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Glioblastoma (GBM) is still a deadly tumor due to its highly infiltrative growth behavior and its resistance to therapy. Evidence is accumulating that sphingosine-1-phosphate (S1P) acts as an important tumor-promoting molecule that is involved in the activation of the S1P receptor subtype 1 (S1PR1). Therefore, we investigated the effect of ACT-209905 (a putative S1PR1 modulator) on the growth of human (primary cells, LN-18) and murine (GL261) GBM cells. The viability and migration of GBM cells were both reduced by ACT-209905. Furthermore, co-culture with monocytic THP-1 cells or conditioned medium enhanced the viability and migration of GBM cells, suggesting that THP-1 cells secrete factors which stimulate GBM cell growth. ACT-209905 inhibited the THP-1-induced enhancement of GBM cell growth and migration. Immunoblot analyses showed that ACT-209905 reduced the activation of growth-promoting kinases (p38, AKT1 and ERK1/2), whereas THP-1 cells and conditioned medium caused an activation of these kinases. In addition, ACT-209905 diminished the surface expression of pro-migratory molecules and reduced CD62P-positive GBM cells. In contrast, THP-1 cells increased the ICAM-1 and P-Selectin content of GBM cells which was reversed by ACT-209905. In conclusion, our study suggests the role of S1PR1 signaling in the growth of GBM cells and gives a partial explanation for the pro-tumorigenic effects that macrophages might have on GBM cells.
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PURPOSE: Cocktails of transporter probe drugs are used in vivo to assess transporter activity and respective drug-drug interactions. An inhibitory effect of components on transporter activities should be ruled out. Here, for a clinically tested cocktail consisting of adefovir, digoxin, metformin, sitagliptin, and pitavastatin, inhibition of major transporters by individual probe substrates was investigated in vitro. METHODS: Transporter transfected HEK293 cells were used in all evaluations. Cell-based assays were applied for uptake by human organic cation transporters 1/2 (hOCT1/2), organic anion transporters 1/3 (hOAT1/3), multidrug and toxin extrusion proteins 1/2K (hMATE1/2K), and organic anion transporter polypeptide 1B1/3 (hOATP1B1/3). For P-glycoprotein (hMDR1) a cell-based efflux assay was used whereas an inside-out vesicle-based assay was used for the bile salt export pump (hBSEP). All assays used standard substrates and established inhibitors (as positive controls). Inhibition experiments using clinically achievable concentrations of potential perpetrators at the relevant transporter expression site were carried out initially. If there was a significant effect, the inhibition potency (Ki) was studied in detail. RESULTS: In the inhibition tests, only sitagliptin had an effect and reduced hOCT1- and hOCT2- mediated metformin uptake and hMATE2K mediated MPP+ uptake by more than 70%, 80%, and 30%, respectively. The ratios of unbound Cmax (observed clinically) to Ki of sitagliptin were low with 0.009, 0.03, and 0.001 for hOCT1, hOCT2, and hMATE2K, respectively. CONCLUSION: The inhibition of hOCT2 in vitro by sitagliptin is in agreement with the borderline inhibition of renal metformin elimination observed clinically, supporting a dose reduction of sitagliptin in the cocktail.
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Metformina , Proteínas de Transporte de Cátions Orgânicos , Humanos , Proteínas de Transporte de Cátions Orgânicos/genética , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Células HEK293 , Transporte Biológico , Fosfato de Sitagliptina/farmacologia , Metformina/metabolismo , Transportador 2 de Cátion Orgânico/metabolismo , Interações MedicamentosasRESUMO
Sphingosine-1-phosphate (S1P) is a versatile signaling lipid involved in the regulation of numerous cellular processes. S1P regulates cellular proliferation, migration, and apoptosis as well as the function of immune cells. S1P is generated from sphingosine (Sph), which derives from the ceramide metabolism. In particular, high concentrations of S1P are present in the blood. This originates mainly from erythrocytes, endothelial cells (ECs), and platelets. While erythrocytes function as a storage pool for circulating S1P, platelets can rapidly generate S1P de novo, store it in large quantities, and release it when the platelet is activated. Platelets can thus provide S1P in a short time when needed or in the case of an injury with subsequent platelet activation and thereby regulate local cellular responses. In addition, platelet-dependently generated and released S1P may also influence long-term immune cell functions in various disease processes, such as inflammation-driven vascular diseases. In this review, the metabolism and release of platelet S1P are presented, and the autocrine versus paracrine functions of platelet-derived S1P and its relevance in various disease processes are discussed. New pharmacological approaches that target the auto- or paracrine effects of S1P may be therapeutically helpful in the future for pathological processes involving S1P.
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Plaquetas , Esfingosina , Plaquetas/metabolismo , Comunicação Celular , Ceramidas/metabolismo , Células Endoteliais/metabolismo , Humanos , Lisofosfolipídeos/metabolismo , Esfingosina/análogos & derivados , Esfingosina/metabolismoRESUMO
The multidrug resistance protein 4 (MRP4) is highly expressed in platelets and several lines of evidence point to an impact on platelet function. MRP4 represents a transporter for cyclic nucleotides as well as for certain lipid mediators. The aim of the present study was to comprehensively characterize the effect of a short-time specific pharmacological inhibition of MRP4 on signaling pathways in platelets. Transport assays in isolated membrane vesicles showed a concentrationdependent inhibition of MRP4-mediated transport of cyclic nucleotides, thromboxane (Tx)B2 and fluorescein (FITC)- labeled sphingosine-1-phosphate (S1P) by the selective MRP4 inhibitor Ceefourin-1. In ex vivo aggregometry studies in human platelets, Ceefourin-1 significantly inhibited platelet aggregation by about 30-50% when ADP or collagen was used as activating agents, respectively. Ceefourin-1 significantly lowered the ADP-induced activation of integrin aIIbb3, indicated by binding of FITC-fibrinogen (about 50% reduction at 50 mM Ceefourin-1), and reduced calcium influx. Furthermore, pre-incubation with Ceefourin-1 significantly increased PGE1- and cinaciguat-induced vasodilatorstimulated phosphoprotein (VASP) phosphorylation, indicating increased cytosolic cAMP as well as cGMP concentrations, respectively. The release of TxB2 from activated human platelets was also attenuated. Finally, selective MRP4 inhibition significantly reduced both the total area covered by thrombi and the average thrombus size by about 40% in a flow chamber model. In conclusion, selective MRP4 inhibition causes reduced platelet adhesion and thrombus formation under flow conditions. This finding is mechanistically supported by inhibition of integrin aIIbb3 activation, elevated VASP phosphorylation and reduced calcium influx, based on inhibited cyclic nucleotide and thromboxane transport as well as possible further mechanisms.
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Plaquetas , Trombose , Transportadores de Cassetes de Ligação de ATP/metabolismo , Difosfato de Adenosina/metabolismo , Difosfato de Adenosina/farmacologia , Plaquetas/metabolismo , Cálcio/metabolismo , Fluoresceína-5-Isotiocianato/metabolismo , Fluoresceína-5-Isotiocianato/farmacologia , Humanos , Integrinas/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos , Nucleotídeos Cíclicos/metabolismo , Nucleotídeos Cíclicos/farmacologia , Transdução de Sinais , Trombose/metabolismo , Tromboxanos/metabolismo , Tromboxanos/farmacologiaRESUMO
Despite comprehensive therapy and extensive research, glioblastoma (GBM) still represents the most aggressive brain tumor in adults. Glioma stem cells (GSCs) are thought to play a major role in tumor progression and resistance of GBM cells to radiochemotherapy. The PIM1 kinase has become a focus in cancer research. We have previously demonstrated that PIM1 is involved in survival of GBM cells and in GBM growth in a mouse model. However, little is known about the importance of PIM1 in cancer stem cells. Here, we report on the role of PIM1 in GBM stem cell behavior and killing. PIM1 inhibition negatively regulates the protein expression of the stem cell markers CD133 and Nestin in GBM cells (LN-18, U-87 MG). In contrast, CD44 and the astrocytic differentiation marker GFAP were up-regulated. Furthermore, PIM1 expression was increased in neurospheres as a model of GBM stem-like cells. Treatment of neurospheres with PIM1 inhibitors (TCS PIM1-1, Quercetagetin, and LY294002) diminished the cell viability associated with reduced DNA synthesis rate, increased caspase 3 activity, decreased PCNA protein expression, and reduced neurosphere formation. Our results indicate that PIM1 affects the glioblastoma stem cell behavior, and its inhibition kills glioblastoma stem-like cells, pointing to PIM1 targeting as a potential anti-glioblastoma therapy.
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Antineoplásicos/farmacologia , Neoplasias Encefálicas/patologia , Glioblastoma/patologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-pim-1/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Cromonas/farmacologia , Cromonas/uso terapêutico , Ensaios de Seleção de Medicamentos Antitumorais , Flavonas/farmacologia , Flavonas/uso terapêutico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Morfolinas/farmacologia , Morfolinas/uso terapêutico , Células-Tronco Neoplásicas/patologia , Proteínas Proto-Oncogênicas c-pim-1/genética , Células Tumorais CultivadasRESUMO
PURPOSE: Periodontitis is an inflammatory disease of the oral cavity with an alarmingly high prevalence within the adult population. The signaling lipid sphingosine-1-phosphate (S1P) plays a crucial role in inflammatory and immunomodulatory responses. In addition to cardiovascular disease, sepsis and tumor entities, S1P has been recently identified as both mediator and biomarker in osteoporosis. We hypothesized that S1P may play a role in periodontitis as an inflammation-prone bone destructive disorder. The goal of our study was to evaluate associations between periodontitis and S1P serum concentrations in the Study of Health in Pomerania (SHIP)-Trend cohort. In addition, we investigated the expression of S1P metabolizing enzymes in inflamed gingival tissue. PATIENTS AND METHODS: We analyzed data from 3371 participants (51.6% women) of the SHIP-Trend cohort. Periodontal parameters and baseline characteristics were assessed. Serum S1P was measured by liquid chromatography tandem mass spectrometry. The expression of S1P metabolizing enzymes was determined by immunofluorescence staining of human gingival tissue. RESULTS: S1P serum concentrations were significantly increased in subjects with both moderate and severe periodontitis, assessed as probing depth and clinical attachment loss. In contrast, no significant association of S1P was seen with caries variables (number and percentage of decayed or filled surfaces). S1P concentrations significantly increased with increasing high-sensitivity C-reactive protein (hs-CRP) levels. Interestingly, inflamed compared to normal human gingival tissue exhibited elevated expression levels of the S1P-generating enzyme sphingosine kinase 1 (SphK1). CONCLUSION: We report an intriguingly significant association of various periodontal parameters with serum levels of the inflammatory lipid mediator S1P. Our data point towards a key role of S1P during periodontitis pathology. Modulation of local S1P levels or its signaling properties may represent a potential future therapeutic strategy to prevent or to retard periodontitis progression and possibly reduce periodontitis-related tooth loss.
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The online version of the original article can be found at https://doi.org/10.1007/s00784-020-03594-w.
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OBJECTIVE: SLC22A4/5 single nucleotide polymorphisms (SNPs) have been reported to affect inflammatory diseases. We report the relationship of these polymorphisms with adiposity and tooth loss as elucidated in a 10-year follow-up study. METHODS: Participants of the Study of Health in Pomerania (SHIP, N = 4105) were genotyped for the polymorphisms c.1507C > T in SLC22A4 (rs1050152) and -207C > G in SLC22A5 (rs2631367) using allele-specific real-time PCR assays. A total of 1817 subjects, 934 female and 883 male aged 30-80 years, underwent follow-up 10 years later (SHIP-2) and were assessed for adiposity and tooth loss. RESULTS: The frequencies of the rarer SLC22A4 TT and SLC22A5 CC alleles were 16.7% and 20.3%, respectively. In women, tooth loss was associated with genotype TT vs. CC with incidence rate ratio IRR = 0.74 (95%C.I. 0.60-0.92) and CC vs. GG IRR = 0.79 (0.65-0.96) for SLC22A4 and SLC22A5 SNPs, respectively. In men, no such associations were observed. In the follow-up examination, the relationship between tooth loss and these SNPs was in parallel with measures of body shape such as BMI, body weight, waist circumference, or body fat accumulation. The association between muscle strength and body fat mass was modified by the genotypes studied. CONCLUSIONS: SLC22A4 c.150C > T and SLC22A5 -207C > G polymorphisms are associated with tooth loss and markers of body shape in women but not in men. CLINICAL RELEVANCE: Tooth loss may be related to obesity beyond inflammatory mechanisms, conceivably with a genetic background.
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Carnitina , Perda de Dente , Adiposidade/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Seguimentos , Predisposição Genética para Doença , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Obesidade/genética , Proteínas de Transporte de Cátions Orgânicos/genética , Polimorfismo de Nucleotídeo Único , Membro 5 da Família 22 de Carreadores de Soluto , Simportadores/genética , Perda de Dente/genéticaRESUMO
The rare PEL-negative phenotype is one of the last blood groups with an unknown genetic basis. By combining whole-exome sequencing and comparative global proteomic investigations, we found a large deletion in the ABCC4/MRP4 gene encoding an ATP-binding cassette (ABC) transporter in PEL-negative individuals. The loss of PEL expression on ABCC4-CRISPR-Cas9 K562 cells and its overexpression in ABCC4-transfected cells provided evidence that ABCC4 is the gene underlying the PEL blood group antigen. Although ABCC4 is an important cyclic nucleotide exporter, red blood cells from ABCC4null/PEL-negative individuals exhibited a normal guanosine 3',5'-cyclic monophosphate level, suggesting a compensatory mechanism by other erythroid ABC transporters. Interestingly, PEL-negative individuals showed an impaired platelet aggregation, confirming a role for ABCC4 in platelet function. Finally, we showed that loss-of-function mutations in the ABCC4 gene, associated with leukemia outcome, altered the expression of the PEL antigen. In addition to ABCC4 genotyping, PEL phenotyping could open a new way toward drug dose adjustment for leukemia treatment.
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Antígenos de Grupos Sanguíneos/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Agregação Plaquetária , Plaquetas/citologia , Plaquetas/metabolismo , Sistemas CRISPR-Cas , Células Eritroides/citologia , Células Eritroides/metabolismo , Deleção de Genes , Humanos , FenótipoRESUMO
The link between thrombocytosis and malignancy has been well known for many years and its associations with worse outcomes have been reported mainly for solid tumors. Besides measuring platelet count, it has become popular to assess platelet function in the context of malignant diseases during the last decade. Malignant gliomas differ tremendously from malignancies outside the central nervous system because they virtually never form distant metastases. This review summarizes the current understanding of the platelet-immune cell communication and its potential role in glioma resistance and progression. Particularly, we focus on platelet-derived proinflammatory modulators, such as sphingosine-1-phosphate (S1P). The multifaceted interaction with immune cells puts the platelet into an interesting perspective regarding the recent advances in immunotherapeutic approaches in malignant glioma.
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Neurosteroids, comprising pregnane, androstane, and sulfated steroids can alter neuronal excitability through interaction with ligand-gated ion channels and other receptors and have therefore a therapeutic potential in several brain disorders. They can be formed in brain cells or are synthesized by an endocrine gland and reach the brain by penetrating the blood-brain barrier (BBB). Especially sulfated steroids such as pregnenolone sulfate (PregS) and dehydroepiandrosterone sulfate (DHEAS) depend on transporter proteins to cross membranes. In this review, we discuss the involvement of ATP-binding cassette (ABC)- and solute carrier (SLC)-type membrane proteins in the transport of these compounds at the BBB and in the choroid plexus (CP), but also in the secretion from neurons and glial cells. Among the ABC transporters, especially BCRP (ABCG2) and several MRP/ABCC subfamily members (MRP1, MRP4, MRP8) are expressed in the brain and known to efflux conjugated steroids. Furthermore, several SLC transporters have been shown to mediate cellular uptake of steroid sulfates. These include members of the OATP/SLCO subfamily, namely OATP1A2 and OATP2B1, as well as OAT3 (SLC22A3), which have been reported to be expressed at the BBB, in the CP and in part in neurons. Furthermore, a role of the organic solute transporter OSTα-OSTß (SLC51A/B) in brain DHEAS/PregS homeostasis has been proposed. This transporter was reported to be localized especially in steroidogenic cells of the cerebellum and hippocampus. To date, the impact of transporters on neurosteroid homeostasis is still poorly understood. Further insights are desirable also with regard to the therapeutic potential of these compounds.
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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.
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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.
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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/fisiologiaRESUMO
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.
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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.
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Antimaláricos/sangue , Eritrócitos/metabolismo , Transportadores de Ânions Orgânicos/sangue , Animais , Antimaláricos/farmacocinética , Cães , Feminino , Voluntários Saudáveis , Humanos , Células Madin Darby de Rim Canino , MasculinoRESUMO
Sphingosine-1-phosphate (S1P) is a versatile lipid signaling molecule and key regulator in vascular inflammation. S1P is secreted by platelets, monocytes, and vascular endothelial and smooth muscle cells. It binds specifically to a family of G-protein-coupled receptors, S1P receptors 1 to 5, resulting in downstream signaling and numerous cellular effects. S1P modulates cell proliferation and migration, and mediates proinflammatory responses and apoptosis. In the vascular barrier, S1P regulates permeability and endothelial reactions and recruitment of monocytes and may modulate atherosclerosis. Only recently has S1P emerged as a critical mediator which directly links the coagulation factor system to vascular inflammation. The multifunctional proteases thrombin and FXa regulate local S1P availability and interact with S1P signaling at multiple levels in various vascular cell types. Differential expression patterns and intracellular signaling pathways of each receptor enable S1P to exert its widespread functions. Although a vast amount of information is available about the functions of S1P and its receptors in the regulation of physiological and pathophysiological conditions, S1P-mediated mechanisms in the vasculature remain to be elucidated. This review summarizes recent findings regarding the role of S1P and its receptors in vascular wall and blood cells, which link the coagulation system to inflammatory responses in the vasculature.
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Coagulação Sanguínea/fisiologia , Inflamação/sangue , Inflamação/imunologia , Lisofosfolipídeos/sangue , Lisofosfolipídeos/imunologia , Receptores de Lisoesfingolipídeo/sangue , Receptores de Lisoesfingolipídeo/imunologia , Esfingosina/análogos & derivados , Coagulação Sanguínea/imunologia , Vasos Sanguíneos/fisiologia , Endotélio Vascular/fisiologia , Humanos , Modelos Cardiovasculares , Modelos Imunológicos , Ativação Plaquetária , Receptores de Trombina/metabolismo , Transdução de Sinais , Esfingosina/sangue , Esfingosina/imunologiaRESUMO
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
We have previously shown that precursors of odorous components characteristic of axillary sweat are hardly detectable or undetectable in individuals carrying the 538G > A SNP in the ABCC11 transporter gene. However, it is unclear, whether ABCC11 is directly involved in the transport of these compounds. To approach this question, transport of peptide-conjugated potential precursors of 3-methyl-3-sulfanylhexanol (3M3SH), a key determinant of axillary malodour, was measured using membrane vesicles of Sf9 insect cells overexpressing human ABCC11. Whilst no ABCC11-mediated transport was detected for the dipeptide precursor Cys-Gly-3M3SH, the glutathione conjugate of 3M3SH (SG-3M3SH) was robustly taken up by ABCC11 at a transport rate of 0.47 pmol/mg/min. Collectively, these results illuminate SG-3M3SH as a putative precursor of 3M3SH, which then may undergo intra-vesicular maturation to generate Cys-Gly-3M3SH. Critically, the apocrine sweat gland was demonstrated to express γ-glutamyl transferase 1 (GGT1) protein, which is known to catalyse the deglutamylation of glutathionyl conjugates. Additionally, we provide evidence that recombinant and isolated hepatic human GGT1 is capable of transforming SG-3M3SH to Cys-Gly-3M3SH in vitro. To sum up, we demonstrate that the functionality of ABCC11 is likely to play an important role in the generation of axillary malodour. Furthermore, we identify GGT1 as a key enzyme involved in the biosynthesis of Cys-Gly-3M3SH.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Glândulas Apócrinas/metabolismo , Hexanóis/metabolismo , Ácidos Sulfanílicos/metabolismo , gama-Glutamiltransferase/metabolismo , Animais , Linhagem Celular , Humanos , OdorantesRESUMO
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.