RESUMO
The solute carrier (SLC) group of membrane transport proteins includes about 400 members organized into more than 50 families. The SLC family that comprises nucleoside-sugar transporters is referred to as SLC35. One of the members of this family is SLC35F1. The function of SLC35F1 is still unknown; however, recent studies demonstrated that SLC35F1 mRNA is highly expressed in the brain and in the kidney. Therefore, we examine the distribution of Slc35f1 protein in the murine forebrain using immunohistochemistry. We could demonstrate that Slc35f1 is highly expressed in the adult mouse brain in a variety of different brain structures, including the cortex, hippocampus, amygdala, thalamus, basal ganglia, and hypothalamus. To examine the possible roles of Slc35f1 and its subcellular localization, we used an in vitro glioblastoma cell line expressing Slc35f1. Co-labeling experiments were performed to reveal the subcellular localization of Slc35f1. Our results indicate that Slc35f1 neither co-localizes with markers for the Golgi apparatus nor with markers for the endoplasmic reticulum. Time-lapse microscopy of living cells revealed that Slc35f1-positive structures are highly dynamic and resemble vesicles. Using super-resolution microscopy, these Slc35f1-positive spots clearly co-localize with the recycling endosome marker Rab11.
Assuntos
Encéfalo/metabolismo , Encéfalo/ultraestrutura , Proteínas Carreadoras de Solutos/metabolismo , Animais , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Células Tumorais Cultivadas , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
Sphingosine-1-phosphate (S1P) is a potent lipid mediator released from activated platelets by an adenosine triphosphate (ATP)-dependent export mechanism. A candidate transport protein is the multidrug resistance protein 4 (MRP4/ABCC4), an ATP-dependent transporter highly expressed in platelets. Furthermore, several statins are known to affect platelet functions and exhibit antithrombotic properties. This study determines the involvement of MRP4 in the transport of S1P and a possible interference by statins. Transport studies in membrane vesicles of Sf9 cells containing recombinant human MRP4 revealed that MRP4 mediates ATP-dependent transport of fluorescein- and tritium-labelled S1P. Also, ATP-dependent S1P transport in platelet membrane vesicles containing endogenous MRP4 was inhibited by the MRP inhibitor MK571 and the MRP4-selective compound Ceefourin-1. Confocal microscopy using fluorescein-labelled S1P as well as boron-dipyrromethene (BODIPY)-labelled sphingosine indicated association of S1P and MRP4 in human platelets. In MRP4-deficient mice, agonist-induced S1P secretion was reduced compared with matched wild-type C57Bl/6 mice and platelet S1P concentrations were lower. Fluvastatin and rosuvastatin interfered with MRP4 function inhibiting ATP-dependent cGMP (cyclic guanosine monophosphate) uptake into MRP4-containing vesicles, inhibited MRP4-mediated S1P transport in vitro and significantly attenuated endogenous S1P release from agonist-activated platelet ex vivo. These data suggest that release of S1P from platelets depends on MRP4 and statins can interfere with this transport process. Potentially, this may be relevant for the pleiotropic anti-inflammatory effects of statins and their effect on modulating atherothrombosis.
Assuntos
Plaquetas/metabolismo , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Lisofosfolipídeos/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Esfingosina/análogos & derivados , Animais , Transporte Biológico , Compostos de Boro , Cromatografia Líquida , Fluvastatina/farmacologia , Voluntários Saudáveis , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Ativação Plaquetária/efeitos dos fármacos , Testes de Função Plaquetária , Proteínas Recombinantes/metabolismo , Rosuvastatina Cálcica/farmacologia , Células Sf9 , Esfingosina/metabolismo , Espectrometria de Massas em TandemRESUMO
The anthracycline-mediated cardiotoxicity is still not completely understood. To examine the impact of cholesterol metabolism and transport in this context, cholesterol and oxysterol levels as well as the expression of the cholesterol transporters ABCA1 and ABCG1 were analyzed in doxorubicin-treated HL-1 murine cardiomyocytes as well as in mouse model for acute doxorubicin-induced cardiotoxicity. Doxorubicin-treated HL-1 cells exhibited enhanced cholesterol (153±20% of control), oxysterol (24S-hydroxycholesterol: 206±29% of control) and cholesterol precursor levels (lathosterol: 122±12% of control; desmosterol: 188±10% of control) indicating enhanced cholesterol synthesis. Moreover, abca1 and abcg1 were upregulated on mRNA, protein and functional level caused by a doxorubicin-mediated activation of the nuclear receptor LXR. In addition, the oxysterols not only induced the abca1 and abcg1 in HL-1 cells but also enhanced the expression of endothelin-1 and transforming growth factor-ß, which have already been identified as important factors in doxorubicin-induced cardiotoxicity. These in vitro findings were verified in a murine model for acute doxorubicin-induced cardiotoxicity, demonstrating elevated cardiac (2.1±0.2vs. 3.6±1.0ng/mg) and systemic cholesterol levels (105.0±8.4vs. 130.0±4.3mg/dl), respectively, as well as enhanced oxysterol levels such as cardiac 24S-hydroxycholesterol (2.1±0.2vs. 3.6±1.0ng/mg). In line with these findings cardiac mRNA expression of abca1 (303% of control) and abcg1 (161% of control) was induced. Taken together, our data demonstrate enhanced cholesterol and oxysterol levels by doxorubicin, resulting in a LXR-dependent upregulation of abca1 and abcg1. In this context, the cytotoxic effects of oxysterols and their impact on cardiac gene expression should be considered as an important factor in doxorubicin-induced cardiotoxicity.
Assuntos
Transportador 1 de Cassete de Ligação de ATP/biossíntese , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/biossíntese , Doxorrubicina/farmacologia , Receptores X do Fígado/fisiologia , Miócitos Cardíacos/metabolismo , Oxisteróis/metabolismo , Animais , Células Cultivadas , Colesterol/metabolismo , Relação Dose-Resposta a Droga , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologiaAssuntos
Acesso à Informação/legislação & jurisprudência , Ensaios Clínicos como Assunto/legislação & jurisprudência , Óxidos N-Cíclicos/efeitos adversos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Piridinas/efeitos adversos , Pesquisa Biomédica/organização & administração , França , Humanos , Organizações , Pesquisadores/organização & administraçãoRESUMO
The multidrug resistance protein 4 (MRP4/ABCC4) has been identified as an important transporter for signalling molecules including cyclic nucleotides and several lipid mediators in platelets and may thus represent a novel target to interfere with platelet function. Besides its localisation in the plasma membrane, MRP4 has been also detected in the membrane of dense granules in resting platelets. In polarised cells it is localised at the basolateral or apical plasma membrane. To date, the mechanism of MRP4 trafficking has not been elucidated; protein interactions may regulate both the localisation and function of this transporter. We approached this issue by searching for interacting proteins by in vitro binding assays, followed by immunoblotting and mass spectrometry, and by visualising their co-localisation in platelets and haematopoietic cells. We identified the PDZ domain containing scaffold proteins ezrin-binding protein 50 (EBP50/NHERF1), postsynaptic density protein 95 (PSD95), and sorting nexin 27 (SNX27), but also the adaptor protein complex 3 subunit ß3A (AP3B1) and the heat shock protein HSP90 as putative interaction partners of MRP4. The knock-down of SNX27, PSD95, and AP3B1 by siRNA in megakaryoblastic leukaemia cells led to a redistribution of MRP4 from intracellular structures to the plasma membrane. Inhibition of HSP90 led to a diminished expression and retention of MRP4 in the endoplasmic reticulum. These results indicate that MRP4 localisation and function are regulated by multiple protein interactions. Changes in the adaptor proteins can hence lead to altered localisation and function of the transporter.
Assuntos
Complexo 3 de Proteínas Adaptadoras/metabolismo , Subunidades beta do Complexo de Proteínas Adaptadoras/metabolismo , Plaquetas/metabolismo , Membrana Celular/metabolismo , Proteína 4 Homóloga a Disks-Large/metabolismo , Leucemia Megacarioblástica Aguda/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Fosfoproteínas/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Complexo 3 de Proteínas Adaptadoras/química , Complexo 3 de Proteínas Adaptadoras/genética , Subunidades beta do Complexo de Proteínas Adaptadoras/química , Subunidades beta do Complexo de Proteínas Adaptadoras/genética , Animais , Plaquetas/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Proteína 4 Homóloga a Disks-Large/química , Proteína 4 Homóloga a Disks-Large/genética , Cães , Células HEK293 , Proteínas de Choque Térmico HSP90/metabolismo , Células HeLa , Humanos , Leucemia Megacarioblástica Aguda/genética , Leucemia Megacarioblástica Aguda/patologia , Macrolídeos/farmacologia , Células Madin Darby de Rim Canino , Proteínas Associadas à Resistência a Múltiplos Medicamentos/química , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Fosfoproteínas/química , Fosfoproteínas/genética , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , Interferência de RNA , Trocadores de Sódio-Hidrogênio/química , Trocadores de Sódio-Hidrogênio/genética , TransfecçãoRESUMO
Important antimalarial drugs, including quinolines, act against blood schizonts by interfering with hemoglobin metabolism. To reach their site of action, these compounds have to cross the plasma membrane of red blood cells (RBCs). Organic cation transporters (OCTs) and organic anion transporting polypeptides (OATPs) are important uptake transporters and interesting candidates for local drug transport. We therefore studied their interaction with antimalarial compounds (quinine, chloroquine, mefloquine, pyrimethamine, artemisinin, and artesunate) and characterized the expression of OATP1A2 and OATP2B1 in RBCs. Competition assays using transporter-overexpressing Madin-Darby canine kidney (MDCKII) cells and the model substrate estrone-3-sulfate identified quinine and chloroquine as potent inhibitors of OATP1A2 function (IC50 quinine: 0.7 ± 1.2 µM; chloroquine: 1.0 ± 1.5 µM), but no or only moderate effects were observed for OATP2B1. Subsequently, quinine was identified as a substrate of OATP1A2 (Km 23.4 µM). The OATP1A2-mediated uptake was sensitive to the OATP1A2-specific inhibitor naringin. Both OATPs were expressed in human RBCs, and ex vivo transport studies demonstrated naringin-sensitive accumulation of quinine in these cells (60 pmol versus 38 pmol/5 × 10(5) RBCs). Additional transport studies using OCT1-3 and organic cation transporter novel type 1 (OCTN1) indicated only significant quinine uptake by OCT1, which was not detected in RBCs. In conclusion, our data demonstrate expression of OATP2B1 and OATP1A2 in RBCs as well as OATP1A2-mediated uptake of quinine. Therefore, modulation of OATP1A2 function may affect quinine uptake into erythrocytes.
Assuntos
Antimaláricos/sangue , Eritrócitos/metabolismo , Transportadores de Ânions Orgânicos/sangue , Animais , Antimaláricos/farmacocinética , Cães , Feminino , Voluntários Saudáveis , Humanos , Células Madin Darby de Rim Canino , MasculinoRESUMO
The clinical efficiency of the highly potent antitumor agent doxorubicin is limited by cardiotoxic effects. In a murine doxorubicin cardiotoxicity model, increased endothelin-1 (ET-1) expression and cardioprotective effects of the dual ET-1 blocker bosentan were demonstrated. To date it is unclear if combined blocking of endothelin A/B receptors is necessary or whether selective inhibition of one of the ET-1 receptors is sufficient for the observed cardioprotection. Therefore, we investigated the impact of dual (bosentan) and single endothelin receptor antagonism through sitaxentan (receptor A blocker) or BQ788 (receptor B blocker) in a murine doxorubicin cardiotoxicity model (C57BL/6N). Simultaneous administration of each endothelin receptor antagonist (ERA) with doxorubicin resulted in a significantly improved hemodynamic performance in comparison to the impaired cardiac function in control mice with bosentan being most effective but closely followed by sitaxentan and also BQ788. This cardioprotection was not caused by diminished doxorubicin levels in heart since the doxorubicin content in cardiac tissue was not altered by ERAs significantly. However, whole transcript expression profiling showed partly different effects of the ERAs on doxorubicin-modulated cardiac gene expression of genes involved in signal transduction (e.g. Stat3, Pim1, Akt1, Plcb2), fibrosis (e.g. Myl4), energy production (e.g. Ant1) or oxidative stress (e.g. Aox1). Furthermore, doxorubicin-mediated gene regulations were verified in the murine cardiomyocyte model HL-1 showing partly reversed expression patterns after co-administration of the ERAs. In summary, our results demonstrate strong cardioprotective effects of blocking ET-1 receptors against the doxorubicin-related cardiomyopathy and provide evidence to potential underlying signaling pathways.
Assuntos
Antibióticos Antineoplásicos/toxicidade , Cardiomiopatias/prevenção & controle , Cardiotônicos/farmacologia , Doxorrubicina/toxicidade , Receptor de Endotelina A/efeitos dos fármacos , Receptor de Endotelina B/efeitos dos fármacos , Animais , Cardiomiopatias/induzido quimicamente , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
BACKGROUND: The efficacy of statins, which are used commonly in primary and secondary prevention of cardiovascular diseases, shows a wide range of interindividual variability. Genetic variants of OATP1B1, a hepatic uptake transporter, can modify access of statins to its therapeutic target, thereby potentially altering drug efficacy. We studied the impact of genetic variants of OATP1B1 on the lipid-lowering efficacy of statins in a population-based setting. MATERIALS AND METHODS: The basis of the analysis was the Study of Health in Pomerania, a cohort of 2732 men and women aged 20-81 years. Included in the statistical analysis to evaluate the impact of OATP1B1 on therapeutic efficacy of statins were 214 individuals diagnosed with dyslipidaemia during initial recruitment and receiving statins during the 5-year follow-up. RESULTS: Analysing the impact of the OATP1B1 genotype, we observed a trend for lower statin-induced total cholesterol reduction in carriers of the SLCO1B1 512C variant. Restricting the analysis to patients receiving simvastatin, pravastatin, lovastatin and fluvastatin indicated a statistically significant association of the OATP1B1 genotype on lipid parameters at the 5-year follow-up. No such effect was observed for atorvastatin. Calculation of achievement of treatment goals according to the NCEP-ATPIII guidelines showed a lower rate of successful treatment when harbouring the mutant allele for patients taking simvastatin (46.7 vs. 73.9%). A similar trend was observed for pravastatin (34.4 vs. 70.4%). CONCLUSION: Genetic variants of OATP1B1 leading to impaired hepatic uptake of statins translated into reduced drug efficacy in a population-based cohort.
Assuntos
Doença das Coronárias/genética , Estudos de Associação Genética , Metabolismo dos Lipídeos/genética , Transportadores de Ânions Orgânicos/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Farmacológicos , Doença das Coronárias/sangue , Doença das Coronárias/tratamento farmacológico , Doença das Coronárias/patologia , Ácidos Graxos Monoinsaturados/administração & dosagem , Feminino , Fluvastatina , Genótipo , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/administração & dosagem , Indóis/administração & dosagem , Metabolismo dos Lipídeos/efeitos dos fármacos , Transportador 1 de Ânion Orgânico Específico do Fígado , Lovastatina/administração & dosagem , Lovastatina/genética , Masculino , Pessoa de Meia-Idade , Pravastatina/administração & dosagem , Pravastatina/genética , Medição de Risco , Sinvastatina/administração & dosagemRESUMO
BACKGROUND: Genetic factors as predictor of the individual outcome of drug therapy is one aim of personalized medicine approaches. CASE PRESENTATION: We report a drug metabolism based analysis of genetic polymorphisms in a Caucasian patient receiving fluvastatin and telmisartan experiencing myotoxicity (myalgia and moderate creatine kinase elevation). CONCLUSIONS: The obtained findings suggest that heterocygocity of cytochrome P450 CYP2C9*3 variant in combination with multidrug resistance-associated protein MRP2-24C > T functions as risk factor predisposing to experience drug-drug interaction combing those drugs.
Assuntos
Benzimidazóis/efeitos adversos , Benzoatos/efeitos adversos , Creatina Quinase/sangue , Citocromo P-450 CYP2C9/genética , Ácidos Graxos Monoinsaturados/efeitos adversos , Heterozigoto , Indóis/efeitos adversos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Adulto , Bloqueadores do Receptor Tipo 1 de Angiotensina II/administração & dosagem , Bloqueadores do Receptor Tipo 1 de Angiotensina II/efeitos adversos , Sequência de Bases , Benzimidazóis/administração & dosagem , Benzoatos/administração & dosagem , Primers do DNA , Quimioterapia Combinada , Ácidos Graxos Monoinsaturados/administração & dosagem , Fluvastatina , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/administração & dosagem , Inibidores de Hidroximetilglutaril-CoA Redutases/efeitos adversos , Indóis/administração & dosagem , Masculino , Proteína 2 Associada à Farmacorresistência Múltipla , TelmisartanRESUMO
Renal tubular handling of urate is realized by a network of uptake and efflux transporters, including members of drug transporter families such as solute carrier proteins and ATP-binding cassette transporters. Solute carrier family 2, member 9 (SLC2A9), is one key factor of this so called "urate transportosome." The aim of the present study was to understand the transcriptional regulation of SLC2A9 and to test whether identified factors might contribute to a coordinated transcriptional regulation of the transporters involved in urate handling. In silico analysis and cell-based reporter gene assays identified a hepatocyte nuclear factor (HNF)4α-binding site in the promoter of SLC2A9 isoform 1, whose activity was enhanced by transient HNF4α overexpression, whereas mutation of the binding site diminished activation. HNF4α overexpression induced endogenous SLC2A9 expression in vitro. The in vivo role of HNF4α in the modulation of renal SLC2A9 gene expression was supported by findings of quantitative real-time RT-PCR analyses and chromatin immunoprecipitation assays. Indeed, mRNA expression of SLC2A9 and HNF4α in human kidney samples was significantly correlated. We also showed that in renal clear cell carcinoma, downregulation of HNF4α mRNA and protein expression was associated with a significant decline in expression of the transporter. Taken together, our data suggest that nuclear receptor family member HNF4α contributes to the transcriptional regulation of SLC2A9 isoform 1. Since HNF4α has previously been assumed to be a modulator of several urate transporters, our findings support the notion that there could be a transcriptional network providing synchronized regulation of the functional network of the urate transportosome.
Assuntos
Proteínas Facilitadoras de Transporte de Glucose/biossíntese , Fator 4 Nuclear de Hepatócito/fisiologia , Transportadores de Ânions Orgânicos/biossíntese , Sítios de Ligação/genética , Carcinoma de Células Renais/patologia , Carcinoma de Células Renais/fisiopatologia , Desdiferenciação Celular , Regulação da Expressão Gênica , Proteínas Facilitadoras de Transporte de Glucose/genética , Células HeLa , Humanos , Transportadores de Ânions Orgânicos/genética , Regiões Promotoras Genéticas/fisiologia , RNA Mensageiro/metabolismo , Transcrição GênicaRESUMO
Multidrug resistance protein 4 (MRP4/ABCC4) has been established as an independent regulator of cyclic AMP (cAMP) levels particularly in vascular smooth muscle cells and in hematopoietic cells. Here, we assessed whether cAMP in turn regulates MRP4. A significant upregulation of MRP4 mRNA and protein by long-term treatment with cAMP-enhancing agents was observed in HeLa cells, smooth muscle cells, and megakaryoblastic leukemia M07e cells. This upregulation was not affected by inhibition of protein kinase A, but could be reverted by inhibitors and siRNA of an alternative cAMP-signaling route involving exchange proteins activated by cyclic AMP (EPAC) and mitogen-activated protein kinases. A selective EPAC activator could equally induce MRP4. The transcriptional regulation was confirmed in a luciferase reporter gene assay using a vector containing a 1494-bp fragment of the promoter region of the MRP4/ABCC4 gene. Our results suggest that enhanced cAMP levels upregulate MRP4 expression, which can result in increased cAMP efflux.
Assuntos
AMP Cíclico/análogos & derivados , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Células Musculares/metabolismo , Músculo Liso/citologia , Transdução de Sinais , Células Cultivadas , AMP Cíclico/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Células HeLa , Humanos , Músculo Liso/metabolismo , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/efeitos dos fármacos , Regulação para CimaRESUMO
BACKGROUND: L-Arginine and its dimethylated derivatives asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) have been associated with cardiovascular (CV) and all-cause mortality in populations at risk. The present study aimed to investigate the prognostic value of L-arginine and its derivatives in the general population. METHODS AND RESULTS: We evaluated 3,952 individuals (1,936 men and 2,016 women) aged 20-81 (median (IQR) 51 (37; 64) years) from the population-based Study of Health in Pomerania (SHIP). Associations of continuous [per standard deviation (SD) increase] and categorized (age- and sex-specific tertiles) serum L-arginine, ADMA, and SDMA concentrations with all-cause and cause-specific mortality were analysed. During a median (IQR) follow-up period of 10.1 (9.3; 10.8) years (38,476 person-years), 426 deaths (10.8%) were observed, including 139 CV deaths (3.5%), and 150 cancer deaths (3.8%). After multivariable adjustment, we revealed a positive association of SDMA with all-cause [hazard ratio (HR) per SD increase: 1.16, 95% confidence interval (CI): 1.07-1.25] and CV mortality [HR: 1.19, 95% CI: 1.05-1.35]. In contrast, we did not observe any association of SDMA with cancer mortality. Neither L-arginine nor ADMA were associated with all-cause or CV mortality. CONCLUSION: SDMA, but not ADMA, is an independent predictor of all-cause and CV mortality in a large population-based cohort of European ancestry.
Assuntos
Arginina/análogos & derivados , Doenças Cardiovasculares/mortalidade , Adulto , Idoso , Idoso de 80 Anos ou mais , Arginina/metabolismo , Feminino , Humanos , Incidência , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
Enhanced proliferation of human coronary artery smooth muscle cells (HCASMCs) and thereby formation of neointima is one of the factors contributing to failure of coronary stents. Even if the use of drug eluting stents (DES) and thereby the local delivery of cytotoxic compounds has significantly improved the clinical outcome, unselective cytotoxic effects are assumed to hamper clinical success. Novel pharmacological approaches are required to enhance cellular selectivity of locally delivered drugs. Cell specific overexpression of a drug transporter could be used to enhance cellular accumulation and therefore cell specificity. In the herein reported study we tested the possibility of cell specific transporter expression to enhance drug effects in HCASMCs. We generated adenoviral constructs to overexpress the organic cation transporter 1 (OCT1) under control of the promoter of SM22α, which had been previously reported as muscle cell specific gene. First the activity of the SM22α-promoter was assessed in various cell types supporting the notion of muscle cell specificity. Subsequently, the activity of the transporter was compared in infected HCAECs and HCASMCs revealing enhanced accumulation of substrate drugs in HCASMCs in presence of the SM22α-promoter. Testing the hypothesis that this kind of targeting might serve as a mechanism for cell-specific drug effects, we investigated the impact on paclitaxel treatment in HCASMC and HCAECs, showing significantly increased antiproliferative activity of this substrate drug on muscle cells. Taken together, our findings suggest that cell-specific expression of transport proteins serves as mechanism governing the uptake of cytotoxic compounds for a selective impact on targeted cells.
Assuntos
Vasos Coronários/metabolismo , Endotélio Vascular/metabolismo , Células Madin Darby de Rim Canino/metabolismo , Proteínas dos Microfilamentos/genética , Proteínas Musculares/genética , Músculo Liso Vascular/metabolismo , Miócitos Cardíacos/metabolismo , Transportador 1 de Cátions Orgânicos/metabolismo , Adenoviridae/genética , Animais , Antineoplásicos Fitogênicos/farmacologia , Western Blotting , Fármacos Cardiovasculares/farmacologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Vasos Coronários/citologia , Vasos Coronários/efeitos dos fármacos , Cães , Sistemas de Liberação de Medicamentos , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Células Madin Darby de Rim Canino/citologia , Células Madin Darby de Rim Canino/efeitos dos fármacos , Camundongos , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Transportador 1 de Cátions Orgânicos/genética , Paclitaxel/farmacologia , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Auto-antibodies against cardiac proteins have been described in patients with dilated cardiomyopathy. Antibodies against the C-terminal part of KChIP2 (anti-KChIP2 [C-12]) enhance cell death of rat cardiomyocytes. The underlying mechanisms are not fully understood. Therefore, we wanted to explore the mechanisms responsible for anti-KChIP2-mediated cell death. Rat cardiomyocytes were treated with anti-KChIP2 (C-12). KChIP2 RNA and protein expressions, nuclear NF-κB, mitochondrial membrane potential Δψm, caspase-3 and -9 activities, necrotic and apoptotic cells, total Ca(2+) and K(+) concentrations, and the effects on L-type Ca(2+) channels were quantified. Anti-KChIP2 (C-12) induced nuclear translocation of NF-κB. Anti-KChIP2 (C-12)-treatment for 2 h significantly reduced KChIP2 mRNA and protein expression. Anti-KChIP2 (C-12) induced nuclear translocation of NF-κB after 1 h. After 6 h, Δψm and caspase-3 and -9 activities were not significantly changed. After 24 h, anti-KChIP2 (C-12)-treated cells were 75 ± 3% necrotic, 2 ± 1% apoptotic, and 13 ± 2% viable. Eighty-six ± 1% of experimental buffer-treated cells were viable. Anti-KChIP2 (C-12) induced significant increases in total Ca(2+) (plus 11 ± 2%) and K(+) (plus 18 ± 2%) concentrations after 5 min. Anti-KChIP2 (C-12) resulted in an increased Ca(2+) influx through L-type Ca(2+) channels. In conclusion, our results suggest that anti-KChIP2 (C-12) enhances cell death of rat cardiomyocytes probably due to necrosis.
Assuntos
Autoanticorpos/farmacologia , Proteínas Interatuantes com Canais de Kv/imunologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Animais , Apoptose/efeitos dos fármacos , Cálcio/metabolismo , Canais de Cálcio Tipo L/metabolismo , Caspase 3/metabolismo , Caspase 9/metabolismo , Morte Celular/efeitos dos fármacos , Células Cultivadas , Proteínas I-kappa B/metabolismo , Proteínas Interatuantes com Canais de Kv/genética , Proteínas Interatuantes com Canais de Kv/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , NF-kappa B/metabolismo , Necrose/tratamento farmacológico , Potássio/metabolismo , Transporte Proteico/efeitos dos fármacos , Ratos , Ratos Wistar , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Molecular transporters that are expressed in brain, especially at the blood-brain barrier (BBB), are increasingly recognized as possible therapeutic targets in the treatment of neurodegenerative disorders such as Alzheimer's disease and Parkinson's disease. Some ATP-binding cassette (ABC) transporters, particularly P-glycoprotein (ABCB1), MRP1 (ABCC1) and BCRP (ABCG2), have been implicated in the clearance of neurotoxic polypeptides that characteristically accumulate in the brain, such as amyloid-ß (Aß) peptides in Alzheimer's disease. Several lines of evidence also implicate lipid transporters of the A-branch of ABC transporters in pathogenesis. Induction of transporters via the activation of specific nuclear receptors may represent a novel approach to restoring diminished BBB function. On the other hand, transporters in the brain capillary endothelium regulate the permeation of therapeutic compounds into the brain. In addition to the export pumps that limit brain entry of exogenous substances, SLC-type uptake transporters, especially of the OCT (SLC22A) family, are of potential relevance in that they mediate not only the uptake of several drugs used for the treatment of neurodegenerative diseases, but also of certain neurotoxins. Here, we summarize recent findings and novel strategies targeting transporters to reduce brain pathology or to improve drug therapy.
Assuntos
Barreira Hematoencefálica/metabolismo , Sistemas de Liberação de Medicamentos , Proteínas de Membrana Transportadoras/metabolismo , Doenças Neurodegenerativas/tratamento farmacológico , Doenças Neurodegenerativas/metabolismo , Preparações Farmacêuticas/administração & dosagem , Animais , Barreira Hematoencefálica/efeitos dos fármacos , HumanosRESUMO
Organic anion transporting polypeptide OATP1B3 is a membrane-bound drug transporter that facilitates cellular entry of a variety of substrates. Most of the previous studies focused on its hepatic expression and function in hepatic drug elimination. In this study, we report expression of OATP1B3 in human pancreatic tissue, with the abundance of the transporter localized in the islets of Langerhans. Transport studies using OATP1B3-overexpressing MDCKII cells revealed significant inhibition of the cellular uptake of the known substrate cholecystokinin-8 in the presence of the insulinotropic antidiabetes compounds tolbutamide, glibenclamide, glimepiride, and nateglinide and identified glibenclamide as a novel substrate of OATP1B3. Sulfonylurea derivatives exert their insulinotropic effect by binding to the SUR1 subunit of the KATP channels inducing insulin secretion in ß-cells. Here, we show that transient overexpression of human OATP1B3 in a murine ß-cell line (MIN6)-which exhibits glucose and glibenclamide-sensitive insulin secretion-significantly enhances the insulinotropic effect of glibenclamide without affecting glucose-stimulated insulin secretion. Taken together, our data provide evidence that the drug transporter OATP1B3 functions as a determinant of the insulinotropic effect of glibenclamide on the tissue level. Changes in transport activity based on drug-drug interactions or genetic variability may therefore influence glibenclamide efficacy.
Assuntos
Glibureto/farmacologia , Hipoglicemiantes/farmacologia , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , Animais , Linhagem Celular , Cães , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Humanos , Transportadores de Ânions Orgânicos Sódio-Independentes/genética , Membro 1B3 da Família de Transportadores de Ânion Orgânico Carreador de SolutoRESUMO
BACKGROUND: Statins, frequently prescribed in lipid-lowering therapies, seem to have additional beneficial effects on periodontitis and tooth loss. If this is true, then chronic treatment with statins should also result in diminished tooth loss as a long-term response. METHODS: A 5-year population-based follow-up study of tooth loss was performed comparing participants treated with statins (n = 134) with those not on the drugs (Study of Health in Pomerania). Negative binomial regression models were used to analyze the count variable of the outcome, including risk factors for tooth loss and measures of cholesterol metabolism. RESULTS: When adjusted for age and sex, statins were associated with reduced tooth loss during the follow-up period (incidence risk ratio [IRR] = 0.70, 95% confidence interval [CI] = 0.50 to 0.99, P = 0.04). When additionally adjusted for risk factors of periodontal breakdown, IRR was 0.72 (95% CI = 0.52 to 1.01). There was significant interaction with low-density lipoprotein cholesterol (LDL-c) at baseline. After stratification by LDL-c, statins were associated with reduced tooth loss, resulting in IRR = 0.89 (95% CI = 0.44 to 1.83) and 0.64 (95% CI = 0.43 to 0.95), P = 0.03, at LDL-c concentrations ≤100 mg/dL and >100 mg/dL (2.58 mmol/L), respectively. The data also showed reduced tooth loss associated with the 5-year reduction in LDL-c levels on a mmol/L basis and independently of statins (IRR = 0.87, 95% CI = 0.80 to 0.96, P = 0.004). CONCLUSION: Long-term treatment with systemically administered statins may have the beneficial effect of protecting against tooth loss.
Assuntos
Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Periodontite/epidemiologia , Perda de Dente/epidemiologia , Adulto , Idoso , Proteína C-Reativa/análise , Colesterol/sangue , HDL-Colesterol/sangue , LDL-Colesterol/sangue , Estudos de Coortes , Assistência Odontológica/estatística & dados numéricos , Escolaridade , Feminino , Seguimentos , Alemanha/epidemiologia , Humanos , Incidência , Renda/estatística & dados numéricos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Perda da Inserção Periodontal/epidemiologia , Bolsa Periodontal/epidemiologia , Vigilância da População , Fatores de Risco , Fumar/epidemiologia , Adulto JovemAssuntos
Aspirina/farmacologia , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Lisofosfolipídeos/metabolismo , Infarto do Miocárdio/tratamento farmacológico , Esfingosina/análogos & derivados , Idoso , Aspirina/administração & dosagem , Doença da Artéria Coronariana/tratamento farmacológico , Doença da Artéria Coronariana/metabolismo , Humanos , Injeções Intravenosas , Pessoa de Meia-Idade , Infarto do Miocárdio/sangue , Inibidores da Agregação Plaquetária/administração & dosagem , Inibidores da Agregação Plaquetária/farmacologia , Esfingosina/metabolismoRESUMO
OBJECTIVE: Data mining represents an alternative approach to identify new predictors of multifactorial diseases. This work aimed at building an accurate predictive model for incident hypertension using data mining procedures. METHODS: The primary study population consisted of 1605 normotensive individuals aged 20-79 years with 5-year follow-up from the population-based study, that is the Study of Health in Pomerania (SHIP). The initial set was randomly split into a training and a testing set. We used a probabilistic graphical model applying a Bayesian network to create a predictive model for incident hypertension and compared the predictive performance with the established Framingham risk score for hypertension. Finally, the model was validated in 2887 participants from INTER99, a Danish community-based intervention study. RESULTS: In the training set of SHIP data, the Bayesian network used a small subset of relevant baseline features including age, mean arterial pressure, rs16998073, serum glucose and urinary albumin concentrations. Furthermore, we detected relevant interactions between age and serum glucose as well as between rs16998073 and urinary albumin concentrations [area under the receiver operating characteristic (AUC 0.76)]. The model was confirmed in the SHIP validation set (AUC 0.78) and externally replicated in INTER99 (AUC 0.77). Compared to the established Framingham risk score for hypertension, the predictive performance of the new model was similar in the SHIP validation set and moderately better in INTER99. CONCLUSION: Data mining procedures identified a predictive model for incident hypertension, which included innovative and easy-to-measure variables. The findings promise great applicability in screening settings and clinical practice.
Assuntos
Mineração de Dados , Hipertensão/epidemiologia , Adulto , Fatores Etários , Idoso , Algoritmos , Teorema de Bayes , Feminino , Alemanha/epidemiologia , Humanos , Hipertensão/etiologia , Masculino , Pessoa de Meia-Idade , Modelos Teóricos , Curva ROC , Distribuição Aleatória , Medição de Risco , Fatores de Risco , Adulto JovemRESUMO
AIM: To identify loci associated with chronic periodontitis through a genome-wide association study (GWAS). MATERIALS AND METHODS: A GWAS was performed in 4032 individuals of two independent cross-sectional studies of West Pomerania (SHIP n = 3365 and SHIP-TREND n = 667) with different periodontal case definitions. Samples were genotyped with the Affymetrix Genome-Wide Human SNP Array 6.0 or the Illumina Human Omni 2.5 array. Imputation of the HapMap as well as the 1000 Genome-based autosomal and X-chromosomal genotypes and short insertions and deletions (INDELs) was performed in both cohorts. Finally, more than 17 million SNPs and short INDELs were analysed. RESULTS: No genome-wide significant associations were found for any periodontitis case definition, regardless of whether individuals aged >60 years where excluded or not. Despite no single SNP association reached genome-wide significance, the proportion of variance explained by additive effects of all common SNPs was around 23% for mean proximal attachment loss. Excluding subjects aged >60 years increased the explained variance to 34%. CONCLUSIONS: No single SNPs were found to be genome-wide significantly associated with chronic periodontitis in this study.