RESUMO
OBJECTIVES: Temozolomide (TMZ) is one of the most commonly used clinical drugs for glioblastoma (GBM) treatment, but its drug sensitivity needs to be improved. Gamabufotalin (CS-6), the primary component of the traditional Chinese medicine "ChanSu," was shown to have strong anti-cancer activity. However, more efforts should be directed towards reducing its toxicity or effective treatment doses. METHODS: Target fishing experiment, Western blotting, PCR, confocal immunofluorescence and molecular cloning techniques were performed to search for possible downstream signalling pathways. In addition, GBM xenografts were used to further determine the potential molecular mechanisms of the synergistic effects of CS-6 and TMZ in vivo. RESULTS: Mechanistic research revealed a negative feedback loop between ATP1A3 and AQP4 through which CS-6 inhibited GBM growth and mediated the synergistic treatment effect of CS-6 and TMZ. In addition, by mutating potential amino acid residues of ATP1A3, which were predicted by modelling and docking to interact with CS-6, we demonstrated that abrogating hydrogen bonding of the amino acid Thr794 interferes with the activation of ATP1A3 by CS-6 and that the Thr794Ala mutation directly affects the synergistic treatment efficacy of CS-6 and TMZ. CONCLUSIONS: As the main potential target of CS-6, ATP1A3 activation critically depends on the hydrogen bonding of Thr794 with CS-6. The combination of CS-6 and TMZ could significantly reduce the therapeutic doses and promote the anti-cancer efficacy of CS-6/TMZ monotherapy.
Assuntos
Aquaporina 4/metabolismo , Bufanolídeos/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioblastoma/tratamento farmacológico , Proteínas de Neoplasias/biossíntese , ATPase Trocadora de Sódio-Potássio/biossíntese , Temozolomida/farmacologia , Animais , Aquaporina 4/genética , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Camundongos , Proteínas de Neoplasias/genética , ATPase Trocadora de Sódio-Potássio/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
A low concentration of boron (B) accelerates the proliferation and differentiation of mammalian osteoblasts. The aim of this study was to investigate the effects of 0.1 mM of B on the membrane function of osteoblastic cells in vitro. Genes involved in cell activity were investigated using gene expression microarray analyses. The Ca2+ influx and efflux were evaluated to demonstrate the activation of L-type Ca2+ channel for the Ca2+ influx, and that of Na+/K+-ATPase for the Ca2+ efflux. A real-time PCR analysis revealed that the messenger RNA (mRNA) expression of four mineralization-related genes was clearly increased after 3 days of culture with a B-supplemented culture medium. Using microarray analyses, five genes involved in cell proliferation and differentiation were upregulated compared to the control group. Regarding the Ca2+ influx, in the nifedipine-pretreated group, the relative fluorescence intensity for 1 min after adding B solution did not increase compared with that for 1 min before addition. In the control group, the relative fluorescence intensity was significantly increased compared with the experimental group (P < 0.05). Regarding the Ca2+ efflux, in the experimental group cultured in 0.1 mM of B-supplemented medium, the relative fluorescence intensity for 10 min after ouabain treatment revealed a significantly lower slope value compared with the control group (P < 0.01). This is the first study to demonstrate the acceleration of Ca2+ flux by B supplementation in osteoblastic cells. Cell membrane stability is related to the mechanism by which a very low concentration of B promotes the proliferation and differentiation of mammalian osteoblastic cells in vitro.
Assuntos
Boro/farmacologia , Cálcio/metabolismo , Osteoblastos/metabolismo , Canais de Cálcio Tipo L/biossíntese , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , ATPase Trocadora de Sódio-Potássio/biossínteseRESUMO
BACKGROUND: Despite growing claims of functional health benefits in folkloric medicine, the safety of chronic/elevated intakes of onion and garlic cannot be assumed. Therefore, this study assesses oral administration of varied doses of onion and garlic on some biomarkers of hepatic and renal functions in rats. METHODS: Animals were divided into five groups: control group received vehicle and extract-treated groups received varied doses of onion or garlic extract (0.5 mL and 1.0 mL/100 g bwt/day) for 6 weeks. RESULTS: Both doses of onion caused marked (p<0.05) increase in hepatic and renal levels of glutathione (GSH), glutathione S-transferase (GST), superoxide dismutase (SOD), catalase (CAT) and marked (p<0.05) decrease in malondialdehyde (MDA). Treatment with low dose of garlic elicited similar trend except in hepatic CAT, renal SOD and GST levels. A high dose of garlic only caused marked (p<0.05) increase in hepatic GST, renal GST, and SOD. Both doses of onion and low dose of garlic significantly (p<0.05) enhanced renal Na+/K+-ATPase activity. Only a high dose of onion caused significant (p<0.05) increase in hepatic aspartate transaminase (AST), alkaline phosphatase (ALP), and decrease in plasma AST activities. CONCLUSIONS: These findings suggest antioxidant enhancing capability for both doses of onion and low dose of garlic, while high dose of garlic elicited pro-oxidant conditions.
Assuntos
Alho , Rim/enzimologia , Fígado/enzimologia , Cebolas , Extratos Vegetais/farmacologia , Animais , Antioxidantes/metabolismo , Aspartato Aminotransferases/biossíntese , Biomarcadores , Catalase/biossíntese , Relação Dose-Resposta a Droga , Glutationa/biossíntese , Glutationa Transferase/biossíntese , Masculino , Malondialdeído/metabolismo , Ratos , Ratos Wistar , ATPase Trocadora de Sódio-Potássio/biossínteseRESUMO
The effect of liquorice root derivatives (LRDs) glycyrrhizic acid (GL) and glycyrrhetinic acid (18ßGA) on salt and water balance and end points of gill ion transport in a freshwater teleost, (rainbow trout) was examined after feeding fish diets containing GL or 18ßGA (0, 5, 50 or 500 µg/g diet) for a two week period. Serum cortisol levels and gill 11ß-hydroxysteroid dehydrogenase type 2 mRNA abundance decreased in fish fed GL but increased (at select doses) in fish fed 18ßGA. At higher doses of GL, gill Na(+)-K(+)-ATPase and H(+)-ATPase activity increased, while cystic fibrosis transmembrane conductance regulator type II mRNA abundance significantly decreased at the lowest dose of GL. End points of gill transcellular ion transport were not significantly altered in fish fed 18ßGA, except for a reduction in Na(+)-K(+)-ATPase activity at a 50 µg/g dose. In contrast, high doses of GL and 18ßGA increased gill transcript abundance of the tight junction protein claudin-31 (cldn-31). Other end points of gill paracellular transport differed in fishes fed LRDs. Tricellulin mRNA abundance was increased by high dose GL and decreased by high dose 18ßGA, and cldn-23a and cldn-27b mRNA abundance significantly decreased in response to GL irrespective of dose. Despite the above observations, systemic end points of salt and water balance (i.e. serum [Na(+)] and [Cl(-)] as well as muscle moisture) were unaffected by LRDs. Therefore data suggest that LRDs can alter end points of ion transport in fishes but that overall salt and water balance need not be perturbed.
Assuntos
Ácido Glicirretínico/administração & dosagem , Ácido Glicirrízico/administração & dosagem , Equilíbrio Hidroeletrolítico/efeitos dos fármacos , Animais , Expressão Gênica/efeitos dos fármacos , Brânquias/efeitos dos fármacos , Brânquias/metabolismo , Ácido Glicirretínico/química , Glycyrrhiza/química , Ácido Glicirrízico/química , Transporte de Íons/efeitos dos fármacos , Oncorhynchus mykiss , RNA Mensageiro/biossíntese , ATPase Trocadora de Sódio-Potássio/biossínteseRESUMO
Diabetes mellitus (DM) is associated with brain alterations that may contribute to cognitive dysfunctions. Chlorogenic acid (CGA) and caffeine (CA), abundant in coffee (CF), are natural compounds that have showed important actions in the brain. The present study aimed to evaluate the effect of CGA, CA, and CF on acetylcholinesterase (AChE), Na(+), K(+)-ATPase, aminolevulinate dehydratase (δ-ALA-D) activities and TBARS levels from cerebral cortex, as well as memory and anxiety in streptozotocin-induced diabetic rats. Animals were divided into eight groups (n = 5-10): control; control/CGA 5 mg/kg; control/CA 15 mg/kg; control/CF 0.5 g/kg; diabetic; diabetic/CGA 5 mg/kg; diabetic/CA 15 mg/kg; and diabetic/CF 0.5 g/kg. Our results demonstrated an increase in AChE activity and TBARS levels in cerebral cortex, while δ-ALA-D and Na(+), K(+)-ATPase activities were decreased in the diabetic rats when compared to control water group. Furthermore, a memory deficit and an increase in anxiety in diabetic rats were observed. The treatment with CGA and CA prevented the increase in AChE activity in diabetic rats when compared to the diabetic water group. CGA, CA, and CF intake partially prevented cerebral δ-ALA-D and Na(+), K(+)-ATPase activity decrease due to diabetes. Moreover, CGA prevented diabetes-induced TBARS production, improved memory, and decreased anxiety. In conclusion, among the compounds studied CGA proved to be a compound which acts better in the prevention of brain disorders promoted by DM.
Assuntos
Comportamento Animal/efeitos dos fármacos , Cafeína/farmacologia , Ácido Clorogênico/farmacologia , Café , Diabetes Mellitus Experimental/tratamento farmacológico , Acetilcolinesterase/biossíntese , Animais , Ansiedade/tratamento farmacológico , Peso Corporal/efeitos dos fármacos , Córtex Cerebral/metabolismo , Masculino , Memória/efeitos dos fármacos , Transtornos da Memória/tratamento farmacológico , Sintase do Porfobilinogênio/biossíntese , Ratos , Ratos Wistar , ATPase Trocadora de Sódio-Potássio/biossíntese , Estreptozocina , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismoRESUMO
In kidney, FXYD proteins regulate Na,K-ATPase in a nephron segment-specific way. FXYD2 is the most abundant renal FXYD but is not expressed in most renal cell lines unless induced by hypertonicity. Expression by transfection of FXYD2a or FXYD2b splice variants in NRK-52E cells reduces the apparent Na(+) affinity of the Na,K-ATPase and slows the cell proliferation rate. Based on RT-PCR, mRNAs for both splice variants were expressed in wild type NRK-52E cells as low abundance species. DNA sequencing of the PCR products revealed a base alteration from C to T in FXYD2b but not FXYD2a from both untreated and hypertonicity-treated NRK-52E cells. The 172CâT sequence change exposed a cryptic KKXX endoplasmic reticulum retrieval signal via a premature stop codon. The truncation affected trafficking of FXYD2b and its association with Na,K-ATPase and blocked its effect on enzyme kinetics and cell growth. The data may be explained by altered splicing or selective RNA editing of FXYD2b, a supplementary process that would ensure that it was inactive even if transcribed and translated, in these cells that normally express only FXYD2a. 172CâT mutation was also identified after mutagenesis of FXYD2b by error-prone PCR coupled with a selection for cell proliferation. Furthermore, the error-prone PCR alone introduced the mutation with high frequency, implying a structural peculiarity. The data confirm truncation of FXYD2b as a potential mechanism to regulate the amount of FXYD2 at the cell surface to control activity of Na,K-ATPase and cell growth.
Assuntos
Retículo Endoplasmático/metabolismo , Sinais Direcionadores de Proteínas/fisiologia , Edição de RNA/fisiologia , RNA Mensageiro/biossíntese , ATPase Trocadora de Sódio-Potássio/biossíntese , Animais , Linhagem Celular Tumoral , Retículo Endoplasmático/genética , Isoenzimas/biossíntese , Isoenzimas/genética , Mutação , RNA Mensageiro/genética , Ratos , ATPase Trocadora de Sódio-Potássio/genéticaRESUMO
Acetate is present in lignocellulosic hydrolysates at growth inhibiting concentrations. Industrial processes based on such feedstock require strains that are tolerant of this and other inhibitors present. We investigated the effect of acetate on Saccharomyces cerevisiae and show that elevated acetate concentrations result in a decreased specific growth rate, an accumulation of cells in the G1 phase of the cell cycle, and an increased cell size. With the cytostat cultivation technology under previously derived optimal operating conditions, several acetate resistant mutants were enriched and isolated in the shortest possible time. In each case, the isolation time was less than 5 days. The independently isolated mutant strains have increased specific growth rates under conditions of high acetate concentrations, high ethanol concentrations, and high temperature. In the presence of high acetate concentrations, the isolated mutants produce ethanol at higher rates and titers than the parental strain and a commercial ethanol producing strain that has been analyzed for comparison. Whole genome microarray analysis revealed gene amplifications in each mutant. In one case, the LPP1 gene, coding for lipid phosphate phosphatase, was amplified. Two mutants contained amplified ENA1, ENA2, and ENA5 genes, which code for P-type ATPase sodium pumps. LPP1 was overexpressed on a plasmid, and the growth data at elevated acetate concentrations suggest that LPP1 likely contributes to the phenotype of acetate tolerance. A diploid cross of the two mutants with the amplified ENA genes grew faster than either individual haploid parent strain when 20 g/L acetate was supplemented to the medium, which suggests that these genes contribute to acetate tolerance in a gene dosage dependent manner.
Assuntos
Acetatos/farmacologia , Adaptação Biológica , Resistência a Medicamentos , Inibidores do Crescimento/farmacologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Animais , Etanol/metabolismo , Etanol/farmacologia , Dosagem de Genes , Perfilação da Expressão Gênica , Temperatura Alta , Análise de Sequência com Séries de Oligonucleotídeos , Fosfatidato Fosfatase/biossíntese , Fosfatidato Fosfatase/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos da radiação , Proteínas de Saccharomyces cerevisiae/biossíntese , Proteínas de Saccharomyces cerevisiae/genética , ATPase Trocadora de Sódio-Potássio/biossíntese , ATPase Trocadora de Sódio-Potássio/genética , Regulação para CimaRESUMO
The teleost gill carries out NaCl uptake in freshwater (FW) and NaCl excretion in seawater (SW). This transformation with salinity requires close regulation of ion transporter capacity and epithelial permeability. This study investigates the regulation of tight-junctional claudins during salinity acclimation in fish. We identified claudin 3- and claudin 4-like immunoreactive proteins and examined their expression and that of select ion transporters by performing Western blot in tilapia (Oreochromis mossambicus) gill during FW and SW acclimation. Transfer of FW tilapia to SW increased plasma osmolality, which was corrected after 4 days, coinciding with increased gill Na+-K+-ATPase and Na+-K+-2Cl(-) cotransporter expression. Gill claudin 3- and claudin 4-like proteins were reduced with exposure to SW. Transfer to FW increased both claudin-like proteins. Immunohistochemistry shows that claudin 3-like protein was localized deep in the FW gill filament, whereas staining was found apically in SW gill. Claudin 4-like proteins are localized predominantly in the filament outer epithelial layer, and staining appears more intense in the gill of FW versus SW fish. In addition, tilapia claudin 28a and 30 genes were characterized, and mRNA expression was found to increase during FW acclimation. These studies are the first to detect putative claudin proteins in teleosts and show their localization and regulation with salinity in gill epithelium. The data indicate that claudins may be important in permeability changes associated with salinity acclimation and possibly the formation of deeper tight junctions in FW gill. This may reduce ion permeability, which is a critical facet of FW osmoregulation.
Assuntos
Brânquias/metabolismo , Proteínas de Membrana/biossíntese , RNA Mensageiro/biossíntese , Salinidade , Tilápia/metabolismo , Sequência de Aminoácidos , Animais , Western Blotting , Água Corporal/metabolismo , Claudina-4 , Clonagem Molecular , DNA Complementar/biossíntese , DNA Complementar/genética , Água Doce , Imuno-Histoquímica , Dados de Sequência Molecular , Músculo Esquelético/metabolismo , Concentração Osmolar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Água do Mar , Simportadores de Cloreto de Sódio-Potássio/biossíntese , ATPase Trocadora de Sódio-Potássio/biossínteseRESUMO
We cloned complementary DNA (cDNA) encoding the Na(+)/K(+)-ATPase (NKA) and the osmotic stress transcription factor 1 (OSTF1) from the kidney and gill, respectively, of the black porgy, Acanthopagrus schlegeli. Black porgy NKA full-length cDNA consists of 3078 base pairs (bp) and encodes a protein of 1025 amino acids; OSTF1 partial cDNA consists of 201 bp. To investigate the osmoregulatory ability of black porgy when black porgy were transferred to freshwater (FW), we examined the expression of NKA and OSTF1 mRNA in osmoregulatory organs, i.e., gill, kidney and intestine, using quantitative polymerase chain reaction (QPCR). To determine the hypoosmotic stressor specificity of the induction of NKA and OSTF1, black porgy were exposed to 30 degrees C water temperature for 24 h. In the gill, NKA mRNA was 4.2 times higher in FW, its expression in the kidney was 5.7 times higher in 10 per thousand seawater (10 per thousand SW) than in SW. In contrast, OSTF1 mRNA in the gill was 3.7 times higher in FW than in SW. The expression of heat shock protein 90 (HSP90) mRNA occurred not only during transfer to FW, but also in high-temperature water in all tested tissues, although the mRNA levels were not significantly different. Plasma osmolality level was decreased and cortisol level was increased when the fish were transferred from SW to FW. These results suggest that NKA and OSTF1 genes play important roles in hormonal regulation in osmoregulatory organs and that these genes are specific to hypoosmotic stress, improving the hyperosmoregulatory ability of black porgy in hypoosmotic environments.
Assuntos
Aclimatação/fisiologia , Proteínas de Peixes/biossíntese , Regulação da Expressão Gênica/fisiologia , Perciformes/metabolismo , ATPase Trocadora de Sódio-Potássio/biossíntese , Fatores de Transcrição/biossíntese , Sequência de Aminoácidos , Animais , Clonagem Molecular , Proteínas de Peixes/genética , Água Doce , Hidrocortisona/metabolismo , Dados de Sequência Molecular , Especificidade de Órgãos/fisiologia , Pressão Osmótica , Perciformes/genética , Água do Mar , ATPase Trocadora de Sódio-Potássio/genética , Fatores de Transcrição/genéticaRESUMO
Cadmium (Cd) induces extensive membrane damage that contributes to the cytotoxic effect of Cd. We studied the effect of diallyl tetrasulfide (DTS) from garlic on Cd-induced changes in lipid peroxidation and membrane-bound enzymes in liver, kidney, and testis of rats. Cadmium exposure (3 mg/kg body weight, s.c) for 3 weeks induced a significant elevation in the levels of lipid peroxidation markers (thiobarbituric acid substances and lipid hydroperoxides) with a significant decrease in the activities of membrane bound ATPases (Na+/K+ ATPase, Ca2+ ATPase, Mg2+ ATPase), the indicators of membrane function in liver, kidney and testis. The oral administration of DTS (40 mg/kg body weight) along with Cd significantly decreased the level of lipid peroxidation and significantly restored the activities of membrane bound ATPases. The results of our study suggest that DTS attenuates lipid peroxidation in tissues and promotes the stability of the membrane by protecting it from Cd-induced alterations.
Assuntos
Adenosina Trifosfatases/biossíntese , Compostos Alílicos/farmacologia , Cloreto de Cádmio/toxicidade , Membrana Celular/efeitos dos fármacos , Peroxidação de Lipídeos/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Sulfetos/farmacologia , Animais , ATPase de Ca(2+) e Mg(2+)/biossíntese , ATPases Transportadoras de Cálcio/biossíntese , Membrana Celular/enzimologia , Alho , Rim/enzimologia , Peróxidos Lipídicos/metabolismo , Fígado/enzimologia , Masculino , Ratos , Ratos Wistar , ATPase Trocadora de Sódio-Potássio/biossíntese , Testículo/enzimologia , Tiobarbitúricos/metabolismoRESUMO
Angiotensin II (ANG II) plays an important role in the development of obstructive nephropathy. Here, we examined the effects of the ANG II receptor type 1 (AT1R) blockade using candesartan on long-term renal molecular and functional changes in response to partial unilateral ureteral obstruction (PUUO). Newborn rats were subjected to severe PUUO or sham operation (Sham) within the first 48 h of life. Candesartan was provided in the drinking water (10 mg.kg(-1).day(-1)) from day 21 of life until 10 wk of age. Renal blood flow (RBF) was evaluated by MRI, glomerular filtration rate (GFR) was measured using the renal clearance of (51)Cr-EDTA, and the renal expression of Na-K-ATPase and the collecting duct water channel aquaporin-2 (AQP2) was examined by immunoblotting and immunocytochemistry. At 10 wk of age, PUUO significantly reduced RBF (0.8 +/- 0.1 vs. 1.6 +/- 0.1 ml.min(-1).100 g body wt(-1); P < 0.05) and GFR (37 +/- 16 vs. 448 +/- 111 microl.min(-1).100 g body wt(-1); P < 0.05) compared with Sham. Candesartan prevented the RBF reduction (PUUO+CAN: 1.6 +/- 0.2 vs. PUUO: 0.8 +/- 0.1 ml.min(-1).100 g body wt(-1); P < 0.05) and attenuated the GFR reduction (PUUO+CAN: 265 +/- 68 vs. PUUO: 37 +/- 16 microl.min(-1).100 g body wt(-1); P < 0.05). PUUO was also associated with a significant downregulation in the expression of Na-K-ATPase (75 +/- 12 vs. 100 +/- 5%, P < 0.05) and AQP2 (52 +/- 15 vs. 100 +/- 4%, P < 0.05), which were also prevented by candesartan (Na-K-ATPase: 103 +/- 8 vs. 100 +/- 5% and AQP2: 74 +/- 13 vs. 100 +/- 4%). These findings were confirmed by immunocytochemistry. Consistent with this, candesartan treatment partly prevented the reduction in solute free water reabsorption and attenuated fractional sodium excretion in rats with PUUO. In conclusion, candesartan prevents or attenuates the reduction in RBF, GFR and dysregulation of AQP2 and Na-K-ATPase in response to congenital PUUO in rats, suggesting that AT1R blockade may protect the neonatally obstructed kidney against development of obstructive nephropathy.
Assuntos
Bloqueadores do Receptor Tipo 1 de Angiotensina II/uso terapêutico , Benzimidazóis/uso terapêutico , Falência Renal Crônica/prevenção & controle , Tetrazóis/uso terapêutico , Obstrução Ureteral/congênito , Obstrução Ureteral/tratamento farmacológico , Aldosterona/sangue , Animais , Animais Recém-Nascidos , Aquaporina 2/biossíntese , Compostos de Bifenilo , Regulação para Baixo/efeitos dos fármacos , Taxa de Filtração Glomerular/efeitos dos fármacos , Hidronefrose/prevenção & controle , NF-kappa B/biossíntese , Ratos , Circulação Renal/efeitos dos fármacos , Sódio/urina , ATPase Trocadora de Sódio-Potássio/biossínteseRESUMO
The Na,K-ATPase generates electrochemical gradients across the plasma membrane that are responsible for numerous cellular and physiological processes. The active Na,K-ATPase is minimally composed of an alpha and a beta subunit and families of isoforms for both subunits exist. Recent studies have identified a physiological role for the rat Na,K-ATPase alpha4 isoform in sperm motility. However, very little is known about the human Na,K-ATPase alpha4 isoform other than its genomic sequence and structure and its mRNA expression pattern. Here, the human alpha4 isoform of the Na,K-ATPase is cloned, expressed, and characterized. Full length cDNAs encoding the putative human alpha4 isoform of the Na,K-ATPase were identified from a number of ESTs and a protein product corresponding to this isoform was shown to be expressed from these cDNAs. The human Na,K-ATPase alpha4 isoform protein was found to be expressed in mature sperm in human testes sections and it is localized specifically to the principle piece of human sperm. In addition, the presence of the Na,K-ATPase alpha4 isoform is absent in immature testes however its expression appears coincident with sexual maturity. And finally, the human Na,K-ATPase alpha4 isoform was shown to be as sensitive to cardiac glycoside inhibition as the human Na,K-ATPase alpha1 isoform. Considering the important role of the rat Na,K-ATPase alpha4 isoform in rat sperm motility, the demonstration that the human alpha4 isoform is a sperm-specific protein localized to the flagellum suggests a role for the human Na,K-ATPase alpha4 isoform in human sperm physiology.
Assuntos
Ouabaína/farmacologia , ATPase Trocadora de Sódio-Potássio/biossíntese , ATPase Trocadora de Sódio-Potássio/genética , Espermatozoides/enzimologia , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA Complementar , Células HeLa , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/biossíntese , Isoenzimas/genética , Masculino , Dados de Sequência Molecular , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , Testículo/enzimologiaRESUMO
The present study was designed to examine whether aqueous extract of steamed root of Rehmannia glutinose (ARR) has an ameliorative effect on renal functional parameters in association with the expressions of aquaporin 2 (AQP 2), Na,K-ATPase, and heme oxygenase-1 (HO-1) in the ischemia-reperfusion induced acute renal failure (ARF) rats. Polyuria caused by down-regulation of renal AQP 2 in the ischemia-induced ARF rats was markedly restored by administration of ARR (200 mg/kg, p.o.) with restoring expression of AQP 2 in the kidney. The expressions of Na,K-ATPase alpha1 and beta1 subunits in the renal medullar and cortex of the ARF rats were also restored in the ARF rats by administration of ARR. On the other hand, administration of ARR lowered the renal expression of HO-1 up-regulated in rats with ischemia-induced ARF. The renal functional parameters including creatinine clearance, urinary sodium excretion, urinary osmolality, and solute-free reabsorption were also markedly restored in ischemia-ARF rats by administration of ARR. Taken together, these data indicate that RSR ameliorates renal defects in rats with ischemia-induced ARF.
Assuntos
Injúria Renal Aguda/prevenção & controle , Fitoterapia , Rehmannia/química , Traumatismo por Reperfusão/prevenção & controle , Injúria Renal Aguda/enzimologia , Injúria Renal Aguda/fisiopatologia , Animais , Aquaporinas/biossíntese , Western Blotting , Heme Oxigenase (Desciclizante)/biossíntese , Heme Oxigenase-1 , Testes de Função Renal , Masculino , Necrose , Extratos Vegetais/uso terapêutico , Ratos , Ratos Sprague-Dawley , Circulação Renal/fisiologia , Traumatismo por Reperfusão/enzimologia , Traumatismo por Reperfusão/fisiopatologia , ATPase Trocadora de Sódio-Potássio/biossíntese , Equilíbrio Hidroeletrolítico/efeitos dos fármacos , Equilíbrio Hidroeletrolítico/fisiologiaRESUMO
The purpose of this study was to compare the expression of BSC-1 (bumetanide-sensitive Na+-K+-2Cl- cotransporter) in kidneys of spontaneously hypertensive rats (SHR) versus Wistar-Kyoto (WKY) rats by immunoblotting and reverse transcription-polymerase chain reaction. To determine the specificity of any observed changes in BSC-1 expression, we also compared expression of the thiazide sensitive Na+-Cl- cotransporter (TSC), the type-3 Na+-H+ exchanger (NHE-3), Na+-K+-ATPase-alpha1, the inwardly rectifying K+ channel (ROMK-1), the type-1 Na+-HCO3- cotransporter (NBC-1), aquaporin-1, and aquaporin-2. Analyses were performed on outer cortex, outer medulla, and inner medulla. BSC-1 protein was detected in outer medulla and was markedly (6-fold) higher in SHR. TSC protein was detected in the cortex and was not overexpressed in SHR. Aquaporin-1 protein was detected in all three regions and was not overexpressed in SHR. Aquaporin-2 and ROMK-1 proteins were detected in all three regions, but were moderately elevated (2-fold) only in the SHR inner medulla. Na+-K+-ATPase and NHE-3 proteins were detected in all three regions. Na+-K+-ATPase-alpha1 was modestly (25%) increased in SHR outer and inner medulla, whereas NHE-3 was moderately (2-fold) increased in the SHR cortex and inner medulla. NBC-1 protein was detected only in the cortex and was higher (2-fold) in SHR. mRNA levels of BSC-1, aquaporin-2, and ROMK-1 were not elevated in SHR, indicating a post-translational mechanism of protein overexpression. High-dose furosemide increased fractional sodium excretion more in SHR than WKY (3-fold). We conclude that increased expression of BSC-1, and to a lesser extent, aquaporin-2, ROMK-1, NHE-3, and NBC-1 may contribute to the pathogenesis of hypertension in the SHR.
Assuntos
Hipertensão/metabolismo , Simportadores de Cloreto de Sódio-Potássio/biossíntese , Animais , Aquaporinas/biossíntese , Aquaporinas/genética , Diuréticos/farmacologia , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Furosemida/farmacologia , Immunoblotting , Medula Renal/metabolismo , Masculino , Canais de Potássio Corretores do Fluxo de Internalização/biossíntese , Canais de Potássio Corretores do Fluxo de Internalização/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Mensageiro/isolamento & purificação , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Simportadores de Sódio-Bicarbonato/biossíntese , Simportadores de Sódio-Bicarbonato/genética , Trocador 3 de Sódio-Hidrogênio , Trocadores de Sódio-Hidrogênio/biossíntese , Trocadores de Sódio-Hidrogênio/genética , Simportadores de Cloreto de Sódio-Potássio/genética , ATPase Trocadora de Sódio-Potássio/biossíntese , ATPase Trocadora de Sódio-Potássio/genética , Membro 1 da Família 12 de Carreador de SolutoRESUMO
Prostaglandins (PGs) play a significant role in the regulation of sodium reabsorption by the kidney, in addition to accumulating during inflammation as well as in several solid tumors. Previously, we presented evidence indicating that prostaglandin E(1) (PGE(1)), a supplement in the serum-free medium for MDCK cells, increases the activity of the Na,K-ATPase in MDCK cells, in addition to its growth stimulatory effect [J. Cell. Physiol. 151 (1992) 337]. This report defines the molecular mechanisms, and signaling pathways responsible for the increased Na,K-ATPase activity. Our results indicate that the increased activity of the Na,K-ATPase in MDCK monolayers treated with either PGE(1) or 8Bromocyclic AMP (8Br-cAMP) can be attributed to an increase in the rate of biosynthesis of the Na,K-ATPase, and an increase in the levels of Na,K-ATPase alpha and beta subunit mRNAs. As beta subunit mRNA increased to a larger extent than alpha subunit mRNA, transient transfection studies were conducted using a human beta1 promoter/luciferase construct [Nucleic Acids Res. 21 (1993) 2619]. While an 8Br-cAMP stimulation was observed (suggesting the involvement of cAMP), our results also suggest that the observed PGE(1) stimulation could be explained by the involvement of Ca(2+) as well protein kinase C (PKC). Consistent with the involvement of Ca(2+), TMB-8 (which inhibits Ca(2+) efflux from intracellular stores) inhibited the PGE(1) stimulation. Moreover, PGE(1) was observed to stimulate the translocation of PKC beta1 from the soluble to the particulate fraction. The translocation of PKC, the PGE(1) stimulation of transcription, and the PGE(1)-mediated increase in the beta subunit mRNA level were all inhibited by the PKC inhibitor Gö6989. These results can be explained by the involvement of two classes of cell surface receptors in mediating the PGE(1) stimulation, including the EP1subtype (which activates phospholipase C), as well as the EP2 subtype (which activates adenylate cyclase).
Assuntos
Alprostadil/metabolismo , Cálcio/metabolismo , AMP Cíclico/metabolismo , Ácido Gálico/análogos & derivados , ATPase Trocadora de Sódio-Potássio/biossíntese , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Alprostadil/farmacologia , Animais , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Linhagem Celular , Cães , Inibidores Enzimáticos/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Ácido Gálico/farmacologia , Túbulos Renais/citologia , Túbulos Renais/metabolismo , Proteína Quinase C/efeitos dos fármacos , Proteína Quinase C/metabolismo , Subunidades Proteicas/genética , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/genética , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Receptores de Prostaglandina E/efeitos dos fármacos , Receptores de Prostaglandina E/metabolismo , Receptores de Prostaglandina E Subtipo EP1 , Receptores de Prostaglandina E Subtipo EP2 , Sódio/metabolismo , ATPase Trocadora de Sódio-Potássio/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/genética , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética , Equilíbrio Hidroeletrolítico/efeitos dos fármacos , Equilíbrio Hidroeletrolítico/fisiologiaRESUMO
In mammals, the gastric H(+)-K(+)-ATPase (HKalpha1) mediates acid secretion in the stomach and kidneys. Like mammals, elasmobranchs also secrete acid from their stomachs, but unlike mammals they primarily use their gills for systemic acid excretion instead of their kidneys. The purpose of this study was to determine if an HKalpha1 orthologue exists in an elasmobranch (Atlantic stingray, Dasyatis sabina), to determine if it is expressed in gills and, if so, to localize its expression and determine if its expression is regulated during hypercapnia or freshwater acclimation. A polyclonal antibody made against an HKalpha1 peptide detected HKalpha1 immunoreactivity in protein isolates and tissue sections of stingray stomachs and gills. Immunohistochemistry demonstrated that HKalpha1 immunoreactivity was present in a subpopulation of epithelial cells in both organs. Double-labeling experiments in the gills showed that HKalpha1 immunoreactivity occurred in Na(+)-K(+)-ATPase-rich cells and not in V-type H(+)-ATPase-rich cells. RT-PCRs were used to deduce the primary sequence of a putative H(+)-K(+)-ATPase from the stomach of Atlantic stingrays. The 3,421-base pair cDNA includes a coding region for a 1,025-amino acid protein that is over 80% identical to HKalpha1 of mammals. RT-PCRs were then used to demonstrate that this transcript is also expressed in the gills. To our knowledge, this is the first H(+)-K(+)-ATPase sequence reported for any elasmobranch and the first full-length sequence for any fish. We also provide the first evidence for its expression in the gills of any fish and demonstrate that its expression increased during freshwater acclimation but not exposure to hypercapnia.
Assuntos
Brânquias/enzimologia , Rajidae/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Western Blotting , Clonagem Molecular , DNA Complementar/análise , DNA Complementar/genética , Regulação Enzimológica da Expressão Gênica/genética , Hipercapnia/metabolismo , Imuno-Histoquímica , Isoenzimas/biossíntese , Isoenzimas/genética , Microssomos/enzimologia , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , ATPase Trocadora de Sódio-Potássio/biossíntese , ATPase Trocadora de Sódio-Potássio/genéticaRESUMO
Post-myocardial infarction (MI) remodeling of cardiac myocytes and the myocardial interstitium results in alteration of gross ventricular geometry and ventricular dysfunction. To investigate the mechanisms of the remodeling process of the heart after large MI, the expression of various genes in viable left ventricle and infarct scar tissue were examined at 16 weeks post-MI. Steady-state expression of Na(+)-K+ ATPase alpha-1 and -2, phospholamban (PLB), alpha-myosin heavy chain (alpha-MHC), ryanodine receptor (Rya) and Ca2+ ATPase (Serca2) mRNAs were decreased in the infarct scar vs noninfarcted sham-operated controls (P < 0.05). On the other hand, Gialpha2 and beta-MHC mRNAs were upregulated (P < 0.05, respectively) in the infarct scar whereas Na(+)-K+ ATPase-beta, Na(+)-Ca2+ exchanger and Gs mRNAs were not altered vs control values. In viable left ventricle, the alpha-1 subunit of Na(+)-K+ ATPase, alpha-3, beta-isoforms, Rya, beta-MHC, Gialpha2, Gs and Na(+)-Ca2+ exchanger were significantly elevated while expression of the alpha-2 subunit of Na(+)-K+ ATPase, PLB and Serca2 were significantly decreased compared to controls. Expression of CK2alpha mRNA was elevated in noninfarcted heart (145 +/- 15%) and diminished in the infarct scar (66 +/- 13%) vs controls. Expression of beta-MHC mRNA was elevated in both viable and infarct scar tissues of experimental hearts (140 +/- 31% and 183 +/- 30% vs. controls, respectively). These results suggest that cardiac genes in the infarcted tissue and viable left ventricle following MI are differentially regulated.
Assuntos
Cicatriz/patologia , Regulação da Expressão Gênica , Ventrículos do Coração/patologia , Infarto do Miocárdio/patologia , Infarto do Miocárdio/cirurgia , Miocárdio/patologia , Animais , Northern Blotting , Caseína Quinase II , DNA Complementar/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Humanos , Hipertrofia Ventricular Esquerda , Masculino , Miocárdio/metabolismo , Cadeias Pesadas de Miosina/biossíntese , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Serina-Treonina Quinases/biossíntese , RNA/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Trocador de Sódio e Cálcio/metabolismo , ATPase Trocadora de Sódio-Potássio/biossíntese , Regulação para CimaRESUMO
We report the case of a caucasian patient with a presentation of a periodic paralysis associated with hypokalaemia disclosing Graves' disease. Major pathophysiologics hypothesis are discused in order to explain relationships between hyperthyroidism and paralysis through a disturbance of the excitability of the muscle fibres. A genetic predisposition explain the high incidence of this affection in asiatic population while it is uncommon in caucasian race. Potassium supplementation is not needed in order to correct hypokalaemia except in case of cardiac disturbances. Treatment by beta-blockers is advisable with the specific treatment of hyperthyroidism.
Assuntos
Doença de Graves/complicações , Paralisia Periódica Hipopotassêmica/etiologia , Tireotoxicose/complicações , Antagonistas Adrenérgicos beta/uso terapêutico , Adulto , Antitireóideos/uso terapêutico , Povo Asiático/genética , Carbimazol/uso terapêutico , Predisposição Genética para Doença , Doença de Graves/diagnóstico , Doença de Graves/tratamento farmacológico , Doença de Graves/fisiopatologia , Humanos , Paralisia Periódica Hipopotassêmica/etnologia , Paralisia Periódica Hipopotassêmica/genética , Paralisia Periódica Hipopotassêmica/fisiopatologia , Canais Iônicos/fisiologia , Masculino , Potenciais da Membrana , Contração Muscular/fisiologia , Propranolol/uso terapêutico , ATPase Trocadora de Sódio-Potássio/biossíntese , ATPase Trocadora de Sódio-Potássio/fisiologia , Hormônios Tireóideos/fisiologia , Tireotoxicose/tratamento farmacológico , Tireotoxicose/fisiopatologia , População Branca/genéticaRESUMO
Short-term aldosterone coordinately regulates the cell-surface expression of luminal epithelial sodium channels (ENaC) and of basolateral Na(+) pumps (Na(+), K(+)-ATPase alpha1-beta1) in aldosterone-sensitive distal nephron (ASDN) cells. To address the question of whether the subcellular localization of the Na(+), K(+)-ATPase and its regulation by aldosterone depend on subunit isoform-specific structures, we expressed the cardiotonic steroid-sensitive human alpha isoforms 1-3 by retroviral transduction in mouse collecting duct mpkCCD(c14) cells. Each of the three exogenous human isoforms could be detected by Western blotting. Immunofluorescence indicated that the exogenous alpha1 subunit to a large extent localizes to the basolateral membrane or close to it, whereas much of the alpha2 subunit remains intracellular. An ouabain-sensitive current carried by exogenous pumps could be detected in apically amphotericin B-permeabilized epithelia expressing human alpha1 and alpha2 subunits, but not the alpha3 subunit. This current displayed a higher apparent Na(+) affinity in pumps containing human alpha2 subunits (10 mM) than in pumps containing human alpha1 (33.2 mM) or endogenous (cardiotonic steroid-resistant) mouse alpha1 subunits (mean: 16.3 mM). A very low mRNA level of the Na(+), K(+)-ATPase gamma subunit (FXYD2) in mpkCCD(c14) cells suggested that this ancillary gene product is not responsible for the relatively low apparent Na(+) affinity measured for a1 subunit-containing pumps. Aldosterone increased the pump current carried by endogenous pumps and by pumps containing the human alpha1 subunit. In contrast, the current carried by pumps with a human alpha2 subunit was not stimulated by the same treatment. In summary, quantitative basolateral localization of the Na(+), K(+)-ATPase and its responsiveness to aldosterone require alpha1 subunit-specific sequences that differentiate this isoform from the alpha2 and alpha3 subunit isoforms.
Assuntos
Aldosterona/fisiologia , Rim/fisiologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Linhagem Celular , Primers do DNA , DNA Complementar/biossíntese , DNA Complementar/genética , Eletrofisiologia , Células Epiteliais/enzimologia , Imunofluorescência , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Immunoblotting , Isoenzimas/fisiologia , Rim/citologia , Túbulos Renais Coletores/enzimologia , Túbulos Renais Coletores/metabolismo , Potenciais da Membrana/fisiologia , Camundongos , Técnicas de Patch-Clamp , Reação em Cadeia da Polimerase Via Transcriptase Reversa , ATPase Trocadora de Sódio-Potássio/biossíntese , ATPase Trocadora de Sódio-Potássio/genética , Estimulação Química , Especificidade por Substrato , Transdução GenéticaRESUMO
Na+,K+-ATPase (pig alpha1,beta1) has been expressed in the methylotrophic yeast Pichia pastoris. A protease-deficient strain was used, recombinant clones were screened for multicopy genomic integrants, and protein expression, and time and temperature of methanol induction were optimized. A 3-liter culture provides 300-500 mg of membrane protein with ouabain binding capacity of 30-50 pmol mg-1. Turnover numbers of recombinant and renal Na+,K+-ATPase are similar, as are specific chymotryptic cleavages. Wild type (WT) and a D369N mutant have been analyzed by Fe2+- and ATP-Fe2+-catalyzed oxidative cleavage, described for renal Na+,K+-ATPase. Cleavage of the D369N mutant provides strong evidence for two Fe2+ sites: site 1 composed of residues in P and A cytoplasmic domains, and site 2 near trans-membrane segments M3/M1. The D369N mutation suppresses cleavages at site 1, which appears to be a normal Mg2+ site in E2 conformations. The results suggest a possible role of the charge of Asp369 on the E1 <--> E2 conformational equilibrium. 5'-Adenylyl-beta,gamma-imidodi-phosphate(AMP-PNP)-Fe2+-catalyzed cleavage of the D369N mutant produces fragments in P (712VNDS) and N (near 440VAGDA) domains, described for WT, but only at high AMP-PNP-Fe2+ concentrations, and a new fragment in the P domain (near 367CSDKTGT) resulting from cleavage. Thus, the mutation distorts the active site. A molecular dynamic simulation of ATP-Mg2+ binding to WT and D351N structures of Ca2+-ATPase (analogous to Asp369 of Na+,K+-ATPase) supplies possible explanations for the new cleavage and for a high ATP affinity, which was observed previously for the mutant. The Asn351 structure with bound ATP-Mg2+ may resemble the transition state of the WT poised for phosphorylation.