RESUMO
Na+/taurocholate cotransporting polypeptide (NTCP) is a member of the solute carrier (SLC) family 10 transporters (gene symbol SLC10A1) and is responsible for the sodium-dependent uptake of bile salts across the basolateral membrane of hepatocytes. In addition to its primary transporter function, NTCP is the high-affinity hepatic receptor for hepatitis B (HBV) and hepatitis D (HDV) viruses and, therefore, is a prerequisite for HBV/HDV virus entry into hepatocytes. The inhibition of HBV/HDV binding to NTCP and internalization of the virus/NTCP receptor complex has become a major concept in the development of new antiviral drugs called HBV/HDV entry inhibitors. Hence, NTCP has emerged as a promising target for therapeutic interventions against HBV/HDV infections in the last decade. In this review, recent findings on protein-protein interactions (PPIs) between NTCP and cofactors relevant for entry of the virus/NTCP receptor complex are summarized. In addition, strategies aiming to block PPIs with NTCP to dampen virus tropism and HBV/HDV infection rates are discussed. Finally, this article suggests novel directions for future investigations evaluating the functional contribution of NTCP-mediated PPIs in the development and progression of HBV/HDV infection and subsequent chronic liver disorders.
Assuntos
Hepatite B , Simportadores , Humanos , Antivirais/farmacologia , Células Hep G2 , Hepatite B/tratamento farmacológico , Hepatite B/metabolismo , Vírus da Hepatite B , Vírus Delta da Hepatite/metabolismo , Hepatócitos/metabolismo , Peptídeos , Simportadores/metabolismo , Ácido Taurocólico/metabolismo , Ácido Taurocólico/uso terapêutico , Internalização do VírusRESUMO
The sodium-dependent organic anion transporter (SOAT, gene symbol SLC10A6) specifically transports 3'- and 17'-monosulfated steroid hormones, such as estrone sulfate and dehydroepiandrosterone sulfate, into specific target cells. These biologically inactive sulfo-conjugated steroids occur in high concentrations in the blood circulation and serve as precursors for the intracrine formation of active estrogens and androgens that contribute to the overall regulation of steroids in many peripheral tissues. Although SOAT expression has been detected in several hormone-responsive peripheral tissues, its quantitative contribution to steroid sulfate uptake in different organs is still not completely clear. Given this fact, the present review provides a comprehensive overview of the current knowledge about the SOAT by summarizing all experimental findings obtained since its first cloning in 2004 and by processing SOAT/SLC10A6-related data from genome-wide protein and mRNA expression databases. In conclusion, despite a significantly increased understanding of the function and physiological significance of the SOAT over the past 20 years, further studies are needed to finally establish it as a potential drug target for endocrine-based therapy of steroid-responsive diseases such as hormone-dependent breast cancer.
Assuntos
Transportadores de Ânions Orgânicos , Transportadores de Ânions Orgânicos/genética , Transportadores de Ânions Orgânicos/metabolismo , Transporte Biológico , Sódio/metabolismoRESUMO
Shrews, insectivorous small mammals, pertain to an ancient mammalian order. We screened 693 European and African shrews for hepatitis B virus (HBV) homologs to elucidate the enigmatic genealogy of HBV. Shrews host HBVs at low prevalence (2.5%) across a broad geographic and host range. The phylogenetically divergent shrew HBVs comprise separate species termed crowned shrew HBV (CSHBV) and musk shrew HBV (MSHBV), each containing distinct genotypes. Recombination events across host orders, evolutionary reconstructions, and antigenic divergence of shrew HBVs corroborated ancient origins of mammalian HBVs dating back about 80 million years. Resurrected CSHBV replicated in human hepatoma cells, but human- and tupaia-derived primary hepatocytes were resistant to hepatitis D viruses pseudotyped with CSHBV surface proteins. Functional characterization of the shrew sodium taurocholate cotransporting polypeptide (Ntcp), CSHBV/MSHBV surface peptide binding patterns, and infection experiments revealed lack of Ntcp-mediated entry of shrew HBV. Contrastingly, HBV entry was enabled by the shrew Ntcp. Shrew HBVs universally showed mutations in their genomic preCore domains impeding hepatitis B e antigen (HBeAg) production and resembling those observed in HBeAg-negative human HBV. Deep sequencing and in situ hybridization suggest that HBeAg-negative shrew HBVs cause intense hepatotropic monoinfections and low within-host genomic heterogeneity. Geographical clustering and low MSHBV/CSHBV-specific seroprevalence suggest focal transmission and high virulence of shrew HBVs. HBeAg negativity is thus an ancient HBV infection pattern, whereas Ntcp usage for entry is not evolutionarily conserved. Shrew infection models relying on CSHBV/MSHBV revertants and human HBV will allow comparative assessments of HBeAg-mediated HBV pathogenesis, entry, and species barriers.
Assuntos
Evolução Molecular , Vírus da Hepatite B/genética , Vírus da Hepatite B/patogenicidade , Modelos Genéticos , Filogenia , Musaranhos/virologia , Proteínas do Envelope Viral/genética , Fatores de Virulência/genética , Animais , Linhagem Celular Tumoral , Hepatite B/genética , Hepatite B/metabolismo , Hepatite B/veterinária , Vírus da Hepatite B/metabolismo , HumanosRESUMO
P-glycoprotein (P-gp) is the gene product of the multidrug resistance gene (MDR1, syn. ABCB1) that normally restricts the transfer of cortisol across the blood-brain barrier. In the absence of P-gp, cortisol access to the hypothalamus is increased and, by feedback inhibition, this finally leads to lower endogenous plasma cortisol levels in dogs with homozygous nt230(del4) MDR1 mutation (MDR1-/- mutant dogs). While a previous study only focused on plasma cortisol levels, the present study used urinary steroid hormone metabolites to analyze cortisol metabolism in MDR1-/- mutant dogs. Morning void urine was collected from 23 MDR1-/- mutant and 16 MDR1+/+ normal dogs and was subjected to targeted GC-MS steroid hormone metabolome analysis. Seven cortisol metabolites, cortisol itself, and 13 other steroid metabolites were detected. In general, all cortisol metabolites were lower in the urine of the MDR1-/- mutant dogs, with allo-tetrahydro-cortisol and ß-cortol reaching the level of significance. In addition, 11-keto-pregnanetriol levels were significantly lower in the urine of the MDR1-/- mutant dogs, indicating that also the 17alpha-OH-progesterone-derived metabolism was altered. In conclusion, the present study provides the first steroid hormone metabolome analysis in the urine of MDR1-/- mutant dogs. Significant differences in the steroid metabolome of MDR1-/- mutant dogs point to a significant role of P-gp for cortisol metabolism and excretion and so indirectly also for hypothalamic-pituitary-adrenal axis regulation in dogs.
Assuntos
Hidrocortisona , Sistema Hipotálamo-Hipofisário , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Animais , Cães , Cromatografia Gasosa-Espectrometria de Massas/veterinária , Metaboloma , Sistema Hipófise-Suprarrenal , EsteroidesRESUMO
OCT1 and OCT2 are polyspecific membrane transporters that are involved in hepatic and renal drug clearance in humans and mice. In this study, we cloned dog OCT1 and OCT2 and compared their function to the human and mouse orthologs. We used liver and kidney RNA to clone dog OCT1 and OCT2. The cloned and the publicly available RNA-Seq sequences differed from the annotated exon-intron structure of OCT1 in the dog genome CanFam3.1. An additional exon between exons 2 and 3 was identified and confirmed by sequencing in six additional dog breeds. Next, dog OCT1 and OCT2 were stably overexpressed in HEK293 cells and the transport kinetics of five drugs were analyzed. We observed strong differences in the transport kinetics between dog and human orthologs. Dog OCT1 transported fenoterol with 12.9-fold higher capacity but 14.3-fold lower affinity (higher KM) than human OCT1. Human OCT1 transported ipratropium with 5.2-fold higher capacity but 8.4-fold lower affinity than dog OCT1. Compared to human OCT2, dog OCT2 showed 10-fold lower transport of fenoterol and butylscopolamine. In conclusion, the functional characterization of dog OCT1 and OCT2 reported here may have implications when using dogs as pre-clinical models as well as for drug therapy in dogs.
Assuntos
Proteínas de Transporte de Cátions Orgânicos , Transportador 1 de Cátions Orgânicos , Animais , Cátions , Clonagem Molecular , Cães , Fenoterol , Células HEK293 , Humanos , Camundongos , Proteínas de Transporte de Cátions Orgânicos/genética , Transportador 1 de Cátions Orgânicos/genética , Transportador 2 de Cátion Orgânico/genética , Especificidade da EspécieRESUMO
Human hepatic bile acid transporter Na+/taurocholate cotransporting polypeptide (NTCP) represents the liver-specific entry receptor for the hepatitis B and D viruses (HBV/HDV). Chronic hepatitis B and D affect several million people worldwide, but treatment options are limited. Recently, HBV/HDV entry inhibitors targeting NTCP have emerged as promising novel drug candidates. Nevertheless, the exact molecular mechanism that NTCP uses to mediate virus binding and entry into hepatocytes is still not completely understood. It is already known that human NTCP mRNA expression is downregulated under cholestasis. Furthermore, incubation of rat hepatocytes with the secondary bile acid taurolithocholic acid (TLC) triggers internalization of the rat Ntcp protein from the plasma membrane. In the present study, the long-term inhibitory effect of TLC on transport function, HBV/HDV receptor function, and membrane expression of human NTCP were analyzed in HepG2 and human embryonic kidney (HEK293) cells stably overexpressing NTCP. Even after short-pulse preincubation, TLC had a significant long-lasting inhibitory effect on the transport function of NTCP, but the NTCP protein was still present at the plasma membrane. Furthermore, binding of the HBV/HDV myr-preS1 peptide and susceptibility for in vitro HDV infection were significantly reduced by TLC preincubation. We hypothesize that TLC rapidly accumulates in hepatocytes and mediates long-lasting trans-inhibition of the transport and receptor function of NTCP via a particular TLC-binding site at an intracellularly accessible domain of NTCP. Physiologically, this trans-inhibition might protect hepatocytes from toxic overload of bile acids. Pharmacologically, it provides an interesting novel NTCP target site for potential long-acting HBV/HDV entry inhibitors.NEW & NOTEWORTHY The hepatic bile acid transporter NTCP is a high-affinity receptor for hepatitis B and D viruses. This study shows that TLC rapidly accumulates in NTCP-expressing hepatoma cells and mediates long-lasting trans-inhibition of NTCP's transporter and receptor function via an intracellularly accessible domain, without substantially affecting its membrane expression. This domain is a promising novel NTCP target site for pharmacological long-acting HBV/HDV entry inhibitors.
Assuntos
Vírus da Hepatite B/efeitos dos fármacos , Hepatite B/tratamento farmacológico , Hepatite D/tratamento farmacológico , Hepatócitos/efeitos dos fármacos , Transportadores de Ânions Orgânicos Dependentes de Sódio/farmacologia , Simportadores/farmacologia , Animais , Ácidos e Sais Biliares/metabolismo , Hepatite B/metabolismo , Hepatócitos/metabolismo , Ratos , Receptores Virais/efeitos dos fármacos , Receptores Virais/metabolismoRESUMO
BACKGROUND: The muscarinic receptor antagonist trospium chloride (TCl) is used for pharmacotherapy of the overactive bladder syndrome. TCl is a hydrophilic positively charged drug. Therefore, it has low permeability through biomembranes and requires drug transporters for distribution and excretion. In humans, the organic cation transporters OCT1 and OCT2 and the multidrug and toxin extrusion MATE1 and MATE2-K carriers showed TCl transport. However, their individual role for distribution and excretion of TCl is unclear. Knockout mouse models lacking mOct1/mOct2 or mMate1 might help to clarify their role for the overall pharmacokinetics of TCl. METHOD: In preparation of such experiments, TCl transport was analyzed in HEK293 cells stably transfected with the mouse carriers mOct1, mOct2, mMate1, and mMate2, respectively. RESULTS: Mouse mOct1, mOct2, and mMate1 showed significant TCl transport with Km values of 58.7, 78.5, and 29.3 µM, respectively. In contrast, mMate2 did not transport TCl but showed MPP+ transport with Km of 60.0 µM that was inhibited by the drugs topotecan, acyclovir, and levofloxacin. CONCLUSION: TCl transport behavior as well as expression pattern were quite similar for the mouse carriers mOct1, mOct2, and mMate1 compared to their human counterparts.
Assuntos
Benzilatos/metabolismo , Proteínas da Membrana Plasmática de Transporte de Catecolaminas/metabolismo , Nortropanos/metabolismo , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Transportador 2 de Cátion Orgânico/metabolismo , Animais , Benzilatos/farmacocinética , Transporte Biológico , Proteínas da Membrana Plasmática de Transporte de Catecolaminas/genética , Células HEK293 , Humanos , Cinética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Antagonistas Muscarínicos/metabolismo , Antagonistas Muscarínicos/farmacocinética , Nortropanos/farmacocinética , Proteínas de Transporte de Cátions Orgânicos/genética , Transportador 2 de Cátion Orgânico/genéticaRESUMO
The solute carrier family SLC10 consists of seven members, including the bile acid transporters Na+/taurocholate co-transporting polypeptide (NTCP) and apical sodium-dependent bile acid transporter (ASBT), the steroid sulfate transporter SOAT as well as four orphan carriers (SLC10A3, SLC10A4, SLC10A5 and SLC10A7). Previously, homodimerization of NTCP, ASBT and SOAT was described and there is increasing evidence that carrier oligomerization is an important regulatory factor for protein sorting and transport function. In the present study, homo- and heterodimerization were systematically analyzed among all SLC10 carriers (except for SLC10A3) using the yeast-two-hybrid membrane protein system. Strong homodimerization occurred for NTCP/NTCP, ASBT/ASBT and SLC10A7/SLC10A7. Heterodimerization was observed for most of the SLC10 carrier combinations. Heterodimerization of NTCP was additionally investigated by co-localization of NTCP-GFP and NTCP-mScarlet with respective SLC10 carrier constructs. NTCP co-localized with SLC10A4, SLC10A5, SOAT and SLC10A7. This co-localization was most pronounced for SLC10A4 and was additionally confirmed by co-immunoprecipitation. Interestingly, SLC10 carrier co-expression decreased the taurocholate transport function of NTCP for most of the analyzed constructs, indicating that SLC10 carrier heterodimerization is of functional relevance. In conclusion, homo- and heterodimerization is a common feature of the SLC10 carriers. The relevance of this finding for regulation and transport function of the SLC10 carriers in vivo needs further investigation.
Assuntos
Transportadores de Ânions Orgânicos Dependentes de Sódio/química , Animais , Transporte Biológico , Dimerização , Humanos , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Sódio/metabolismoRESUMO
BACKGROUND: Although the new isoxazoline drug fluralaner (Bravecto®) is generally well tolerated in dogs, adverse drug reactions involving neurological dysfunction occurred in individual dogs. However, most of these cases are documented inadequately and none of them is reported and discussed in the literature. As isoxazoline drugs target neuronal chloride channels with a clear preference for invertebrates, they are considered to have a good safety profile. However, pharmacodynamic effects in the nervous system of vertebrates cannot be ruled out completely. CASE PRESENTATION: A seven-month-old female Kooikerhondje dog was treated with Bravecto® at the recommended dose. About 24 h after administration, the dog exhibited signs of neurological toxicity, including generalized ataxia, myoclonic jerks, tremor of head and body, muscle twitching and oral dysphagia. All symptoms were transient and the dog fully recovered without any treatment after 10 h. CONCLUSION: This case report describes transient occurrence of neurological dysfunction after administration of Bravecto®. It may help to better classify adverse drug reactions after application of isoxazoline drugs and documents a good prognosis even after occurrence of severe neurological dysfunction in the present case.
Assuntos
Doenças do Sistema Nervoso Central/veterinária , Doenças do Cão/induzido quimicamente , Inseticidas/efeitos adversos , Isoxazóis/efeitos adversos , Animais , Doenças do Sistema Nervoso Central/induzido quimicamente , Cães , FemininoRESUMO
BACKGROUND: Porcine urinary bladders are widely used for uro-pharmacological examinations due to their resemblance to the human organ. However, characterisations of the porcine urothelium at the molecular level are scarce up to now. As it has become clear over the last years that this tissue plays an important role in the signaling-pathways of the bladder, we examined whether the transporter and receptor pattern (with focus on the transmitter acetylcholine) is comparable to the human urothelium. With regard to in vitro studies, we also investigated if there is a difference between the native tissue and cultivated primary urothelial cells in culture. METHODS: Urothelium from German Landrace and Göttingen Minipig bladders was collected. One part of the German Landrace tissue was used for cultivation, and different passages of the urothelial cells were collected. The actual mRNA expression of different transporters and receptors was examined via quantitative real-time PCR. These included the vesicular acetylcholine transporter (VAChT), the choline acetyl transferase (ChAT), organic cation transporters 1-3 (OCT1-3), organic anion transporting polypeptide 1A2 (OATP1A2), P-glycoprotein (ABCB1), the carnitine acetyl-transferase (CarAT), as well as the muscarinic receptors 1-5 (M1-5). RESULTS: There is a strong qualitative resemblance between the human and the porcine urothelium with regard to the investigated cholinergic receptors, enzymes and transporters. CarAT, OCT1-3, OATP1A2 and ABCB1 could be detected in the urothelium of both pig races. Moreover, all 5 M-receptors were prominent with an emphasis on M2 and M3. VAChT and ChAT could not be detected at all. Cultures of the derived urothelial cells showed decreased expression of all targets apart from ABCB1 and CarAT. CONCLUSIONS: Based on the expression pattern of receptors, transporters and enzymes of the cholinergic system, the porcine urinary bladder can be regarded as a good model for pharmacological studies. However, cultivation of primary urothelial cells resulted in a significant drop in mRNA expression of the targets. Therefore, it can be concluded that the intact porcine urothelium, or the whole pig bladder, may be appropriate models for studies with anticholinergic drugs, whereas cultivated urothelial cells have some limitation due to significant changes in the expression levels of relevant targets.
Assuntos
Neurônios Colinérgicos/metabolismo , Transportadores de Ânions Orgânicos/metabolismo , Receptores Muscarínicos/metabolismo , Bexiga Urinária/metabolismo , Urotélio/metabolismo , Animais , Células Cultivadas , Transportadores de Ânions Orgânicos/genética , Receptores Muscarínicos/genética , Especificidade da Espécie , Suínos , Porco Miniatura , Bexiga Urinária/citologia , Urotélio/citologiaRESUMO
BACKGROUND & AIMS: All known hepatitis B virus (HBV) genotypes occur in humans and hominoid Old World non-human primates (NHPs). The divergent woolly monkey HBV (WMHBV) forms another orthohepadnavirus species. The evolutionary origins of HBV are unclear. METHODS: We analysed sera from 124 Brazilian monkeys collected during 2012-2016 for hepadnaviruses using molecular and serological tools, and conducted evolutionary analyses. RESULTS: We identified a novel orthohepadnavirus species in capuchin monkeys (capuchin monkey hepatitis B virus [CMHBV]). We found CMHBV-specific antibodies in five animals and high CMHBV concentrations in one animal. Non-inflammatory, probably chronic infection was consistent with an intact preCore domain, low genetic variability, core deletions in deep sequencing, and no elevated liver enzymes. Cross-reactivity of antisera against surface antigens suggested antigenic relatedness of HBV, CMHBV, and WMHBV. Infection-determining CMHBV surface peptides bound to the human HBV receptor (human sodium taurocholate co-transporting polypeptide), but preferentially interacted with the capuchin monkey receptor homologue. CMHBV and WMHBV pseudotypes infected human hepatoma cells via the human sodium taurocholate co-transporting polypeptide, and were poorly neutralised by HBV vaccine-derived antibodies, suggesting that cross-species infections may be possible. Ancestral state reconstructions and sequence distance comparisons associated HBV with humans, whereas primate hepadnaviruses as a whole were projected to NHP ancestors. Co-phylogenetic analyses yielded evidence for co-speciation of hepadnaviruses and New World NHP. Bayesian hypothesis testing yielded strong support for an association of the HBV stem lineage with hominoid ancestors. Neither CMHBV nor WMHBV was likely the ancestor of the divergent human HBV genotypes F/H found in American natives. CONCLUSIONS: Our data suggest ancestral co-speciation of hepadnaviruses and NHP, and an Old World origin of the divergent HBV genotypes F/H. The identification of a novel primate hepadnavirus offers new perspectives for urgently needed animal models of chronic hepatitis B. LAY SUMMARY: The origins of HBV are unclear. The new orthohepadnavirus species from Brazilian capuchin monkeys resembled HBV in elicited infection patterns and could infect human liver cells using the same receptor as HBV. Evolutionary analyses suggested that primate HBV-related viruses might have emerged in African ancestors of New World monkeys millions of years ago. HBV was associated with hominoid primates, including humans and apes, suggesting evolutionary origins of HBV before the formation of modern humans. HBV genotypes found in American natives were divergent from those found in American monkeys, and likely introduced along prehistoric human migration. Our results elucidate the evolutionary origins and dispersal of primate HBV, identify a new orthohepadnavirus reservoir, and enable new perspectives for animal models of hepatitis B.
Assuntos
Cebus/virologia , Evolução Molecular , Vírus da Hepatite B/genética , Vírus da Hepatite B/isolamento & purificação , Orthohepadnavirus/genética , Orthohepadnavirus/isolamento & purificação , Sequência de Aminoácidos , Animais , Teorema de Bayes , Brasil , Especiação Genética , Genoma Viral , Hepatite B/veterinária , Hepatite B/virologia , Antígenos da Hepatite B/química , Antígenos da Hepatite B/genética , Antígenos da Hepatite B/imunologia , Vírus da Hepatite B/classificação , Interações entre Hospedeiro e Microrganismos/genética , Humanos , Modelos Genéticos , Doenças dos Macacos/virologia , Transportadores de Ânions Orgânicos Dependentes de Sódio/fisiologia , Orthohepadnavirus/classificação , Filogenia , Primatas/virologia , Receptores Virais/fisiologia , Simportadores/fisiologia , Internalização do VírusRESUMO
The hepatitis B virus (HBV), family Hepadnaviridae, is one of most relevant human pathogens. HBV origins are enigmatic, and no zoonotic reservoirs are known. Here, we screened 3,080 specimens from 54 bat species representing 11 bat families for hepadnaviral DNA. Ten specimens (0.3%) from Panama and Gabon yielded unique hepadnaviruses in coancestral relation to HBV. Full genome sequencing allowed classification as three putative orthohepadnavirus species based on genome lengths (3,149-3,377 nt), presence of middle HBV surface and X-protein genes, and sequence distance criteria. Hepatic tropism in bats was shown by quantitative PCR and in situ hybridization. Infected livers showed histopathologic changes compatible with hepatitis. Human hepatocytes transfected with all three bat viruses cross-reacted with sera against the HBV core protein, concordant with the phylogenetic relatedness of these hepadnaviruses and HBV. One virus from Uroderma bilobatum, the tent-making bat, cross-reacted with monoclonal antibodies against the HBV antigenicity determining S domain. Up to 18.4% of bat sera contained antibodies against bat hepadnaviruses. Infectious clones were generated to study all three viruses in detail. Hepatitis D virus particles pseudotyped with surface proteins of U. bilobatum HBV, but neither of the other two viruses could infect primary human and Tupaia belangeri hepatocytes. Hepatocyte infection occurred through the human HBV receptor sodium taurocholate cotransporting polypeptide but could not be neutralized by sera from vaccinated humans. Antihepadnaviral treatment using an approved reverse transcriptase inhibitor blocked replication of all bat hepadnaviruses. Our data suggest that bats may have been ancestral sources of primate hepadnaviruses. The observed zoonotic potential might affect concepts aimed at eradicating HBV.
Assuntos
Quirópteros/virologia , Hepadnaviridae/genética , Hepadnaviridae/patogenicidade , Zoonoses/virologia , Animais , Sequência de Bases , Linhagem Celular Tumoral , Reações Cruzadas/imunologia , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Genoma/genética , Vírus da Hepatite B/genética , Hepatócitos/virologia , Humanos , Immunoblotting , Hibridização In Situ , Dados de Sequência Molecular , Análise de Sequência de DNA , Especificidade da Espécie , TupaiidaeRESUMO
BACKGROUND: SLC10A4 belongs to the solute carrier family SLC10 whose founding members are the Na(+)/taurocholate co-transporting polypeptide (NTCP, SLC10A1) and the apical sodium-dependent bile acid transporter (ASBT, SLC10A2). These carriers maintain the enterohepatic circulation of bile acids between the liver and the gut. SLC10A4 was identified as a novel member of the SLC10 carrier family with the highest phylogenetic relationship to NTCP. The SLC10A4 protein was detected in synaptic vesicles of cholinergic and monoaminergic neurons of the peripheral and central nervous system, suggesting a transport function for any kind of neurotransmitter. Therefore, in the present study, we performed systematic transport screenings for SLC10A4 and also aimed to identify the vesicular sorting domain of the SLC10A4 protein. RESULTS: We detected a vesicle-like expression pattern of the SLC10A4 protein in the neuronal cell lines SH-SY5Y and CAD. Differentiation of these cells to the neuronal phenotype altered neither SLC10A4 gene expression nor its vesicular expression pattern. Functional transport studies with different neurotransmitters, bile acids and steroid sulfates were performed in SLC10A4-transfected HEK293 cells, SLC10A4-transfected CAD cells and in Xenopus laevis oocytes. For these studies, transport by the dopamine transporter DAT, the serotonin transporter SERT, the choline transporter CHT1, the vesicular monoamine transporter VMAT2, the organic cation transporter Oct1, and NTCP were used as positive control. SLC10A4 failed to show transport activity for dopamine, serotonin, norepinephrine, histamine, acetylcholine, choline, acetate, aspartate, glutamate, gamma-aminobutyric acid, pregnenolone sulfate, dehydroepiandrosterone sulfate, estrone-3-sulfate, and adenosine triphosphate, at least in the transport assays used. When the C-terminus of SLC10A4 was replaced by the homologous sequence of NTCP, the SLC10A4-NTCP chimeric protein revealed clear plasma membrane expression in CAD and HEK293 cells. But this chimera also did not show any transport activity, even when the N-terminal domain of SLC10A4 was deleted by mutagenesis. CONCLUSIONS: Although different kinds of assays were used to screen for transport function, SLC10A4 failed to show transport activity for a series of neurotransmitters and neuromodulators, indicating that SLC10A4 does not seem to represent a typical neurotransmitter transporter such as DAT, SERT, CHT1 or VMAT2.
Assuntos
Transporte Biológico/fisiologia , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Proteínas de Transporte de Ânions , Western Blotting , Linhagem Celular Tumoral , Imunofluorescência , Células HEK293 , Humanos , Proteínas de Membrana/genética , Camundongos , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Oócitos , Reação em Cadeia da Polimerase em Tempo Real , Simportadores , Transfecção , Proteínas de Transporte Vesicular/genética , Xenopus laevisRESUMO
Soy isoflavones (IF) are phytoestrogens, which interact with estrogen receptors. They are extensively metabolized by glucuronosyltransferases and sulfotransferases, leading to the modulation of their estrogenic activity. It can be assumed that this biotransformation also has a crucial impact on the uptake of IF by active or passive cellular transport mechanisms, but little is known about the transport of IF phase II metabolites into the cell. Therefore, transport assays for phase II metabolites of daidzein (DAI) were carried out using HEK293 cell lines transfected with five human candidate carriers, i.e., organic anion transporter OAT4, sodium-dependent organic anion transporter (SOAT), Na(+)-taurocholate cotransporting polypeptide (NTCP), apical sodium-dependent bile acid transporter ASBT, and organic anion transporting polypeptide OATP2B1. Cellular uptake was monitored by UHPLC-DAD. DAI monosulfates were transported by the carriers NTCP and SOAT in a sodium-dependent manner, while OAT4-HEK293 cells revealed a partly sodium-dependent transport for these compounds. In contrast, DAI-7,4'-disulfate was only taken up by NTCP-HEK293 cells. DAI-7-glucuronide, but not DAI-4'-glucuronide, was transported exclusively by OATP2B1 in a sodium-independent manner. DAI-7-glucuronide-4'-sulfate, DAI-7-glucoside, and DAI were no substrate of any of the tested carriers. In addition, the inhibitory potency of the DAI metabolites toward estrone-sulfate (E1S) uptake of the above-mentioned carriers was determined. In conclusion, human SOAT, NTCP, OATP2B1, and OAT4 were identified as carriers for the DAI metabolites. Several metabolites were able to inhibit carrier-dependent E1S uptake. These findings might contribute to a better understanding of the bioactivity of IF especially in case of hormone-related cancers.
Assuntos
Isoflavonas/farmacocinética , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Fitoestrógenos/farmacocinética , Simportadores/metabolismo , Transporte Biológico , Cromatografia Líquida de Alta Pressão/métodos , Células HEK293 , Humanos , Isoflavonas/metabolismo , Transportadores de Ânions Orgânicos/metabolismo , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , Esterol O-Aciltransferase/metabolismoRESUMO
BACKGROUND & AIMS: The human liver bile acid transporter Na(+)/taurocholate cotransporting polypeptide (NTCP) has recently been identified as liver-specific receptor for infection of hepatitis B virus (HBV), which attaches via the myristoylated preS1 (myr-preS1) peptide domain of its large surface protein to NTCP. Since binding of the myr-preS1 peptide to NTCP is an initiating step of HBV infection, we investigated if this process interferes with the physiological bile acid transport function of NTCP. METHODS: HBV infection, myr-preS1 peptide binding, and bile acid transport assays were performed with primary Tupaia belangeri (PTH) and human (PHH) hepatocytes as well as NTCP-transfected human hepatoma HepG2 cells allowing regulated NTCP expression, in the presence of various bile acids, ezetimibe, and myr-preS1 peptides. RESULTS: The myr-preS1 peptide of HBV inhibited bile acid transport in PTH and PHH as well as in NTCP-expressing HEK293 and HepG2 cells. Inversely, HBV infection of PTH, PHH, and NTCP-transfected HepG2 cells was inhibited in a concentration-dependent manner by taurine and glycine conjugates of cholic acid and ursodeoxycholic acid as well as by ezetimibe. In NTCP-HepG2 cells and PTH, NTCP expression, NTCP transport function, myr-preS1 peptide binding, and HBV infection followed comparable kinetics. CONCLUSIONS: Myr-preS1 virus binding to NTCP, necessary for productive HBV infection, interferes with the physiological bile acid transport function of NTCP. Therefore, HBV infection via NTCP may be lockable by NTCP substrates and NTCP-inhibiting drugs. This opens a completely new way for an efficient management of HBV infection by the use of NTCP-directed drugs.
Assuntos
Ácidos e Sais Biliares/metabolismo , Proteínas de Transporte/metabolismo , Vírus da Hepatite B/fisiologia , Hepatite B , Hepatócitos/fisiologia , Glicoproteínas de Membrana/metabolismo , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Simportadores/metabolismo , Animais , Transporte Biológico , Células Hep G2 , Hepatite B/metabolismo , Hepatite B/virologia , Humanos , Tupaia , Proteínas Virais/metabolismo , Internalização do VírusRESUMO
Synthesis and secretion of bile acids (BA) is a key physiological function of the liver. In pathological conditions like portosystemic shunt, hepatic insufficiency, hepatitis, or cirrhosis BA metabolism and secretion are disturbed. Quantification of total serum BA is an established diagnostic method to assess the general liver function and allows early detection of abnormalities, liver disease progression and guidance of treatment decisions. To date, data on comparative BA profiles in dogs are limited. However, BA profiles might be even better diagnostic parameters than total BA concentrations. On this background, the present study analyzed and compared individual BA profiles in serum, plasma, urine, and feces of 10 healthy pups and 40 adult healthy dogs using ultra-high performance liquid chromatography coupled to electrospray ionization mass spectrometry. Sample preparation was performed by solid-phase extraction for serum, plasma, and urine samples or by protein precipitation with methanol for the feces samples. For each dog, 22 different BA, including unconjugated BA and their glycine and taurine conjugates, were analyzed. In general, there was a great interindividual variation for the concentrations of single BA, mostly exemplified by the fact that cholic acid (CA) was by far the most prominent BA in blood and urine samples of some of the dogs (adults and pups), while in others, CA was under the detection limit. There were no significant age-related differences in the BA profiles, but pups showed generally lower absolute BA concentrations in serum, plasma, and urine. Taurine-conjugated BA were predominant in the serum and plasma of both pups (68%) and adults (74-75%), while unconjugated BA were predominant in the urine and feces of pups (64 and 95%, respectively) and adults (68 and 99%, respectively). The primary BA chenodeoxycholic acid and taurocholic acid and the secondary BA deoxycholic acid and lithocholic acid were the most robust analytes for potential diagnostic purpose. In conclusion, this study reports simultaneous BA profiling in dog serum, plasma, urine, and feces and provides valuable diagnostic data for subsequent clinical studies in dogs with different kinds of liver diseases.
RESUMO
Cellular entry of the hepatitis B and D viruses (HBV/HDV) requires binding of the viral surface polypeptide preS1 to the hepatobiliary transporter Na+-taurocholate co-transporting polypeptide (NTCP). This interaction can be blocked by bulevirtide (BLV, formerly Myrcludex B), a preS1 derivative and approved drug for treating HDV infection. Here, to elucidate the basis of this inhibitory function, we determined a cryo-EM structure of BLV-bound human NTCP. BLV forms two domains, a plug lodged in the bile salt transport tunnel of NTCP and a string that covers the receptor's extracellular surface. The N-terminally attached myristoyl group of BLV interacts with the lipid-exposed surface of NTCP. Our structure reveals how BLV inhibits bile salt transport, rationalizes NTCP mutations that decrease the risk of HBV/HDV infection, and provides a basis for understanding the host specificity of HBV/HDV. Our results provide opportunities for structure-guided development of inhibitors that target HBV/HDV docking to NTCP.
Assuntos
Hepatite B , Lipopeptídeos , Simportadores , Humanos , Vírus da Hepatite B/fisiologia , Antivirais/uso terapêutico , Receptores Virais/metabolismo , Ácidos e Sais Biliares/metabolismo , Vírus Delta da Hepatite/fisiologia , Simportadores/metabolismo , Internalização do Vírus , Hepatócitos/metabolismoRESUMO
The plasma membrane protein CaRch1p of Candida albicans, homologous to the human solute carrier protein SLC10A7, is involved in the regulation of calcium homeostasis. C. albicans cells lacking CaRCH1 are hypersensitive to high extracellular Ca(2+) concentrations and show increased tolerance to ketoconazole (KCZ). We assume a higher basal Ca(2+) influx in the rch1/rch1 mutant strain at low extracellular Ca(2+) concentrations, which is not detrimental to C. albicans cells but may be sufficient to activate calcineurin, finally resulting in an increased tolerance to KCZ. However, at 8 µg/ml KCZ plus 3 mm Ca(2+) the rch1/rch1 mutant and the wild-type strains showed identical growth. By further increasing the Ca(2+) concentration to 30 mm, this phenotype was completely reversed and the rch1/rch1 mutant strain became extremely sensitive to 8 µg/ml KCZ, probably due to synergistic toxic effects of Ca(2+) and KCZ under these conditions. Furthermore, we aimed to clarify whether CaRch1p interacts with the Cch1p component of the voltage-gated calcium influx channel Cch1p/Mid1p in C. albicans cells. As disruption of the two alleles of CCH1 in the rch1/rch1 mutant strain did not alter its hypersensitivity to high extracellular Ca(2+) , and as this phenotype was completely abolished by low amounts of Mg(2+) in the rch1/rch1 mutant as well as in the cch1/cch1 rch1/rch1 double mutant, we conclude that CaRch1p is a functional component of the low-affinity calcium uptake system (LACS) system and does not functionally interact with Cch1p.