RESUMO
BACKGROUND: Bicarbonate transport has crucial roles in regulating intracellular pH (pHi) in a variety of cells. The purpose of this study was to evaluate its participation in the regulation of pHi in resting and stimulated human neutrophils. METHODS: Freshly isolated human neutrophils acidified by an ammonium prepulse were used in this study. RESULTS: We demonstrated that resting neutrophils have a bicarbonate transport mechanism that prevents acidification when the Na(+)/H(+) exchanger is blocked by EIPA. Neutrophils acidified by an ammonium prepulse showed an EIPA-resistant recovery of pHi that was inhibited by the blocker of the anionic transporters SITS or the Na(+)/HCO3(-) cotransporter (NBC) selective inhibitor S0859, and abolished when sodium was removed from the extracellular medium. In western blot and RT-PCR analysis the expression of NBCe2 but not NBCe1 or NBCn1 was detected in neutrophils Acidified neutrophils increased the EIPA-insensitive pHi recovery rate when its activity was stimulated with fMLF/ cytochalasin B. This increase in the removal of acid equivalents was insensitive to the blockade of the NADPH oxidase with DPI. CONCLUSION: It is concluded that neutrophils have an NBC that regulates basal pHi and is modulated by chemotactic agents.
Assuntos
Neutrófilos/metabolismo , Simportadores de Sódio-Bicarbonato/metabolismo , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-dissulfônico/farmacologia , Amilorida/análogos & derivados , Amilorida/farmacologia , Cloreto de Amônio/farmacologia , Benzamidas/farmacologia , Bicarbonatos/farmacologia , Citocalasina B/farmacologia , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Transporte de Íons/efeitos dos fármacos , N-Formilmetionina Leucil-Fenilalanina/farmacologia , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/metabolismo , Neutrófilos/efeitos dos fármacos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/metabolismo , Simportadores de Sódio-Bicarbonato/genética , Trocadores de Sódio-Hidrogênio/metabolismo , Sulfonamidas/farmacologiaRESUMO
Although nitrate (NO3(-)) but not ammonium (NH4(+)) improves plant tolerance to oxygen deficiency, the mechanisms involved in this phenomenon are just beginning to be understood. By using gas chromatography-mass spectrometry, we investigated the metabolic fate of (15)NO3(-) and (15)NH4(+) in soybean plants (Glycine max L. Merril cv. IAC-23) subjected to root hypoxia. This stress reduced the uptake of (15)NO3(-) and (15)NH4(+) from the medium and decreased the overall assimilation of these nitrogen sources into amino acids in roots and leaves. Root (15)NO3(-) assimilation was more affected by hypoxia than that of (15)NH4(+), resulting in enhanced nitrite and nitric oxide release in the solution. However, (15)NO3(-) was translocated in substantial amounts by xylem sap and considerable (15)NO3(-) assimilation into amino acids also occurred in the leaves, both under hypoxia and normoxia. By contrast, (15)NH4(+) assimilation occurred predominantly in roots, resulting in accumulation of mainly (15)N-alanine in this tissue during hypoxia. Analysis of lactate levels suggested higher fermentation in roots from NH4(+)-treated plants compared to the NO3(-) treatment. Thus, foliar NO3(-) assimilation may be relevant to plant tolerance to oxygen deficiency, since it would economize energy expenditure by hypoxic roots. Additionally, the involvement of nitric oxide synthesis from nitrite in the beneficial effect of NO3(-) is discussed.
Assuntos
Glycine max/metabolismo , Nitratos/metabolismo , Nitrogênio/metabolismo , Oxigênio/metabolismo , Raízes de Plantas/metabolismo , Adaptação Fisiológica , Trifosfato de Adenosina/metabolismo , Alanina/metabolismo , Cloreto de Amônio/metabolismo , Cloreto de Amônio/farmacologia , Transporte Biológico , Hipóxia Celular , Meios de Cultura/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Nitratos/farmacologia , Óxido Nítrico/metabolismo , Floema/metabolismo , Folhas de Planta/metabolismo , Raízes de Plantas/efeitos dos fármacos , Compostos de Amônio Quaternário/metabolismo , Glycine max/efeitos dos fármacos , Xilema/metabolismoRESUMO
Considering that stimulation of melanogenesis may lead to alterations of cellular responses, besides melanin production, our main goal was to study the cellular effects of melanogenesis stimulation of B16-F10 melanoma cells. Our results show increased levels of the reactive oxygen species after 15 h of melanogenesis stimulation. Following 48 h of melanogenesis stimulation, proliferation was inhibited (by induction of cell cycle arrest in the G1 phase) and the expression levels of p21 mRNA were increased. In addition, melanogenesis stimulation did not induce cellular senescence. Proteomic analysis demonstrated the involvement of proteins from other pathways besides those related to the cell cycle, including protein disulfide isomerase A3, heat-shock protein 70, and fructose biphosphate aldolase A (all up-regulated), and lactate dehydrogenase (down-regulated). In RT-qPCR experiments, the levels of pyruvate kinase M2 mRNA dropped, whereas the levels of ATP synthase (beta-F1) mRNA increased. These data indicate that melanogenesis stimulation of B16-F10 cells leads to alterations in metabolism and cell cycle progression that may contribute to an induction of cell quiescence, which may provide a mechanism of resistance against cellular injury promoted by melanin synthesis.
Assuntos
Melaninas/biossíntese , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Cloreto de Amônio/farmacologia , Animais , Apoptose , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/fisiologia , Proliferação de Células , Inibidor de Quinase Dependente de Ciclina p21/genética , Pontos de Checagem da Fase G1 do Ciclo Celular , Expressão Gênica , Genes Supressores de Tumor , Melanoma Experimental/genética , Camundongos , Proteínas de Neoplasias/metabolismo , Proteoma/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Tirosina/farmacologiaRESUMO
Serratia marcescens is an opportunistic human pathogen that represents a growing problem for public health, particularly in hospitalized or immunocompromised patients. However, little is known about factors and mechanisms that contribute to S. marcescens pathogenesis within its host. In this work, we explore the invasion process of this opportunistic pathogen to epithelial cells. We demonstrate that once internalized, Serratia is able not only to persist but also to multiply inside a large membrane-bound compartment. This structure displays autophagic-like features, acquiring LC3 and Rab7, markers described to be recruited throughout the progression of antibacterial autophagy. The majority of the autophagic-like vacuoles in which Serratia resides and proliferates are non-acidic and have no degradative properties, indicating that the bacteria are capable to either delay or prevent fusion with lysosomal compartments, altering the expected progression of autophagosome maturation. In addition, our results demonstrate that Serratia triggers a non-canonical autophagic process before internalization. These findings reveal that S. marcescens is able to manipulate the autophagic traffic, generating a suitable niche for survival and proliferation inside the host cell.
Assuntos
Autofagia , Serratia marcescens/fisiologia , Vacúolos/microbiologia , Cloreto de Amônio/farmacologia , Androstadienos/farmacologia , Animais , Células CHO , Linhagem Celular , Cricetinae , Células Epiteliais/microbiologia , Técnica Indireta de Fluorescência para Anticorpo , Gentamicinas/farmacologia , Humanos , Macrolídeos/farmacologia , Microscopia Confocal , Serratia marcescens/efeitos dos fármacos , WortmaninaRESUMO
In this study, we describe the presence of apoptosis, associated with a mitochondrial dysfunction in the hippocampus of animals in an experimental model defined as minimal hepatic encephalopathy (MHE). This experimental model was studied after 10 days of induced portal vein calibrated stricture, leading to portal hypertension and to a moderate hyperammonemia, without the presence of other evident central nervous system changes. The molecular mechanisms here proposed indicate the presence of apoptotic intrinsic pathways that point to hippocampal mitochondria as an important mediator of apoptosis in this experimental model. In this model of MHE, the presence of DNA fragmentation is documented by 2.3-times increased number of TUNEL-positive cells. These findings together with a higher ratio of the Bcl-2 family members Bax/Bcl-xL in the outer mitochondrial membrane of the MHE animals together with 11% of cytochrome c release indicate the presence of apoptosis in this experimental model. A detailed analysis of the hippocampal mitochondrial physiology was performed after mitochondrial isolation. The determination of the respiratory rate in the presence of malate plus glutamate and ADP showed a 45% decrease in respiratory control in MHE animals as compared with the sham group. A marked decrease of cytochrome oxidase (complex IV of the electron transport chain) was also observed, showing 46% less activity in hippocampal mitochondria from MHE animals. In addition, mitochondria from these animals showed less ability to maintain membrane potential (ΔΨ (m)) which was 13% lower than the sham group. Light scattering experiments showed that mitochondria from MHE animals were more sensitive to swell in the presence of increased calcium concentrations as compared with the sham group. In addition, in vitro studies performed in mitochondria from sham animals showed that mitochondrial permeability transition (MPT) could be a mitochondrial mediator of the apoptotic signaling in the presence of NH(4) (+) and calcium.
Assuntos
Apoptose , Encefalopatia Hepática/fisiopatologia , Hipocampo/patologia , Mitocôndrias/metabolismo , Cloreto de Amônio/metabolismo , Cloreto de Amônio/farmacologia , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Cálcio/farmacologia , Cálcio/fisiologia , Constrição Patológica/patologia , Fragmentação do DNA , Modelos Animais de Doenças , Complexo I de Transporte de Elétrons/metabolismo , Complexo II de Transporte de Elétrons/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Encefalopatia Hepática/complicações , Hiperamonemia/etiologia , Masculino , Potencial da Membrana Mitocondrial , Mitocôndrias/efeitos dos fármacos , Dilatação Mitocondrial , Consumo de Oxigênio , Permeabilidade , Veia Porta/patologia , Ratos , Ratos Endogâmicos WKYRESUMO
BACKGROUND: GlnR is an atypical response regulator found in actinomycetes that modulates the transcription of genes in response to changes in nitrogen availability. We applied a global in vivo approach to identify the GlnR regulon of Streptomyces venezuelae, which, unlike many actinomycetes, grows in a diffuse manner that is suitable for physiological studies. Conditions were defined that facilitated analysis of GlnR-dependent induction of gene expression in response to rapid nitrogen starvation. Microarray analysis identified global transcriptional differences between glnR+ and glnR mutant strains under varying nitrogen conditions. To differentiate between direct and indirect regulatory effects of GlnR, chromatin immuno-precipitation (ChIP) using antibodies specific to a FLAG-tagged GlnR protein, coupled with microarray analysis (ChIP-chip), was used to identify GlnR binding sites throughout the S. venezuelae genome. RESULTS: GlnR bound to its target sites in both transcriptionally active and apparently inactive forms. Thirty-six GlnR binding sites were identified by ChIP-chip analysis allowing derivation of a consensus GlnR-binding site for S. venezuelae. GlnR-binding regions were associated with genes involved in primary nitrogen metabolism, secondary metabolism, the synthesis of catabolic enzymes and a number of transport-related functions. CONCLUSIONS: The GlnR regulon of S. venezuelae is extensive and impacts on many facets of the organism's biology. GlnR can apparently bind to its target sites in both transcriptionally active and inactive forms.
Assuntos
Proteínas de Bactérias/fisiologia , Genoma Bacteriano , Nitrogênio/metabolismo , Streptomyces/genética , Transativadores/fisiologia , Cloreto de Amônio/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Sítios de Ligação , Imunoprecipitação da Cromatina , Análise em Microsséries , Ligação Proteica , Transativadores/genética , Transativadores/metabolismo , Transcrição GênicaRESUMO
BACKGROUND/AIMS: It has been widely accepted that chloride ions moving along chloride channels act to dissipate the electrical gradient established by the electrogenic transport of H(+) ions performed by H(+)-ATPase into subcellular vesicles. Largely known in intracellular compartments, this mechanism is also important at the plasma membrane of cells from various tissues, including kidney. The present work was performed to study the modulation of plasma membrane H(+)-ATPase by chloride channels, in particular, CFTR and ClC-5 in kidney proximal tubule. METHODS AND RESULTS: Using in vivo stationary microperfusion, it was observed that ATPase-mediated HCO(3)(-) reabsorption was significantly reduced in the presence of the Cl(-) channels inhibitor NPPB. This effect was confirmed in vitro by measuring the cell pH recovery rates after a NH(4)Cl pulse in immortalized rat renal proximal tubule cells, IRPTC. In these cells, even after abolishing the membrane potential with valinomycin, ATPase activity was seen to be still dependent on Cl(-). siRNA-mediated CFTR channels and ClC-5 chloride-proton exchanger knockdown significantly reduced H(+)-ATPase activity and V-ATPase B2 subunit expression. CONCLUSION: These results indicate a role of chloride in modulating plasma membrane H(+)-ATPase activity in proximal tubule and suggest that both CFTR and ClC-5 modulate ATPase activity.
Assuntos
Canais de Cloreto/fisiologia , Regulador de Condutância Transmembrana em Fibrose Cística/fisiologia , Túbulos Renais Proximais/enzimologia , ATPases Vacuolares Próton-Translocadoras/metabolismo , Cloreto de Amônio/farmacologia , Animais , Antibacterianos/farmacologia , Bicarbonatos/metabolismo , Linhagem Celular , Canais de Cloreto/genética , Canais de Cloreto/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Nitrobenzoatos/farmacologia , Interferência de RNA , RNA Interferente Pequeno , Ratos , Valinomicina/farmacologiaRESUMO
Arthrospiraplatensis was cultivated in minitanks at 13 klux, using a mixture of KNO(3) and NH(4)Cl as nitrogen source. Fed-batch daily supply of NH(4)Cl at exponentially-increasing feeding rate allowed preventing ammonia toxicity and nitrogen deficiency, providing high maximum cell concentration (X(m)) and high-quality biomass (21.85 mg chlorophyll g cells(-1); 20.5% lipids; 49.8% proteins). A central composite design combined to response surface methodology was utilized to determine the relationships between responses (X(m), cell productivity and nitrogen-to-cell conversion factor) and independent variables (KNO(3) and NH(4)Cl concentrations). Under optimum conditions (15.5mM KNO(3); 14.1mM NH(4)Cl), X(m) was 4327 mg L(-1), a value almost coincident with that obtained with only 25.4mM KNO(3), but more than twice that obtained with 21.5mM NH(4)Cl. A 30%-reduction of culture medium cost can be estimated when compared to KNO(3)-batch runs, thus behaving as a cheap alternative for the commercial production of this cyanobacterium.
Assuntos
Cloreto de Amônio/farmacologia , Biotecnologia/métodos , Nitratos/farmacologia , Nitrogênio/farmacologia , Compostos de Potássio/farmacologia , Spirulina/efeitos dos fármacos , Spirulina/crescimento & desenvolvimento , Amônia/análise , Biomassa , Carbono/farmacologia , Clorofila/análise , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Lipídeos/análise , Análise Multivariada , Nitratos/análise , Proteínas/análise , Análise de Regressão , Spirulina/citologia , Fatores de TempoRESUMO
Diuron, a substituted urea herbicide, is carcinogenic to the urinary bladder of rats at high dietary levels. Its proposed carcinogenic mode of action (MOA) includes urothelial cytotoxicity and necrosis followed by regenerative cell proliferation and sustained urothelial hyperplasia. Cytotoxicity could be induced either by urinary solids or by chemical toxicity by diuron and/or metabolites excreted in the urine. Diuron was not genotoxic in a previous single-cell gel (comet) assay, but possible cross-linking activity remained to be evaluated. The present study explored the MOA of diuron and the effect of urinary acidification on the development of urothelial lesions. Male Wistar rats were fed diuron (2500 ppm, about 130 mg/kg of body weight) either with or without NH(4)Cl 10,000 ppm to acidify the urine. Reversibility of urothelial changes was also examined. The animals were euthanized after 15, 25, or 30 weeks. Diuron-fed rats had urinary amorphous precipitate and magnesium ammonium phosphate crystals similar to control animals. Groups treated with diuron + NH(4)Cl showed decreased urinary pH and reduced amounts of urinary crystals and precipitate. Urothelial necrosis and simple hyperplasia were observed by light microscopy and scanning electron microscopy both in diuron- and in diuron + NH(4)Cl-treated groups. Cytotoxicity and proliferative changes were mostly reversible. A modified comet assay developed in vitro with Chinese hamster ovary cells showed that diuron did not induce DNA cross-links. These data suggest that cytotoxicity with consequent regenerative cell proliferation is the predominant MOA for diuron rat urothelial carcinogenesis, the cytotoxicity being chemically induced and not due to urinary solids.
Assuntos
Carcinógenos/toxicidade , Proliferação de Células/efeitos dos fármacos , Diurona/toxicidade , Herbicidas/toxicidade , Regeneração/efeitos dos fármacos , Neoplasias da Bexiga Urinária/induzido quimicamente , Bexiga Urinária/efeitos dos fármacos , Cloreto de Amônio/farmacologia , Animais , Peso Corporal/efeitos dos fármacos , Células CHO , Ensaio Cometa , Cricetinae , Cricetulus , Dano ao DNA , Concentração de Íons de Hidrogênio , Hiperplasia , Compostos de Magnésio/urina , Masculino , Necrose , Fosfatos/urina , Ratos , Ratos Wistar , Estruvita , Fatores de Tempo , Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/urina , Cálculos Urinários/induzido quimicamente , Cálculos Urinários/urina , Urotélio/efeitos dos fármacos , Urotélio/patologiaRESUMO
ClC-2 chloride channel is present in the brain and some transporting epithelia where its function is poorly understood. We have now demonstrated that the surface channels are rapidly internalised and approximately the 70% of the surface membrane protein recycles after 4- to 8-min internalisation. Endocytosis of ClC-2 was dependent upon tyrosine 179 located within an endocytic motif. Rapid recycling accompanied by an even faster internalisation could account for the abundant presence of ClC-2 in intracellular membranous structures. At least a proportion of ClC-2 resides in lipid rafts. Use of beta-cyclodextrin led to an increase in cell surface channel, but, surprisingly, a decrease in functionally active channels. We suggest that ClC-2 requires residing in beta-cyclodextrin sensitive clusters with other molecules in order to remain active. Regulation of ClC-2 trafficking to and within the membrane could be a means of modulating its activity.
Assuntos
Membrana Celular/metabolismo , Canais de Cloreto/metabolismo , Endocitose/fisiologia , Endossomos/metabolismo , Transporte Proteico/fisiologia , Tirosina/genética , Motivos de Aminoácidos/fisiologia , Substituição de Aminoácidos/fisiologia , Cloreto de Amônio/farmacologia , Androstadienos/farmacologia , Canais de Cloro CLC-2 , Linhagem Celular , Canais de Cloreto/efeitos dos fármacos , Canais de Cloreto/genética , Endocitose/efeitos dos fármacos , Endossomos/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Hemaglutininas/genética , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/fisiologia , Cinética , Microdomínios da Membrana/efeitos dos fármacos , Microdomínios da Membrana/fisiologia , Potenciais da Membrana/fisiologia , Transporte Proteico/efeitos dos fármacos , Proteínas Recombinantes de Fusão/fisiologia , Transfecção , Wortmanina , beta-Ciclodextrinas/farmacologiaRESUMO
Long-term effects of angiotensin II (Ang II) on vacuolar H(+)-ATPase were studied in a SV40-transformed cell line derived from rat proximal tubules (IRPTC). Using pH(i) measurements with the fluorescent dye BCECF, the hormone increased Na(+)-independent pH recovery rate from an NH(4)Cl pulse from 0.066 +/- 0.014 pH U/min (n = 7) to 0.14 +/- 0.021 pH U/min (n = 13; p < 0.05) in 10 h Ang II (10(-9) M)-treated cells. The increased activity of H(+)-ATPase did not involve changes in mRNA or protein abundance of the B2 subunit but increased cell surface expression of the V-ATPase. Inhibition of tyrosine kinase by genistein blocked Ang II-dependent stimulation of H(+)-ATPase. Inhibition of phosphatidylinositol-3-kinase (PI3K) by wortmannin and of p38 mitogen-activated protein kinase (MAPK) by SB 203580 also blocked this effect. Thus, long-term exposure of IRPTC cells to Ang II causes upregulation of H(+)-ATPase activity due, at least in part, to increased B2 cell surface expression. This regulatory pathway is dependent on mechanisms involving tyrosine kinase, p38 MAPK, and PI3K activation.
Assuntos
Angiotensina II/farmacologia , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/efeitos dos fármacos , ATPases Vacuolares Próton-Translocadoras/metabolismo , Cloreto de Amônio/farmacologia , Androstadienos/farmacologia , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Animais , Soluções Tampão , Linhagem Celular Transformada , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Cicloeximida/farmacologia , Dactinomicina/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Expressão Gênica/efeitos dos fármacos , Genisteína/farmacologia , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Imidazóis/farmacologia , Túbulos Renais Proximais/enzimologia , Losartan/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/farmacologia , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Transporte Proteico/efeitos dos fármacos , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Piridinas/farmacologia , Ratos , Fatores de Tempo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologia , ATPases Vacuolares Próton-Translocadoras/genética , Wortmanina , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The effects of aldosterone on the intracellular pH recovery rate (pHirr) via Na+/H+ exchanger and on the [Ca2+]i were investigated in isolated rat S3 segment. Aldosterone [10(-12), 10(-10), or 10(-8) M with 1-h, 15- or 2-min preincubation (pi)] caused a dose-dependent increase in the pHirr, but aldosterone (10(-6) M with 1-h, 15- or 2-min pi) decreased it (these effects were prevented by HOE694 but not by S3226). After 1 min of aldosterone pi, there was a transient and dose-dependent increase of the [Ca2+]i and after 6-min pi there was a new increase of [Ca2+]i that persisted after 1 h. Spironolactone, actinomycin D, or cycloheximide did not affect the effects of aldosterone (15- or 2-min pi) but inhibited the effects of aldosterone (1-h pi) on pHirr and on [Ca2+]i. RU 486 prevented the stimulatory effect of aldosterone (10(-12) M, 15- or 2-min pi) on both parameters and maintained the inhibitory effect of aldosterone (10(-6) M, 15- or 2-min pi) on the pHirr but reversed its stimulatory effect on the [Ca2+]i to an inhibitory effect. The data indicate a genomic (1 h, via MR) and a nongenomic action (15 or 2 min, probably via GR) on [Ca2+]i and on the basolateral NHE1 and are compatible with stimulation of the NHE1 by increases in [Ca2+]i in the lower range (at 10(-12) M aldosterone) and inhibition by increases at high levels (at 10(-6) M aldosterone) or decreases in [Ca2+]i (at 10(-6) M aldosterone plus RU 486).
Assuntos
Aldosterona/farmacologia , Sinalização do Cálcio/fisiologia , Túbulos Renais Proximais/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Cloreto de Amônio/farmacologia , Animais , Cálcio/metabolismo , Cicloeximida/farmacologia , Citosol/metabolismo , Dactinomicina/farmacologia , Expressão Gênica/efeitos dos fármacos , Guanidinas/farmacologia , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Técnicas In Vitro , Túbulos Renais Proximais/efeitos dos fármacos , Masculino , Metacrilatos/farmacologia , Mifepristona/farmacologia , Ratos , Ratos Wistar , Receptores de Glucocorticoides/genética , Receptores de Mineralocorticoides/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sódio/farmacologia , Trocador 1 de Sódio-Hidrogênio , Trocadores de Sódio-Hidrogênio/antagonistas & inibidores , Trocadores de Sódio-Hidrogênio/efeitos dos fármacos , Espironolactona/farmacologia , Sulfonas/farmacologiaRESUMO
Current methods for dengue virus quantitation are either time consuming, technically demanding or costly. As an alternative, the commercial enzyme immunoassay Platelia Dengue NS1 AG (Bio-Rad Laboratories) was used to monitor semiquantitatively dengue virus replication in cultured cells. The presence of NS1 protein was evaluated in supernatants from Vero and C6/36 HT cells infected with dengue virus. The amount of NS1 detected in the supernatants of infected cells was proportional to the initial MOI used and to the time of post infection harvest. This immunoassay was also able to detect the presence of NS1 in the supernatants of infected human macrophages. Inhibition of dengue virus replication in C6/36 HT cells treated with lysosomotropic drugs was readily monitored with the use of this assay. These results suggest that the Platelia Dengue NS1 AG kit can be used as a fast and reliable surrogate method for the relative quantitation of dengue virus replication in cultured cells.
Assuntos
Vírus da Dengue/isolamento & purificação , Técnicas Imunoenzimáticas/métodos , Replicação Viral , Aedes , Cloreto de Amônio/farmacologia , Animais , Linhagem Celular , Células Cultivadas , Chlorocebus aethiops , Cricetinae , Vírus da Dengue/efeitos dos fármacos , Vírus da Dengue/fisiologia , Humanos , Macrófagos/metabolismo , Macrófagos/virologia , Camundongos , Células Vero , Proteínas não Estruturais Virais/análise , Ensaio de Placa Viral , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacosRESUMO
BACKGROUND: We have previously shown that chronic metabolic acidosis, induced in rats by NH(4)Cl feeding, leads to nephron hypertrophy and to a decreased water-salt reabsorption by the kidneys. Since mitochondria are the main source of metabolic energy that drives ion transport in kidney tubules, we examined energy-linked functions (respiration, electrochemical membrane potential and coupling between respiration and ADP phosphorylation) in mitochondria isolated from rat kidney and liver at 48 h after metabolic acidosis induced by NH(4)Cl. METHODS: Mitochondria isolated from the kidneys and liver of metabolic acidotic rats, induced by NH(4)Cl, was used to study of the oxygen consumption by Clark-type electrode, mitochondrial electrical transmembrane potential estimated by the safranine O method and the variations in free medium Ca(2+) concentrations examined by absorbance spectrum of Arsenazo III set at the 675-685 nm wavelength pair. RESULTS: Whole kidney and liver mitochondria isolated from 48 h acidotic rats presented higher resting respiration, lower respiratory control and a lower ADP/O ratio than controls. These differences in mitochondrial coupling, between respiration and oxidative phosphorylation (ATP synthesis), were totally corrected when experiments were carried out in the presence of carboxyatractyloside, GDP and BSA, indicating that mitochondrial uncoupling proteins are more active in acidotic rat kidneys. Interestingly, determination of Ca(2+) transport demonstrated a faster rate of initial Ca(2+) uptake by acidotic kidney mitochondria, which resulted in a lower concentration of extra-mitochondrial Ca(2+) under steady-state conditions (Ca(2+) set point) when compared with control mitochondria. In contrast, there were no significant differences in the rates of Na(+) or ruthenium red induced Ca(2+) efflux. CONCLUSIONS: We suggest that the mild uncoupling and higher Ca(2+) accumulation represents an adaptation of the mitochondria to cope with conditions of oxidative stress and high cytosolic Ca(2+), which are associated with a decreased efficiency of oxidative phosphorylation that may explain, at least in part, the striking natriuresis observed under chronic acidosis. Finally, there were no changes in Ca(2+) transport or coupling in liver mitochondria isolated from the acidotic rats.
Assuntos
Acidose/induzido quimicamente , Cloreto de Amônio/farmacologia , Cálcio/metabolismo , Rim/metabolismo , Mitocôndrias/metabolismo , Animais , Transporte Biológico , Túbulos Renais/metabolismo , Fígado/metabolismo , Potencial da Membrana Mitocondrial , Modelos Biológicos , Fosforilação Oxidativa , Oxigênio/metabolismo , Consumo de Oxigênio , Ratos , Ratos WistarRESUMO
Azospirillum brasilense is a nitrogen-fixing, root-colonizing bacterium that brings about plant-growth-promoting effects mainly because of its ability to produce phytohormones. Ethylenediamine (EDA)-resistant mutants of A. brasilense were isolated and screened for their higher ability to decrease acetylene and release ammonia in the medium. One of the mutants showed considerably higher levels of acetylene decrease and ammonia excretion. Nitrogenase activity of this mutant was relatively resistant to inhibition by NH(4)Cl. Adenosine triphosphate ribosylation of dinitrogenase reductase in the mutant did not increase even in presence of 10 mM NH(4)Cl. Although the mutant showed decreased glutamine synthetase (GS) activity, neither the levels of GS synthesized by the mutant nor the NH (4) (+) -binding site in the GS differed from those of the parent. The main reason for the release of ammonia by the mutant seems to be the fixation of higher levels of nitrogen than its GS can assimilate, as well as higher levels of adenylylation of GS, which may decrease ammonia assimilation.
Assuntos
Amônia/metabolismo , Apirase/metabolismo , Azospirillum brasilense/genética , Azospirillum brasilense/metabolismo , Glutamato-Amônia Ligase/metabolismo , Adenosina Difosfato Ribose/metabolismo , Cloreto de Amônio/farmacologia , Azospirillum brasilense/efeitos dos fármacos , Azospirillum brasilense/crescimento & desenvolvimento , Dinitrogenase Redutase/metabolismo , Farmacorresistência Bacteriana , Etilenodiaminas/farmacologia , Mutação , Fixação de NitrogênioRESUMO
Human sperm are endowed with putative voltage-dependent calcium channels (VDCC) that produce measurable increases in intracellular calcium concentration ([Ca(2+)](i)) in response to membrane depolarization with potassium. These channels are blocked by nickel, inactivate in 1-2 min in calcium-deprived medium, and are remarkably stimulated by NH(4)Cl, suggesting a role for intracellular pH (pH(i)). In a previous work, we showed that calcium permeability through these channels increases approximately onefold during in vitro "capacitation," a calcium-dependent process that sperm require to fertilize eggs. In this work, we have determined the pH(i) dependence of sperm VDCC. Simultaneous depolarization and pH(i) alkalinization with NH(4)Cl induced an [Ca(2+)](i) increase that depended on the amount of NH(4)Cl added. VDCC stimulation as a function of pH(i) showed a sigmoid curve in the 6.6-7.2 pH(i) range, with a half-maximum stimulation at pH approximately 7.00. At higher pH(i) (> or =7.3), a further stimulation occurred. Calcium release from internal stores did not contribute to the stimulating effect of pH(i) because the [Ca(2+)](i) increase induced by progesterone, which opens a calcium permeability pathway that does not involve gating of VDCC, was unaffected by ammonium. The ratio of pH(i)-stimulated-to-nonstimulated calcium influx was nearly constant at different test depolarization values. Likewise, depolarization-induced calcium influx in pH(i)-stimulated and nonstimulated cells was equally blocked by nickel. In our capacitating conditions pH(i) increased 0.11 pH units, suggesting that the calcium influx stimulation observed during sperm capacitation might be partially caused by pH(i) alkalinization. Additionally, a calcium permeability pathway triggered exclusively by pH(i) alkalinization was detected.
Assuntos
Cálcio/metabolismo , Concentração de Íons de Hidrogênio , Capacitação Espermática/fisiologia , Espermatozoides/metabolismo , Cloreto de Amônio/farmacologia , Cálcio/farmacocinética , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/metabolismo , Estimulação Elétrica , Humanos , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Níquel/farmacologiaRESUMO
BACKGROUND: Both dietary phosphorus restriction and the ingestion of ammonium chloride (NH(4)Cl) given to rats on a high-phosphorus diet have been shown to preserve renal function in the azotaemic rat. Parathyroidectomy also has been reported to preserve renal function and, in addition, to prevent kidney hypertrophy in the remnant kidney model. Our goals were (i) to evaluate in azotaemic rats the effect of dietary phosphorus on renal function in a shorter time frame than previously studied and (ii) to determine whether NH(4)Cl administration (a) enhances the renoprotective effect of dietary phosphorus restriction and (b) improves renal function in the absence of parathyroid hormone (PTH). METHODS: High (H; 1.2%), normal (N; 0.6%) and low (L; <0.05%) phosphorus diets (PD) were given for 30 days to 5/6 nephrectomized rats. In each dietary group, one-half of the rats were given NH(4)Cl in the drinking water. The six groups were HPD + NH(4)Cl, HPD, NPD + NH(4)Cl, NPD, LPD + NH(4)Cl and LPD. The effect of NH(4)Cl administration was also evaluated in 5/6 nephrectomized, parathyroidectomized (PTX) rats on NPD. RESULTS: In each of the three dietary phosphorus groups, creatinine and urea clearances were greater (P<0.01) in rats receiving NH(4)Cl. Neither creatinine nor urea clearance was reduced by high dietary phosphorus. Urine calcium excretion was greatest in the LPD group and was increased (P < or = 0.001) in all three groups by NH(4)Cl ingestion. An inverse correlation was present between plasma calcium and phosphorus in the parathyroid intact (r = -0.79, P<0.001) and PTX groups (r = -0.46, P = 0.02). In PTX rats, NH(4)Cl ingestion increased (P < or = 0.01) creatinine and urea clearances and both an increasing plasma calcium concentration (r = 0.67, P<0.001) and urine calcium excretion (r = 0.73, P<0.001) increased urine phosphorus excretion. CONCLUSIONS: At 30 days of renal failure (i) NH(4)Cl ingestion increased creatinine and urea clearances, irrespective of dietary phosphorus; (ii) high urine calcium excretion, induced by dietary phosphorus restriction and NH(4)Cl ingestion, did not adversely affect renal function; (iii) high dietary phosphorus did not decrease renal function; (iv) the absence of PTH did not preserve renal function or prevent NH(4)Cl from improving renal function; and (v) both an increasing plasma calcium concentration and urine calcium excretion resulted in an increase in urine phosphorus excretion in PTX rats.
Assuntos
Cloreto de Amônio/farmacologia , Diuréticos/farmacologia , Rim/efeitos dos fármacos , Fósforo/farmacologia , Insuficiência Renal/fisiopatologia , Uremia/fisiopatologia , Animais , Cálcio/sangue , Cálcio/urina , Creatinina/urina , Masculino , Nefrectomia , Paratireoidectomia , Ratos , Ratos Sprague-Dawley , Insuficiência Renal/sangue , Uremia/sangueRESUMO
BACKGROUND: Kidney hypertrophy is stimulated by both partial nephrectomy and NH(4)Cl administration. Also, parathyroidectomy (PTX) has been reported to prevent kidney hypertrophy induced by a high protein diet. Our goal was to determine in the azotaemic rat: (i) the combined effects of NH(4)Cl administration and dietary phosphorus on the development of kidney hypertrophy and calcium deposition in the kidney and (ii) whether the absence of parathyroid hormone (PTH) affected the development of kidney hypertrophy and calcium deposition. METHODS: High (HPD, 1.2%), normal (NPD, 0.6%) or low (LPD, <0.05%) phosphorus diets were given to 5/6 nephrectomized rats for 30 days. In each dietary group, one-half of the rats were given NH(4)Cl in the drinking water. The six groups of rats were: (i) HPD + NH(4)Cl; (ii) HPD; (iii) NPD + NH(4)Cl; (iv) NPD; (v) LPD + NH(4)Cl and (vi) LPD. In a separate study, PTX was performed to determine whether PTH affected renal hypertrophy in 5/6 nephrectomized rats given NH(4)Cl. RESULTS: Both with and without NH(4)Cl (+/-NH(4)Cl), kidney weight was greatest (P<0.05) in the HPD groups. In each dietary phosphorus group, kidney weight was greater (P<0.05) in the NH(4)Cl group. In both the +/-NH(4)Cl groups, kidney calcium content was greatest (P<0.05) in the HPD group, but was less (P<0.05) in the NPD and HPD groups given NH(4)Cl. An inverse correlation was present between creatinine clearance and kidney calcium content (r = -0.51, P<0.001). When factored for kidney weight, creatinine clearance was less (P<0.05) in the HPD group in both the +/-NH(4)Cl groups, but was greater in the HPD + NH(4)Cl than in the HPD group. In PTX rats, kidney weight was greater (P<0.05) and kidney calcium deposition was less (P<0.05) in rats given NH(4)Cl. CONCLUSIONS: In azotaemic rats studied for 30 days, NH(4)Cl administration induced kidney hypertrophy. A HPD also induced kidney hypertrophy. The effects on kidney calcium deposition were divergent for which NH(4)Cl administration decreased and a HPD increased calcium deposition. The inverse correlation between kidney calcium content and creatinine clearance suggests that kidney calcium deposition is harmful to renal function. When factored for kidney weight, the lower creatinine clearance in the high phosphorus group suggests that kidney hypertrophy does not completely compensate for the harmful effects of a HPD. This result also suggests that a longer study would probably result in more rapid deterioration in the high phosphorus group. In PTX rats, the absence of PTH did not prevent NH(4)Cl from inducing kidney hypertrophy and reducing kidney calcium deposition. In conclusion, NH(4)Cl and dietary phosphorus each independently affect kidney growth and calcium deposition in the growing rat with renal failure.
Assuntos
Cloreto de Amônio/farmacologia , Cálcio/metabolismo , Diuréticos/farmacologia , Rim/metabolismo , Rim/patologia , Fósforo/farmacologia , Insuficiência Renal/patologia , Uremia/patologia , Animais , Creatinina/metabolismo , Progressão da Doença , Hipertrofia , Masculino , Nefrectomia , Paratireoidectomia , Ratos , Ratos Sprague-Dawley , Insuficiência Renal/metabolismo , Uremia/metabolismoRESUMO
Hyperammonemia is a common finding in children with methylmalonic acidemia, an inherited metabolic disease characterized by mental retardation, convulsions, and accumulation of methylmalonic acid (MMA). Although it has been suggested that MMA induces convulsions through succinate dehydrogenase (SDH) inhibition, very little is known about the contribution of hyperammonemia to the development of convulsions in these patients. In the present study we investigated the effects of ammonium ions on the convulsant action of MMA, MMA-induced inhibition of striatal succinate dehydrogenase, and the striatal content of thiobarbituric acid-reactive substances (TBARS). Adult rats were injected with ammonium acetate (1.5 mmol/kg, sc) or sodium acetate (1.5 mmol/kg, sc), followed 5 min later by buffered MMA (3 micromol/microl) or NaCl (4.5 micromol/microl) injected into the striatum. The animals were observed in an open field for the appearance of convulsive episodes. After 30 min of behavioral evaluation, the animals were sacrificed and had their striatal TBARS content measured. Ammonium acetate pretreatment caused no behavioral effects per se, but potentiated MMA-induced convulsions and increased basal TBARS content and MMA-induced TBARS production in the striatum. Ammonium chloride had no effect on basal succinate dehydrogenase activity and did not alter MMA-induced inhibition of SDH in vitro. These results suggest that ammonia potentiates MMA-induced behavioral effects through a mechanism that does not involve further succinate dehydrogenase inhibition, but may involve facilitation of MMA-induced oxidative damage and provide evidence that ammonia and MMA may have mutually additive toxicity.
Assuntos
Amônia/farmacologia , Ácido Metilmalônico/toxicidade , Convulsões/fisiopatologia , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismo , Acetatos/farmacologia , Cloreto de Amônio/farmacologia , Animais , Comportamento Animal/efeitos dos fármacos , Corpo Estriado/química , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Sinergismo Farmacológico , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/toxicidade , Masculino , Ratos , Ratos Wistar , Convulsões/induzido quimicamente , Succinato Desidrogenase/antagonistas & inibidores , Succinato Desidrogenase/metabolismoRESUMO
The modulation by Na(+), K(+), NH(4)(+) and ATP of the (Na(+),K(+))-ATPase in a microsomal fraction from Callinectes danae gills was analyzed. ATP was hydrolyzed at high-affinity binding sites at a maximal rate of V=35.4+/-2.1 Umg(-1) and K(0.5)=54.0+/-3.6 nM, obeying cooperative kinetics (n(H)=3.6). At low-affinity sites, the enzyme hydrolyzed ATP obeying Michaelis-Menten kinetics with K(M)=55.0+/-3.0 microM and V=271.5+/-17.2 Umg(-1). This is the first demonstration of a crustacean (Na(+),K(+))-ATPase with two ATP hydrolyzing sites. Stimulation by sodium (K(0.5)=5.80+/-0.30 mM), magnesium (K(0.5)=0.48+/-0.02 mM) and potassium ions (K(0.5)=1.61+/-0.06 mM) exhibited site-site interactions, while that by ammonium ions obeyed Michaelis-Menten kinetics (K(M)=4.61+/-0.27 mM). Ouabain (K(I)=147.2+/-7.microM) and orthovanadate (K(I)=11.2+/-0.6 microM) completely inhibited ATPase activity, indicating the absence of contaminating ATPase and/or neutral phosphatase activities. Ammonium and potassium ions synergistically stimulated the enzyme, increasing specific activities up to 90%, suggesting that these ions bind to different sites on the molecule. The presence of each ion modulates enzyme stimulation by the other. The modulation of (Na(+),K(+))-ATPase activity by ammonium ions, and the excretion of NH(4)(+) in benthic crabs are discussed.