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1.
Sci Total Environ ; 753: 141403, 2021 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-32889313

RESUMO

Chronic or repeated exposure to environmental contaminants may result in allostatic overload, a physiological situation in which the costs of coping affect long-term survival and reproductive output. Continuous measurements in Otra, the largest river in southern Norway, show the occurrence of repeated 24-48 h episodes of acidification. This work investigates the impact of repeated short acidification episodes on a unique land-locked population of normally anadromous Atlantic salmon ("Bleke"). This was done by recording physiological measures of stress and allostatic load in fish exposed for 7 days to continuous or repeated episodes of simulated environmental acidification or untreated Otra water (controls). A standardized acute stress test was performed after these different exposure regimes, with brain and blood samples taken before (baseline) or after the stress test. Treatment effects on stress coping ability were assessed by neuroendocrine indicators, including telencephalic serotonergic activity and plasma cortisol. Continuous exposure to acidification resulted in increased baseline plasma Cl- and Na+ and elevated baseline plasma cortisol compared to episodic exposed fish. However, both episodic and continuous acidification resulted in similar increase in gill Al, indicating similar impact on gill permeability of these two exposures. This suggests a lower impact on the electrolyte homeostasis in episodic compared to continuous exposure and that this effect is not directly related to the effects of Al complexes binding to the gills. Furthermore, there were no treatment induced differences on stress coping ability, suggesting that episodic exposure to the sublethal concentrations of Al in pH 5.5 in the present study do not result in higher allostatic load than in control or continuous exposed Bleke.


Assuntos
Salmo salar , Animais , Brânquias/metabolismo , Homeostase , Concentração de Íons de Hidrogênio , Noruega , ATPase Trocadora de Sódio-Potássio/metabolismo
2.
An Acad Bras Cienc ; 92(4): e20201080, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33146236

RESUMO

Cardiac glycosides have been found to have an anti-viral effect. This was noted in the past during various epidemics including MERS and SARS. It is due to their inhibitory effect on the Na, K-ATPase membrane pump. Furthermore, they exhibit anti-inflammatory properties. These preclinical observations may prove useful in further clinical utility of these well-known compounds in the current COVID-19 pandemic.


Assuntos
Glicosídeos Cardíacos , Infecções por Coronavirus , Pandemias , Pneumonia Viral , Betacoronavirus , Glicosídeos Cardíacos/farmacologia , Humanos , ATPase Trocadora de Sódio-Potássio/metabolismo
3.
Zhonghua Yi Xue Za Zhi ; 100(38): 3014-3017, 2020 Oct 20.
Artigo em Chinês | MEDLINE | ID: mdl-33086454

RESUMO

Objective: To investigate the effect of Na(+)/K(+)-ATPase inhibitor ouabain on the proliferation and division of liver cancer HepG2 cells, and to explore the anticancer mechanism. Methods: HepG2 cells were exposed with different concentrations of ouabain (0.1, 1, 10 µmol/L) for 24 h, the proliferation ability was appraised using CCK-8, and the HepG2 cells was as a control group. The status of chromosome separation was detected with cell immunofluorescence (ICC) coupled to confocal microscope. The expression levels of AURKA, mTOR, p-mTOR, ERK and p-ERK protein were analyzed using western blot. Results: After treating with 0.1, 1 and 10 µmol/L of ouabain for 24 h, the inhibitory rate of cells were (23.5±4.57)%, (49.80±5.32)%, and (72.10±5.62)%, respectively. Ouabain could significantly inhibit the proliferation of HepG2, and presented in a dose-dependent manner(F=32.8, P<0.05). The ICC results showed that the chromosome separation disorders occurred in HepG2 cells treated with 1 µmol/L for 24 h, and the spindle diameter of HepG2 cells with ouabain treatment was decreased significantly compared with the control group(t=9.58, P<0.05). The results of western blot showed that the expression levels of AURKA, p-mTOR and p-ERK expressions in HepG2 cells treated with 1 µmol/L of ouabain were significantly decreased compared with the control group(F=16.26, 8.32, 33.59, P<0.05). Ouabain inhibited the growth of hepatocellular carcinoma cells in nude mice(F=370.20, P<0.05). Conclusion: Ouabain can induce chromosome division disorder and inhibit the proliferation in liver cancer HepG2 cells by inhibiting AURKA signaling pathway.


Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Animais , Camundongos , Camundongos Nus , Ouabaína/farmacologia , Transdução de Sinais , ATPase Trocadora de Sódio-Potássio/metabolismo
4.
Chemosphere ; 261: 128051, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33113650

RESUMO

The potential toxicity of copper nanoparticles (CuNPs) to early stages of fishes is not fully understood, and little is known about their effects on ionocytes and associated functions. This study used zebrafish embryos as a model to investigate the toxic effects of CuNPs on two subtypes of ionocytes. Zebrafish embryos were exposed to 0.1, 1, and 3 mg L-1 CuNPs for 96 h. After exposure, whole-body Na+ and Ca2+ contents were significantly reduced at ≥0.1 mg L-1, while the K+ content had decreased at ≥1 mg L-1. H+ and NH4+ excretion by the skin significantly decreased at ≥1 mg L-1. The number of living ionocytes labeled with rhodamine-123 had significantly decreased with ≥0.1 mg L-1 CuNPs. The ionocyte subtypes of H+-ATPase-rich (HR) and Na+/K+-ATPase-rich (NaR) cells were labeled by immunostaining and had decreased with ≥1 mg L-1. Shrinkage of the apical opening of ionocytes was revealed by scanning electronic microscopy. Functional impairment was also reflected by changes in gene expressions, including ion transporters/channels and Ca2+-regulatory hormones. This study shows that CuNP exposure can impair two subtypes of ionocytes and their associated functions, including Na+/Ca2+ uptake and H+/NH4+ excretion in zebrafish embryos.


Assuntos
Amônia/metabolismo , Cobre/toxicidade , Embrião não Mamífero/efeitos dos fármacos , Nanopartículas/toxicidade , Poluentes Químicos da Água/toxicidade , Peixe-Zebra/metabolismo , Ácidos/metabolismo , Animais , Transporte Biológico , Cálcio/metabolismo , Cobre/metabolismo , Embrião não Mamífero/metabolismo , Canais Iônicos/metabolismo , Íons/metabolismo , Nanopartículas/metabolismo , Pele/metabolismo , Sódio/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Poluentes Químicos da Água/metabolismo , Proteínas de Peixe-Zebra/metabolismo
5.
Signal Transduct Target Ther ; 5(1): 218, 2020 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-33011739

Assuntos
Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Glicosídeos Cardíacos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Animais , Antivirais/química , Betacoronavirus/patogenicidade , Produtos Biológicos/química , Produtos Biológicos/farmacologia , Bufanolídeos/química , Bufanolídeos/farmacologia , Glicosídeos Cardíacos/química , Sobrevivência Celular/efeitos dos fármacos , Chlorocebus aethiops , Cloroquina/química , Cloroquina/farmacologia , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/virologia , Digoxina/química , Digoxina/farmacologia , Ensaios de Triagem em Larga Escala , Interações Hospedeiro-Patógeno/genética , Humanos , Janus Quinases/antagonistas & inibidores , Janus Quinases/genética , Janus Quinases/metabolismo , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fator 2 Relacionado a NF-E2/antagonistas & inibidores , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , NF-kappa B/antagonistas & inibidores , NF-kappa B/genética , NF-kappa B/metabolismo , Pandemias , Fenantrenos/química , Fenantrenos/farmacologia , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/virologia , Transdução de Sinais , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , ATPase Trocadora de Sódio-Potássio/genética , ATPase Trocadora de Sódio-Potássio/metabolismo , Células Vero , Replicação Viral/efeitos dos fármacos
6.
Yakugaku Zasshi ; 140(9): 1095-1099, 2020.
Artigo em Japonês | MEDLINE | ID: mdl-32879241

RESUMO

The lens of the eye is an avascular and anuclear tissue that serves to focus objects on the retina. Cataract is opacity within the clear lens that changes the transparency and refractive index of the lens causing significant visual impairments. These impairments can severely restrict the ability to carry out daily activities. Cataracts is common among elderly person occurring in more than 80% of patients aged 80 or older. Notably, we have recently identified key compounds that are effective against cataract formation. Presbyopia is also an ocular disease that typically develops in people over the age of 45 while affecting almost 100% of people over the age of 65. Recent research suggests that age-related changes in hydrostatic pressure of the lens controlled by Na/K ATPase contribute to the development of presbyopia. In the lens, Na/K ATPase has been shown to be regulated by transient receptor potential cation channels, vanilloid 1 (TRPV1) and 4, thus suggesting the potential role of TRPV1 and TRPV4 in the development of presbyopia. This review article summarizes data obtained from our laboratory with my colleagues highlighting the critical role of aquaporin 0 (AQP0) in maintaining a healthy lens redox environment, key molecules that delay the onset of cataract in vivo, as well as potential mechanisms of lens hydrostatic pressure control that may be associated with presbyopia.


Assuntos
Aquaporinas/fisiologia , Catarata/tratamento farmacológico , Catarata/etiologia , Desenvolvimento de Medicamentos , Proteínas do Olho/fisiologia , Pressão Hidrostática , Cristalino/metabolismo , Oxirredução , Presbiopia/etiologia , ATPase Trocadora de Sódio-Potássio/metabolismo , ATPase Trocadora de Sódio-Potássio/fisiologia , Canais de Cátion TRPV/fisiologia
7.
J Toxicol Sci ; 45(9): 549-558, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32879254

RESUMO

Trimethyltin chloride (TMT) is a stabilizer by-product in the process of manufacturing plastic, which is a kind of very strong toxic substance, and has acute, cumulative and chronic toxicity. TMT may cause bradycardia in patients with occupational poisoning, the mechanism of which has not been reported. This study explored the mechanism of TMT resulting in bradycardia of C57BL/6 mice. TMT was administered to mice to measure heart rate, serum succinate dehydrogenase (SDH) level, and myocardial Na+/K+-ATPase activity and expression. The effects of TMT on myocardial apoptosis were observed by changing the expressions of caspase-3, Bax and Bcl-2 in myocardium. It was found that the heart rate and SDH activity in serum of mice gradually decreased with the increase of TMT dose compared with the control group. The activity and the expression of Na+/K+-ATPase in the heart tissue of mice exposed to TMT was measured and gradually decreased with the increase of dose and time. We measured the expression of Bcl-2, Bax, caspase-3 and cleaved caspase-3 in the heart tissues of TMT exposed mice and found that the expressions of Bax, caspase-3 and cleaved caspase-3 increased and the expressions of Bcl-2 decreased in the heart tissues of the TMT-exposed mice at different doses. With the extension of TMT exposure time, the expression of Bax and caspase-3 increased and the expression of Bcl-2 decreased in the heart tissues of TMT exposed mice. Our findings suggest the mechanisms of TMT resulting in bradycardia may be associated with the inhibited activity and decreased content of Na+/K+-ATPase, thus further leading to the changes of Bcl-2, Bax, caspase-3 and cleaved caspase-3 in the mice's ventricular tissues.


Assuntos
Apoptose/efeitos dos fármacos , Bradicardia/etiologia , Miocárdio/metabolismo , Miocárdio/patologia , ATPase Trocadora de Sódio-Potássio/genética , ATPase Trocadora de Sódio-Potássio/metabolismo , Compostos de Trimetilestanho/toxicidade , Animais , Apoptose/genética , Bradicardia/genética , Caspase 3/genética , Caspase 3/metabolismo , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/genética , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo
8.
PLoS One ; 15(8): e0237328, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32790707

RESUMO

α-Synuclein (αSyn) fibrils spread from one neuronal cell to another. This prion-like phenomenon is believed to contribute to the progression of the pathology in Parkinson's disease and other synucleinopathies. The binding of αSyn fibrils originating from affected cells to the plasma membrane of naïve cells is key in their prion-like propagation propensity. To interfere with this process, we designed polypeptides derived from proteins we previously showed to interact with αSyn fibrils, namely the molecular chaperone Hsc70 and the sodium/potassium pump NaK-ATPase and assessed their capacity to bind αSyn fibrils and/or interfere with their take-up by cells of neuronal origin. We demonstrate here that polypeptides that coat αSyn fibrils surfaces in such a way that they are changed affect αSyn fibrils binding to the plasma membrane components and/or their take-up by cells. Altogether our observations suggest that the rationale design of αSyn fibrils polypeptide binders that interfere with their propagation between neuronal cells holds therapeutic potential.


Assuntos
Neurônios/efeitos dos fármacos , Peptídeos/farmacologia , Agregação Patológica de Proteínas/tratamento farmacológico , alfa-Sinucleína/metabolismo , Sequência de Aminoácidos , Amiloide/antagonistas & inibidores , Amiloide/metabolismo , Animais , Linhagem Celular , Proteínas de Choque Térmico HSC70/química , Proteínas de Choque Térmico HSC70/metabolismo , Proteínas de Choque Térmico HSC70/farmacologia , Humanos , Camundongos , Modelos Moleculares , Neurônios/metabolismo , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/metabolismo , Peptídeos/química , Príons/antagonistas & inibidores , Príons/metabolismo , Agregados Proteicos/efeitos dos fármacos , Agregação Patológica de Proteínas/metabolismo , ATPase Trocadora de Sódio-Potássio/química , ATPase Trocadora de Sódio-Potássio/metabolismo , ATPase Trocadora de Sódio-Potássio/farmacologia
9.
Aquat Toxicol ; 226: 105561, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32688145

RESUMO

In the aquatic environment, metals are present as mixtures, therefore studies on mixture toxicity are crucial to thoroughly understand their toxic effects on aquatic organisms. Common carp (Cyprinus carpio) were used to assess the effects of short-term Cu(II) and Cd(II) mixtures, using a fixed concentration of one of the metals, representing 25 % of its individual 96h-LC50 (concentration lethal for 50 % of the population) combined with a variable concentration of the other metal corresponding to 10, 25 or 50 % of its 96h-LC50, and vice versa. Our results showed a fast Cu and Cd bioaccumulation, with the percentage of increase in the order gill > liver > carcass. An inhibitory effect of Cu on Cd uptake was observed; higher Cu concentrations at fixed Cd levels resulted in a decreased accumulation of Cd. The presence of the two metal ions resulted in losses of total Na, K and Ca. Fish tried to compensate for the Na loss through the induction of the genes coding for Na+/K+-ATPase and H+-ATPase. Additionally, a counterintuitive induction of the gene encoding the high affinity copper transporter (CTR1) occurred, while a downregulation was expected to prevent further metal ion uptake. An induction of defensive mechanisms, both metal ion binding protein and anti-oxidant defences, was observed. Despite the metal accumulation and electrolyte loss, the low mortality suggest that common carp is able to cope with these metal levels, at least during a one-week exposure.


Assuntos
Bioacumulação/efeitos dos fármacos , Cádmio/toxicidade , Carpas/metabolismo , Cobre/toxicidade , Homeostase/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Animais , Cádmio/metabolismo , Carpas/genética , Cobre/metabolismo , Transportador de Cobre 1/genética , Transportador de Cobre 1/metabolismo , Eletrólitos/metabolismo , Brânquias/efeitos dos fármacos , Brânquias/metabolismo , Transporte de Íons , Dose Letal Mediana , Potássio/metabolismo , Sódio/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Poluentes Químicos da Água/metabolismo
10.
Am J Physiol Regul Integr Comp Physiol ; 319(2): R148-R155, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32663032

RESUMO

Naked mole-rats (NMRs) are mammalian champions of hypoxia tolerance that enter metabolic suppression to survive in low oxygen environments. Common physiological mechanisms used by animals to suppress metabolic rate include downregulating energy metabolism (ATP supply) as well as ion pumps (primary cellular ATP consumers). A recent goldfish study demonstrated that remodeling of membrane lipids may mediate these responses, but it is unknown if NMR employs the same strategies; therefore, we aimed to test the hypotheses that these fossorial mammals 1) downregulate the activity of key enzymes of glycolysis, tricarboxylic acid (TCA) cycle, and ß-oxidation, 2) inhibit sodium-potassium-ATPase, and 3) alter membrane lipids in response to chronic hypoxia. We found that NMRs exposed to 11% oxygen for 4 wk had a lower metabolic rate by 34%. This suppression occurs concurrently with tissue-specific 25-99% decreases in metabolic enzymes activities, a 77% decrease in brain sodium/potassium-ATPase activity, and widespread changes in membrane cholesterol abundance. By reducing glycolytic and ß-oxidation fluxes, NMRs decrease the supply of acetyl-CoA to the TCA cycle. By contrast, there is a 94% upregulation of citrate synthase in the heart, possibly to support circulation and thus oxygen supply to other organs. Taken together, these responses may reflect a coordinated physiological response to hypoxia, but a clear functional link between changes in membrane composition and enzyme activities could not be established. Nevertheless, this is the first demonstration that hypometabolic NMRs alter the lipid composition of their membranes in response to chronic in vivo exposure to hypoxia.


Assuntos
Membrana Celular/metabolismo , Colesterol/metabolismo , Metabolismo Energético/fisiologia , Hipóxia/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Encéfalo/metabolismo , Ratos-Toupeira , Oxigênio/metabolismo
11.
Biochem Pharmacol ; 180: 114122, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32592721

RESUMO

An unprecedented biological function of natural cardenolides independent of their membrane target Na+/K+-ATPase is disclosed. Previously, we reported that cardenolides impart anti-transmissible gastroenteritis coronavirus (anti-TGEV) activity through the targeting of Na+/K+-ATPase and its associated PI3K_PDK1_RSK2 signaling. Swine testis cells with Na+/K+-ATPase α1 knocked down exhibited decreased susceptibility to TGEV infectivity and attenuated PI3K_PDK1_RSK2 signaling. Herein, we further explored a Na+/K+-ATPase-independent signaling axis induced by natural cardenolides that also afforded significant anti-coronaviral activity for porcine TGEV and human HCoV-OC43. Using pharmacological inhibition and gene silencing techniques, we found that this anti-TGEV or anti-HCoV-OC43 activity was caused by JAK1 proteolysis and mediated through upstream activation of Ndfip1/2 and its effector NEDD4. This study provides novel insights into the pharmacological effects of natural cardenolides, and is expected to inform their future development as antiviral agents.


Assuntos
Antivirais/farmacologia , Cardenolídeos/farmacologia , Coronavirus Humano OC43/efeitos dos fármacos , Janus Quinase 1/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Vírus da Gastroenterite Transmissível/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Animais , Proteínas de Transporte/metabolismo , Linhagem Celular , Regulação para Baixo/efeitos dos fármacos , Feminino , Técnicas de Silenciamento de Genes , Humanos , Leupeptinas , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Ubiquitina-Proteína Ligases Nedd4/metabolismo , Ouabaína/farmacologia , Fosforilação , Inibidores de Proteases/farmacologia , Proteólise , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Suínos
12.
Am J Chin Med ; 48(3): 651-678, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32349518

RESUMO

Cinobufagin is a Na+/K+-ATPase (NKA) inhibitor with excellent anticancer effects to prolong the survival of patients. The purpose of the present study was to clarify the underlying mechanism of the anticancer effects of cinobufagin using overexpression or inhibition of aurora kinase A (AURKA) signaling. First, high expression of Na+/K+-ATPase alpha 1 subunit (ATP1A1) and AURAK resulted in increased malignant transformation in hepatocellular carcinoma (HCC) patients using the cancer genome atlas (TCGA) data and tissue samples. After treatment with cinobufagin, we successfully screened 202, 249, and 335 changing expression proteins in Huh-7 cells under normal, overexpression, and inhibition of AURKA using tandem mass tags (TMT)-labeled quantitative proteomics coupled to 2D liquid chromatography-tandem mass spectrometry (LC-MS/MS). Bioinformatics analysis revealed that these molecules were closely associated with chromosome segregation, DNA damage, and regulation of translation processes. We further confirmed that cinobufagin induced DNA damage and chromosome segregation disorders and suppresses translational processing in oncogenes by decreasing the expression of AURKA, mechanistic target of rapamycin kinase (mTOR), p-mTOR, p-extracellular regulated protein kinases (ERK), eukaryotic translation initiation factor 4E (eIF4E), and p-eIF4E, while increasing the expression of p-eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1) (S65, T37, T46, T45) and increasing the interaction between eIF4 and 4E-BP1. Our results suggested that cinobufagin performed an antitumor effects in liver cancer cells by inhibiting the AURKA-mTOR-eIF4E axis.


Assuntos
Antineoplásicos Fitogênicos , Aurora Quinase A/metabolismo , Bufanolídeos/farmacologia , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Fatores de Transcrição/metabolismo , Segregação de Cromossomos/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Oncogenes/genética , ATPase Trocadora de Sódio-Potássio/metabolismo , Células Tumorais Cultivadas
13.
Bull Environ Contam Toxicol ; 104(6): 755-762, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32394051

RESUMO

We examined copper accumulation in the hemolymph, gills and hepatopancreas, and hemolymph osmolality, Na+ and Cl- concentrations, together with gill Na+/K+-ATPase and carbonic anhydrase activities, after dietary copper delivery (0, 100 or 500 Cu µg g-1) for 12 days in a fiddler crab, Minuca rapax. In contaminated crabs, copper concentration decreased in the hemolymph and hepatopancreas, but increased in the gills. Hemolymph osmolality and gill Na+/K+-ATPase activity increased while hemolymph [Na+] and [Cl-] and gill carbonic anhydrase activity decreased. Excretion likely accounts for the decreased hemolymph and hepatopancreas copper titers. Dietary copper clearly affected osmoregulatory ability and hemolymph Na+ and Cl- regulation in M. rapax. Gill copper accumulation decreased carbonic anhydrase activity, suggesting that dietary copper affects acid-base balance. Elevated gill Na+/K+-ATPase activity appears to compensate for the ion-regulatory disturbance. These effects of dietary copper illustrate likely impacts on semi-terrestrial species that feed on metal-contaminated sediments.


Assuntos
Bioacumulação , Braquiúros/efeitos dos fármacos , Cobre/metabolismo , Osmorregulação/efeitos dos fármacos , Poluentes Químicos da Água/metabolismo , Animais , Braquiúros/metabolismo , Cobre/análise , Exposição Dietética , Brânquias/efeitos dos fármacos , Brânquias/metabolismo , Hemolinfa/metabolismo , Hepatopâncreas/metabolismo , Transporte de Íons , Concentração Osmolar , Alimentos Marinhos , ATPase Trocadora de Sódio-Potássio/metabolismo , Poluentes Químicos da Água/análise
14.
Invest Ophthalmol Vis Sci ; 61(5): 1, 2020 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-32392309

RESUMO

Purpose: Mutations in the RS1 gene, which encodes retinoschisin, cause X-linked juvenile retinoschisis, a retinal dystrophy in males. Retinoschisin specifically interacts with the retinal sodium-potassium adenosine triphosphatase (Na/K-ATPase), a transmembrane ion pump. Na/K-ATPases also bind cardiac glycosides, which control the activity of the pump and have been linked to disturbances in retinal homeostasis. In this study, we investigated the crosstalk between retinoschisin and cardiac glycosides at the retinal Na/K-ATPase and the consequences of this interplay on retinal integrity. Methods: The effect of cardiac glycosides (ouabain and digoxin) on the binding of retinoschisin to the retinal Na/K-ATPase was investigated via western blot and immunocytochemistry. Also, the influence of retinoschisin on the binding of cardiac glycosides was analyzed via enzymatic assays, which quantified cardiac glycoside-sensitive Na/K-ATPase pump activity. Moreover, retinoschisin-dependent binding of tritium-labeled ouabain to the Na/K-ATPase was determined. Finally, a reciprocal effect of retinoschisin and cardiac glycosides on Na/K-ATPase localization and photoreceptor degeneration was addressed using immunohistochemistry in retinoschisin-deficient murine retinal explants. Results: Cardiac glycosides displaced retinoschisin from the retinal Na/K-ATPase; however, retinoschisin did not affect cardiac glycoside binding. Notably, cardiac glycosides reduced the capacity of retinoschisin to regulate Na/K-ATPase localization and to protect against photoreceptor degeneration. Conclusions: Our findings reveal opposing effects of retinoschisin and cardiac glycosides on retinal Na/K-ATPase binding and on retinal integrity, suggesting that a fine-tuned interplay between both components is required to maintain retinal homeostasis. This observation provides new insight into the mechanisms underlying the pathological effects of cardiac glycoside treatment on retinal integrity.


Assuntos
Digoxina/metabolismo , Proteínas do Olho/metabolismo , Ouabaína/metabolismo , Retinosquise/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Células Cultivadas , Modelos Animais de Doenças , Humanos , Imuno-Histoquímica , Camundongos Endogâmicos C57BL , Ligação Proteica , Transdução de Sinais
15.
Arch Biochem Biophys ; 688: 108403, 2020 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-32418893

RESUMO

Myopia is a main cause of preventable or treatable visual impairment, it has become a major public health issue due to its increasingly high prevalence worldwide. Currently, it is confirmed that the development of myopia is associated with the disorders of accommodation. As a dominant factor for accommodation, ciliary muscle contraction/relaxation can regulate the physiological state of the lens and play a crucial role in the development of myopia. To investigate the relationship between myopia and ciliary muscle, the guinea pigs were randomly divided into a normal control (NC) group and a negative lens-induced myopia (LIM) group, and the animals in each group were further randomly assigned into 2-week (n = 18) and 4-week (n = 21) subgroups in accordance with the duration of myopic induction of 2 and 4 weeks, respectively. In the present study, right eyes of the animals in LIM group were covered with -6.0 D lenses to induce myopia. Next, we performed the haematoxylin and eosin (H&E) staining to observe the pathological change of ciliary muscle, determined the contents of adenosine triphosphate (ATP) and lactate acid (LA), and measured the Na+/K+-ATPase expression and activity in ciliary muscles in both NC and LIM groups. Moreover, we also analyzed the potassium ion (K+) flux in ciliary muscles from 4-week NC and LIM guinea pigs. As a result, we found that the arrangements of ciliary muscles in LIM guinea pigs were broken, dissolved or disorganized; the content of ATP decreased, whereas the content of LA increased in ciliary muscles from LIM guinea pigs. Monitoring of K+ flux in ciliary muscles from LIM guinea pigs demonstrated myopia-triggered K+ influx. Moreover, we also noted a decreased expression of Na+/K+-ATPase (Atp1a1) at both mRNA and protein levels and reduced activity in ciliary muscles from LIM guinea pigs. Overall, our results will facilitate the understanding of the mechanism associated with inhibitory Na+/K+-ATPase in lens-induced myopia and which consequently lead to the disorder of microenvironment within ciliary muscles from LIM guinea pigs, paving the way for a promising adjuvant approach in treating myopia in clinical practice.


Assuntos
Olho/metabolismo , Homeostase/fisiologia , Músculo Liso/metabolismo , Miopia/metabolismo , Potássio/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Olho/patologia , Cobaias , Ácido Láctico/metabolismo , Masculino , Músculo Liso/patologia , Miopia/patologia , RNA Mensageiro/metabolismo , ATPase Trocadora de Sódio-Potássio/genética , ATPase Trocadora de Sódio-Potássio/metabolismo
16.
Nat Commun ; 11(1): 2248, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32382046

RESUMO

During high-frequency network activities, fast-spiking, parvalbumin-expressing basket cells (PV+-BCs) generate barrages of fast synaptic inhibition to control the probability and precise timing of action potential (AP) initiation in principal neurons. Here we describe a subcellular specialization that contributes to the high speed of synaptic inhibition mediated by PV+-BCs. Mapping of hyperpolarization-activated cyclic nucleotide-gated (HCN) channel distribution in rat hippocampal PV+-BCs with subcellular patch-clamp methods revealed that functional HCN channels are exclusively expressed in axons and completely absent from somata and dendrites. HCN channels not only enhance AP initiation during sustained high-frequency firing but also speed up the propagation of AP trains in PV+-BC axons by dynamically opposing the hyperpolarization produced by Na+-K+ ATPases. Since axonal AP signaling determines the timing of synaptic communication, the axon-specific expression of HCN channels represents a specialization for PV+-BCs to operate at high speed.


Assuntos
Neurônios GABAérgicos/metabolismo , Interneurônios/metabolismo , Potenciais de Ação/fisiologia , Animais , Axônios , Dendritos/metabolismo , Masculino , Ratos , Ratos Wistar , Transdução de Sinais/fisiologia , ATPase Trocadora de Sódio-Potássio/metabolismo
17.
Nat Commun ; 11(1): 2137, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32358514

RESUMO

The light-driven sodium-pumping rhodopsin KR2 from Krokinobacter eikastus is the only non-proton cation active transporter with demonstrated potential for optogenetics. However, the existing structural data on KR2 correspond exclusively to its ground state, and show no sodium inside the protein, which hampers the understanding of sodium-pumping mechanism. Here we present crystal structure of the O-intermediate of the physiologically relevant pentameric form of KR2 at the resolution of 2.1 Å, revealing a sodium ion near the retinal Schiff base, coordinated by N112 and D116 of the characteristic NDQ triad. We also obtained crystal structures of D116N and H30A variants, conducted metadynamics simulations and measured pumping activities of putative pathway mutants to demonstrate that sodium release likely proceeds alongside Q78 towards the structural sodium ion bound between KR2 protomers. Our findings highlight the importance of pentameric assembly for sodium pump function, and may be used for rational engineering of enhanced optogenetic tools.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Flavobacteriaceae/metabolismo , ATPase Trocadora de Sódio-Potássio/química , ATPase Trocadora de Sódio-Potássio/metabolismo , Cristalografia por Raios X , Escherichia coli/metabolismo , Simulação de Dinâmica Molecular , Dobramento de Proteína , Rodopsina/química , Rodopsina/metabolismo , Sódio/metabolismo , Difração de Raios X
18.
J Therm Biol ; 88: 102526, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32126001

RESUMO

Sea bream (Sparus aurata Linneaus) was acclimated to three salinity concentrations, viz. 5 (LSW), 38 (SW) and 55psµ (HSW) and three water temperatures regimes (12, 19 and 26 °C) for five weeks. Osmoregulatory capacity parameters (plasma osmolality, sodium, chloride, cortisol, and branchial and renal Na+,K+-ATPase activities) were also assessed. Salinity and temperature affected all of the parameters tested. Our results indicate that environmental temperature modulates capacity in sea bream, independent of environmental salinity, and set points of plasma osmolality and ion concentrations depend on both ambient salinity and temperature. Acclimation to extreme salinity resulted in stress, indicated by elevated basal plasma cortisol levels. Response to salinity was affected by ambient temperature. A comparison between branchial and renal Na+,K+-ATPase activities appears instrumental in explaining salinity and temperature responses. Sea bream regulate branchial enzyme copy numbers (Vmax) in hyperosmotic media (SW and HSW) to deal with ambient temperature effects on activity; combinations of high temperatures and salinity may exceed the adaptive capacity of sea bream. Salinity compromises the branchial enzyme capacity (compared to basal activity at a set salinity) when temperature is elevated and the scope for temperature adaptation becomes smaller at increasing salinity. Renal Na+,K+-ATPase capacity appears fixed and activity appears to be determined by temperature.


Assuntos
Osmorregulação/fisiologia , Salinidade , Dourada/fisiologia , Temperatura , Animais , Cloretos , Brânquias/metabolismo , Hidrocortisona/sangue , Rim/metabolismo , Dourada/sangue , Sódio , ATPase Trocadora de Sódio-Potássio/metabolismo , Água
19.
Biochim Biophys Acta Bioenerg ; 1861(7): 148190, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32194062

RESUMO

Krokinobacter rhodopsin 2 (KR2) was discovered as the first light-driven sodium pumping rhodopsin (NaR) in 2013, which contains unique amino acid residues on C-helix (N112, D116, and Q123), referred to as an NDQ motif. Based on the recent X-ray crystal structures of KR2, the sodium transport pathway has been investigated by various methods. However, due to complicated structural information around the protonated Schiff base (PRSB) region in the dark state and lack of structural information in the intermediates with sodium bound in KR2, detailed sodium pump mechanism is still unclear. Here we applied comprehensive low-temperature light-induced difference FTIR spectroscopy on isotopically labeled KR2 WT and site-directed mutant proteins (N112A, D116E, R109A, and R109K). We assigned the N-D stretching vibration of the PRSB at 2095 cm-1 and elucidate the hydrogen bonding interaction with D116 (a counter ion for the PRSB). We also assigned strongly hydrogen-bonded water (2333 cm-1) near R109 and D251, and found that presence of a positive charge at the position of R109 is prerequisite for the pumping function of KR2.


Assuntos
Luz , Retinaldeído/química , Rodopsina/química , Bases de Schiff/química , ATPase Trocadora de Sódio-Potássio/metabolismo , Cristalografia por Raios X , Flavobacteriaceae/metabolismo , Ligação de Hidrogênio , Isomerismo , Modelos Moleculares , Mutagênese Sítio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Isótopos de Nitrogênio , Espectroscopia de Infravermelho com Transformada de Fourier , Vibração , Água/química
20.
Am J Physiol Gastrointest Liver Physiol ; 318(5): G931-G945, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32174134

RESUMO

Helicobacter pylori infection always induces gastritis, which may progress to ulcer disease or cancer. The mechanisms underlying mucosal injury by the bacteria are incompletely understood. Here, we identify a novel pathway for H. pylori-induced gastric injury, the impairment of maturation of the essential transport enzyme and cell adhesion molecule, Na-K-ATPase. Na-K-ATPase comprises α- and ß-subunits that assemble in the endoplasmic reticulum (ER) before trafficking to the plasma membrane. Attachment of H. pylori to gastric epithelial cells increased Na-K-ATPase ubiquitylation, decreased its surface and total levels, and impaired ion balance. H. pylori did not alter degradation of plasmalemma-resident Na-K-ATPase subunits or their mRNA levels. Infection decreased association of α- and ß-subunits with ER chaperone BiP and impaired assembly of α/ß-heterodimers, as was revealed by quantitative mass spectrometry and immunoblotting of immunoprecipitated complexes. The total level of BiP was not altered, and the decrease in interaction with BiP was not observed for other BiP client proteins. The H. pylori-induced decrease in Na-K-ATPase was prevented by BiP overexpression, stopping protein synthesis, or inhibiting proteasomal, but not lysosomal, protein degradation. The results indicate that H. pylori impairs chaperone-assisted maturation of newly made Na-K-ATPase subunits in the ER independently of a generalized ER stress and induces their ubiquitylation and proteasomal degradation. The decrease in Na-K-ATPase levels is also seen in vivo in the stomachs of gerbils and chronically infected children. Further understanding of H. pylori-induced Na-K-ATPase degradation will provide insights for protection against advanced disease.NEW & NOTEWORTHY This work provides evidence that Helicobacter pylori decreases levels of Na-K-ATPase, a vital transport enzyme, in gastric epithelia, both in acutely infected cultured cells and in chronically infected patients and animals. The bacteria interfere with BiP-assisted folding of newly-made Na-K-ATPase subunits in the endoplasmic reticulum, accelerating their ubiquitylation and proteasomal degradation and decreasing efficiency of the assembly of native enzyme. Decreased Na-K-ATPase expression contributes to H. pylori-induced gastric injury.


Assuntos
Retículo Endoplasmático/enzimologia , Células Epiteliais/enzimologia , Mucosa Gástrica/enzimologia , Gastrite/enzimologia , Proteínas de Choque Térmico/metabolismo , Infecções por Helicobacter/enzimologia , Helicobacter pylori/patogenicidade , ATPase Trocadora de Sódio-Potássio/metabolismo , Células Cultivadas , Retículo Endoplasmático/microbiologia , Estabilidade Enzimática , Células Epiteliais/microbiologia , Mucosa Gástrica/microbiologia , Gastrite/genética , Gastrite/microbiologia , Infecções por Helicobacter/genética , Infecções por Helicobacter/microbiologia , Interações Hospedeiro-Patógeno , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Dobramento de Proteína , Proteólise , ATPase Trocadora de Sódio-Potássio/genética , Ubiquitinação
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