Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 51
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
J Hepatol ; 68(6): 1114-1122, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29428874

RESUMO

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 Vírus
2.
Endocrinology ; 158(10): 3307-3318, 2017 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-28938430

RESUMO

The thyroid hormone (TH) analog eprotirome (KB2115) was developed to lower cholesterol through selective activation of the TH receptor (TR) ß1 in the liver. Interestingly, eprotirome shows low uptake in nonhepatic tissues, explaining its lipid-lowering action without adverse extrahepatic thyromimetic effects. Clinical trials have shown marked decreases in serum cholesterol levels. We explored the transport of eprotirome across the plasma membrane by members of three TH transporter families: monocarboxylate transporters MCT8 and MCT10; Na-independent organic anion transporters 1A2, 1B1, 1B3, 1C1, 2A1, and 2B1; and Na-dependent organic anion transporters SLC10A1 to SLC10A7. Cellular transport was studied in transfected COS1 cells using [14C]eprotirome and [125I]TH analogs. Of the 15 transporters tested initially, the liver-specific bile acid transporter SLC10A1 showed the highest eprotirome uptake (greater than a sevenfold induction after 60 minutes) as well as TRß1-mediated transcriptional activity. Uptake of eprotirome by SLC10A1 was Na+ dependent and saturable with a Michaelis constant of 8 µM. Eprotirome transport was inhibited by known substrates for SLC10A1 (e.g., cholate and taurocholate), and by TH analogs such as triiodothyropropionic acid and triiodothyroacetic acid. However, no significant SLC10A1-mediated transport was observed of these [125I]TH analogs. We also studied the plasma disappearance and biliary excretion of [14C]eprotirome injected in control and Slc10a1 knockout mice. Although eprotirome is also transported by mouse Slc10a1, the pharmacokinetics of eprotirome were not affected by Slc10a1 deficiency. In conclusion, we have demonstrated that the liver-specific bile acid transporter SLC10A1 effectively transports eprotirome. However, Slc10a1 does not appear to be critical for the liver targeting of this TH analog in mice. Therefore, the importance of SLC10A1 for liver uptake of eprotirome in humans remains to be elucidated.


Assuntos
Anilidas/farmacologia , Anilidas/farmacocinética , Anticolesterolemiantes , Fígado/efeitos dos fármacos , Transportadores de Ânions Orgânicos Dependentes de Sódio/fisiologia , Simportadores/fisiologia , Animais , Transporte Biológico , Células COS , Membrana Celular/metabolismo , Humanos , Fígado/metabolismo , Camundongos , Camundongos Knockout , Terapia de Alvo Molecular , Transportadores de Ácidos Monocarboxílicos/metabolismo , Transportadores de Ânions Orgânicos/metabolismo , Transportadores de Ânions Orgânicos Dependentes de Sódio/deficiência , Transportadores de Ânions Orgânicos Dependentes de Sódio/genética , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sódio/farmacologia , Simportadores/deficiência , Simportadores/genética , Hormônios Tireóideos/metabolismo , Transfecção
3.
Clin Res Hepatol Gastroenterol ; 41(5): 509-515, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28336180

RESUMO

The enterohepatic circulation of bile acids (BAs) is governed by specific transporters expressed in the liver and the intestine and plays a critical role in the digestion of fats and oils. During this process, the majority of the BAs secreted from the liver is reabsorbed in intestinal epithelial cells via the apical sodium-dependent bile acid transporter (ASBT/SLC10A2) and then transported into the portal vein. Previous studies revealed that regulation of the ASBT involves BAs and cholesterol. In addition, abnormal ASBT expression and function might lead to some diseases associated with disorders in the enterohepatic circulation of BAs and cholesterol homeostasis, such as diarrhoea and gallstones. However, decreasing cholesterol or BAs by partly inhibiting ASBT-mediated transport might be used for treatments of hypercholesterolemia, cholestasis and diabetes. This review mainly discusses the regulation of the ASBT by BAs and cholesterol and its relevance to diseases and treatment.


Assuntos
Ácidos e Sais Biliares/fisiologia , Colesterol/fisiologia , Circulação Êntero-Hepática/fisiologia , Homeostase , Transportadores de Ânions Orgânicos Dependentes de Sódio/fisiologia , Simportadores/fisiologia , Humanos
4.
Yao Xue Xue Bao ; 52(2): 189-97, 2017 Feb.
Artigo em Chinês | MEDLINE | ID: mdl-29979499

RESUMO

Bile acids play critical roles in the regulation of metabolism and absorption of lipids. The ileal apical sodium-dependent bile acid transporter (ASBT) located at the enterocyte brush border is responsible for the reuptake of bile acids and the maintenance of bile acid homeostasis. Recently, a number of investigations have been made concerning the regulation and control of ASBT and the relationship between ASBT and intestinal inflammation, tumorigenesis, diabetes mellitus and hyperlipemia, which suggests ASBT as a potential therapeutic target of these diseases. In this review, advances in the study of above-mentioned issues were summarized.


Assuntos
Ácidos e Sais Biliares/fisiologia , Íleo/fisiologia , Transportadores de Ânions Orgânicos Dependentes de Sódio/fisiologia , Simportadores/fisiologia , Transporte Biológico , Homeostase , Humanos , Intestinos/fisiopatologia
5.
J Hepatol ; 64(3): 556-64, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26576481

RESUMO

BACKGROUND & AIMS: Hepatitis B virus (HBV) is a major human pathogen restricted to hepatocytes. Expression of the specific receptor human sodium taurocholate cotransporting polypeptide (hNTCP) in mouse hepatocytes renders them susceptible to hepatitis delta virus (HDV), a satellite of HBV; however, HBV remains restricted at an early stage of replication. This study aims at clarifying whether this restriction is caused by the lack of a dependency factor or the activity of a restriction factor. METHODS: Six hNTCP-expressing mouse and human cell lines were generated and functionally characterized. By fusion with replication-supporting but non-infectable HepG2 cells, we analysed the ability of these heterokaryonic cells to fully support HBV replication by HBcAg expression and HBsAg/HBeAg secretion. RESULTS: While hNTCP expression in three mouse cell lines and the non-hepatic human HeLa cells conferred susceptibility to HDV, HBV replication was still restricted. Upon fusion of refractive cells to HepG2 cells, all heterokaryonic cells supported receptor-mediated infection with HBV. hNTCP was provided by the mouse cells and replication competence came from the HepG2 cell line. Transfection of a covalently closed circular DNA (cccDNA)-like molecule into non-susceptible cells promoted gene expression, indicating that the limiting step is upstream of cccDNA formation. CONCLUSIONS: In addition to the expression of hNTCP, establishment of HBV infection in mouse and non-hepatocytic human cell lines requires supplementation with a dependency factor and is not limited by a restriction factor. This result opens new avenues for the development of a fully permissive immunocompetent HBV mouse model.


Assuntos
Vírus da Hepatite B/fisiologia , Transportadores de Ânions Orgânicos Dependentes de Sódio/fisiologia , Simportadores/fisiologia , Replicação Viral , Animais , Linhagem Celular , Células Hep G2 , Vírus Delta da Hepatite/fisiologia , Hepatócitos/virologia , Humanos , Camundongos , Transportadores de Ânions Orgânicos Dependentes de Sódio/genética , Simportadores/genética
6.
PLoS One ; 10(8): e0134764, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26241473

RESUMO

Enhanced sodium excretion is associated with intrarenal oxidative stress. The present study evaluated whether oxidative stress caused by high sodium (HS) may be involved in calcium oxalate crystal formation. Male rats were fed a sodium-depleted diet. Normal-sodium and HS diets were achieved by providing drinking water containing 0.3% and 3% NaCl, respectively. Rats were fed a sodium-depleted diet with 5% hydroxyl-L-proline (HP) for 7 and 42 days to induce hyperoxaluria and/or calcium oxalate deposition. Compared to normal sodium, HS slightly increased calcium excretion despite diuresis; however, the result did not reach statistical significance. HS did not affect the hyperoxaluria, hypocalciuria or supersaturation caused by HP; however, it increased calcium oxalate crystal deposition soon after 7 days of co-treatment. Massive calcium oxalate formation and calcium crystal excretion in HS+HP rats were seen after 42 days of treatment. HP-mediated hypocitraturia was further exacerbated by HS. Moreover, HS aggravated HP-induced renal injury and tubular damage via increased apoptosis and oxidative stress. Increased urinary malondialdehyde excretion, in situ superoxide production, NAD(P)H oxidase and xanthine oxidase expression and activity, and decreased antioxidant enzyme expression or activity in the HS+HP kidney indicated exaggerated oxidative stress. Interestingly, this redox imbalance was associated with reduced renal osteopontin and Tamm-Horsfall protein expression (via increased excretion) and sodium-dependent dicarboxylate cotransporter NaDC-1 upregulation. Collectively, our results demonstrate that a HS diet induces massive crystal formation in the hyperoxaluric kidney; this is not due to increased urinary calcium excretion but is related to oxidative injury and loss of anticrystallization defense.


Assuntos
Oxalato de Cálcio/química , Hiperoxalúria/metabolismo , Cálculos Renais/etiologia , Túbulos Renais/metabolismo , Natriurese/fisiologia , Estresse Oxidativo/efeitos dos fármacos , Sódio na Dieta/farmacologia , Animais , Antioxidantes/metabolismo , Biomarcadores , Citratos/urina , Creatinina/urina , Cristalização , Transportadores de Ácidos Dicarboxílicos/genética , Transportadores de Ácidos Dicarboxílicos/fisiologia , Dieta Hipossódica , Diurese/efeitos dos fármacos , Indução Enzimática , Regulação da Expressão Gênica , Hidroxiprolina/toxicidade , Hiperoxalúria/induzido quimicamente , Hiperoxalúria/genética , Cálculos Renais/metabolismo , Cálculos Renais/urina , Túbulos Renais/efeitos dos fármacos , Túbulos Renais/patologia , Masculino , Transportadores de Ânions Orgânicos Dependentes de Sódio/genética , Transportadores de Ânions Orgânicos Dependentes de Sódio/fisiologia , Osteopontina/genética , Osteopontina/fisiologia , Ratos , Ratos Wistar , Sódio na Dieta/administração & dosagem , Sódio na Dieta/toxicidade , Superóxidos/metabolismo , Simportadores/genética , Simportadores/fisiologia , Uromodulina/genética , Uromodulina/fisiologia
7.
J Hepatol ; 63(3): 697-704, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26022694

RESUMO

BACKGROUND & AIMS: Regulation of bile acid homeostasis in mammals is a complex process regulated via extensive cross-talk between liver, intestine and intestinal microbiota. Here we studied the effects of gut microbiota on bile acid homeostasis in mice. METHODS: Bile acid homeostasis was assessed in four mouse models. Germfree mice, conventionally-raised mice, Asbt-KO mice and intestinal-specific Gata4-iKO mice were treated with antibiotics (bacitracin, neomycin and vancomycin; 100 mg/kg) for five days and subsequently compared with untreated mice. RESULTS: Attenuation of the bacterial flora by antibiotics strongly reduced fecal excretion and synthesis of bile acids, but increased the expression of the bile acid synthesis enzyme CYP7A1. Similar effects were seen in germfree mice. Intestinal bile acid absorption was increased and accompanied by increases in plasma bile acid levels, biliary bile acid secretion and enterohepatic cycling of bile acids. In the absence of microbiota, the expression of the intestinal bile salt transporter Asbt was strongly increased in the ileum and was also expressed in more proximal parts of the small intestine. Most of the effects of antibiotic treatment on bile acid homeostasis could be prevented by genetic inactivation of either Asbt or the transcription factor Gata4. CONCLUSIONS: Attenuation of gut microbiota alters Gata4-controlled expression of Asbt, increasing absorption and decreasing synthesis of bile acids. Our data support the concept that under physiological conditions microbiota stimulate Gata4, which suppresses Asbt expression, limiting the expression of this transporter to the terminal ileum. Our studies expand current knowledge on the bacterial control of bile acid homeostasis.


Assuntos
Ácidos e Sais Biliares/metabolismo , Fator de Transcrição GATA4/fisiologia , Microbioma Gastrointestinal/fisiologia , Absorção Intestinal , Transportadores de Ânions Orgânicos Dependentes de Sódio/fisiologia , Simportadores/fisiologia , Animais , Antibacterianos/farmacologia , Colesterol 7-alfa-Hidroxilase/genética , Homeostase , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/análise
8.
Toxicol Sci ; 146(2): 363-73, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26001962

RESUMO

Among the perfluoroalkyl sulfonates (PFASs), perfluorohexane sulfonate (PFHxS), and perfluorooctane sulfonate (PFOS) have half-lives of several years in humans, mainly due to slow renal clearance and potential hepatic accumulation. Both compounds undergo enterohepatic circulation. To determine whether transporters involved in the enterohepatic circulation of bile acids are also involved in the disposition of PFASs, uptake of perfluorobutane sulfonate (PFBS), PFHxS, and PFOS was measured using freshly isolated human and rat hepatocytes in the absence or presence of sodium. The results demonstrated sodium-dependent uptake for all 3 PFASs. Given that the Na(+)/taurocholate cotransporting polypeptide (NTCP) and the apical sodium-dependent bile salt transporter (ASBT) are essential for the enterohepatic circulation of bile acids, transport of PFASs was investigated in stable CHO Flp-In cells for human NTCP or HEK293 cells transiently expressing rat NTCP, human ASBT, and rat ASBT. The results demonstrated that both human and rat NTCP can transport PFBS, PFHxS, and PFOS. Kinetics with human NTCP revealed Km values of 39.6, 112, and 130 µM for PFBS, PFHxS, and PFOS, respectively. For rat NTCP Km values were 76.2 and 294 µM for PFBS and PFHxS, respectively. Only PFOS was transported by human ASBT whereas rat ASBT did not transport any of the tested PFASs. Human OSTα/ß was also able to transport all 3 PFASs. In conclusion, these results suggest that the long half-live and the hepatic accumulation of PFOS in humans are at least, in part, due to transport by NTCP and ASBT.


Assuntos
Ácidos Alcanossulfônicos/farmacocinética , Fluorcarbonetos/farmacocinética , Transportadores de Ânions Orgânicos Dependentes de Sódio/fisiologia , Ácidos Sulfônicos/farmacocinética , Simportadores/fisiologia , Animais , Hepatócitos/metabolismo , Humanos , Ratos
9.
Clin Res Hepatol Gastroenterol ; 38(6): 661-3, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25178832

RESUMO

Several recent papers support the view that Na-taurocholate cotransporting polypeptide (NTCP), the sinusoidal bile acid transporter of hepatocytes, is a cell surface receptor enabling entry into these cells of the hepatitis B virus (HBV). This major advance in the understanding of the HBV life cycle paves the way to new therapeutic strategies aimed at blocking HBV entry into hepatocytes.


Assuntos
Vírus da Hepatite B/fisiologia , Transportadores de Ânions Orgânicos Dependentes de Sódio/fisiologia , Simportadores/fisiologia , Animais , Hepatite B/tratamento farmacológico , Humanos , Transportadores de Ânions Orgânicos Dependentes de Sódio/antagonistas & inibidores , Simportadores/antagonistas & inibidores
10.
Drug Metab Dispos ; 41(8): 1522-8, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23695864

RESUMO

Silibinin has been reported to be a promising compound for hepatitis C treatment of nonresponders to standard treatment. Although administered silibinin is well tolerated, increased serum bilirubin levels have been observed during high-dose i.v. silibinin therapy. The mechanism of silibinin-induced hyperbilirubinemia in humans, however, has not been identified so far. The aim of this study was to investigate the effect of silibinin on hepatocellular uptake and efflux transport systems for organic anions to elucidate the cause of silibinin-induced hyperbilirubinemia. Therefore, the effect of silibinin on transport activity of the hepatocellular uptake transporters organic anion-transporting polypeptides (OATPs) OATP1B1, OATP1B3, and OATP2B1, as well as Na(+)-taurocholate cotransporting polypeptide (NTCP) and of the efflux transporters multidrug resistance-associated protein 2 (MRP2) and bile-salt export pump (BSEP) was studied. The effect of silibinin on OATPs and NTCP function was studied in stable transfected Chinese hamster ovary cells using the radiolabeled model substrates estrone-3-sulfate and dehydroepiandrosteronesulfate for OATPs and taurocholate for NTCP. Interaction of silibinin with MRP2 and BSEP was measured in vesicles isolated from Sf21 or Sf9 insect cells expressing these transporters using either estradiol-17ß-glucuronide or taurocholate as substrates. OATP1B1, OATP1B3, and OATP2B1 were inhibited by silibinin, with OATP1B1 being inhibited by (a) complex mechanism(s). An inhibitory effect was also seen for MRP2. In contrast, the bile acid transporters NTCP and BSEP were not affected by silibinin. We concluded that silibinin-induced hyperbilirubinemia may be caused by an inhibition of the bilirubin-transporting OATPs and the efflux-transporter MRP2.


Assuntos
Fígado/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/antagonistas & inibidores , Transportadores de Ânions Orgânicos/antagonistas & inibidores , Silimarina/farmacologia , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/fisiologia , Animais , Transporte Biológico , Células CHO , Cricetinae , Cricetulus , Estradiol/metabolismo , Fígado/efeitos dos fármacos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/fisiologia , Transportadores de Ânions Orgânicos/fisiologia , Transportadores de Ânions Orgânicos Dependentes de Sódio/fisiologia , Silibina , Simportadores/fisiologia , Ácido Taurocólico/metabolismo
11.
Mol Aspects Med ; 34(2-3): 252-69, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23506869

RESUMO

The solute carrier (SLC) family 10 (SLC10) comprises influx transporters of bile acids, steroidal hormones, various drugs, and several other substrates. Because the seminal transporters of this family, namely, sodium/taurocholate cotransporting polypeptide (NTCP; SLC10A1) and the apical sodium-dependent bile acid transporter (ASBT; SLC10A2), were primarily bile acid transporters, the term "sodium bile salt cotransporting family" was used for the SLC10 family. However, this notion became obsolete with the finding of other SLC10 members that do not transport bile acids. For example, the sodium-dependent organic anion transporter (SOAT; SLC10A6) transports primarily sulfated steroids. Moreover, NTCP was shown to also transport steroids and xenobiotics, including HMG-CoA inhibitors (statins). The SLC10 family contains four additional members, namely, P3 (SLC10A3; SLC10A3), P4 (SLC10A4; SLC10A4), P5 (SLC10A5; SLC10A5) and SLC10A7 (SLC10A7), several of which were unknown or considered hypothetical until approximately a decade ago. While their substrate specificity remains undetermined, great progress has been made towards their characterization in recent years. Explicitly, SLC10A4 may participate in vesicular storage or exocytosis of neurotransmitters or mastocyte mediators, whereas SLC10A5 and SLC10A7 may be involved in solute transport and SLC10A3 may have a role as a housekeeping protein. Finally, the newly found role of bile acids in glucose and energy homeostasis, via the TGR5 receptor, sheds new light on the clinical relevance of ASBT and NTCP. The present mini-review provides a brief summary of recent progress on members of the SLC10 family.


Assuntos
Ácidos e Sais Biliares/metabolismo , Modelos Moleculares , Família Multigênica/genética , Transportadores de Ânions Orgânicos Dependentes de Sódio/genética , Transportadores de Ânions Orgânicos Dependentes de Sódio/fisiologia , Conformação Proteica , Simportadores/genética , Simportadores/fisiologia , Variação Genética , Hormônios Esteroides Gonadais/metabolismo , Humanos , Modelos Biológicos , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Simportadores/metabolismo
12.
Biochem J ; 444(3): 497-502, 2012 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-22530691

RESUMO

Candida albicans RCH1 (regulator of Ca(2+) homoeostasis 1) encodes a protein of ten TM (transmembrane) domains, homologous with human SLC10A7 (solute carrier family 10 member 7), and Rch1p localizes in the plasma membrane. Deletion of RCH1 confers hypersensitivity to high concentrations of extracellular Ca(2+) and tolerance to azoles and Li(+), which phenocopies the deletion of CaPMC1 (C. albicans PMC1) encoding the vacuolar Ca(2+) pump. Additive to CaPMC1 mutation, lack of RCH1 alone shows an increase in Ca(2+) sensitivity, Ca(2+) uptake and cytosolic Ca(2+) level. The Ca(2+) hypersensitivity is abolished by cyclosporin A and magnesium. In addition, deletion of RCH1 elevates the expression of CaUTR2 (C. albicans UTR2), a downstream target of the Ca(2+)/calcineurin signalling. Mutational and functional analysis indicates that the Rch1p TM8 domain, but not the TM9 and TM10 domains, are required for its protein stability, cellular functions and subcellular localization. Therefore Rch1p is a novel regulator of cytosolic Ca(2+) homoeostasis, which expands the functional spectrum of the vertebrate SLC10 family.


Assuntos
Cálcio/metabolismo , Candida albicans/fisiologia , Membrana Celular/fisiologia , Proteínas Fúngicas/fisiologia , Homeostase/fisiologia , Transportadores de Ânions Orgânicos Dependentes de Sódio/fisiologia , Simportadores/fisiologia , Cálcio/fisiologia , Candida albicans/genética , Membrana Celular/genética , Citosol/metabolismo , Citosol/fisiologia , Proteínas Fúngicas/genética , Homeostase/genética , Humanos , Família Multigênica , Mutação
13.
J Vet Pharmacol Ther ; 35(3): 209-15, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21623837

RESUMO

Mammary epithelial cells express a diversity of membrane transporters including members of organic cation and organic anion (OAT) transporter subfamilies. Four mammal OAT isoforms have been identified: OAT-1, OAT-2, OAT-3, and OAT-4. The pharmacological significance of OAT isoforms has been emphasized because of their role in the movement of a wide variety of substrates across epithelial barriers. The present study identified (molecularly and functionally) bovine OAT isoforms in bovine mammary epithelial (BME-UV) cells. mRNA expression levels of all tested transporters in BME-UV cells were less than expression levels of the corresponding transporters in bovine kidney. Directionality in the flux of P-aminohippuric acid and acetylsalicylate, compounds known to interact with OAT-1 and OAT-2, respectively, across BME-UV monolayers was not observed at the concentrations used in this study. Directionality was, however, observed in the flux of estrone sulfate (EsS). Adding probenecid, penicillin G or nonradiolabeled EsS to the apical donor compartment significantly increased the apical-to-basolateral flux of EsS across the BME-UV monolayer. These results suggest that BME-UV cells express an organic anion transport system, making it a potentially useful model to study the role of this transport system in the mammary epithelial barrier.


Assuntos
Glândulas Mamárias Animais/citologia , Transportadores de Ânions Orgânicos/fisiologia , Animais , Bovinos , Células Cultivadas , Epitélio/metabolismo , Feminino , Glândulas Mamárias Animais/metabolismo , Glândulas Mamárias Animais/fisiologia , Proteína 1 Transportadora de Ânions Orgânicos/genética , Proteína 1 Transportadora de Ânions Orgânicos/fisiologia , Transportadores de Ânions Orgânicos/genética , Transportadores de Ânions Orgânicos Dependentes de Sódio/genética , Transportadores de Ânions Orgânicos Dependentes de Sódio/fisiologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiologia , Reação em Cadeia da Polimerase em Tempo Real
14.
Free Radic Biol Med ; 50(1): 27-36, 2011 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-20974251

RESUMO

To test whether severe ascorbic acid deficiency in macrophages affects progression of early atherosclerosis, we used fetal liver cell transplantation to generate atherosclerosis-prone apolipoprotein E-deficient (apoE(-/-)) mice that selectively lacked the ascorbate transporter (SVCT2) in hematopoietic cells, including macrophages. After 13 weeks of chow diet, apoE(-/-) mice lacking the SVCT2 in macrophages had surprisingly less aortic atherosclerosis, decreased lesion macrophage numbers, and increased macrophage apoptosis compared to control-transplanted mice. Serum lipid levels were similar in both groups. Peritoneal macrophages lacking the SVCT2 had undetectable ascorbate; increased susceptibility to H(2)O(2)-induced mitochondrial dysfunction and apoptosis; decreased expression of genes for COX-2, IL1ß, and IL6; and decreased lipopolysaccharide-stimulated NF-κB and antiapoptotic gene expression. These changes were associated with decreased expression of both the receptor for advanced glycation end products and HIF-1α, either or both of which could have been the proximal cause of decreased macrophage activation and apoptosis in ascorbate-deficient macrophages.


Assuntos
Deficiência de Ácido Ascórbico/genética , Aterosclerose/genética , Aterosclerose/prevenção & controle , Macrófagos Peritoneais/metabolismo , Transportadores de Ânions Orgânicos Dependentes de Sódio/genética , Simportadores/genética , Animais , Apolipoproteínas E/genética , Ácido Ascórbico/efeitos adversos , Ácido Ascórbico/metabolismo , Deficiência de Ácido Ascórbico/metabolismo , Deficiência de Ácido Ascórbico/patologia , Aterosclerose/etiologia , Aterosclerose/patologia , Células Cultivadas , Progressão da Doença , Feminino , Predisposição Genética para Doença/prevenção & controle , Macrófagos Peritoneais/patologia , Macrófagos Peritoneais/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Especificidade de Órgãos/genética , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Transportadores de Ânions Orgânicos Dependentes de Sódio/fisiologia , Transportadores de Sódio Acoplados à Vitamina C , Simportadores/metabolismo , Simportadores/fisiologia , Fatores de Tempo
15.
Biochim Biophys Acta ; 1808(6): 1454-61, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21073858

RESUMO

Citric acid cycle intermediates, including succinate and citrate, are absorbed across the apical membrane by the NaDC1 Na+/dicarboxylate cotransporter located in the kidney and small intestine. The secondary structure model of NaDC1 contains 11 transmembrane helices (TM). TM7 was shown previously to contain determinants of citrate affinity, and Arg-349 at the extracellular end of the helix is required for transport. The present study involved cysteine scanning mutagenesis of 26 amino acids in TM7 and the associated loops. All of the mutants were well expressed on the plasma membrane, but many had low or no transport activity: 6 were inactive and 7 had activity less than 25% of the parental. Three of the mutants had notable changes in functional properties. F336C had increased transport activity due to an increased Vmax for succinate. The conserved residue F339C had very low transport activity and a change in substrate selectivity. G356C in the putative extracellular loop was the only cysteine mutant that was affected by the membrane-impermeant cysteine reagent, MTSET. However, direct labeling of G356C with MTSEA-biotin gave a weak signal, indicating that this residue is not readily accessible to more bulky reagents. The results suggest that the amino acids of TM7 are functionally important because their replacement by cysteine had large effects on transport activity. However, most of TM7 does not appear to be accessible to the extracellular fluid and is likely to be an outer helix in contact with the lipid bilayer.


Assuntos
Transportadores de Ácidos Dicarboxílicos/química , Transportadores de Ânions Orgânicos Dependentes de Sódio/química , Estrutura Secundária de Proteína , Simportadores/química , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Transporte Biológico/efeitos dos fármacos , Células COS , Membrana Celular/metabolismo , Transportadores de Ácidos Dicarboxílicos/genética , Transportadores de Ácidos Dicarboxílicos/fisiologia , Células HeLa , Humanos , Immunoblotting , Cinética , Mesilatos , Dados de Sequência Molecular , Mutação , Transportadores de Ânions Orgânicos Dependentes de Sódio/genética , Transportadores de Ânions Orgânicos Dependentes de Sódio/fisiologia , Coelhos , Homologia de Sequência de Aminoácidos , Ácido Succínico/metabolismo , Reagentes de Sulfidrila/farmacologia , Simportadores/genética , Simportadores/fisiologia
16.
J Nephrol ; 23 Suppl 16: S49-56, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-21170889

RESUMO

Hypocitraturia is a known risk factor for kidney stone formation. By forming soluble complexes with calcium, citrate prevents crystal nucleation, aggregation and growth; therefore, the presence of citrate in the urine reduces the risk for calcium stone formation. Ingested citrate is rapidly metabolized, and plasma citrate levels vary little, so changes in filtered load do not significantly influence urinary citrate excretion. Changes in urinary citrate excretion are predominantly influenced by the rate of citrate absorption from the glomerular filtrate and metabolism by the proximal tubule cell. The former is mediated by the apical membrane cotransporter NaDC1, and the latter is mediated by both cytoplasmic and mitochondrial metabolism. Acid-base status is the most important physiological determinant of urinary citrate excretion, by modulating the activities of NaDC1 and cytoplasmic (ATP citrate lyase) and mitochondrial (m-aconitase) enzymes involved in citrate metabolism. Following an acid load, both the transport and metabolic processes are up-regulated leading to hypocitraturia; in contrast, an alkaline load increases citrate excretion, by regulating only the mitochondrial metabolic process.


Assuntos
Ácido Cítrico/metabolismo , Rim/metabolismo , Animais , Ácido Cítrico/urina , Transportadores de Ácidos Dicarboxílicos/fisiologia , Humanos , Mitocôndrias/metabolismo , Transportadores de Ânions Orgânicos Dependentes de Sódio/fisiologia , Simportadores/fisiologia
17.
Mol Cell Biochem ; 343(1-2): 217-22, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20549544

RESUMO

Endothelial dysfunction is an early manifestation of atherosclerosis caused in part by oxidized LDL (oxLDL). Since vitamin C, or ascorbic acid, prevents several aspects of endothelial dysfunction, the effects of oxLDL on oxidative stress and regulation of the ascorbate transporter, SVCT2, were studied in cultured EA.hy926 endothelial cells. Cells cultured for 18 h with 0.2 mg/ml oxLDL showed increased lipid peroxidation that was prevented by a single addition of 0.25 mM ascorbate at the beginning of the incubation. This protection caused a decrease in intracellular ascorbate, but no change in the cell content of GSH. In the absence of ascorbate, oxLDL increased SVCT2 protein and function during 18 h in culture. Although culture of the cells with ascorbate did not affect SVCT2 protein expression, the oxLDL-induced increase in SVCT2 protein expression was prevented by ascorbate. These results suggest that up-regulation of endothelial cell SVCT2 expression and function may help to maintain intracellular ascorbate during oxLDL-induced oxidative stress, and that ascorbate in turn can prevent this effect.


Assuntos
Ácido Ascórbico/metabolismo , Lipoproteínas LDL/fisiologia , Transportadores de Ânions Orgânicos Dependentes de Sódio/fisiologia , Simportadores/fisiologia , Regulação para Cima/fisiologia , Ácido Ascórbico/farmacologia , Linhagem Celular , Peroxidação de Lipídeos/efeitos dos fármacos , Estresse Oxidativo , Transportadores de Sódio Acoplados à Vitamina C
18.
Free Radic Biol Med ; 49(5): 821-9, 2010 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-20541602

RESUMO

The sodium-dependent vitamin C transporter (SVCT2) is responsible for the transport of vitamin C into cells in multiple organs, from either the blood or the cerebrospinal fluid. Mice null for SVCT2 (SVCT2(-/-)) do not survive past birth but the cause of death has not yet been ascertained. After mating of SVCT2(+/-) males and SVCT2(+/-) females, fewer SVCT2(-/-) and SVCT2(+/-) progeny were observed than would be expected according to Mendelian ratios. Vitamin C levels in SVCT2(-/-), SVCT2(+/-), and SVCT2(+/+) were genotype-dependent. SVCT2(-/-) fetuses had significantly lower vitamin C levels than littermates in placenta, cortex, and lung, but not in liver (the site of vitamin C synthesis). Low vitamin C levels in placenta and cortex were associated with elevations in several markers of oxidative stress: malondialdehyde, isoketals, F(2)-isoprostanes, and F(4)-neuroprostanes. Oxidative stress was not elevated in fetal SVCT2(-/-) lung tissue despite low vitamin C levels. In addition to the expected severe hemorrhage in cortex, we also found hemorrhage in the brain stem, which was accompanied by cell loss. We found evidence of increased apoptosis in SVCT2(-/-) mice and disruption of the basement membrane in fetal brain. Together these data show that SVCT2 is critical for maintaining vitamin C levels in fetal and placental tissues and that the lack of SVCT2, and the resulting low vitamin C levels, results in fetal death and, in SVCT2(-/-) mice that survive the gestation period, in oxidative stress and cell death.


Assuntos
Ácido Ascórbico/sangue , Transportadores de Ânions Orgânicos Dependentes de Sódio/genética , Estresse Oxidativo/genética , Simportadores/genética , Animais , Encéfalo/embriologia , Encéfalo/metabolismo , Encéfalo/patologia , Morte Celular/genética , Regulação para Baixo/genética , Feminino , Genótipo , Masculino , Malondialdeído/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Transportadores de Ânions Orgânicos Dependentes de Sódio/fisiologia , Placenta/metabolismo , Placenta/patologia , Gravidez , Transportadores de Sódio Acoplados à Vitamina C , Simportadores/metabolismo , Simportadores/fisiologia , Distribuição Tecidual , Regulação para Cima/genética
19.
J Clin Invest ; 120(4): 1069-83, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20200446

RESUMO

Levels of the necessary nutrient vitamin C (ascorbate) are tightly regulated by intestinal absorption, tissue accumulation, and renal reabsorption and excretion. Ascorbate levels are controlled in part by regulation of transport through at least 2 sodium-dependent transporters: Slc23a1 and Slc23a2 (also known as Svct1 and Svct2, respectively). Previous work indicates that Slc23a2 is essential for viability in mice, but the roles of Slc23a1 for viability and in adult physiology have not been determined. To investigate the contributions of Slc23a1 to plasma and tissue ascorbate concentrations in vivo, we generated Slc23a1-/- mice. Compared with wild-type mice, Slc23a1-/- mice increased ascorbate fractional excretion up to 18-fold. Hepatic portal ascorbate accumulation was nearly abolished, whereas intestinal absorption was marginally affected. Both heterozygous and knockout pups born to Slc23a1-/- dams exhibited approximately 45% perinatal mortality, and this was associated with lower plasma ascorbate concentrations in dams and pups. Perinatal mortality of Slc23a1-/- pups born to Slc23a1-/- dams was prevented by ascorbate supplementation during pregnancy. Taken together, these data indicate that ascorbate provided by the dam influenced perinatal survival. Although Slc23a1-/- mice lost as much as 70% of their ascorbate body stores in urine daily, we observed an unanticipated compensatory increase in ascorbate synthesis. These findings indicate a key role for Slc23a1 in renal ascorbate absorption and perinatal survival and reveal regulation of vitamin C biosynthesis in mice.


Assuntos
Ácido Ascórbico/metabolismo , Rim/metabolismo , Transportadores de Ânions Orgânicos Dependentes de Sódio/fisiologia , Mortalidade Perinatal , Simportadores/fisiologia , Absorção , Animais , Animais Recém-Nascidos , Ácido Ascórbico/administração & dosagem , Feminino , Intestino Delgado/metabolismo , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Transportadores de Ânions Orgânicos Dependentes de Sódio/genética , Polimorfismo de Nucleotídeo Único , Gravidez , Transportadores de Sódio Acoplados à Vitamina C , Simportadores/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA