RESUMO
Cardiac glycosides, such as digoxin and digitoxin, are compounds that interact with Na+ /K+ -ATPase to induce anti-neoplastic effects; however, these cardiac glycosides have narrow therapeutic index. Thus, semi-synthetic analogs of digitoxin with modifications in the sugar moiety has been shown to be an interesting approach to obtain more selective and more effective analogs than the parent natural product. Therefore, the aim of this study was to assess the cytotoxic potential of novel digitoxigenin derivatives, digitoxigenin-α-L-rhamno-pyranoside (1) and digitoxigenin-α-L-amiceto-pyranoside (2), in cervical carcinoma cells (HeLa) and human diploid lung fibroblasts (Wi-26-VA4). In addition, we studied the anticancer mechanisms of action of these compounds by comparing its cytotoxic effects with the potential to modulate the activity of three P-type ATPases; Na+ /K+ -ATPase, sarco/endoplasmic reticulum Ca2+ -ATPase (SERCA), and plasma membrane Ca2+ -ATPase (PMCA). Briefly, the results showed that compounds 1 and 2 were more cytotoxic and selectivity for HeLa tumor cells than the nontumor cells Wi-26-VA4. While the anticancer cytotoxicity in HeLa cells involves the modulation of Na+ /K+ -ATPase, PMCA and SERCA, the modulation of these P-type ATPases was completely absent in Wi-26-VA4 cells, which suggest the importance of their role in the cytotoxic effect of compounds 1 and 2 in HeLa cells. Furthermore, the compound 2 inhibited directly erythrocyte ghosts PMCA and both compounds were more cytotoxic than digitoxin in HeLa cells. These results provide a better understanding of the mode of action of the synthetic cardiac glycosides and highlights 1 and 2 as potential anticancer agents.
Assuntos
Membrana Celular/enzimologia , Digitoxigenina , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Membrana Celular/genética , Digitoxigenina/análogos & derivados , Digitoxigenina/farmacologia , Células HeLa , Humanos , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , ATPase Trocadora de Sódio-Potássio/genéticaRESUMO
The accumulation of Ca2+ and its subsequent increase in oxidative stress is proposed to be involved in selective dysfunctionality of dopaminergic neurons, the main cell type affected in Parkinson's disease. To test the in vivo impact of Ca2+ increment in dopaminergic neurons physiology, we downregulated the plasma membrane Ca2+ ATPase (PMCA), a pump that extrudes cytosolic Ca2+ , by expressing PMCARNAi in Drosophila melanogaster dopaminergic neurons. In these animals, we observed major locomotor alterations paralleled to higher cytosolic Ca2+ and increased levels of oxidative stress in mitochondria. Interestingly, although no overt degeneration of dopaminergic neurons was observed, evidences of neuronal dysfunctionality were detected such as increases in presynaptic vesicles in dopaminergic neurons and in the levels of dopamine in the brain, as well as presence of toxic effects when PMCA was downregulated in the eye. Moreover, reduced PMCA levels were found in a Drosophila model of Parkinson's disease, Parkin knock-out, expanding the functional relevance of PMCA reduction to other Parkinson's disease-related models. In all, we have generated a new model to study motor abnormalities caused by increments in Ca2+ that lead to augmented oxidative stress in a dopaminergic environment, added to a rise in synaptic vesicles and dopamine levels.
Assuntos
Doença de Parkinson , ATPases Transportadoras de Cálcio da Membrana Plasmática , Animais , Cálcio/metabolismo , Neurônios Dopaminérgicos/metabolismo , Regulação para Baixo , Drosophila melanogaster , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismoRESUMO
Lysophosphatidic acid (LPA) acts through the activation of G protein-coupled receptors, in a Ca2+-dependent manner. We show the effects of LPA on the plasma membrane Ca2+-ATPase (PMCA) from kidney proximal tubule cells. The Ca2+-ATPase activity was inhibited by nanomolar concentrations of LPA, with maximal inhibition (~50%) obtained with 20 nM LPA. This inhibitory action on PMCA activity was blocked by Ki16425, an antagonist for LPA receptors, indicating that this lipid acts via LPA1 and/or LPA3 receptor. This effect is PKC-dependent, since it is abolished by calphostin C and U73122, PKC, and PLC inhibitors, respectively. Furthermore, the addition of 10-8 M PMA, a well-known PKC activator, mimicked PMCA modulation by LPA. We also demonstrated that the PKC activation leads to an increase in PMCA phosphorylation. These results indicate that LPA triggers LPA1 and/or LPA3 receptors at the BLM, inducing PKC-dependent phosphorylation with further inhibition of PMCA. Thus, LPA is part of the regulatory lipid network present at the BLM and plays an important role in the regulation of intracellular Ca2+ concentration that may result in significant physiological alterations in other Ca2+-dependent events ascribed to the renal tissue.
Assuntos
Cálcio/metabolismo , Membrana Celular/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Lisofosfolipídeos/farmacologia , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , Receptores de Ácidos Lisofosfatídicos/genética , Animais , Fracionamento Celular , Membrana Celular/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Estrenos/farmacologia , Regulação da Expressão Gênica , Transporte de Íons/efeitos dos fármacos , Isoxazóis/farmacologia , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/metabolismo , Naftalenos/farmacologia , Fosforilação/efeitos dos fármacos , ATPases Transportadoras de Cálcio da Membrana Plasmática/antagonistas & inibidores , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Cultura Primária de Células , Propionatos/farmacologia , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Pirrolidinonas/farmacologia , Receptores de Ácidos Lisofosfatídicos/antagonistas & inibidores , Receptores de Ácidos Lisofosfatídicos/metabolismo , Transdução de Sinais , Suínos , Acetato de Tetradecanoilforbol/farmacologia , Fosfolipases Tipo C/antagonistas & inibidores , Fosfolipases Tipo C/genética , Fosfolipases Tipo C/metabolismoRESUMO
Populations in sub-Saharan Africa have historically been exposed to intense selection from chronic infection with falciparum malaria. Interestingly, populations with the highest malaria intensity can be identified by the increased occurrence of endemic Burkitt Lymphoma (eBL), a pediatric cancer that affects populations with intense malaria exposure, in the so called "eBL belt" in sub-Saharan Africa. However, the effects of intense malaria exposure and sub-Saharan populations' genetic histories remain poorly explored. To determine if historical migrations and intense malaria exposure have shaped the genetic composition of the eBL belt populations, we genotyped ~4.3 million SNPs in 1,708 individuals from Ghana and Northern Uganda, located on opposite sides of eBL belt and with ≥ 7 months/year of intense malaria exposure and published evidence of high incidence of BL. Among 35 Ghanaian tribes, we showed a predominantly West-Central African ancestry and genomic footprints of gene flow from Gambian and East African populations. In Uganda, the North West population showed a predominantly Nilotic ancestry, and the North Central population was a mixture of Nilotic and Southern Bantu ancestry, while the Southwest Ugandan population showed a predominant Southern Bantu ancestry. Our results support the hypothesis of diverse ancestral origins of the Ugandan, Kenyan and Tanzanian Great Lakes African populations, reflecting a confluence of Nilotic, Cushitic and Bantu migrations in the last 3000 years. Natural selection analyses suggest, for the first time, a strong positive selection signal in the ATP2B4 gene (rs10900588) in Northern Ugandan populations. These findings provide important baseline genomic data to facilitate disease association studies, including of eBL, in eBL belt populations.
Assuntos
Linfoma de Burkitt/genética , Fluxo Gênico , Malária Falciparum/genética , Seleção Genética , Adolescente , África Subsaariana , Idoso , Linfoma de Burkitt/epidemiologia , Estudos de Casos e Controles , Criança , Pré-Escolar , Doenças Endêmicas , Feminino , Genética Populacional , Estudo de Associação Genômica Ampla , Gana/epidemiologia , Migração Humana , Humanos , Incidência , Lactente , Recém-Nascido , Malária Falciparum/epidemiologia , Masculino , Pessoa de Meia-Idade , Modelos Genéticos , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , Polimorfismo de Nucleotídeo Único , Uganda/epidemiologiaRESUMO
LCA and 1,25(OH)2D3 are vitamin D receptor ligands with different binding affinity. The secosteroid stimulates intestinal Ca2+ absorption. Whether LCA alters this process remains unknown. The aim of our work was to determine the effect of LCA on intestinal Ca2+ absorption in the absence or presence of NaDOC, bile acid that inhibits the cation transport. The data show that LCA by itself did not alter intestinal Ca2+ absorption, but prevented the inhibitory effect of NaDOC. The concomitant administration of LCA avoided the reduction of intestinal alkaline phosphatase activity caused by NaDOC. In addition, LCA blocked a decrease caused by NaDOC on gene and protein expression of molecules involved in the transcellular pathway of intestinal Ca2+ absorption. The oxidative stress and apoptosis triggered by NaDOC were abrogated by LCA co-treatment. In conclusion, LCA placed in the intestinal lumen protects intestinal Ca2+ absorption against the inhibitory effects caused by NaDOC. LCA avoids the reduction of the transcellular Ca2+ movement, apparently by blocking the oxidative stress and apoptosis triggered by NaDOC, normalizing the gene and protein expression of molecules involved in Ca2+ movement. Therefore, LCA might become a possible treatment to improve intestinal calcium absorption under oxidant conditions.
Assuntos
Cálcio/metabolismo , Ácido Desoxicólico/antagonistas & inibidores , Duodeno/efeitos dos fármacos , Enterócitos/efeitos dos fármacos , Absorção Intestinal/efeitos dos fármacos , Ácido Litocólico/farmacologia , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Animais , Animais Recém-Nascidos , Apoptose/efeitos dos fármacos , Calcitriol/metabolismo , Galinhas , Ácido Desoxicólico/farmacologia , Duodeno/metabolismo , Enterócitos/citologia , Enterócitos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Absorção Intestinal/fisiologia , Transporte de Íons/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Estresse Oxidativo , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Carbonilação Proteica/efeitos dos fármacos , Receptores de Calcitriol/genética , Receptores de Calcitriol/metabolismo , Trocador de Sódio e Cálcio/genética , Trocador de Sódio e Cálcio/metabolismoRESUMO
BACKGROUND: Resistant hypertension (RHTN), defined by lack of blood pressure (BP) control despite treatment with at least 3 antihypertensive drugs, increases cardiovascular risk compared with controlled hypertension. Yet, there are few data on genetic variants associated with RHTN. METHODS AND RESULTS: We used a gene-centric array containing ≈50 000 single-nucleotide polymorphisms (SNPs) to identify polymorphisms associated with RHTN in hypertensive participants with coronary artery disease (CAD) from INVEST-GENES (the INnternational VErapamil-SR Trandolapril STudy-GENEtic Substudy). RHTN was defined as BP≥140/90 on 3 drugs, or any BP on 4 or more drugs. Logistic regression analysis was performed in European Americans (n=904) and Hispanics (n=837), using an additive model adjusted for age, gender, randomized treatment assignment, body mass index, principal components for ancestry, and other significant predictors of RHTN. Replication of the top SNP was conducted in 241 European American women from WISE (Women's Ischemia Syndrome Evaluation), where RHTN was defined similarly. To investigate the functional effect of rs12817819, mRNA expression was measured in whole blood. We found ATP2B1 rs12817819 associated with RHTN in both INVEST European Americans (P-value=2.44×10(-3), odds ratio=1.57 [1.17 to 2.01]) and INVEST Hispanics (P=7.69×10(-4), odds ratio=1.76 [1.27 to 2.44]). A consistent trend was observed at rs12817819 in WISE, and the INVEST-WISE meta-analysis result reached chip-wide significance (P=1.60×10(-6), odds ratio=1.65 [1.36 to 1.95]). Expression analyses revealed significant differences in ATP2B1 expression by rs12817819 genotype. CONCLUSIONS: The ATP2B1 rs12817819 A allele is associated with increased risk for RHTN in hypertensive participants with documented CAD or suspected ischemic heart disease. CLINICAL TRIAL REGISTRATION URL: www.clinicaltrials.gov; Unique identifiers: NCT00133692 (INVEST), NCT00000554 (WISE).
Assuntos
Pressão Sanguínea/genética , Resistência a Medicamentos/genética , Hipertensão/genética , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , Polimorfismo de Nucleotídeo Único , Idoso , Anti-Hipertensivos/uso terapêutico , Pressão Sanguínea/efeitos dos fármacos , Quimioterapia Combinada , Feminino , Estudos de Associação Genética , Marcadores Genéticos , Predisposição Genética para Doença , Hispânico ou Latino/genética , Humanos , Hipertensão/tratamento farmacológico , Hipertensão/etnologia , Hipertensão/fisiopatologia , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Razão de Chances , Fenótipo , Porto Rico/epidemiologia , Fatores de Risco , Estados Unidos/epidemiologia , População Branca/genéticaRESUMO
The transport of calcium to the extracellular space carried out by plasma membrane Ca(2+) pumps (PMCAs) is essential for maintaining low Ca(2+) concentrations in the cytosol of eukaryotic cells. The activity of PMCAs is controlled by autoinhibition. Autoinhibition is relieved by the binding of Ca(2+)-calmodulin to the calmodulin-binding autoinhibitory sequence, which in the human PMCA is located in the C-terminal segment and results in a PMCA of high maximal velocity of transport and high affinity for Ca(2+). Autoinhibition involves the intramolecular interaction between the autoinhibitory domain and a not well defined region of the molecule near the catalytic site. Here we show that the fusion of GFP to the C terminus of the h4xb PMCA causes partial loss of autoinhibition by specifically increasing the Vmax. Mutation of residue Glu(99) to Lys in the cytosolic portion of the M1 transmembrane helix at the other end of the molecule brought the Vmax of the h4xb PMCA to near that of the calmodulin-activated enzyme without increasing the apparent affinity for Ca(2+). Altogether, the results suggest that the autoinhibitory interaction of the extreme C-terminal segment of the h4 PMCA is disturbed by changes of negatively charged residues of the N-terminal region. This would be consistent with a recently proposed model of an autoinhibited form of the plant ACA8 pump, although some differences are noted.
Assuntos
Cálcio/metabolismo , Membrana Celular/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Calmodulina/metabolismo , Catálise , Citosol/metabolismo , Deleção de Genes , Glutamina/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Humanos , Lisina/metabolismo , Mutação , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , Ligação Proteica , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/metabolismoRESUMO
Introducción. La hipertensión arterial es una enfermedad multifactorial influenciada por componentes genéticos y ambientales, cuya prevalencia varía entre grupos étnicos. Se han llevado a cabo numerosos estudios en genes de sistemas reguladores de la presión arterial, como el sistema renina-angiotensinaaldosterona, el sistema nervioso simpático, los factores endoteliales, y el balance de sodio, mostrando resultados incongruentes entre poblaciones. Objetivos. Evaluar el efecto de variantes en los genes AGT , AGTR1 , ACE , ADRB2 , DRD1 , ADD1 , ADD2 , ATP2B1 , TBXA2R y PTGS2 y del componente ancestral individual, sobre la hipertensión arterial y las cifras de presión arterial en una muestra de población antioqueña. Materiales y métodos. Se genotipificaron 107 casos y 253 controles para 12 variantes en los genes AGT , AGTR1 , ACE , ADRB2 , DRD1 , ADD1 , ADD2 , ATP2B1 , TBXA2R y PTGS2 , y para 20 marcadores informativos de ascendencia. Se evaluó la asociación de los polimorfismos y sus interacciones, y de la composición genética ancestral con hipertensión y cifras de presión arterial. Resultados. Los genes ADD2 , rs4852706 (OR=3,0; p=0,023); DRD1 , rs686 (OR=0,38; p=0,012) y ADRB2 , rs1042718 (OR=10,0; p=0,008); y combinaciones genotípicas de DRD1 con AGTR1 ; de AGT con ADD1 ; y de ADD1 con ATP2B1 y PTGS2 , se asociaron con hipertensión arterial. El componente ancestral amerindio se asoció con disminución en la presión arterial diastólica. Conclusiones. Variantes en los genes ADD2 , DRD1 , ADRB2 , AGTR1 , AGT , ADD1 , ATP2B1 y PTGS2 , individualmente o en su interacción, se encuentran asociadas con hipertensión. El componente ancestral amerindio tiene un efecto sobre las cifras de presión arterial.
Introduction: Hypertension is a multifactorial disease influenced by genetic and environmental components, with its prevalence varying across ethnic groups. Manifold studies on blood pressure regulatory system genes have been carried out -such as the renin-angiotensin-aldosterone system, the sympathetic nervous system, endothelial factor, and sodium balance-, but the results yielded were inconsistent among populations. Objectives: To evaluate the effect of both variants in genes AGT, AGTR1, ACE, ADRB2, DRD1, ADD1, ADD2, ATP2B1, TBXA2R PTGS2, and the result of the individual ancestry component on hypertension and blood pressure levels among population in Antioquia. Methods and materials: 107 cases and 253 controls were genotyped for 12 variants on genes AGT, AGTR1, ACE, ADRB2, DRD1, ADD1, ADD2, ATP2B1, TBXA2R y PTGS2, and for 20 ancestry informative markers. The association of polymorphisms and their interactions, and the association of ancestral genetic composition with hypertension and blood pressure levels were examined. Results: Genes ADD2, rs4852706 (OR=3.0; p=0.023); DRD1, rs686 (OR=0.38; p=0.012) and ADRB2, rs1042718 (OR=10.0; p=0.008); as well as genotypic combinations of DRD1 and AGTR1; AGT and ADD1; and ADD1 to ATP2B1 and PTGS2 were associated to hypertension. The Amerindian ancestry component was associated to some decrease in diastolic blood pressure. Conclusion: Variants on genes ADD2, DRD1, ADRB2, AGTR1, AGT, ADD1, ATP2B1 and PTGS2 individually or interacting, are associated to hypertension. The Amerindian ancestry component has an effect on blood pressure.
Assuntos
Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Hipertensão/genética , Angiotensinogênio/genética , Pressão Sanguínea/genética , Proteínas de Ligação a Calmodulina/genética , Colômbia , /genética , Peptidil Dipeptidase A/genética , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , Receptor Tipo 1 de Angiotensina/genética , /genética , Receptores de Dopamina D1/genética , /genética , Fatores de RiscoRESUMO
INTRODUCTION: Hypertension is a multifactorial disease influenced by genetic and environmental components, with its prevalence varying across ethnic groups. Manifold studies on blood pressure regulatory system genes have been carried out -such as the renin-angiotensin-aldosterone system, the sympathetic nervous system, endothelial factor, and sodium balance-, but the results yielded were inconsistent among populations. OBJECTIVES: To evaluate the effect of both variants in genes AGT, AGTR1, ACE, ADRB2, DRD1, ADD1, ADD2, ATP2B1, TBXA2R PTGS2, and the result of the individual ancestry component on hypertension and blood pressure levels among population in Antioquia. METHODS AND MATERIALS: 107 cases and 253 controls were genotyped for 12 variants on genes AGT, AGTR1, ACE, ADRB2, DRD1, ADD1, ADD2, ATP2B1, TBXA2R y PTGS2, and for 20 ancestry informative markers. The association of polymorphisms and their interactions, and the association of ancestral genetic composition with hypertension and blood pressure levels were examined. RESULTS: Genes ADD2, rs4852706 (OR=3.0; p=0.023); DRD1, rs686 (OR=0.38; p=0.012) and ADRB2, rs1042718 (OR=10.0; p=0.008); as well as genotypic combinations of DRD1 and AGTR1; AGT and ADD1; and ADD1 to ATP2B1 and PTGS2 were associated to hypertension. The Amerindian ancestry component was associated to some decrease in diastolic blood pressure. CONCLUSION: Variants on genes ADD2, DRD1, ADRB2, AGTR1, AGT, ADD1, ATP2B1 and PTGS2 individually or interacting, are associated to hypertension. The Amerindian ancestry component has an effect on blood pressure.
Assuntos
Hipertensão/genética , Adulto , Angiotensinogênio/genética , Pressão Sanguínea/genética , Proteínas de Ligação a Calmodulina/genética , Colômbia , Ciclo-Oxigenase 2/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Peptidil Dipeptidase A/genética , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , Receptor Tipo 1 de Angiotensina/genética , Receptores Adrenérgicos beta 2/genética , Receptores de Dopamina D1/genética , Receptores de Tromboxano A2 e Prostaglandina H2/genética , Fatores de RiscoRESUMO
Aspergillus fumigatus is a primary and opportunistic pathogen, as well as a major allergen, of mammals. The Ca(+2)-calcineurin pathway affects virulence, morphogenesis and antifungal drug action in A. fumigatus. Here, we investigated three components of the A. fumigatus Ca(+2)-calcineurin pathway, pmcA,-B, and -C, which encode calcium transporters. We demonstrated that CrzA can directly control the mRNA accumulation of the pmcA-C genes by binding to their promoter regions. CrzA-binding experiments suggested that the 5'-CACAGCCAC-3' and 5'-CCCTGCCCC-3' sequences upstream of pmcA and pmcC genes, respectively, are possible calcineurin-dependent response elements (CDREs)-like consensus motifs. Null mutants were constructed for pmcA and -B and a conditional mutant for pmcC demonstrating pmcC is an essential gene. The ΔpmcA and ΔpmcB mutants were more sensitive to calcium and resistant to manganese and cyclosporin was able to modulate the sensitivity or resistance of these mutants to these salts, supporting the interaction between calcineurin and the function of these transporters. The pmcA-C genes have decreased mRNA abundance into the alveoli in the ΔcalA and ΔcrzA mutant strains. However, only the A. fumigatus ΔpmcA was avirulent in the murine model of invasive pulmonary aspergillosis.