 59% in drug-induced cholestasis patients, drug-induced hepatocellular injury patients and healthy controls, respectively; P<0.05). The in-vitro transport activity of the V444A and the D676Y BSEP constructs was similar, whereas the G855R mutation was nonfunctional. CONCLUSION: In summary, our data support a role of ABCB11 and ABCB4 mutations and polymorphisms in drug-induced cholestasis. Genotyping of selected patients with acquired cholestasis might help to identify individuals with a genetic predisposition.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B/genetics , ATP-Binding Cassette Transporters/genetics , Chemical and Drug Induced Liver Injury , Cholestasis/chemically induced , Mutation , Polymorphism, Genetic , ATP Binding Cassette Transporter, Subfamily B, Member 11 , Adolescent , Adult , Aged , Aged, 80 and over , Amino Acid Sequence , Cholestasis/genetics , Cholestasis/pathology , Female , Genetic Predisposition to Disease , Humans , Liver Diseases/genetics , Liver Diseases/pathology , Male , Middle Aged , Molecular Sequence Data , Protein Structure, Secondary , Taurocholic Acid/metabolism , White PeopleABSTRACT
UNLABELLED: Intrahepatic cholestasis of pregnancy (ICP) is defined as pruritus and elevated bile acid serum concentrations in late pregnancy. Splicing mutations have been described in the multidrug resistance p-glycoprotein 3 (MDR3, ABCB4) gene in up to 20% of ICP women. Pedigrees studied were not large enough for linkage analysis. Ninety-seven family members of a woman with proven ICP were asked about pruritus in earlier pregnancies, birth complications and symptomatic gallstone disease. The familial cholestasis type 1 (FIC1, ATP8B1) gene, bile salt export pump (BSEP, ABCB11) and MDR3 gene were analyzed in 55 relatives. We identified a dominant mode of inheritance with female restricted expression and a new intronic MDR3 mutation c.3486+5G>A resulting in a 54 bp (3465-3518) inframe deletion via cryptic splicing site activation. Linkage analysis of the ICP trait versus this intragenic MDR3 variant yielded a LOD score of 2.48. A Bayesian analysis involving MDR3, BSEP, FIC1 and an unknown locus gave a posterior probability of >0.9966 in favor of MDR3 as causative ICP locus. During the episode of ICP the median gamma-glutamyl transpeptidase (gamma-GT) activity was 10 U/l (95% CI, 6.9 to 14.7 U/l) in the index woman. Four stillbirths were reported in seven heterozygous women (22 pregnancies) and none in five women (14 pregnancies) without MDR3 mutation. Symptomatic gallstone disease was more prevalent in heterozygous relatives (7/21) than in relatives without the mutation (1/34), (P = 0.00341). CONCLUSION: This study demonstrates that splicing mutations in the MDR3 gene can cause ICP with normal gamma-GT and may be associated with stillbirths and gallstone disease.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B/genetics , ATP-Binding Cassette Transporters/genetics , Cholestasis, Intrahepatic/genetics , Genetic Linkage , Mutation/genetics , Pregnancy Complications/genetics , ATP Binding Cassette Transporter, Subfamily B/analysis , ATP Binding Cassette Transporter, Subfamily B/chemistry , ATP-Binding Cassette Transporters/analysis , ATP-Binding Cassette Transporters/chemistry , Adult , Amino Acid Sequence , Base Sequence , DNA/genetics , Exons/genetics , Female , Genetic Predisposition to Disease , Genotype , Humans , Molecular Sequence Data , Pedigree , Phenotype , Pregnancy , Stillbirth/geneticsABSTRACT
In the present study we isolated two splice variants of organic anion transporting polypeptide 3A1 (OATP3A1_v1 and OATP3A1_v2) from human brain. OATP3A1_v2 lacks 18 amino acids (aa) at the COOH-terminal end (692 aa) but is otherwise similar in sequence to OATP3A1_v1 (710 aa). OATP3A1_v1 exhibits a wide tissue distribution, with expression in testis, various brain regions, heart, lung, spleen, peripheral blood leukocytes, and thyroid gland, whereas OATP3A1_v2 is predominantly expressed in testis and brain. On the cellular and subcellular levels OATP3A1_v1 could be immunolocalized in testicular germ cells, the basolateral plasma membrane of choroid plexus epithelial cells, and neuroglial cells of the gray matter of human frontal cortex. Immunolocalization of OATP3A1_v2 included Sertoli cells in testis, apical and/or subapical membranes in choroid plexus epithelial cells, and neurons (cell bodies and axons) of the gray and white matter of human frontal cortex. The rodent ortholog Oatp3a1 was also widely distributed in rat brain, and its localization included somatoneurons as well as astroglial cells. Transport studies in cRNA-injected Xenopus laevis oocytes and in stably transfected Chinese hamster ovary FlpIn cells revealed a similar broad substrate specificity for both splice variants. Transported substrates include prostaglandin (PG)E(1) and PGE(2), thyroxine, and the cyclic oligopeptides BQ-123 (endothelin receptor antagonist) and vasopressin. These studies provide further evidence for the involvement of OATPs in oligopeptide transport. They specifically suggest that OATP3A1 variants might be involved in the regulation of extracellular vasopressin concentration in human brain and thus might influence the neuromodulation of neurotransmission by cerebral neuropeptides such as vasopressin.
Subject(s)
Alternative Splicing , Brain/metabolism , Organic Anion Transporters/physiology , Alprostadil/metabolism , Amino Acid Sequence , Animals , CHO Cells , Cricetinae , Cricetulus , Dinoprostone/metabolism , Endothelin Receptor Antagonists , Humans , Molecular Sequence Data , Organ Specificity , Organic Anion Transporters/genetics , Organic Anion Transporters/metabolism , Rats , Vasopressins/metabolism , Xenopus laevisABSTRACT
Canalicular secretion of bile salts mediated by the bile salt export pump Bsep constitutes the major driving force for the generation of bile flow. Bsep is a member of the B-family of the super family of ATP-binding cassette transporters and is classified as ABCB11. Bsep has a narrow substrate specificity, which is largely restricted to bile salts. Bsep is extensively regulated at the transcriptional and posttranscriptional level, which directly modulates canalicular bile formation. Pathophysiological alterations of Bsep by either inherited mutations or acquired processes such as inhibition by drugs or disease-related down regulation may lead to a wide spectrum of mild to severe forms of liver disease. Furthermore, many genetic variants of Bsep are known, some of which potentially render individuals susceptible to acquired forms of liver disease.
Subject(s)
ATP-Binding Cassette Transporters/physiology , Bile Acids and Salts/metabolism , ATP Binding Cassette Transporter, Subfamily B, Member 11 , ATP-Binding Cassette Transporters/antagonists & inhibitors , ATP-Binding Cassette Transporters/genetics , Amino Acid Sequence , Animals , Bile Canaliculi/physiology , Cholestasis, Intrahepatic/genetics , Gene Expression Regulation , Humans , Liver/embryology , Mice , Molecular Sequence Data , RNA-Binding Proteins/physiology , Rats , Transcription, GeneticABSTRACT
The liver has a large regenerative capacity in response to injury. However, in severe cases of liver injury, its regenerative capacity may prove insufficent and the liver injury may progress to liver failure, and in such situations liver transplantation is the only treatment option. An alternative, less invasive approach may be transplantation of hepatocytes or hepatocyte precursor cells. In the adult liver two candidate progenitor cells have been identified: oval cells and small hepatocytes. The former are induced by liver injury under conditions preventing cell division of mature hepatocytes, while the latter are present in small numbers in normal liver. Both cell types have the capacity to expand and differentiate into hepatocytes. In recent years evidence has been presented that bone-marrow derived stem cells can also be expanded and differentiated into hepatocyte progenitor cells. Such cells may be a source for hepatocyte transplantation and hence have the potential to offer a novel therapy for liver failure.
Subject(s)
Bone Marrow Cells , Hepatocytes/cytology , Stem Cells/cytology , Animals , Bone Marrow Transplantation , Cell Differentiation , Cells, Cultured/cytology , Humans , Kidney Failure, Chronic/surgery , Mice , Models, Animal , Stem Cell TransplantationABSTRACT
Drug-induced liver injury is an important clinical problem with significant morbidity and mortality. Whereas for most hepatocellular forms of drug-induced hepatic injury the underlying pathophysiological mechanism is poorly understood, there is increasing evidence that cholestatic forms of drug-induced liver damage result from a drug- or metabolite-mediated inhibition of hepatobiliary transporter systems. In addition to their key role in determining hepatic drug exposure and clearance, the coordinated action of these transport systems is essential for bile formation and the biliary secretion of cholephilic compounds and xenobiotics. Any drug-mediated functional disturbance of these processes can lead to an intracellular accumulation of potentially harmful bile constituents and result in the development of cholestatic liver cell damage. In addition to direct drug-mediated inhibition of hepatocellular transport, function of these transporters can be altered by pre-existing hepatic disease and genetic factors, which contribute to the development of drug-induced cholestasis in susceptible individuals. This review summarizes current knowledge about the function of hepatobiliary uptake and efflux systems and discusses factors that might predispose to drug-induced cholestasis.
Subject(s)
Bile/metabolism , Biliary Tract/drug effects , Cholestasis/chemically induced , Liver/drug effects , Organic Anion Transporters/drug effects , Animals , Bile Acids and Salts/metabolism , Biliary Tract/physiology , Biological Transport/drug effects , Cholestasis/genetics , Cholestasis/physiopathology , Humans , Liver/physiology , Organic Anion Transporters/physiology , Risk FactorsABSTRACT
Organic anion transporting polypeptide (OATP) superfamily member 2B1 (OATP2B1) mediates the uptake of steroid hormone precursors and selected drugs in the placenta, liver, mammary gland, brain, and intestine. This action is modulated by sulfhydryl reagents. Common to all OATPs is a large extracellular loop between transmembrane domains IX and X with 10 conserved cysteines. To elucidate the structure-function relationship of this cysteine rich ectodomain, a truncated OATP2B1 lacking 10 extracellular cysteines (OATP2B1(Delta489-557)) and 10 OATP2B1 mutants containing individual Cys-to-Ala substitutions were generated and expressed in CHO-K1 cells. The immunolocalization, cell-surface expression, transport activity, and free cysteine labeling with N-biotinoylaminoethylmethane-thiosulfonate of mutant proteins and wild-type OATP2B1 were compared. OATP2B1(Delta489-557) accumulated intracellularly. Nine Cys-to-Ala substitutions, C489A, C495A, C504A, C516A, C520A, C539A, C541A, C553A, and C557A, were misprocessed, appearing predominantly as core-glycosylated, 60-kDa proteins and as 180-kDa complexes. Only C493A was a fully glycosylated 75-kDa protein expressed at the cell surface. Thapsigargin partially corrected the misprocessing of mutants. Compared with OATP2B1, C493A and C557A transported estrone-3-sulfate and dehydroepiandrosterone sulfate less efficiently, whereas all other mutants were functionally impaired. MTSEA labeled free cysteines in all Cys-to-Ala mutants but not in OATP2B1, suggesting that all 10 extracellular cysteines are normally disulfide-bonded. Our findings show that the trafficking and function of OATP2B1 is vulnerable to changes in the cysteine residues of extracellular loop IX-X.
Subject(s)
Cysteine/metabolism , Liver-Specific Organic Anion Transporter 1/chemistry , Liver-Specific Organic Anion Transporter 1/metabolism , Organic Anion Transporters/chemistry , Organic Anion Transporters/metabolism , Animals , Biological Transport/drug effects , CHO Cells , Cricetinae , Cricetulus , Cross-Linking Reagents , Disulfides/metabolism , Glycosylation/drug effects , Humans , Microscopy, Confocal , Mutation/drug effects , Mutation/genetics , Peptides/chemistry , Peptides/metabolism , Prostaglandins A/pharmacology , Structure-Activity Relationship , Thapsigargin/pharmacologyABSTRACT
Interindividual variability in hepatic canalicular transporter expression might predispose to the development of hepatic disorders such as acquired forms of intrahepatic cholestasis. We therefore investigated expression patterns of bile salt export pump (BSEP, ABCB11), multidrug resistance protein 3 (MDR3, ABCB4), multidrug resistance associated protein 2 (MRP2, ABCC2) and multidrug resistance protein 1 (MDR1, ABCB1) in healthy liver tissue of a white population. Protein expression levels were correlated with specific single nucleotide polymorphisms (SNPs) in the corresponding transporter genes. Hepatic protein expression levels from 110 individuals undergoing liver resection were assessed by Western blot analysis of liver plasma membranes enriched in canalicular marker enzymes. Each individual was genotyped for the following synonymous (s) and nonsynonymous (ns) SNPs: ABCB11: (ns:1457T>C and 2155A>G), ABCB4: (ns:3826A>G) and ABCC2 (ns:1286G>A,3600T>A and 4581G>A) and ABCB1 (ns:2677G>T/A and s:3435C>T). Transporter expression followed unimodal distribution. However, of all tested individuals 30% exhibited a high expression and 32% a low or very low expression phenotype for at least one of the four investigated transport proteins. Transporter expression levels did not correlate with age, sex, underlying liver disease, or presurgery medication. However, low BSEP expression was associated with the 1457C-allele in ABCB11 (P = .167) and high MRP2 expression was significantly correlated with the 3600A and 4581A ABCC2 variants (P = .006). In conclusion, the results demonstrate a considerable interindividual variability of canalicular transporter expression in normal liver. Furthermore, data suggest a polymorphic transporter expression pattern, which might constitute a risk factor for the development of acquired forms of cholestatic liver diseases.
Subject(s)
ATP-Binding Cassette Transporters/genetics , Bile Canaliculi/metabolism , DNA/genetics , Gene Expression , Liver/metabolism , Adolescent , Adult , Age Factors , Aged , Aged, 80 and over , Alleles , Biomarkers/metabolism , Blotting, Western , Cholestasis, Intrahepatic/metabolism , Cholestasis, Intrahepatic/surgery , Female , Hepatectomy , Humans , Liver/surgery , Male , Middle Aged , Multidrug Resistance-Associated Protein 2 , Polymerase Chain Reaction , Prognosis , Sex FactorsABSTRACT
BACKGROUND/AIMS: Inherited dysfunction of the bile salt export pump BSEP (ABCB11) causes a progressive and a benign form of familial intrahepatic cholestasis, denominated as PFIC2 and BRIC2, respectively. We functionally characterized novel ABCB11 mutations encountered in two patients with a PFIC2 and a BRIC2 phenotype, respectively. METHODS: BSEP expression was determined in liver biopsies by immunohistochemistry. ABCB11 mutations were functionally characterized by taurocholate transport in SF9 cells transfected with human ABCB11. RESULTS: The PFIC2 patient was compound heterozygous for a splicing mutation in intron 4 ((+3)A > C) combined with an early stop codon at position 930 (R930X), while the BRIC2 patient was compound heterozygous for two nonsynonymous mutations in exon 9 (E297G) and exon 12 (R432T), respectively. Hepatic BSEP expression was absent in PFIC2 and preserved in BRIC2. In BRIC2, taurocholate transport was decreased to 13% and 20% of reference levels for R432T and E297G, respectively. CONCLUSIONS: The intron 4 (+3)A > C, R930X and R432T represent previously undescribed mutations of the ABCB11 gene that confer a PFIC2 and a BRIC2 phenotype, respectively. By combining functional in-vitro characterization with immunohistochemical detection of variant BSEP we provide direct evidence for the role of ABCB11 mutations in the pathogenesis of different forms of intrahepatic cholestasis.
Subject(s)
ATP-Binding Cassette Transporters/genetics , Cholestasis, Intrahepatic/genetics , Gene Expression Regulation , Mutation , ATP Binding Cassette Transporter, Subfamily B, Member 11 , ATP-Binding Cassette Transporters/chemistry , ATP-Binding Cassette Transporters/metabolism , Amino Acid Sequence , Base Sequence , Bile Acids and Salts/metabolism , Biological Transport , DNA Primers , Female , Humans , Male , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Pedigree , Protein Conformation , Recombinant Proteins/chemistry , Recombinant Proteins/metabolismABSTRACT
The secretion of bile is the result of active hepatocellular transport processes, most of which occur across the canalicular membrane of liver cells. Disturbance of the function and/or expression of these transporters leads to the intracellular accumulation of toxic bile acids, thereby promoting cholestatic liver cell injury. Genetically determined alterations of hepatobiliary transporter function are increasingly recognized as important risk factors for an individual's susceptibility to develop cholestasis. It has become evident that, besides the established pathogenic role of mutations in canalicular transporter genes in progressive and benign forms of familial intrahepatic cholestasis, genetics may also play an important role in acquired cholestatic syndromes, such as intrahepatic cholestasis of pregnancy or drug-induced cholestasis. This overview summarizes the physiologic function and regulation of human hepatobiliary transport systems and discusses the impact of their genetic variations for the pathophysiology of different cholestatic syndromes.
Subject(s)
Cholestasis/metabolism , Liver/metabolism , Bile Acids and Salts/metabolism , Biological Transport, Active , Cholangitis, Sclerosing/metabolism , Female , Hepatocytes/metabolism , Humans , Liver Cirrhosis, Biliary/metabolism , Male , Pregnancy , Pregnancy Complications/metabolismABSTRACT
OBJECTIVE: During biliary cirrhosis in rats, organic anion-transporting peptides (Oatps) and ATP-dependent multidrug resistance-associated protein 2 (Mrp2) that are likely to transport the contrast agent Gd-BOPTA through hepatocytes are down-regulated. However, the consequences of such down-regulation on the signal intensity (SI) enhancement are unknown. Consequently, the aim of our study was to measure the hepatic SI enhancement during Gd-BOPTA perfusion as well as the Oatp and Mrp2 expression in normal and cirrhotic livers. MATERIALS AND METHODS: The hepatic SI enhancement during Gd-BOPTA perfusion was measured in livers isolated from normal rats and rats that had a bile duct ligation (BDL) 15, 30, and 60 days before the perfusion. Hepatic injury and transporter expression were measured in control and cirrhotic rats. RESULTS: BDL induced a severe hepatic injury that increased over time with a down-regulation of the transporter expression. The extracellular space (assessed by Gd-DTPA perfusion) increased with the severity of the disease. Gd-BOPTA-induced SI enhancement remained similar in BDL-15 and BDL-30 rats than in control rats but significantly decreased in severe cirrhosis (BDL-60 rats). In comparison, the Mn-DPDP-induced SI enhancement decreases proportionally to the severity of the disease. CONCLUSION: During biliary cirrhosis, Gd-BOPTA-induced SI enhancement could not be related to the hepatic expression of transporters.