RESUMO
PURPOSE: Identifying factors that determine concentrations of antiretroviral drugs in CD4 cells are important for improving therapeutic efficacy. Experimental models indicate that the nucleoside reverse transcriptase inhibitor lamivudine is transported by the organic cation transporters 1 and 2 (OCT1 and OCT2, respectively). Here, we tested whether OCT1 and OCT2 contribute to the uptake of lamivudine into native CD4 cells of human immunodeficiency virus (HIV)-infected individuals. METHODS: CD4 cells obtained by non-activated cell sorting from 35 individuals with HIV-1 infection were incubated with lamivudine (10 µM, 30 min), and intracellular concentrations of lamivudine and its active metabolite lamivudine triphosphate were determined by liquid chromatography tandem mass spectrometry. The expression of OCT1 and OCT2 mRNA was measured by quantitative real-time polymerase chain reaction (PCR). A model of OCT2-transfected CD4 cells was established for mechanistic investigations. RESULTS: Intracellular concentrations of lamivudine and its active metabolite lamivudine triphosphate showed strong linear correlations with each other and with the CD4 mRNA expression of OCT1 and OCT2 (r > 0.80). Coincubation with protease inhibitors (ritonavir, nelfinavir) that inhibit OCT1 and OCT2 yielded decreased intracellular concentrations of lamivudine and lamivudine triphosphate. Incubation of CD4 cells from healthy donors transfected with an OCT2 expression vector yielded increased concentrations of lamivudine and lamivudine triphosphate. CONCLUSION: Our studies indicate a role of OCT1 and OCT2 for the cellular accumulation of lamivudine in HIV-infected individuals.
Assuntos
Linfócitos T CD4-Positivos/efeitos dos fármacos , Citidina Trifosfato/análogos & derivados , Didesoxinucleotídeos/farmacocinética , Infecções por HIV/tratamento farmacológico , Lamivudina/análogos & derivados , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Transportador 1 de Cátions Orgânicos/metabolismo , Adulto , Terapia Antirretroviral de Alta Atividade , Linfócitos T CD4-Positivos/metabolismo , Citidina Trifosfato/administração & dosagem , Citidina Trifosfato/farmacocinética , Didesoxinucleotídeos/administração & dosagem , Feminino , Inibidores da Protease de HIV/administração & dosagem , Inibidores da Protease de HIV/farmacocinética , HIV-1/efeitos dos fármacos , Humanos , Lamivudina/administração & dosagem , Lamivudina/farmacocinética , Masculino , Pessoa de Meia-Idade , Nelfinavir/farmacologia , Proteínas de Transporte de Cátions Orgânicos/genética , Transportador 1 de Cátions Orgânicos/genética , Transportador 2 de Cátion Orgânico , Fosforilação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Ritonavir/farmacologia , Espectrometria de Massas em Tandem , Transfecção , Adulto JovemRESUMO
Ergothioneine (ET) is a unique natural antioxidant which mammalia acquire exclusively from their food. Recently, we have discovered an ET transporter (ETT; gene symbol SLC22A4). The existence of a specific transporter suggests a beneficial role for ET; however, the precise physiological purpose of ET is still unclear. A conspicuous site of high extracellular ET accumulation is boar seminal plasma. Here, we have investigated whether ETT is responsible for specific accumulation of ET in the boar reproductive tract. The putative ETT from pig (ETTp) was cloned and validated by functional expression in 293 cells. The highest levels of ETTp mRNA were detected by real-time RT-PCR in seminal vesicles, eye, and kidney; much less was present in bulbourethral gland, testis, and prostate. By contrast, there was virtually no ETT mRNA in rat seminal vesicles. ET content in boar reproductive tissues, determined by LC-MS/MS, closely matched the ETT expression profile. Thus, strong and specific expression of ETTp in boar seminal vesicles explains high accumulation of ET in this gland and hence also in seminal plasma. Previous reports suggest that the glutathione (GSH) content of seminal plasma correlates directly with ET content; however, a comprehensive analysis across several species is not available. We have measured ET and GSH in seminal plasma from human, boar, bull, stallion, and rabbit by LC-MS/MS. GSH levels in seminal plasma do not correlate with ET levels. This suggests that the function of ET, at least in this extracellular context, does not depend on redox cycling with GSH.
Assuntos
Ergotioneína/metabolismo , Glutationa/metabolismo , Proteínas de Transporte de Cátions Orgânicos/biossíntese , RNA Mensageiro/metabolismo , Sêmen/metabolismo , Glândulas Seminais/metabolismo , Sus scrofa/metabolismo , Animais , Bovinos , Galinhas , Clonagem Molecular , DNA Complementar/genética , Células HEK293 , Cavalos , Humanos , Masculino , Proteínas de Transporte de Cátions Orgânicos/genética , Oxirredução , Plasmídeos , Coelhos , Ratos Wistar , Reação em Cadeia da Polimerase em Tempo Real , Especificidade da Espécie , Sus scrofa/genética , Simportadores , TransfecçãoAssuntos
Doença de Crohn/metabolismo , Ergotioneína/análise , Íleo/metabolismo , Proteínas de Transporte de Cátions Orgânicos/genética , Adulto , Doença de Crohn/genética , Feminino , Predisposição Genética para Doença , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Simportadores , Adulto JovemRESUMO
This study aimed to quantify the inhibition of cytochrome P450 (CYP3A), CYP2D6, and P-glycoprotein in human immunodeficiency virus (HIV)-infected patients receiving an antiretroviral therapy (ART) containing ritonavir boosted lopinavir, and to identify factors influencing ritonavir and lopinavir pharmacokinetics. We measured activities of CYP3A, CYP2D6, and P-glycoprotein in 28 patients before and during ART using a cocktail phenotyping approach. Activities, demographics, and genetic polymorphisms in CYP3A, CYP2D6, and P-glycoprotein were tested as covariates. Oral midazolam clearance (overall CYP3A activity) decreased to 0.19-fold (90% confidence interval (CI), 0.15-0.23), hepatic midazolam clearance and intestinal midazolam availability changed to 0.24-fold (0.20-0.29) and 1.12-fold (1.00-1.26), respectively. In CYP2D6 extensive metabolizers, the plasma ratio AUC(dextromethorphan)/AUC(dextrorphan) increased to 2.92-fold (2.31-3.69). Digoxin area under the curve (AUC)(0-12) (P-glycoprotein activity) increased to 1.81-fold (1.56-2.09). Covariates had no major influence on lopinavir and ritonavir pharmacokinetics. In conclusion, CYP3A, CYP2D6, and P-glycoprotein are profoundly inhibited in patients receiving ritonavir boosted lopinavir. The covariates investigated are not useful for a priori dose selection.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Citocromo P-450 CYP2D6/metabolismo , Citocromo P-450 CYP3A/metabolismo , Infecções por HIV/metabolismo , Inibidores da Protease de HIV/administração & dosagem , Pirimidinonas/administração & dosagem , Ritonavir/administração & dosagem , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Adulto , Citocromo P-450 CYP2D6/genética , Citocromo P-450 CYP3A/genética , Sinergismo Farmacológico , Feminino , Infecções por HIV/tratamento farmacológico , Infecções por HIV/genética , Inibidores da Protease de HIV/sangue , Inibidores da Protease de HIV/farmacocinética , Humanos , Mucosa Intestinal/metabolismo , Intestinos/efeitos dos fármacos , Intestinos/enzimologia , Fígado/efeitos dos fármacos , Fígado/enzimologia , Fígado/metabolismo , Lopinavir , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Pirimidinonas/sangue , Pirimidinonas/farmacocinética , Ritonavir/sangue , Ritonavir/farmacocinéticaRESUMO
The extraneuronal monoamine transporter (EMT) corresponds to the classical steroid-sensitive monoamine transport mechanism that was first described as "uptake2" in rat heart with noradrenaline as substrate. The organic cation transporters OCT1 and OCT2 are related to EMT. The three carriers share basic structural and functional characteristics. Hence, EMT, OCT1 and OCT2 constitute a group referred to as non-neuronal monoamine transporters or organic cation transporters. After a brief general introduction, this review focuses on the critical analysis of substrate specificity. We calculate from the available literature and compare consensus transport efficiency (clearance) data for human and rat EMT, OCT1 and OCT2, expressed in transfected cell lines. From the plethora of inhibitors that have been tested, the casual observer likely gets the impression that these carriers indiscriminately transport very many compounds. However, our knowledge about actual substrates is rather limited. 1-Methyl-4-phenylpyridinium (MPP+) is an excellent substrate for all three carriers, with clearances typically in the range of 20-50 microl min(-1) mg protein(-1). The second-best general substrate is tyramine with a transport efficiency (TE) range relative to MPP+ of 20%-70%. The TEs of OCT1 and OCT2 for dopamine, noradrenaline, adrenaline and 5-HT in general are rather low, in the range relative to MPP+ of 5%-15%. This suggests that OCT1 and OCT2 are not primarily dedicated to transport these monoamine transmitters; only EMT may play a significant role in catecholamine inactivation. For many substrates, such as tetraethylammonium, histamine, agmatine, guanidine, cimetidine, creatinine, choline and acetylcholine, the transport efficiencies are markedly different among the carriers.
Assuntos
Proteínas de Transporte de Cátions Orgânicos/metabolismo , Transportador 1 de Cátions Orgânicos/metabolismo , 1-Metil-4-fenilpiridínio/metabolismo , Animais , Transporte Biológico , Catecolaminas/metabolismo , Linhagem Celular , Humanos , Camundongos , Camundongos Knockout , Proteínas de Transporte de Cátions Orgânicos/química , Proteínas de Transporte de Cátions Orgânicos/genética , Transportador 1 de Cátions Orgânicos/química , Transportador 1 de Cátions Orgânicos/genética , Transportador 2 de Cátion Orgânico , Conformação Proteica , Ratos , Transfecção , Tiramina/metabolismoAssuntos
Inibidores da Agregação Plaquetária/farmacologia , Ticlopidina/análogos & derivados , Adulto , Área Sob a Curva , Cromatografia Líquida , Clopidogrel , Resistência a Medicamentos , Feminino , Humanos , Espectrometria de Massas , Inibidores da Agregação Plaquetária/farmacocinética , Ticlopidina/farmacocinética , Ticlopidina/farmacologiaRESUMO
BACKGROUND: Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme in the metabolism of the chemotherapeutic drug 5-fluorouracil (5-FU). Application of 5-FU is restricted by a narrow therapeutic index because of severe toxicity of WHO grades III-IV. The exon 14-skipping mutation (c.1905+1G>A) accounts for approximately a quarter of all severely toxic cases. However, numerous other polymorphisms have been identified within the DPYD gene in affected patients, and the pathophysiological significance of most of them is unclear. PATIENT AND METHODS: We report a patient with advanced caecum cancer who twice received 950 mg 5-FU and 45 mg folinic acid as adjuvant by bolus injection. 2 days after onset of chemotherapy, the patient developed a multiple organ dysfunction exhibiting a cardiogenic shock with severe left ventricular insufficiency, marked reduction of renal function, and beginning hepatic encephalopathy with somnolence, myoclonus, and a seizure. In order to investigate a possible defect within the DPYD gene direct sequencing of all 23 exons was carried out. RESULTS: Genotyping revealed a rare c.1601G>A polymorphism which causes a change in the protein sequence (S534N). Data regarding the clinical relevance are ambiguous. The polymorphism has been detected together with an intronic mutation and both polymorphisms have consistently been reported with reduced enzyme activity. CONCLUSION: The present case provides further evidence of an etiologic role of the c.1601G>A mutation for DPD deficiency and the occurrence of severe 5-FU-related toxicity and underlines the value of comprehensive pharmakogenetic diagnostics with respect to the dihydropyrimidine dehydrogenase.
Assuntos
Neoplasias do Ceco/tratamento farmacológico , Neoplasias do Ceco/enzimologia , Deficiência da Di-Hidropirimidina Desidrogenase , Fluoruracila/efeitos adversos , Fluoruracila/uso terapêutico , Doenças Metabólicas/diagnóstico , Doenças Metabólicas/enzimologia , Insuficiência de Múltiplos Órgãos/induzido quimicamente , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/enzimologia , Adenocarcinoma/genética , Adulto , Antineoplásicos/efeitos adversos , Antineoplásicos/uso terapêutico , Neoplasias do Ceco/genética , Di-Hidrouracila Desidrogenase (NADP)/análise , Di-Hidrouracila Desidrogenase (NADP)/genética , Predisposição Genética para Doença/genética , Humanos , Masculino , Doenças Metabólicas/complicações , Doenças Metabólicas/genética , Doenças Raras/complicações , Doenças Raras/diagnóstico , Doenças Raras/enzimologia , Doenças Raras/genética , Resultado do TratamentoRESUMO
1. The study was designed to test the hypothesis that aspirin may stimulate nitric oxide (NO) release from vascular endothelium, a pivotal factor for maintenance of vascular homeostasis. 2. Clinical evidence suggests that low-dose aspirin may improve vascular endothelial function. Since other cyclooxygenase (COX) inhibitors showed no beneficial vascular effects, aspirin may exhibit a vasculoprotective, COX-independent mechanism. 3. Luminal NO release was monitored in real time on dissected porcine coronary arteries (PCA) by an amperometric, NO-selective sensor. Additionally, endothelial NO synthase (eNOS) activity was measured in EA.hy 926 cell homogenates by an l-[(3)H]citrulline/l-[(3)H]arginine conversion assay. Superoxide scavenging capacity was assessed by lucigenin-enhanced luminescence. 4. Aspirin induced an immediate concentration-dependent NO release from PCA with an EC(50) of 50 nm and potentiated the NO stimulation by the receptor-dependent agonist substance P. These effects were independent of an increase in intracellular calcium and could be mimicked by stimulation with acetylating aspirin derivatives. The aspirin metabolite salicylic acid or the reversible cyclooxygenase inhibitor indomethacin failed to modulate NO release. Incubation of soluble eNOS for 15 min with 100 microm aspirin or acetylating aspirin analogues increased the l-[(3)H]citrulline yield by 40-80%, while salicylic acid had no effect. Aspirin and salicylic acid showed a similar, but only modest, magnitude and velocity of superoxide scavenging. 5. Our findings demonstrate that therapeutically relevant concentrations of aspirin elicit NO release from vascular endothelium. This effect appears to be due to a direct acetylation of the eNOS protein, but is independent of COX inhibition or inhibition of superoxide-mediated NO degradation.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Aspirina/farmacologia , Ácido Egtázico/análogos & derivados , Endotélio Vascular/metabolismo , Óxido Nítrico/metabolismo , Animais , Arteriosclerose/tratamento farmacológico , Arteriosclerose/fisiopatologia , Células Cultivadas , Quelantes/farmacologia , Citrulina/metabolismo , Vasos Coronários/efeitos dos fármacos , Vasos Coronários/metabolismo , Ácido Egtázico/farmacologia , Endotélio Vascular/efeitos dos fármacos , Sequestradores de Radicais Livres/farmacologia , Homeostase/efeitos dos fármacos , Humanos , Técnicas In Vitro , Oxidantes/metabolismo , Substância P/farmacologia , Superóxidos/metabolismo , SuínosAssuntos
Aspirina/uso terapêutico , Inibidores de Ciclo-Oxigenase/uso terapêutico , Neoplasias/prevenção & controle , Anti-Inflamatórios não Esteroides/uso terapêutico , Análise Custo-Benefício , Estudos Epidemiológicos , Humanos , Mesalamina/uso terapêutico , Neoplasias/epidemiologia , Prevenção Primária/métodos , Ensaios Clínicos Controlados Aleatórios como Assunto , Medição de RiscoRESUMO
AIMS/HYPOTHESIS: Chronic exposure to high concentrations of glucose has consistently been demonstrated to impair endothelium-dependent, nitric oxide (NO)-mediated vasodilation. In contrast, several clinical investigations have reported that acute exposure to high glucose, alone or in combination with insulin, triggers vasodilation. The aim of this study was to examine whether elevated glucose itself stimulates endothelial NO formation or enhances insulin-mediated endothelial NO release. METHODS: We measured NO release and vessel tone ex vivo in porcine coronary conduit arteries (PCAs). Intracellular Ca(2+) was monitored in porcine aortic endothelial cells (PAECs) by fura-2 fluorescence. Expression of the Na(+)/glucose cotransporter-1 (SGLT-1) was assayed in PAECs and PCA endothelium by RT-PCR. RESULTS: Stimulation of PCAs with D: -glucose, but not the osmotic control L: -glucose, induced a transient increase in NO release (EC(50) approximately 10 mmol/l), mediated by a rise in intracellular Ca(2+) levels due to an influx from the extracellular space. This effect was abolished by inhibitors of the plasmalemmal Na(+)/Ca(2+) exchanger (dichlorobenzamil) and the SGLT-1 (phlorizin), which was found to be expressed in aortic and coronary endothelium. Alone, D: -glucose did not relax PCA, but did augment the effect of insulin on NO release and vasodilation. CONCLUSIONS/INTERPRETATION: An increased supply of extracellular D: -glucose appears to enhance the activity of the endothelial isoform of nitric oxide synthase by increasing intracellular Na(+) concentrations via SGLT-1, which in turn stimulates an extracellular Ca(2+) influx through the Na(+)/Ca(2+) exchanger. This mechanism may be responsible for glucose-enhanced, insulin-dependent increases in tissue perfusion (including coronary blood-flow), thus accelerating glucose extraction from the blood circulation to limit the adverse vascular effects of prolonged hyperglycaemia.
Assuntos
Amilorida/análogos & derivados , Endotélio Vascular/fisiologia , Glucose/farmacologia , Insulina/farmacologia , Amilorida/farmacologia , Animais , Aorta , Endotélio Vascular/efeitos dos fármacos , Cinética , Tono Muscular/efeitos dos fármacos , Tono Muscular/fisiologia , Óxido Nítrico/metabolismo , Florizina/farmacologia , Sacarose/farmacologia , Superóxidos/metabolismo , SuínosRESUMO
The aim of this work was to characterise the intestinal absorption of organic cations, by testing the possibility of involvement of known members of the amphiphilic solute facilitator (ASF) family in this process. For that purpose, the characteristics of the uptake of 1-methyl-4-phenylpyridinium, a model organic cation, at the brush-border membrane of Caco-2 cells were compared with those of the extraneuronal monoamine transporter (EMT)-mediated transport. Uptake of [3H]MPP+ by Caco-2 and 293hEMT cells showed pH-dependence: it was significantly reduced (to 86% and 62% of control, respectively) when the pH of the extracellular medium was decreased to 6.2, and increased (to 116% and 136% of control, respectively) when the extracellular pH was increased to 8.2. Uptake of [3H]MPP+ by Caco-2 cells and 293hEMT cells showed potential-dependence: substitution of KCl for NaCl in the incubation medium resulted in a reduction in the inward transport of [3H]MPP+ (to 70% and 40% of control, respectively). Uptake of [3H]MPP+ by Caco-2 and 293hEMT cells showed only little dependence on Na+: substitution of NaCl of the incubation media with LiCl resulted in a small decrease (of 19% and 14%, respectively) in [3H]MPP+ uptake. However, when NaCl was substituted with choline chloride, a significant reduction in [3H]MPP+ uptake by Caco-2 and 2931hEMT cells (of 56% and 68%, respectively) was observed. The effect of various compounds on initial rates of [3H]MPP+ uptake into Caco-2 and 293hEMT cells was tested. All compounds tested interacted with the specific [3H]MPP+ uptake in both cell lines. There was no correlation between the IC50s in relation to inhibition of [3H]MPP+ uptake into Caco-2 cells and into 293hEMT cells. Reverse transcriptase-polymerase chain reaction indicates that mRNA of hEMT and of the human organic cation transporter 1 (hOCT1) are present in Caco-2 cells. In conclusion, our results suggest that uptake of organic cations at the brush-border membrane of Caco-2 cells may occur through two distinct Na+-independent transporters belonging to the ASF family: hEMT and hOCT1.
Assuntos
Cátions/farmacocinética , Mucosa Intestinal/metabolismo , Proteínas de Transporte de Cátions Orgânicos , 1-Metil-4-fenilpiridínio/farmacocinética , Transporte Biológico/efeitos dos fármacos , Células CACO-2 , Proteínas de Transporte/efeitos dos fármacos , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular , Clonidina/farmacologia , Corticosterona/farmacologia , DNA Recombinante , Relação Dose-Resposta a Droga , Expressão Gênica , Histamina/farmacologia , Humanos , Concentração de Íons de Hidrogênio , Intestinos/citologia , Intestinos/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Proteínas de Membrana/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Transportador 1 de Cátions Orgânicos , Plasmídeos/genética , Quinolinas/farmacologia , Compostos de Quinolínio/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sódio/farmacologia , Tetraetilamônio/farmacologia , Fatores de Tempo , Transfecção , TrítioRESUMO
Several transmembrane transporters of organic compounds are regulated by phosphorylation/dephosphorylation mechanisms. The aim of this study was to investigate the possible regulation of the human extraneuronal monoamine transporter, hEMT, by these mechanisms. The experiments were performed using HEK293 cells stably transfected with pcDNA3hEMT (293hEMT). The characteristics of hEMT-mediated uptake of [3H]1-methyl-4-phenylpyridinium ([3H]MPP+) were studied by incubating the cells at 37 degrees C for 1 min with 200 nM [3H]MPP+. Uptake of [3H]MPP+ by 293hEMT cells was not affected or only slightly reduced by modulators of protein kinase A, protein kinase C, or protein kinase G. It was not affected by an inhibitor of protein tyrosine kinase and was reduced by mitogen-activated protein kinase inhibitors. Uptake of [3H]MPP+ by 293hEMT cells was independent of extracellular Ca2+ and strongly reduced by Ca2+/calmodulin pathway inhibitors. Uptake of [3H]MPP+ by 293hEMT cells was strongly reduced in the presence of non-selective phosphodiesterase inhibitors (IBMX, caffeine, theophylline). The effect of IBMX was independent of extracellular Ca2+ its IC50 was found to be 82.0 microM (66.2-101.6 microM; n=4), and its inhibitory effect resulted from a significant decrease in the maximal velocity of [3H]MPP+ uptake, with no change in the Michaelis-Menten constant. [3H]MPP+ uptake was reduced by 8-methoxy-methyl-IBMX, a selective inhibitor of the Ca2+/calmodulin-dependent phosphodiesterase (PDE1), but not by zaprinast, a selective inhibitor of PDE5. Uptake of [3H]MPP+ by 293hEMT cells was strongly reduced by protein tyrosine phosphatase inhibitors, by an alkaline phosphatase inhibitor and, by contrast. showed an increase in the presence of exogenous alkaline phosphatase. In conclusion, these results suggest that hEMT is regulated by phosphorylation/dephosphorylation mechanisms, being active in the dephosphorylated state.
Assuntos
Monoaminas Biogênicas/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Transporte de Cátions Orgânicos , Sistemas do Segundo Mensageiro/fisiologia , 1-Metil-4-fenilpiridínio/farmacocinética , Monoaminas Biogênicas/biossíntese , Proteínas de Transporte/biossíntese , Linhagem Celular Transformada , Embrião de Mamíferos , Ativadores de Enzimas/farmacologia , Inibidores Enzimáticos/farmacologia , Humanos , Rim/citologia , Fosforilação/efeitos dos fármacos , Sistemas do Segundo Mensageiro/efeitos dos fármacos , TransfecçãoRESUMO
Non-neuronal monoamine transporters OCT1, OCT2, and EMT, which are all members of the amphiphilic solute facilitator family, control signal transmission by removing released transmitters, such as dopamine, noradrenaline, adrenaline, 5-hydroxytryptamine, and histamine, from the extracellular space. In the current study, we have isolated human EMT (gene symbol SLC22A3) and OCT2 (SLC22A2) genes and report the gene and promoter organization. Both genes consist of 11 coding exons, with consensus GT/AG splice sites and conserved intron locations. The EMT gene is 77 kb, and the OCT2 gene is 45 kb in size. For the EMT gene, two transcription start points were identified by inverse polymerase chain reaction based on mRNA from Caki-1 cells. The EMT promoter, located within a CpG island, lacks a consensus TATA box but contains a prototypical initiator element and a number of potential binding sites for ubiquitous transcription factors Sp1 and NF-1. In contrast, the OCT2 promoter is not associated with a CpG island, contains a putative TATA box, and potential binding sites for specific transcription factors, such as HFH-8 and IK2. Since EMT and OCT2 may play important roles in catecholamine homeostasis and, as such, are candidate genes in human disease, the present results provide a basis for the analysis of genetic variation and the regulation of transcription.
Assuntos
Monoaminas Biogênicas/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte de Cátions Orgânicos , Composição de Bases , Sequência de Bases , Proteínas de Transporte/metabolismo , Éxons , Biblioteca Genômica , Humanos , Íntrons , Dados de Sequência Molecular , Família Multigênica , Transportador 2 de Cátion Orgânico , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas/genética , Análise de Sequência de DNA , Homologia de Sequência do Ácido Nucleico , Transcrição Gênica/genéticaRESUMO
The recently identified transport proteins organic cation transporter 1 (OCT1), OCT2, and extraneuronal monoamine transporter (EMT) accept dopamine, noradrenaline, adrenaline, and 5-hydroxytryptamine as substrates and hence qualify as non-neuronal monoamine transporters. In the present study, selective transport substrates were identified that allow, by analogy to receptor agonists, functional discrimination of these transporters. To contrast efficiency of solute transport, stably transfected 293 cell lines, each expressing a single transporter, were examined side by side in uptake experiments with radiolabeled substrates. Normalized uptake rates indicate that tetraethylammonium, with a rate of about 0.5 relative to 1-methyl-4-phenylpyridinium (MPP+), is a good substrate for OCT1 and OCT2. It was not, however, accepted as substrate by EMT. Choline was transported exclusively by OCT1, with a rate of about 0.5 relative to MPP+. Histamine was a good substrate with a rate of about 0.6 relative to MPP+ for OCT2 and EMT, but was not transported by OCT1. Guanidine was an excellent substrate for OCT2, with a rate as high as that of MPP+. Transport of guanidine by OCT1 was low, and transport by EMT was negligible. With the guanidine derivatives cimetidine and creatinine, a pattern strikingly similar to guanidine was observed. Collectively, these substrates reveal key differences in solute recognition and turnover and thus challenge the concept of "polyspecific" organic cation transporters. In addition, our data, when compared with previous studies, suggest that OCT2 corresponds to the organic cation/H+ antiport mechanism in renal brush-border membrane vesicles, and that EMT corresponds to the guanidine/H+ antiport mechanism in membrane vesicles from placenta and intestine.
Assuntos
Proteínas de Transporte/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras , Neuropeptídeos , Proteínas de Transporte de Cátions Orgânicos , Sequência de Aminoácidos , Animais , Transporte Biológico , Proteínas de Transporte/química , Proteínas de Transporte/genética , Células Cultivadas , DNA Complementar/genética , Guanidina/análogos & derivados , Guanidina/metabolismo , Histamina/metabolismo , Humanos , Dados de Sequência Molecular , Transportador 1 de Cátions Orgânicos , Transportador 2 de Cátion Orgânico , Ratos , Homologia de Sequência de Aminoácidos , Proteínas Vesiculares de Transporte de Aminas BiogênicasRESUMO
Bone morphogenetic proteins (BMPs), growth and differentiation factor 5 (GDF5) and glial cell line-derived neurotrophic factor (GDNF) are members of the transforming growth factor-beta superfamily that have been implicated in tissue growth and differentiation. Several BMPs are expressed in embryonic and adult brain. We show now that BMP-2, -6 and -7 and GDF5 are expressed in the embryonic rat hindbrain raphe. To start to define roles for BMPs in the regulation of serotonergic (5-HT) neuron development, we have generated serum-free cultures of 5-HT neurons isolated from the embryonic (E14) rat raphe. Addition of saturating concentrations (10 ng/mL) of BMP-6 and GDF5 augmented numbers of tryptophan hydroxylase (TpOH) -immunoreactive neurons and cells specifically taking up 5, 7-dihydroxytryptamine (5,7-DHT) by about two-fold. Alterations in 5-HT neuron numbers were due to the induction of serotonergic markers rather than increased survival, as shown by the efficacy of short-term treatments. Importantly, BMP-7 selectively induced 5, 7-DHT uptake without affecting TpOH immunoreactivity. BMP-6 and -7 also promoted DNA synthesis and increased numbers of cells immunoreactive for vimentin and glial fibrillary acidic protein (GFAP). Pharmacological suppression of cell proliferation or glial development abolished the induction of serotonergic markers by BMP-6 and -7, suggesting that BMPs act indirectly by stimulating synthesis or release of glial-derived serotonergic differentiation factors. Receptor bodies for the neurotrophin receptor trkB, but not trkC, abolished the BMP-mediated effects on serotonergic development, suggesting that the glia-derived factor is probably brain-derived neurotrophic factor (BDNF) or neurotrophin-4. In support of this notion, we detected increased levels of BDNF mRNA in BMP-treated cultures. Together, these data suggest both distinct and overlapping roles of several BMPs in regulating 5-HT neuron development.
Assuntos
Proteínas Morfogenéticas Ósseas/farmacologia , Fator Neurotrófico Derivado do Encéfalo/fisiologia , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Serotonina/metabolismo , 5,7-Di-Hidroxitriptamina/farmacocinética , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Sobrevivência Celular/fisiologia , Células Cultivadas , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Neuroglia/fisiologia , Neurônios/metabolismo , Fenótipo , Núcleos da Rafe/embriologia , Ratos , Ratos Wistar , Rombencéfalo/embriologia , Células-Tronco/fisiologia , Triptofano Hidroxilase/farmacocinéticaRESUMO
The recently cloned apical renal transport system for organic cations (OCT2) exists in dopamine-rich tissues such as kidney and some brain areas (Gründemann, D., Babin-Ebell, J., Martel, F., Ording, N., Schmidt, A., and Schömig, E. (1997) J. Biol. Chem. 272, 10408-10413). The study at hand was performed to answer the question of whether OCT2 accepts dopamine and other monoamine transmitters as substrate. 293 cells were stably transfected with the OCT2r cDNA resulting in the 293OCT2r cell line. Expression of OCT2r in 293 cells induces specific transport of tritiated dopamine, noradrenaline, adrenaline, and 5-hydroxytryptamine (5-HT). Initial rates of specific 3H-dopamine, 3H-noradrenaline, 3H-adrenaline, and 3H-5-HT transport were saturable, the Km values being 2.1, 4.4, 1.9, and 3.6 mmol/liter. The corresponding Vmax values were 3.9, 1.0, 0. 59, and 2.5 nmol min-1.mg of protein-1, respectively. 1, 1'-diisopropyl-2,4'-cyanine (disprocynium24), a known inhibitor of OCT2 with a potent eukaliuric diuretic activity, inhibited 3H-dopamine uptake into 293OCT2r cells with an Ki of 5.1 (2.6, 9.9) nmol/liter. In situ hybridization reveals that, within the kidney, the OCT2r mRNA is restricted to the outer medulla and deep portions of the medullary rays indicating selective expression in the S3 segment of the proximal tubule. These findings open the possibility that OCT2r plays a role in renal dopamine handling.
Assuntos
Proteínas de Transporte/metabolismo , Dopamina/metabolismo , Proteínas de Transporte de Cátions Orgânicos , Animais , Monoaminas Biogênicas/metabolismo , Transporte Biológico/efeitos dos fármacos , Proteínas de Transporte/genética , Proteínas de Transporte/isolamento & purificação , Cátions/metabolismo , Cocaína/farmacologia , Corticosterona/farmacologia , Hibridização In Situ , Isoproterenol/análogos & derivados , Isoproterenol/farmacologia , Rim/química , Transportador 2 de Cátion Orgânico , Quinolinas/farmacologia , RNA Mensageiro/isolamento & purificação , Ratos , Proteínas Recombinantes/metabolismo , Reserpina/farmacologia , Especificidade por Substrato , TransfecçãoRESUMO
1. Liver and kidney extract adrenaline and noradrenaline from the circulation by a mechanism which does not seem to be one of the classical catecholamine transporters. The hypothesis that OCT1 is involved the organic cation transporter type 1 which exists in rat kidney and liver-was tested. 2. Based on human embryonic kidney cells (293), we constructed a cell line which stably expresses OCT1r (293OCT1r cells). Transfection with OCT1 resulted in a transport activity not only for prototypical known substrates of OCT1 such as 3H-1-methyl-4-phenylpyridinium and 14C-tetraethylammonium but also for the catecholamines 3H-adrenaline, 3H-noradrenaline (3H-NA) and 3H-dopamine (3H-DA), the indoleamine 3H-5-hydroxytryptamine (3H-5HT) as well as the indirect sympathomimetic 14C-tyramine. 3. For 3H-DA, 3H-5HT and 3H-NA, at non-saturating concentrations, the rate constants for inwardly directed substrate flux (kin) were 6.9+/-0.8, 3.1+/-0.2, and 1.2+/-0.1 microl min(-1) mg protein(-1). In wild type cells (293WT) the corresponding kin's were considerably lower, being 0.94+/-0.40, 0.47+/-0.08 and 0.23+/-0.05 microl min(-1) mg protein ' (n=12). The indirectly determined half-saturating concentrations of DA, 5HT, and NA were 1.1 (95% c.i.: 0.8, 1.4), 0.65 (0.49, 0.86), and 2.8 (2.1, 3.7) mmol l(-1) (n=3). 4. Specific 3H-DA uptake in 293OCT1r cells was resistant to cocaine (1 micromoll(-1)), 3H-5HT uptake was resistant to citalopram (300 nmol l(-1)) and 3H-NA uptake was resistant to desipramine (100 nmoll(-1)), corticosterone (1 micromol l(-1)), and reserpine (10 nmoll(-1)) which rules out the involvement of classical transporters for biogenic amines. 5. The findings demonstrate that OCTI efficiently transports catecholamines and other biogenic amines and support the hypothesis that OCT1 is responsible for hepatic and renal inactivation of circulating catecholamines.