RESUMO
It has been reported that phaseolin, the major storage globulin of the common bean (Phaseolus vulgaris), is toxic to Callosobruchus maculatus larvae, an Old World bruchid beetle that is not capable of infesting this New World edible bean. It has also been demonstrated that vicilin, the major storage globulin found in cowpea (Vigna unguiculata) seeds, is absorbed through receptor-mediated endocytosis in the insect midgut. A putative vicilin receptor has been purified and showed high homology to α-tocopherol transfer protein. However, the ingestion of a variant vicilin purified from C. maculatus resistant seeds inhibits transcytosis, resulting in the accumulation of vicilins in the midgut cells and ultimately antibiosis. In the present work, we studied the cellular up-take of phaseolin in C. maculatus larvae with the aim of discovering if this protein is also capable of inhibiting endocytic traffic in the enterocytes. FITC-labelled vicilin and FITC-labelled phaseolin were incorporated into the diet of the larvae at a physiological concentration of 0.5% w/w. The fate of labelled and non-labelled globulins was monitored by confocal microscopy. Here we demonstrated that phaseolin is also endocytosed by enterocytes causing an accumulation of endocytic vesicles in the midgut when compared to the ingestion of vicilin obtained from a susceptible V. unguiculata cultivar. From the results obtained for HNE, MDA and TBARS, a pro-oxidative scenario was established in the intestinal epithelial cells of the larvae, which may explain the deleterious effect observed in larvae developing inside P. vulgaris seeds.
Assuntos
Besouros/metabolismo , Intestinos , Estresse Oxidativo/efeitos dos fármacos , Proteínas de Plantas/farmacologia , Vesículas Secretórias/metabolismo , Animais , Transporte Biológico Ativo/efeitos dos fármacos , LarvaRESUMO
OBJECTIVES: KPC-producing strains present a wide range of carbapenem minimum inhibitory concentrations (MICs). This variation may be due to differential expression of blaKPC and porin genes, efflux pump activity and the production of extended-spectrum ß-lactamases and/or AmpC ß-lactamases. The aim of this study was to determine the role of efflux pumps inhibited by phenylalanine-arginine ß-naphthylamide (PAßN) in resistance to carbapenems in Chilean clinical isolates of blaKPC-harbouring Klebsiella pneumoniae. METHODS: MICs were determined by the agar dilution method for imipenem, meropenem, ertapenem and ciprofloxacin in the presence and absence of PAßN (25mg/L) in 17 carbapenem-resistant KPC-producing K. pneumoniae strains. Outer protein membrane (OMP) profiles were determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Expression levels of the ompK35 and ompK36 genes were also determined by real-time quantitative reverse transcription PCR (qRT-PCR). RESULTS: No contribution of PAßN-inhibited efflux pumps to carbapenem resistance was found, unlike ciprofloxacin resistance. However, a ≥4-fold increase in the MIC of at least one carbapenem was observed in 13 isolates in the presence of PAßN. Additionally, decreased gene expression of ompK35 and ompK36 in the presence of PAßN was detected, however no obvious differences in porin band intensity were observed by SDS-PAGE. CONCLUSIONS: The presence of PAßN resulted in an increase in carbapenem MICs unrelated to efflux pump inhibition, and a decrease in the expression of ompK35 and ompK36 genes without an obvious difference in OMP profiles observed by SDS-PAGE. Therefore, additional factors are responsible for the increase in carbapenem MIC in the presence of PAßN.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Carbapenêmicos/farmacologia , Dipeptídeos/metabolismo , Inibidores Enzimáticos/metabolismo , Klebsiella pneumoniae/enzimologia , Proteínas de Membrana Transportadoras/metabolismo , beta-Lactamases/metabolismo , Proteínas da Membrana Bacteriana Externa/análise , Proteínas da Membrana Bacteriana Externa/genética , Transporte Biológico Ativo/efeitos dos fármacos , Enterobacteriáceas Resistentes a Carbapenêmicos/efeitos dos fármacos , Enterobacteriáceas Resistentes a Carbapenêmicos/enzimologia , Enterobacteriáceas Resistentes a Carbapenêmicos/isolamento & purificação , Chile , Eletroforese em Gel de Poliacrilamida , Perfilação da Expressão Gênica , Humanos , Infecções por Klebsiella/microbiologia , Klebsiella pneumoniae/química , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/isolamento & purificação , Testes de Sensibilidade Microbiana , Reação em Cadeia da Polimerase em Tempo RealRESUMO
BACKGROUND: V-ATPase interactions with cholesterol enriched membrane microdomains have been related to metastasis in a variety of cancers, but the underlying mechanism remains at its beginnings. It has recently been reported that the inhibition of this H+ pump affects cholesterol mobilization to the plasma membrane. METHODS: Inhibition of melanoma cell migration and invasiveness was assessed by wound healing and Transwell assays in murine cell lines (B16F10 and Melan-A). V-ATPase activity was measured in vitro by ATP hydrolysis and H+ transport in membrane vesicles, and intact cell H+ fluxes were measured by using a non-invasive Scanning Ion-selective Electrode Technique (SIET). RESULTS: Cholesterol depletion by 5mM MßCD was found to be inhibitory to the hydrolytic and H+ pumping activities of the V-ATPase of melanoma cell lines, as well as to the migration and invasiveness capacities of these cells. Nearly the same effects were obtained using concanamycin A, a specific inhibitor of V-ATPase, which also promoted a decrease of the H+ efflux in live cells at the same extent of MßCD. CONCLUSIONS: We found that cholesterol depletion significantly affects the V-ATPase activity and the initial metastatic processes following a profile similar to those observed in the presence of the V-ATPase specific inhibitor, concanamycin. GENERAL SIGNIFICANCE: The results shed new light on the functional role of the interactions between V-ATPases and cholesterol-enriched microdomains of cell membranes that contribute with malignant phenotypes in melanoma.
Assuntos
Colesterol/metabolismo , Melanoma Experimental/tratamento farmacológico , Lipídeos de Membrana/metabolismo , Microdomínios da Membrana/efeitos dos fármacos , Invasividade Neoplásica/prevenção & controle , Proteínas de Neoplasias/antagonistas & inibidores , ATPases Vacuolares Próton-Translocadoras/antagonistas & inibidores , Trifosfato de Adenosina/metabolismo , Animais , Transporte Biológico Ativo/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Macrolídeos/farmacologia , Melanoma Experimental/enzimologia , Melanoma Experimental/patologia , Fluidez de Membrana/efeitos dos fármacos , Camundongos , Proteínas de Neoplasias/metabolismo , Prótons , ATPases Vacuolares Próton-Translocadoras/metabolismo , beta-Ciclodextrinas/farmacologiaRESUMO
The objectives of this study were to evaluate tetrahydropyridine derivatives as efflux inhibitors and to understand the mechanism of action of the compounds by in silico studies. Minimum inhibitory concentration (MIC) determination, fluorometric methods and docking simulations were performed. The compounds NUNL02, NUNL09 and NUNL10 inhibited efflux, and NUNL02 is very likely a substrate of the transporter protein AcrB. Docking studies suggested that the mechanism of action could be by competition with substrate for binding sites and protein residues. We showed for the first time the potential of tetrahydropyridines as efflux inhibitors and highlighted compound NUNL02 as an AcrB-specific inhibitor. Docking studies suggested that competition is the putative mechanism of action of these compounds.
Assuntos
Antibacterianos/metabolismo , Transporte Biológico Ativo/efeitos dos fármacos , Inibidores Enzimáticos/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/efeitos dos fármacos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Piridinas/metabolismo , Antibacterianos/química , Inibidores Enzimáticos/química , Proteínas de Escherichia coli/química , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Proteínas Associadas à Resistência a Múltiplos Medicamentos/química , Ligação Proteica , Piridinas/químicaRESUMO
This study aimed to determine in the canine corpus luteum throughout the dioestrus (1) the influence of insulin on glucose uptake; (2) the regulation of genes potentially involved; and (3) the influence of hypoxia on glucose transporter expression and steroidogenesis, after treatment with cobalt chloride (CoCl2). Glucose uptake by luteal cells increased 2.7 folds (P < 0.05) in response to insulin; a phenomenon related to increased expression of glucose transporter (GLUT) 4 and phosphorylation of protein kinase B (AKT). The gene expression of insulin receptor and SLC2A4 (codifier of GLUT4) genes after insulin stimulation increased on day 20 post ovulation (p.o.) and declined on day 40 p.o. (P < 0.05). Regarding potentially involved molecular mechanisms, the nuclear factor kappa B gene RELA was upregulated on days 30/40 p.o., when SLC2A4 mRNA was low, and the interleukin 6 (IL6) gene was upregulated in the first half of dioestrus, when SLC2A4 mRNA was high. CoCl2 in luteal cell cultures increased the hypoxia-inducible factor HIF1A/HIF1A and the SLC2A4/GLUT4 expression, and decreased progesterone (P4) production and hydroxyl-delta-5-steroid dehydrogenase 3 beta (HSD3B) mRNA expression (P < 0.05). This study shows that the canine luteal cells are responsive to insulin, which stimulates glucose uptake in AKT/GLUT4-mediated pathway; that may be related to local activity of RELA and IL6. Besides, the study reveals that luteal cells under hypoxia activate HIF1A-modulating luteal function and insulin-stimulated glucose uptake. These data indicate that insulin regulates luteal cells' glucose disposal, participating in the maintenance and functionality of the corpus luteum.
Assuntos
Corpo Lúteo/metabolismo , Cães/metabolismo , Insulina/metabolismo , Animais , Transporte Biológico Ativo/efeitos dos fármacos , Cobalto/farmacologia , Manutenção do Corpo Lúteo/genética , Manutenção do Corpo Lúteo/metabolismo , Cães/genética , Feminino , Expressão Gênica/efeitos dos fármacos , Glucose/metabolismo , Transportador de Glucose Tipo 1/genética , Transportador de Glucose Tipo 1/metabolismo , Transportador de Glucose Tipo 4/genética , Transportador de Glucose Tipo 4/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Insulina/fisiologia , Interleucina-6/genética , Interleucina-6/metabolismo , Células Lúteas/efeitos dos fármacos , Células Lúteas/metabolismo , Gravidez , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismoRESUMO
In this review we discuss the structure and functions of the aspartate/glutamate carriers (AGC1-aralar and AGC2-citrin). Those proteins supply the aspartate synthesized within mitochondrial matrix to the cytosol in exchange for glutamate and a proton. A structure of an AGC carrier is not available yet but comparative 3D models were proposed. Moreover, transport assays performed by using the recombinant AGC1 and AGC2, reconstituted into liposome vesicles, allowed to explore the kinetics of those carriers and to reveal their specific transport properties. AGCs participate to a wide range of cellular functions, as the control of mitochondrial respiration, calcium signaling and antioxydant defenses. AGC1 might also play peculiar tissue-specific functions, as it was found to participate to cell-to-cell metabolic symbiosis in the retina. On the other hand, AGC1 is involved in the glutamate-mediated excitotoxicity in neurons and AGC gene or protein alterations were discovered in rare human diseases. Accordingly, a mice model of AGC1 gene knock-out presented with growth delay and generalized tremor, with myelinisation defects. More recently, AGC was proposed to play a crucial role in tumor metabolism as observed from metabolomic studies showing that the asparate exported from the mitochondrion by AGC1 is employed in the regeneration of cytosolic glutathione. Therefore, given the central role of AGCs in cell metabolism and human pathology, drug screening are now being developed to identify pharmacological modulators of those carriers. This article is part of a Special Issue entitled: Mitochondrial Channels edited by Pierre Sonveaux, Pierre Maechler and Jean-Claude Martinou.
Assuntos
Ácido Aspártico/metabolismo , Proteínas de Ligação ao Cálcio/fisiologia , Ácido Glutâmico/metabolismo , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/fisiologia , Transportadores de Ânions Orgânicos/fisiologia , Sequência de Aminoácidos , Animais , Transporte Biológico Ativo/efeitos dos fármacos , Proteínas de Ligação ao Cálcio/antagonistas & inibidores , Proteínas de Ligação ao Cálcio/genética , Bovinos , Sequência Consenso , Humanos , Malatos/metabolismo , Camundongos , Proteínas de Transporte da Membrana Mitocondrial/antagonistas & inibidores , Proteínas de Transporte da Membrana Mitocondrial/deficiência , Proteínas de Transporte da Membrana Mitocondrial/genética , Modelos Moleculares , NAD/metabolismo , Proteínas de Neoplasias/fisiologia , Especificidade de Órgãos , Transportadores de Ânions Orgânicos/antagonistas & inibidores , Transportadores de Ânions Orgânicos/genética , Oxirredução , Conformação Proteica , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Within its mammalian host, Leishmania resides and replicates as an intracellular parasite. The direct activity of antileishmanials must therefore depend on intracellular drug transport, metabolism, and accumulation within the host cell. In this study, we explored the role of human macrophage transporters in the intracellular accumulation and antileishmanial activity of miltefosine (MLF), the only oral drug available for the treatment of visceral and cutaneous leishmaniasis (CL). Membrane transporter gene expression in primary human macrophages infected in vitro with Leishmania Viannia panamensis and exposed to MLF showed modulation of ABC and solute liquid carrier transporters gene transcripts. Among these, ABCA3, a lipid transporter, was significantly induced after exposure to MLF, and this induction was confirmed in primary macrophages from CL patients. Functional validation of MLF as a substrate for ABCA3 was performed by shRNA gene knockdown (KD) in THP-1 monocytes. Intracellular accumulation of radiolabeled MLF was significantly higher in ABCA3(KD) macrophages. ABCA3(KD) resulted in increased cytotoxicity induced by MLF exposure. ABCA3 gene expression inversely correlated with intracellular MLF content in primary macrophages from CL patients. ABCA3(KD) reduced parasite survival during macrophage infection with an L. V. panamensis strain exhibiting low in vitro susceptibility to MLF. Confocal microscopy showed ABCA3 to be located in the cell membrane of resting macrophages and in intracellular compartments in L. V. panamensis-infected cells. These results provide evidence of ABCA3 as an MLF efflux transporter in human macrophages and support its role in the direct antileishmanial effect of this alkylphosphocholine drug.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Leishmania/metabolismo , Leishmaniose/tratamento farmacológico , Macrófagos/metabolismo , Macrófagos/parasitologia , Fosforilcolina/análogos & derivados , Transportadores de Cassetes de Ligação de ATP/genética , Transporte Biológico Ativo/efeitos dos fármacos , Transporte Biológico Ativo/genética , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Humanos , Leishmania/genética , Leishmaniose/genética , Leishmaniose/metabolismo , Macrófagos/patologia , Fosforilcolina/farmacocinética , Fosforilcolina/farmacologiaRESUMO
Methylphenidate (MPH, Ritalin©) is widely used in the treatment of Attention Deficit Hyperactivity Disorder and recently as a drug of abuse. Although the effect of MPH has been studied in brain regions such as striatum and prefrontal cortex (PFC), the hippocampus has received relatively little attention. It is known that MPH increases the TBS-dependent Long Term Potentiation (LTP) in the CA1 area. However, the cellular and molecular mechanisms involved in this process are still unknown. Using field potential recordings and western blot analysis in rat hippocampal slices of young rats, we found that acute application of MPH enhances LTP in CA3-CA1 synapses in a dose-dependent manner with an EC50 of 73.44±6.32 nM. Using specific antagonists and paired-pulse facilitation protocols, we observed that the MPH-dependent increase of LTP involves not only ß-adrenergic receptors activation but also post-synaptic D1/D5 dopamine receptors. The inhibition of PKA with PKI, suppressed the facilitation of LTP induced by MPH consistent with an involvement of the adenyl cyclase-cAMP-PKA dependent cascade downstream of the activation of D1/D5 receptors. In addition, samples of CA1 areas taken from slices potentiated with MPH presented an increase in the phosphorylation of the Ser845 residue of the GluA1 subunit of AMPA receptors compared to control slices. This effect was reverted by SCH23390, antagonist of D1/D5 receptors, and PKI. Moreover, we found an increase of surface-associated functional AMPA receptors. We propose that MPH increases TBS-dependent LTP in CA3-CA1 synapses through a polysynaptic mechanism involving activation of ß-adrenergic and D1/D5 dopaminergic receptors and promoting the trafficking and insertion of functional AMPA receptors to the plasma membrane.
Assuntos
Região CA1 Hipocampal/efeitos dos fármacos , Região CA1 Hipocampal/fisiologia , Estimulantes do Sistema Nervoso Central/farmacologia , Potenciação de Longa Duração/efeitos dos fármacos , Potenciação de Longa Duração/fisiologia , Metilfenidato/farmacologia , Animais , Transporte Biológico Ativo/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Técnicas de Patch-Clamp , Fosforilação/efeitos dos fármacos , Ratos Sprague-Dawley , Receptores de AMPA/metabolismo , Receptores Adrenérgicos beta/metabolismo , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D5/metabolismo , Transdução de Sinais/efeitos dos fármacos , Técnicas de Cultura de TecidosRESUMO
OBJECTIVES: To evaluate the occurrence of systemic and renal abnormalities in the offspring of Wistar rats exposed to tenofovir disoproxil fumarate (DF) during pregnancy. METHODS: Female Wistar rats received a standard diet, with or without addition of tenofovir DF (100 mg/kg diet), 1 week before mating and during pregnancy. Offspring from the tenofovir DF group were placed with an untreated foster mother during breastfeeding and compared with offspring from rats maintained on a standard diet during mating and pregnancy (control). Control and tenofovir DF were followed up at 3 and 6 months of age. Monthly body weight and systolic blood pressure (SBP), glomerular counts, renal function, biochemical parameters, angiotensin II, renal renin angiotensin aldosterone system (RAAS) and renal sodium transporters were analysed. RESULTS: Tenofovir DF offspring showed lower birth weight compared with the control group. After the third month, growth among the tenofovir DF group experienced a rapid catch-up. SBP increased progressively after the second month of age in the tenofovir DF group. Nephron number did not differ between the groups; however, the tenofovir DF group showed glomerular structural changes. Plasma aldosterone was higher in the tenofovir DF group, associated with a significant increase in renal expression of RAAS. The tenofovir DF rats showed up-regulation of renal sodium transporters and consequently lower urinary sodium excretion. CONCLUSIONS: This is the first demonstration using an experimental model that maternal exposure to tenofovir DF during gestation results in overactivation of RAAS, up-regulation of renal sodium transporters and hypertension in the offspring.
Assuntos
Adenina/análogos & derivados , Antivirais/administração & dosagem , Antivirais/efeitos adversos , Hipertensão/induzido quimicamente , Organofosfonatos/administração & dosagem , Organofosfonatos/efeitos adversos , Adenina/administração & dosagem , Adenina/efeitos adversos , Animais , Transporte Biológico Ativo/efeitos dos fármacos , Feminino , Modelos Animais , Gravidez , Ratos Wistar , Sistema Renina-Angiotensina/efeitos dos fármacos , Sódio/metabolismo , TenofovirRESUMO
The effect of addition of the nitric oxide donor S-nitrosoglutathione (GSNO) on the Zn nutritional status was evaluated in hydroponically-cultured wheat plants (Triticum aestivum cv. Chinese Spring). Addition of GSNO in Zn-deprived plants did not modify biomass accumulation but accelerated leaf senescence in a mode concomitant with accelerated decrease of Zn allocation to shoots. In well-supplied plants, Zn concentration in both roots and shoots declined due to long term exposure to GSNO. A further evaluation of net Zn uptake rate (ZnNUR) during the recovery of long-term Zn-deprivation unveiled that enhanced Zn-accumulation was partially blocked when GSNO was present in the uptake medium. This effect on uptake was mainly associated with a change of Zn translocation to shoots. Our results suggest a role for GSNO in the modulation of Zn uptake and in root-to-shoot translocation during the transition from deficient to sufficient levels of Zn-supply.
Assuntos
Gasotransmissores/farmacologia , Óxido Nítrico/farmacologia , Compostos Nitrosos/farmacologia , Triticum/metabolismo , Zinco/metabolismo , Transporte Biológico Ativo/efeitos dos fármacos , Folhas de Planta/metabolismo , Brotos de Planta/metabolismoRESUMO
The lprG-p55 operon of Mycobacterium tuberculosis and Mycobacterium bovis is involved in the transport of toxic compounds. P55 is an efflux pump that provides resistance to several drugs, while LprG is a lipoprotein that modulates the host's immune response against mycobacteria. The knockout mutation of this operon severely reduces the replication of both mycobacterial species during infection in mice and increases susceptibility to toxic compounds. In order to gain insight into the function of LprG in the Mycobacterium avium complex, in this study, we assayed the effect of the deletion of lprG gene in the D4ER strain of Mycobacterium avium subsp. avium. The replacement of lprG gene with a hygromycin cassette caused a polar effect on the expression of p55. Also, a twofold decrease in ethidium bromide susceptibility was observed and the resistance to the antibiotics rifampicin, amikacin, linezolid, and rifabutin was impaired in the mutant strain. In addition, the mutation decreased the virulence of the bacteria in macrophages in vitro and in a mice model in vivo. These findings clearly indicate that functional LprG and P55 are necessary for the correct transport of toxic compounds and for the survival of MAA in vitro and in vivo.
Assuntos
Antituberculosos , Proteínas de Bactérias , Farmacorresistência Bacteriana , Lipoproteínas , Camundongos Endogâmicos BALB C , Mycobacterium avium , Óperon , Fatores de Virulência , Animais , Antituberculosos/farmacocinética , Antituberculosos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Transporte Biológico Ativo/efeitos dos fármacos , Transporte Biológico Ativo/genética , Farmacorresistência Bacteriana/efeitos dos fármacos , Farmacorresistência Bacteriana/genética , Lipoproteínas/genética , Lipoproteínas/metabolismo , Camundongos , Viabilidade Microbiana/efeitos dos fármacos , Viabilidade Microbiana/genética , Mycobacterium avium/genética , Mycobacterium avium/metabolismo , Mycobacterium avium/patogenicidade , Tuberculose/tratamento farmacológico , Tuberculose/genética , Tuberculose/metabolismo , Tuberculose/veterinária , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
Repeated acetaminophen (AP) administration modulates intestinal P-glycoprotein (P-gp) expression. Whether AP can modulate P-gp activity in a short-term fashion is unknown. We investigated the acute effect of AP on rat intestinal P-gp activity in vivo and in vitro. In everted intestinal sacs, AP inhibited serosal-mucosal transport of rhodamine 123 (R123), a prototypical P-gp substrate. R123 efflux plotted against R123 concentration adjusted well to a sigmoidal curve. Vmax decreased 50% in the presence of AP, with no modification in EC50, or slope, ruling out the possibility of inhibition to be competitive. Inhibition by AP was absent at 0°C, consistent with interference of the active transport of R123 by AP. Additionally, AP showed no effect on normal localization of P-gp at the apical membrane of the enterocyte and neither affected paracellular permeability. Consistent with absence of a competitive inhibition, two further strategies strongly suggested that AP is not a P-gp substrate. First, serosal-mucosal transport of AP was not affected by the classical P-gp inhibitors verapamil or Psc 833. Second, AP accumulation was not different between P-gp knock-down and wild-type HepG2 cells. In vivo intestinal absorption of digoxin, another substrate of P-gp, was assessed in the presence or absence of AP (100 µM). Portal digoxin concentration was increased by 214%, in average, by AP, as compared with digoxin alone. In conclusion, AP inhibited P-gp activity, increasing intestinal absorption of digoxin, a prototypical substrate. These results suggest that therapeutic efficacy of P-gp substrates can be altered if coadministered with AP.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Acetaminofen/farmacologia , Transporte Biológico Ativo/efeitos dos fármacos , Intestinos/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Ciclosporinas/farmacologia , Digoxina/farmacologia , Enterócitos/efeitos dos fármacos , Enterócitos/metabolismo , Células Hep G2 , Humanos , Absorção Intestinal/efeitos dos fármacos , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Masculino , Permeabilidade/efeitos dos fármacos , Ratos , Ratos Wistar , Rodamina 123/farmacologia , Verapamil/farmacologiaRESUMO
The primary objective of this work was to evaluate the capability of curcumin, a natural compound found in the Curcuma longa plant, to sensitize a clinical isolate of Candida albicans, which was found to have a high resistance to fluconazole. In addition, we assessed whether the resistance of this isolate was the result of the existence of efflux pumps, which could confer a multiple drug resistance phenotype. To evaluate azole resistance, we used the Clinical Laboratory Standard Institute (CLSI) MIC assays procedures with minor modifications. For evaluation of synergistic interaction of curcumin and fluconazole, checkerboard experiments were employed. Nile red and Rhodamine 6G accumulation assays were used to evaluate efflux pump activity. Curcumin was found to have a great capability to inhibit fluconazole resistance of the isolate of C. albicans. It was capable of restoring its sensitivity to this azole when used at 11 µM. Analysis with different azoles and the two indicated dyes showed that an efflux pump could be acting and contributing to the resistance of this isolate to fluconazole. The results suggest that a major facilitator superfamily (MFS) transporter might be involved in this process.
Assuntos
Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Curcumina/farmacologia , Farmacorresistência Fúngica Múltipla/efeitos dos fármacos , Fluconazol/farmacologia , Transporte Biológico Ativo/efeitos dos fármacos , Curcuma/química , Humanos , Testes de Sensibilidade Microbiana , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/farmacologiaRESUMO
Although the biological effects of thyroid hormones are mediated by nuclear receptors (genomic mechanisms), interactions with receptors associated with the plasma membrane (non-genomic mechanisms) of target cells are not clear. In this study we investigated the rapid stimulatory effect of thyroxine (T(4)) on (45)Ca(2+) uptake as well as ionic currents and intracellular messengers involved in the stimulatory action of T(4) in amino acid accumulation in immature rat testes. Results indicated that 10(-9)M or 10(-6)M T(4) was able to increase immediately (45)Ca(2+) uptake after 60s of hormone exposure. These results indicate for the first time that voltage-dependent Ca(2+) channels and ATP-dependent K(+) channels can be seen as a set-point in the stimulatory effect of T(4) on amino acid accumulation. Apamin-sensitive small-conductance Ca(2+)-activated K(+) channels (SK(Ca)) and chloride channels were shown to be partially involved in this mechanism. The amino acid accumulation triggered by the PKC pathway suggests a functional link between different ion channel activities and the stimulatory effect of T(4) on amino acid accumulation. In conclusion, we show in this study a rapid and stimulatory effect of T(4) on calcium uptake and on amino acid accumulation, both events initiated at the plasma membrane, which strongly characterizes a non-genomic effect of T(4) in immature rat testes.
Assuntos
Aminoácidos Neutros/metabolismo , Cálcio/metabolismo , Membrana Celular/efeitos dos fármacos , Testículo/metabolismo , Tiroxina/farmacologia , Animais , Animais Recém-Nascidos , Transporte Biológico Ativo/efeitos dos fármacos , Canais de Cálcio/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , Canais de Cloreto/metabolismo , Masculino , Canais de Potássio Cálcio-Ativados/metabolismo , Ratos , Ratos Wistar , Testículo/efeitos dos fármacos , Testículo/patologiaRESUMO
Local anesthetics have myotoxic effects and inhibit Ca-ATPase activity and Ca transport in skeletal muscles. Such effects have not been fully elucidated in masticatory muscles. We tested the hypothesis that local anesthetics increase myoplasmic calcium in masticatory muscles by inhibiting Ca-ATPase at a concentration similar to that of dental cartridges. The effects of lidocaine and bupivacaine on Ca-ATPase from rabbit masseter and medial pterygoid muscles were tested with radioisotopic and colorimetric methods. Bupivacaine had an action similar to that of lidocaine on Ca-ATPase activity, but less effect on calcium transport. The pre-exposure of the membranes to the anesthetics enhanced the Ca-ATPase activity in the absence of calcium ionophore, supporting their permeabilizing effect. The results demonstrate that amide-type anesthetics do not inhibit calcium binding, but do reduce calcium transport and enzyme phosphorylation by ATP, and suggest that the myoplasmic calcium increase induced by lidocaine and bupivacaine might promote masticatory muscle contraction and eventual rigidity.
Assuntos
Anestésicos Locais/toxicidade , Músculos da Mastigação/efeitos dos fármacos , Músculos da Mastigação/enzimologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/antagonistas & inibidores , Animais , Transporte Biológico Ativo/efeitos dos fármacos , Bupivacaína/toxicidade , Cálcio/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/toxicidade , Membranas Intracelulares/efeitos dos fármacos , Lidocaína/toxicidade , Fosforilação/efeitos dos fármacos , CoelhosRESUMO
The effect of the cholestatic estrogens ethynylestradiol (EE) and estradiol 17beta-D-glucuronide (E2-17G) on expression and activity of intestinal multidrug resistant-associated protein 2 (Mrp2, Abcc2) was studied in rats. Expression and localization of Mrp2 were evaluated by Western blotting, real-time polymerase chain reaction, and confocal immunofluorescence microscopy. Mrp2 transport activity toward dinitrophenyl-S-glutathione (DNP-SG) was assessed in vitro in intestinal sacs. EE, administered subcutaneously at a 5 mg/kg b.wt. dose, for 5 consecutive days, produced a marked decrease in Mrp2 expression at post-transcriptional level, without affecting its normal localization at the apical membrane of the enterocyte. This effect was selective because expression of other ATP-binding cassette proteins such as breast cancer resistance protein and Mrp3 were not affected and that of multidrug resistance protein 1 was only minimally impaired. Consistent with down-regulation of expression of Mrp2, a significant impairment in serosal to mucosal transport of DNP-SG and in protection against absorption of this same compound were registered. Simultaneous administration of EE with spironolactone (200 micromol/kg b.wt./day for 3 days), an Mrp2 inducer, prevented these alterations, confirming down-regulation of expression of Mrp2 by EE as a major component of functional changes. Incorporation of E2-17G (30 microM) in the serosal medium of intestinal sacs decreased serosal to mucosal transport of DNP-SG, probably because of competitive inhibition, without affecting normal Mrp2 expression or localization. Our data indicate impairment of function of intestinal Mrp2 by both cholestatic estrogens, although through a different mechanism. This finding represents an aggravation of deteriorated hepatic Mrp2 function that could further increase bioavailability of specific xenobiotics after oral exposure.
Assuntos
Colestase/metabolismo , Estrogênios/farmacologia , Expressão Gênica/efeitos dos fármacos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Transporte Biológico Ativo/efeitos dos fármacos , Biomarcadores/metabolismo , Peso Corporal/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Estradiol/análogos & derivados , Estradiol/farmacologia , Mucosa Intestinal/metabolismo , Masculino , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Ratos , Ratos WistarRESUMO
BACKGROUND: Considering that sunflower production is expanding to arid regions, tolerance to abiotic stresses as drought, low temperatures and salinity arises as one of the main constrains nowadays. Differential organ-specific sunflower ESTs (expressed sequence tags) were previously generated by a subtractive hybridization method that included a considerable number of putative abiotic stress associated sequences. The objective of this work is to analyze concerted gene expression profiles of organ-specific ESTs by fluorescence microarray assay, in response to high sodium chloride concentration and chilling treatments with the aim to identify and follow up candidate genes for early responses to abiotic stress in sunflower. RESULTS: Abiotic-related expressed genes were the target of this characterization through a gene expression analysis using an organ-specific cDNA fluorescence microarray approach in response to high salinity and low temperatures. The experiment included three independent replicates from leaf samples. We analyzed 317 unigenes previously isolated from differential organ-specific cDNA libraries from leaf, stem and flower at R1 and R4 developmental stage. A statistical analysis based on mean comparison by ANOVA and ordination by Principal Component Analysis allowed the detection of 80 candidate genes for either salinity and/or chilling stresses. Out of them, 50 genes were up or down regulated under both stresses, supporting common regulatory mechanisms and general responses to chilling and salinity. Interestingly 15 and 12 sequences were up regulated or down regulated specifically in one stress but not in the other, respectively. These genes are potentially involved in different regulatory mechanisms including transcription/translation/protein degradation/protein folding/ROS production or ROS-scavenging. Differential gene expression patterns were confirmed by qRT-PCR for 12.5% of the microarray candidate sequences. CONCLUSION: Eighty genes isolated from organ-specific cDNA libraries were identified as candidate genes for sunflower early response to low temperatures and salinity. Microarray profiling of chilling and NaCl-treated sunflower leaves revealed dynamic changes in transcript abundance, including transcription factors, defense/stress related proteins, and effectors of homeostasis, all of which highlight the complexity of both stress responses. This study not only allowed the identification of common transcriptional changes to both stress conditions but also lead to the detection of stress-specific genes not previously reported in sunflower. This is the first organ-specific cDNA fluorescence microarray study addressing a simultaneous evaluation of concerted transcriptional changes in response to chilling and salinity stress in cultivated sunflower.
Assuntos
Adaptação Fisiológica/fisiologia , Temperatura Baixa , Regulação da Expressão Gênica de Plantas , Helianthus/genética , Helianthus/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Cloreto de Sódio/farmacologia , Adaptação Fisiológica/efeitos dos fármacos , Adaptação Fisiológica/genética , Transporte Biológico Ativo/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genes de Plantas/genética , Helianthus/efeitos dos fármacos , Fotossíntese/efeitos dos fármacos , Fotossíntese/fisiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Transcrição Gênica/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacosRESUMO
The effect of methylmalonate (MMA) on mitochondrial succinate oxidation has received great attention since it could present an important role in energy metabolism impairment in methylmalonic acidaemia. In the present work, we show that while millimolar concentrations of MMA inhibit succinate-supported oxygen consumption by isolated rat brain or muscle mitochondria, there is no effect when either a pool of NADH-linked substrates or N,N,N',N'-tetramethyl-p-phenylendiamine (TMPD)/ascorbate were used as electron donors. Interestingly, the inhibitory effect of MMA, but not of malonate, on succinate-supported brain mitochondrial oxygen consumption was minimized when nonselective permeabilization of mitochondrial membranes was induced by alamethicin. In addition, only a slight inhibitory effect of MMA was observed on succinate-supported oxygen consumption by inside-out submitochondrial particles. In agreement with these observations, brain mitochondrial swelling experiments indicate that MMA is an important inhibitor of succinate transport by the dicarboxylate carrier. Under our experimental conditions, there was no evidence of malonate production in MMA-treated mitochondria. We conclude that MMA inhibits succinate-supported mitochondrial oxygen consumption by interfering with the uptake of this substrate. Although succinate generated outside the mitochondria is probably not a sig-nificant contributor to mitochondrial energy generation, the physiopathological implications of MMA-induced inhibition of substrate transport by the mitochondrial dicarboxylate carrier are discussed.
Assuntos
Ácido Metilmalônico/farmacologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Consumo de Oxigênio/efeitos dos fármacos , Ácido Succínico/farmacologia , Animais , Transporte Biológico Ativo/efeitos dos fármacos , Transportadores de Ácidos Dicarboxílicos/antagonistas & inibidores , Regulação para Baixo/efeitos dos fármacos , Feminino , Malonatos/metabolismo , Técnicas de Cultura de Órgãos , Ratos , Ratos Wistar , Succinato Desidrogenase/metabolismo , Ácido Succínico/metabolismo , Ácido Succínico/farmacocinéticaRESUMO
Stroke syndromes are a major cause of disability in middle and later life resulting in severe neuronal degeneration and loss of brain functions. In situations with energy failure, glutamate transport is impaired and high levels of this amino acid accumulate on the synaptic cleft. Our group has showed that guanosine exerts neuroprotection against neurotoxicity situations. The aim of this work is draw a post-ischemic profile of glutamate uptake and cell damage using an oxygen and glucose deprivation model (OGD) in hippocampal slices from young (P10) and adult (P60) rats, analyzing guanosine effect. OGD decreases glutamate uptake in both ages and recovery times, although decrease in cell viability was only observed 1 and 3 h after OGD in young and adult animals, respectively. Guanosine partially protected cell damage from 1 h in P10 and at 3 h in P60 rats and avoided glutamate uptake decrease from P10 rats at 3 h. The impairment of glutamate transporters since immediately after the insult observed here is probably due to an energetic failure; loss of cell viability was only observed from 1 h after OGD. The mechanism by which guanosine acts in the 'ischemic' model used here is still unknown, but evidence leads to its antiapoptotic effect.
Assuntos
Envelhecimento/metabolismo , Ácido Glutâmico/metabolismo , Guanosina/metabolismo , Hipocampo/crescimento & desenvolvimento , Hipocampo/metabolismo , Hipóxia-Isquemia Encefálica/metabolismo , Sistema X-AG de Transporte de Aminoácidos/efeitos dos fármacos , Sistema X-AG de Transporte de Aminoácidos/metabolismo , Animais , Animais Recém-Nascidos , Transporte Biológico Ativo/efeitos dos fármacos , Transporte Biológico Ativo/fisiologia , Infarto Encefálico/tratamento farmacológico , Infarto Encefálico/metabolismo , Infarto Encefálico/fisiopatologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Metabolismo Energético/efeitos dos fármacos , Metabolismo Energético/fisiologia , Guanosina/farmacologia , Hipocampo/fisiopatologia , Hipóxia-Isquemia Encefálica/tratamento farmacológico , Hipóxia-Isquemia Encefálica/fisiopatologia , Masculino , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fármacos Neuroprotetores/metabolismo , Fármacos Neuroprotetores/farmacologia , Técnicas de Cultura de Órgãos , Ratos , Ratos WistarRESUMO
Olfactory sensory neurons respond to odorants increasing Ca(2+) concentrations in their chemosensory cilia. Calcium enters the cilia through cAMP-gated channels, activating Ca(2+)-dependent chloride or potassium channels. Calcium also has a fundamental role in odour adaptation, regulating cAMP turnover rate and the affinity of the cyclic nucleotide-gated channels for cAMP. It has been shown that a Na(+)/Ca(2+) exchanger (NCX) extrudes Ca(2+) from the cilia. Here we confirm previous evidence that olfactory cilia also express plasma membrane Ca(2+)-ATPase (PMCA), and show the first evidence supporting a role in Ca(2+) removal. Both transporters were detected by immunoblot of purified olfactory cilia membranes. The pump was also revealed by immunocytochemistry and immunohistochemistry. Inside-out cilia membrane vesicles transported Ca(2+) in an ATP-dependent fashion. PMCA activity was potentiated by luminal Ca(2+) (K(0.5) = 670 nm) and enhanced by calmodulin (CaM; K(0.5) = 31 nm). Both carboxyeosin (CE) and calmidazolium reduced Ca(2+) transport, as expected for a CaM-modulated PMCA. The relaxation time constant (tau) of the Ca(2+)-dependent Cl(-) current (272 +/- 78 ms), indicative of luminal Ca(2+) decline, was increased by CE (2181 +/- 437 ms), by omitting ATP (666 +/- 49 ms) and by raising pH (725 +/- 65 ms), suggesting a role of the pump on Ca(2+) clearance. Replacement of external Na(+) by Li(+) had a similar effect (tau = 442 +/- 8 ms), confirming the NCX involvement in Ca(2+) extrusion. The evidence suggests that both Ca(2+) transporters contribute to re-establish resting Ca(2+) levels in the cilia following olfactory responses.