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1.
J Recept Signal Transduct Res ; 39(3): 226-234, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31509043

RESUMO

Cardiotonic steroids (CTS) are steroidal drugs, processed from the seeds and dried leaves of the genus Digitalis as well as from the skin and parotid gland of amphibians. The most commonly known CTS are ouabain, digoxin, digoxigenin and bufalin. CTS can be used for safer medication of congestive heart failure and other related conditions due to promising pharmacological and medicinal properties. Ouabain isolated from plants is widely utilized in in vitro studies to specifically block the sodium potassium (Na+/K+-ATPase) pump. For checking, whether ouabain derivatives are robust inhibitors of Na+/K+-ATPase pump, molecular docking simulation was performed between ouabain and its derivatives using YASARA software. The docking energy falls within the range of 8.470 kcal/mol to 7.234 kcal/mol, in which digoxigenin was found to be the potential ligand with the best docking energy of 8.470 kcal/mol. Furthermore, pharmacophore modeling was applied to decipher the electronic features of CTS. Molecular dynamics simulation was also employed to determine the conformational properties of Na+/K+-ATPase-ouabain and Na+/K+-ATPase-digoxigenin complexes with the plausible structural integrity through conformational ensembles for 100 ns which promoted digoxigenin as the most promising CTS for treating conditions of congestive heart failure patients.


Assuntos
Glicosídeos Cardíacos/farmacologia , Simulação de Acoplamento Molecular , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , Difusão , Digoxina/química , Digoxina/farmacologia , Ligações de Hidrogênio , Ligantes , Modelos Biológicos , Ouabaína/química , Ouabaína/farmacologia , Relação Quantitativa Estrutura-Atividade , Reprodutibilidade dos Testes , ATPase Trocadora de Sódio-Potássio/metabolismo
2.
Cell Physiol Biochem ; 53(4): 638-647, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31556253

RESUMO

BACKGROUND/AIMS: Prolonged hyperosmotic shrinkage evokes expression of osmoprotective genes via nuclear factor NFAT5-mediated pathway and activates Na+ influx via hypertonicity-induced cation channels (HICC). In human umbilical vein endothelial cells (HUVEC) elevation of intracellular sodium concentration ([Na+]i) triggers transcription of dozens of early response genes (ERG). This study examined the role of monovalent cations in the expression of Na+i-sensitive ERGs in iso- and hyperosmotically shrunken HUVEC. METHODS: Cell volume was measured by 3D reconstruction of cell shape and as 14C-urea available space. Intracellular Na+ and K+ content was measured by flame atomic absorption spectrometry. ERG transcription was estimated by RT-PCR. RESULTS: Elevation of medium osmolality by 150 mM mannitol or cell transfer from hypo- to isosmotic medium decreased cell volume by 40-50%. Hyperosmotic medium increased [Na+]i by 2-fold whereas isosmotic shrinkage had no impact on this parameter. Hyperosmotic but not isosmotic shrinkage increased up-to 5-fold the content of EGR1, FOS, ATF3, ZFP36 and JUN mRNAs. Expression of these ERGs triggered by hyperosmotic shrinkage and Na+,K+-ATPase inhibition by 0.1 µM ouabain exhibited positive correlation (R2=0.9383, p=0.0005). Isosmotic substitution of NaCl by N-methyl-D-glucamine abolished an increment of [Na+]i and ERG expression triggered by mannitol addition. CONCLUSION: Augmented expression of ERGs in hyperosmotically shrunken HUVEC is mediated by elevation of [Na+]i.


Assuntos
Tamanho Celular , Sódio/metabolismo , Tamanho Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Proteína 1 de Resposta de Crescimento Precoce/genética , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Meglumina/farmacologia , Ouabaína/farmacologia , Potássio/metabolismo , Cloreto de Sódio/farmacologia , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , ATPase Trocadora de Sódio-Potássio/metabolismo , Tristetraprolina/genética , Tristetraprolina/metabolismo
3.
PLoS Pathog ; 15(8): e1007963, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31381610

RESUMO

Human respiratory syncytial virus (RSV) is the leading viral cause of acute pediatric lower respiratory tract infections worldwide, with no available vaccine or effective antiviral drug. To gain insight into virus-host interactions, we performed a genome-wide siRNA screen. The expression of over 20,000 cellular genes was individually knocked down in human airway epithelial A549 cells, followed by infection with RSV expressing green fluorescent protein (GFP). Knockdown of expression of the cellular ATP1A1 protein, which is the major subunit of the Na+,K+-ATPase of the plasma membrane, had one of the strongest inhibitory effects on GFP expression and viral titer. Inhibition was not observed for vesicular stomatitis virus, indicating that it was RSV-specific rather than a general effect. ATP1A1 formed clusters in the plasma membrane very early following RSV infection, which was independent of replication but dependent on the attachment glycoprotein G. RSV also triggered activation of ATP1A1, resulting in signaling by c-Src-kinase activity that transactivated epidermal growth factor receptor (EGFR) by Tyr845 phosphorylation. ATP1A1 signaling and activation of both c-Src and EGFR were found to be required for efficient RSV uptake. Signaling events downstream of EGFR culminated in the formation of macropinosomes. There was extensive uptake of RSV virions into macropinosomes at the beginning of infection, suggesting that this is a major route of RSV uptake, with fusion presumably occurring in the macropinosomes rather than at the plasma membrane. Important findings were validated in primary human small airway epithelial cells (HSAEC). In A549 cells and HSAEC, RSV uptake could be inhibited by the cardiotonic steroid ouabain and the digitoxigenin derivative PST2238 (rostafuroxin) that bind specifically to the ATP1A1 extracellular domain and block RSV-triggered EGFR Tyr845 phosphorylation. In conclusion, we identified ATP1A1 as a host protein essential for macropinocytic entry of RSV into respiratory epithelial cells, and identified PST2238 as a potential anti-RSV drug.


Assuntos
Pinocitose , Infecções por Vírus Respiratório Sincicial/complicações , Vírus Sincicial Respiratório Humano/patogenicidade , Infecções Respiratórias/prevenção & controle , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , Proteínas Virais/metabolismo , Internalização do Vírus , Células A549 , Cardiotônicos/farmacologia , Digitoxigenina/química , Digitoxigenina/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/enzimologia , Células Epiteliais/virologia , Receptores ErbB/metabolismo , Ensaios de Triagem em Larga Escala , Humanos , Ouabaína/farmacologia , RNA Interferente Pequeno/genética , Infecções por Vírus Respiratório Sincicial/virologia , Sistema Respiratório/efeitos dos fármacos , Sistema Respiratório/enzimologia , Sistema Respiratório/virologia , Infecções Respiratórias/epidemiologia , Infecções Respiratórias/virologia , Transdução de Sinais , ATPase Trocadora de Sódio-Potássio/genética , Proteínas Virais/genética
4.
Eur J Med Chem ; 180: 417-429, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31325787

RESUMO

Oleandrin, the major biologically active constituent of shrub Nerium oleander preparations of which have been used in traditional Mediterranean and Asian medicine, attracts a great deal of attention due to its pronounced anticancer activity. The synthesis of oleandrigenin model, 16ß-hydroxy-3ß-methoxy-5α-card-20(22)-enolide 16-acetate, from androstenolone acetate through 17ß-(3-furyl)-intermediates has been developed. Several related 17ß-(butenolidyl)- and 17ß-(furyl)-androstane derivatives were synthesized and tested for in vitro cytotoxic and Na+/K+-ATP-ase inhibitory activities. Comparison of Na+/K+-ATP-ase inhibitory and cytotoxic activity underlines complex nature of the relationship.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Cardenolídeos/farmacologia , Inibidores Enzimáticos/farmacologia , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , Antineoplásicos Fitogênicos/síntese química , Antineoplásicos Fitogênicos/química , Cardenolídeos/síntese química , Cardenolídeos/química , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Conformação Molecular , Nerium/química , ATPase Trocadora de Sódio-Potássio/metabolismo , Relação Estrutura-Atividade
5.
J Immunol Res ; 2019: 1094520, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31236418

RESUMO

Cardiotonic steroids, such as ouabain and digoxin, are known to bind to Na+/K+-ATPase and to promote several biological activities, including anti-inflammatory activity. However, there are still no reports in the literature about inflammation and marinobufagenin, a cardiotonic steroid from the bufadienolide family endogenously found in mammals. Therefore, the aim of this work was to analyze, in vivo and in vitro, the role of marinobufagenin in acute inflammation. Swiss mice were treated with 0.56 mg/kg of marinobufagenin intraperitoneally (i.p.) and zymosan (2 mg/mL, i.p.) was used to induce peritoneal inflammation. Peritoneal fluid was collected and used for counting cells by optical microscopy and proinflammatory cytokine quantification (IL-1ß, IL-6, and TNF-α) by immunoenzymatic assay (ELISA). Zymosan stimulation, as expected, induced increased cell migration and proinflammatory cytokine levels in the peritoneum. Marinobufagenin treatment reduced polymorphonuclear cell migration and IL-1ß and IL-6 levels in the peritoneal cavity, without interfering in TNF-α levels. In addition, the effect of marinobufagenin was evaluated using peritoneal macrophages stimulated by zymosan (0.2 mg/mL) in vitro. Marinobufagenin treatment at different concentrations (10, 100, 1000, and 10000 nM) showed no cytotoxic effect on peritoneal macrophages. Interestingly, the lowest concentration, which did not inhibit Na+/K+-ATPase activity, attenuated proinflammatory cytokines IL-1ß, IL-6, and TNF-α levels. To investigate the putative mechanism of action of marinobufagenin, the expression of surface molecules (TLR2 and CD69) and P-p38 MAPK were also evaluated, but no significant effect was observed. Thus, our results suggest that marinobufagenin has an anti-inflammatory role in vivo and in vitro and reveals a novel possible endogenous function of this steroid in mammals.


Assuntos
Bufanolídeos/farmacologia , Quimiotaxia de Leucócito/efeitos dos fármacos , Citocinas/biossíntese , Inibidores Enzimáticos/farmacologia , Mediadores da Inflamação/metabolismo , Neutrófilos/efeitos dos fármacos , Neutrófilos/fisiologia , Animais , Biomarcadores , Sobrevivência Celular/efeitos dos fármacos , Citotoxicidade Imunológica/efeitos dos fármacos , Feminino , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/metabolismo , Camundongos , Fosforilação , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores
6.
Environ Pollut ; 252(Pt B): 1288-1300, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31252126

RESUMO

Glyphosate (GLY)-based herbicide, one of the most widely used herbicides, might cause a series of environmental problems and pose a toxicological risk to aquatic organisms. However, data on the potential hazard and toxicity mechanism of GLY to fish gills are relatively scarce. In this study, a subacute toxicity test of common carp (Cyprinus carpio L.) treated with commercial GLY at 52.08 and 104.15 mg L-1 for 7 d was conducted. The results revealed that GLY exposure significantly inhibited Na+/K+-ATPase and increased AST and ALT activities in the fish gills. The biochemical assays results revealed that GLY treatment remarkably altered the transcriptional levels of HSP70 and HSP90; inhibited the activities of SOD, CAT, GPx, GR, and T-AOC; reduced the contents of GSH, but remarkably promoted MDA and PC contents, suggesting that GLY exposure induced oxidative stress and lipids and proteins damage in the carp gills. Further research revealed that GLY exposure also promoted expression of NF-κB, iNOS, IL-1ß, IL-6, IL-8, and TNF-α; altered the levels of IL-10 and TGF-ß, indicating that GLY exposure induced inflammatory response in the fish gills. Additionally, we found that GLY exposure activated apaf-1 and bax and inhibited bcl-2, induced caspase-9 and caspase-3 expression and caused remarkable histological damage in the fish gills. These results may further enriches the toxicity mechanistic theory of GLY to fish gills, which may be useful for the risk assessment of GLY and aquatic organism protection.


Assuntos
Carpas/metabolismo , Brânquias/lesões , Glicina/análogos & derivados , Herbicidas/toxicidade , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , Transcrição Genética/efeitos dos fármacos , Animais , Caspase 3/metabolismo , Caspase 9/metabolismo , Brânquias/efeitos dos fármacos , Glicina/toxicidade , Interleucina-10/metabolismo , NF-kappa B/metabolismo , Estresse Oxidativo/efeitos dos fármacos
7.
Kidney Blood Press Res ; 44(1): 1-11, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30808844

RESUMO

BACKGROUND/AIMS: Dopamine (DA) is a natriuretic hormone that inhibits renal sodium reabsorption, being Angiotensin II (Ang II) its powerful counterpart. These two systems work together to maintain sodium homeostasis and consequently, the blood pressure (BP) within normal limits. We hypothesized that L-tyrosine (L-tyr) or L-dihydroxyphenylalanine (L-dopa) could inhibit the Na+/K+-ATPase activity. We also evaluated whether L-tyr treatment modulates Tyrosine Hydroxylase (TH). METHODS: Experiments involved cultured LLCPK1 cells treated with L-tyr or L-dopa for 30 minutes a 37°C. In experiments on the effect of Dopa Descarboxylase (DDC) inhibition, cells were pre incubated for 15 minutes with 3-Hydroxybenzylhydrazine dihydrochloride (HBH), and them L-dopa was added for 30 minutes. Na+/K+-ATPase activity was quantified colorimetrically. We used immunoblotting and immunocytochemistry to identify the enzymes TH, DDC and the dopamine receptor D1R in LLCPK1 cells. TH activity was accessed by immunoblotting (increase in the phosphorylation). TH and DDC activities were also evaluated by the modulation of the Na+/K+-ATPase activity, which can be ascribed to the synthesis of dopamine. RESULTS: LLCPK1 cells express the required machinery for DA synthesis: the enzymes TH, and (DDC) as well as its receptor D1R, were detected in control steady state cells. Cells treated with L-tyr or L-dopa showed an inhibition of the basolateral Na+/K+-ATPase activity. We can assume that DA formed in the cytoplasm from L-tyr or L-dopa led to inhibition of the Na+/K+-ATPase activity compared to control. L-tyr treatment increases TH phosphorylation at Ser40 by 100%. HBH, a specific DDC inhibitor; BCH, a LAT2 inhibitor; and Sch 23397, a specific D1R antagonist, totally suppressed the inhibition of Na+/K+-ATPase activity due to L-dopa or L-tyr administration, as indicated in the figures. CONCLUSION: The results indicate that DA formed mainly from luminal L-tyr or L-dopa uptake by LAT2, can inhibit the Na+/K+-ATPase. In addition, our results showed for the very first time that TH activity is also significantly increased when the cells were exposed to L-tyr.


Assuntos
Dopamina/biossíntese , Rim/citologia , Serina/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismo , Tirosina/farmacologia , Animais , Linhagem Celular , Dopa Descarboxilase , Rim/metabolismo , Fosforilação/efeitos dos fármacos , Receptores de Dopamina D1 , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , Suínos , Tirosina 3-Mono-Oxigenase/efeitos dos fármacos
8.
Nat Prod Res ; 33(23): 3426-3431, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29781304

RESUMO

This study investigated the antioxidant activity of Cuphea glutinosa (CG) and its effect on Na+, K+-ATPase from cardiac muscle. The ethanolic extract showed higher antioxidant capacity compared to aqueous and ethyl acetate fraction. Ethyl acetate fraction showed ß-sitosterol-3-O-ß-glucoside, kaempferol, quercetin, isoquercetin, gallic acid methyl ester, and gallic acid. The ethanolic extract also reduced the Na+,K+-ATPase activity. CG presented a promising antioxidant activity and inhibitory effect on the Na+, K+-ATPase activity, supporting biochemical evidences the popular use of this plant in the treatment of heart failure.


Assuntos
Antioxidantes/isolamento & purificação , Cuphea/química , Compostos Fitoquímicos/química , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , Animais , Antioxidantes/química , Brasil , Coração/efeitos dos fármacos , Insuficiência Cardíaca/tratamento farmacológico , Quempferóis/isolamento & purificação , Miocárdio , Extratos Vegetais/química , Quercetina/isolamento & purificação
9.
Mol Cell Biochem ; 450(1-2): 175-185, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29922947

RESUMO

The aim of this study was to examine and compare the effects of the acute administration of verapamil or amlodipine as representatives of the calcium channel blockers or nicorandil as a representative of the mitochondrial ATP-dependent potassium (KATP) channel opener to cardiac contractility, coronary flow, and oxidative stress markers on ischemia/reperfusion injury in the isolated rat heart. The hearts of adult male Wistar albino rats (n = 60 total, 12 per group) were divided into five groups, two controls (preconditioning with Krebs-Henseleit solution) and three experimental depending on acute administrated pharmacological agents (0,63 µmol/L of verapamil, 0,1 µmol/L of amlodipine, and 200 µmol/L of nicorandil). After stabilization and 5 min of preconditioning in experimental groups, hearts from I/R control and all experimental groups underwent global ischemia (20 min) and reperfusion (30 min). Hearts from sham group were continuously followed for 50 min, after stabilization period. Cardiodynamic parameters and coronary flow were recorded at the end of stabilization (S), at the last minute of pharmacological preconditioning (P) and at intervals of 5 min after global ischemia, during reperfusion, or in case of sham group during 20-50 min after stabilization. At the same intervals, we collected coronary venous effluent from which we spectrophotometrically measured the parameters of oxidative stress: the index of lipid peroxidation, superoxide anion radical, hydrogen peroxide, and nitrite. In summary, our findings clearly indicate that the blocking of the calcium channel or the activation of KATP may mediate the protective effect of myocardial preconditioning. The ex vivo results showed that all examined drugs after ischemia and reperfusion have beneficial cardioprotective properties associated with lower values of major pro-oxidative molecules. Obtained effects seem to be the most convincible in case of nicorandil.


Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/metabolismo , Contração Miocárdica/efeitos dos fármacos , Traumatismo por Reperfusão Miocárdica/metabolismo , Miocárdio/metabolismo , ATPase Trocadora de Sódio-Potássio , Animais , Masculino , Traumatismo por Reperfusão Miocárdica/patologia , Miocárdio/patologia , Nicorandil/farmacologia , Técnicas de Cultura de Órgãos , Ratos , Ratos Wistar , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , ATPase Trocadora de Sódio-Potássio/metabolismo
10.
Artigo em Inglês | MEDLINE | ID: mdl-30267892

RESUMO

We provide a kinetic characterization of (Na+, K+)-ATPase activity in a posterior gill microsomal fraction from a hololimnetic population of the diadromous Amazon River shrimp Macrobrachium amazonicum. Sucrose density gradient centrifugation reveals two distinct membrane fractions showing considerable (Na+, K+)ATP-ase activity, but also containing other microsomal ATPases. Only a single immune-reactive (Na+, K+)-ATPase with Mr of ≈110 kDa is present that hydrolyzes ATP with VM = 130.3 ±â€¯4.8 nmol Pi min-1 mg protein-1 and K0.5 = 0.065 ±â€¯0.00162 mmol L-1, exhibiting site-site interactions. Stimulation by Na+ (VM = 127.5 ±â€¯5.3 nmol Pi min-1 mg protein-1, K0.5 = 5.3 ±â€¯0.42 mmol L-1), Mg2+ (VM = 130.6 ±â€¯6.8 nmol Pi min-1 mg protein-1, K0.5 = 0.33 ±â€¯0.042 mmol L-1), K+ (VM = 126.7 ±â€¯7.7 nmol Pi min-1 mg protein-1, K0.5 = 0.65 ±â€¯0.0079 mmol L-1) and NH4+ (VM = 134.5 ±â€¯8.6 nmol Pi min-1 mg protein-1, K0.5 = 1.28 ±â€¯0.44 mmol L-1) also obeys cooperative kinetics. Ouabain (KI = 0.18 ±â€¯0.058 mmol L-1) inhibits total ATPase activity by ≈70%. This study reveals considerable differences in the kinetic characteristics of the gill (Na+, K+)-ATPase in a hololimnetic population that appear to result from the adaptation of diadromous Macrobrachium amazonicum populations to different limnic habitats.


Assuntos
Proteínas de Artrópodes/metabolismo , Microssomos/enzimologia , Palaemonidae/enzimologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Proteínas de Artrópodes/antagonistas & inibidores , Biocatálise , Brasil , Inibidores Enzimáticos/farmacologia , Brânquias/enzimologia , Brânquias/crescimento & desenvolvimento , Brânquias/fisiologia , Microssomos/efeitos dos fármacos , Ouabaína/farmacologia , Palaemonidae/citologia , Palaemonidae/crescimento & desenvolvimento , Palaemonidae/fisiologia , Rios , Tolerância ao Sal , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores
11.
Neurosci Res ; 146: 54-64, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30296459

RESUMO

Hyperactivity and impulsivity are common symptoms in several psychiatric disorders. Although dysfunction of Na+, K+-ATPase has been reported to be associated with the psychiatric disorders, it is not clear whether inhibition of Na+, K+-ATPase causes behavioral effects, including hyperactivity and impulsivity, in mice. Here, we evaluated the effect of intracerebroventricular (icv) injection of ouabain, an inhibitor of Na+, K+-ATPase, on hyperactivity and impulsivity in mice. At seven days after icv injection, ouabain-injected mice displayed the increase in the distance traveled in the open field arena in the open field test and the increase in the number of head-dipping behavior in the cliff avoidance test. Chlorpromazine or haloperidol, typical antipsychotics, reduced the hyperactivity and impulsivity in ouabain-injected mice. On the other hand, neither lithium carbonate nor valproate, established mood-stabilizing drugs, improved hyperactivity and impulsivity in our mouse model. Furthermore, ouabain-injected mice exhibited the increase in the number of c-fos-positive cells in the nucleus accumbens and the prefrontal cortex but not in the ventral tegmental area, which was reduced by haloperidol. These results suggest that the dysfunction of Na+, K+-ATPase causes hyperactivity and impulsivity via hyperactivation of dopamine D2 receptor-mediated signaling pathway, causing disturbed neuronal circuits in mice.


Assuntos
Antagonistas dos Receptores de Dopamina D2/farmacologia , Ouabaína/farmacologia , Receptores de Dopamina D2/metabolismo , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Clorpromazina/farmacologia , Modelos Animais de Doenças , Genes fos/efeitos dos fármacos , Genes fos/fisiologia , Haloperidol/farmacologia , Comportamento Impulsivo/efeitos dos fármacos , Comportamento Impulsivo/fisiologia , Infusões Intraventriculares , Locomoção/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neuroglia/efeitos dos fármacos , Córtex Pré-Frontal/metabolismo , Transdução de Sinais/efeitos dos fármacos
12.
Toxicon ; 158: 63-68, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30529380

RESUMO

Cardiac glycosides (CGs) are secondary compounds found in plants and amphibians and are widely distributed in nature with potential cardiovascular action. Their mechanism is based on the blockage of the heart's sodium potassium ATPase, with a positive inotropic effect. Some of the most well-known CGs are digoxin, ouabain, oleandrin, and bufalin. They have similar chemical structures: a lactone ring, steroid ring, and sugar moiety. Digoxin, ouabain, and oleandrin are classified as cardenolides, consisting of a lactone ring with five carbons, while bufalin is classified as bufodienolides, with a six-carbon ring. Small structural differences determine variations in the toxicokinetics and toxicodynamics of such substances. Most case reports of poisoning caused by CGs are associated with cardiovascular toxicity, causing a variety of arrhythmias and lesions in the heart tissue. Experimental studies also describe important similarities among different CGs, especially regarding species sensitivity. Recent studies furthermore focus on their antineoplastic potential, with controversial results. Data from research studies and case reports were reviewed to identify the main characteristics of the CGs, including toxicokinetics, toxicodynamics, clinical signs, electrocardiographic, pathological findings, antineoplastic potential and the main techniques used for diagnostic purposes.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Glicosídeos Cardíacos/química , Glicosídeos Cardíacos/toxicidade , Animais , Bufonidae , Glicosídeos Cardíacos/farmacocinética , Glicosídeos Cardíacos/farmacologia , Neoplasias/tratamento farmacológico , Plantas/química , Envenenamento/diagnóstico , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , Toxicocinética
13.
J Chem Ecol ; 45(1): 50-60, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30523520

RESUMO

Cardenolides are classically studied steroidal defenses in chemical ecology and plant-herbivore coevolution. Although milkweed plants (Asclepias spp.) produce up to 200 structurally different cardenolides, all compounds seemingly share the same well-characterized mode of action, inhibition of the ubiquitous Na+/K+ ATPase in animal cells. Over their evolutionary radiation, milkweeds show a quantitative decline of cardenolide production and diversity. This reduction is contrary to coevolutionary predictions and could represent a cost-saving strategy, i.e. production of fewer but more toxic cardenolides. Here we test this hypothesis by tandem cardenolide quantification using HPLC (UV absorption of the unsaturated lactone) and a pharmacological assay (in vitro inhibition of a sensitive Na+/K+ ATPase) in a comparative study of 16 species of Asclepias. We contrast cardenolide concentrations in leaf tissue to the subset of cardenolides present in exuding latex. Results from the two quantification methods were strongly correlated, but the enzymatic assay revealed that milkweed cardenolide mixtures often cause stronger inhibition than equal amounts of a non-milkweed reference cardenolide, ouabain. Cardenolide concentrations in latex and leaves were positively correlated across species, yet latex caused 27% stronger enzyme inhibition than equimolar amounts of leaf cardenolides. Using a novel multiple regression approach, we found three highly potent cardenolides (identified as calactin, calotropin, and voruscharin) to be primarily responsible for the increased pharmacological activity of milkweed cardenolide mixtures. However, contrary to an expected trade-off between concentration and toxicity, later-diverging milkweeds had the lowest amounts of these potent cardenolides, perhaps indicating an evolutionary response to milkweed's diverse community of specialist cardenolide-sequestering insect herbivores.


Assuntos
Asclepias/fisiologia , Borboletas/fisiologia , Cardenolídeos/metabolismo , Herbivoria , Látex/metabolismo , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , Animais , Asclepias/química , Asclepias/genética , Borboletas/efeitos dos fármacos , Borboletas/enzimologia , Cardenolídeos/análise , Cardenolídeos/toxicidade , Inibidores Enzimáticos/análise , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/toxicidade , Látex/química , Látex/toxicidade , Filogenia , Folhas de Planta/química , Folhas de Planta/genética , Folhas de Planta/fisiologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Suínos
14.
J Biol Chem ; 294(1): 269-280, 2019 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-30409907

RESUMO

The cerebellar ataxia, areflexia, pes cavus, optic atrophy, and sensorineural hearing loss (CAPOS) syndrome is caused by the single mutation E818K of the α3-isoform of Na+,K+-ATPase. Here, using biochemical and electrophysiological approaches, we examined the functional characteristics of E818K, as well as of E818Q and E818A mutants. We found that these amino acid substitutions reduce the apparent Na+ affinity at the cytoplasmic-facing sites of the pump protein and that this effect is more pronounced for the lysine and glutamine substitutions (3-4-fold) than for the alanine substitution. The electrophysiological measurements indicated a more conspicuous, ∼30-fold reduction of apparent Na+ affinity for the extracellular-facing sites in the CAPOS mutant, which was related to an accelerated transition between the phosphoenzyme intermediates E1P and E2P. The apparent affinity for K+ activation of the ATPase activity was unaffected by these substitutions, suggesting that primarily the Na+-specific site III is affected. Furthermore, the apparent affinities for ATP and vanadate were WT-like in E818K, indicating a normal E1-E2 equilibrium of the dephosphoenzyme. Proton-leak currents were not increased in E818K. However, the CAPOS mutation caused a weaker voltage dependence of the pumping rate and a stronger inhibition by cytoplasmic K+ than the WT enzyme, which together with the reduced Na+ affinity of the cytoplasmic-facing sites precluded proper pump activation under physiological conditions. The functional deficiencies could be traced to the participation of Glu-818 in an intricate hydrogen-bonding/salt-bridge network, connecting it to key residues involved in Na+ interaction at site III.


Assuntos
Trifosfato de Adenosina/metabolismo , Ataxia Cerebelar/metabolismo , Deformidades Congênitas do Pé/metabolismo , Perda Auditiva Neurossensorial/metabolismo , Potenciais da Membrana , Mutação de Sentido Incorreto , Atrofia Óptica/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Trifosfato de Adenosina/genética , Substituição de Aminoácidos , Animais , Ataxia Cerebelar/genética , Deformidades Congênitas do Pé/genética , Perda Auditiva Neurossensorial/genética , Humanos , Atrofia Óptica/genética , Domínios Proteicos , Reflexo Anormal/genética , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , ATPase Trocadora de Sódio-Potássio/genética , Vanadatos/farmacologia , Xenopus laevis
15.
Cancer Biol Ther ; 20(1): 52-64, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30183476

RESUMO

Small cell lung cancer (SCLC) is a malignant neuroendocrine tumor with very high mortality. Effective new therapy for advanced SCLC patients is urgently needed. By screening a FDA-approved drug library, we identified a cardiac glycoside (CG), namely digoxin (an inhibitor of cellular Na+/K+ ATPase pump), which was highly effective in inhibiting SCLC cell growth. Intriguing findings showed that NaCl supplement markedly enhanced the anti-tumor activities of digoxin in both in vitro and in vivo models of SCLC. Subsequent analysis revealed that this novel combination of digoxin/NaCl caused an up-regulation of intracellular Na+ and Ca2+ levels with an induction of higher resting membrane potential of SCLC cells. We also found that this combination lead to morphological shrinking of SCLC cells, together with high levels of cytochrome C release. Lastly, our data revealed that NaCl supplement was able to induce the expression of ATP1A1 (a Na+/K+ ATPase subunit), in which contributes directly to the increased sensitivity of SCLC cells to digoxin. Thus, this is the first demonstration that NaCl is a potent supplement necessitating superior anti-cancer effects of digoxin for SCLC. Further, our study suggests that digoxin treatment could need to be combined with NaCl supplement in future clinical trial of SCLC, particularly where low Na+ is often present in SCLC patients.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Digoxina/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Cloreto de Sódio/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linhagem Celular Tumoral , Digoxina/uso terapêutico , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Ensaios de Triagem em Larga Escala , Humanos , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/patologia , Potenciais da Membrana/efeitos dos fármacos , Carcinoma de Pequenas Células do Pulmão/sangue , Carcinoma de Pequenas Células do Pulmão/patologia , Sódio/sangue , Cloreto de Sódio/uso terapêutico , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores
16.
Cell Death Dis ; 10(1): 6, 2018 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-30584244

RESUMO

Na+/K+ ATPase (NKA) is important in maintaining cellular functions. We found that loss of NKA activities in NKAα1+/- mice is associated with increased susceptibility to ischemic injuries following transient middle cerebral artery occlusion (tMCAO). This is corroborated by the neuroprotective effects of an antibody raised against an extracellular DR region (897DVEDSYGQQWTYEQR911, sequence number as in rat) of NKAα subunit (DR-Ab) in both preventive and therapeutic settings. DR-Ab protects cortical neurons against glutamate-induced toxicity by stimulating activities of NKA and Na+/Ca2+ exchanger (NCX), which resulted in accelerated Ca2+ extrusion. DR-Ab also enhanced the association between NKA and GluR2 and therefore reduced the internalization of both proteins from membrane induced by glutamate toxicity. The mechanism appears to involve suppression of GluR2 phosphorylation through PKCα/PICK pathway. Our data indicate that DR-region of NKA may be a novel therapeutic target for drug development for the treatment of ischemic stroke.


Assuntos
Anticorpos/farmacologia , Córtex Cerebral/metabolismo , Neurônios/metabolismo , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , Acidente Vascular Cerebral/tratamento farmacológico , Animais , Córtex Cerebral/patologia , Modelos Animais de Doenças , Ácido Glutâmico/efeitos adversos , Ácido Glutâmico/farmacologia , Camundongos , Neurônios/patologia , Peptídeos/química , Receptores de AMPA/genética , Receptores de AMPA/metabolismo , Trocador de Sódio e Cálcio/genética , Trocador de Sódio e Cálcio/metabolismo , ATPase Trocadora de Sódio-Potássio/genética , ATPase Trocadora de Sódio-Potássio/metabolismo , Acidente Vascular Cerebral/genética , Acidente Vascular Cerebral/metabolismo , Acidente Vascular Cerebral/patologia
17.
Life Sci ; 215: 198-206, 2018 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-30439376

RESUMO

AIMS: Sphingosine-1-phosphate (S1P) has been implicated lately in inflammatory bowel disease which has diarrhea as one of its symptoms. Diarrhea is due to altered water movements as a result of altered electrolyte transport, and in particular sodium. Sodium movements are geared by the sodium gradient established by the Na+/K+ ATPase. The aim of this work was to investigate if S1P can modulate the activity of the ATPase, using Caco-2 cells as a model and the S1P analogue, FTY720P. MATERIALS AND METHODS: The activity of the ATPase was assayed by measuring the amount of inorganic phosphate liberated in presence and absence of ouabain. Protein expression of the various S1P receptors was studied by western blot analysis. KEY FINDINGS: Caco-2 cells were found to express mainly S1PR2 and S1PR3. FTY720P (7.5 nM) reduced significantly the activity of the Na+/K+ ATPase when applied for 15 min. This inhibitory effect disappeared in presence of JTE-013, a specific blocker of S1PR2, and indomethacin, an inhibitor of cyclooxygenase enzymes, and was mimicked by CYM5520, a S1PR2 agonist and by exogenous PGE2. The inhibitory effect of PGE2 did not appear when EP3 receptors were blocked or when a nitric oxide scavenger was added. RpcAMP, a PKA inhibitor, reduced the activity of the Na+/K+ ATPase, while dbcAMP, a PKA activator was without any effect and when added, abrogated the effect of PGE2. SIGNIFICANCE: It was concluded that FTY720P inhibits the Na+/K+ ATPase via activation of S1PR2 and generation of PGE2 nitric oxide.


Assuntos
Dinoprostona/metabolismo , Óxido Nítrico/metabolismo , Organofosfatos/farmacologia , Receptores de Lisoesfingolipídeo/metabolismo , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , Esfingosina/análogos & derivados , Western Blotting , Células CACO-2 , Humanos , Indometacina/farmacologia , Lisofosfolipídeos/metabolismo , Pirazóis/farmacologia , Piridinas/farmacologia , Pirróis/farmacologia , Transdução de Sinais/efeitos dos fármacos , ATPase Trocadora de Sódio-Potássio/metabolismo , Esfingosina/metabolismo , Esfingosina/farmacologia
18.
Toxins (Basel) ; 10(11)2018 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-30400202

RESUMO

As a black widow spider, Latrodectus tredecimguttatus has poisonous components not only in venomous glands but also in eggs. Our previous work had carried out a transcriptome analysis of the spider eggs in an attempt to probe into the molecular basis of the egg toxicity. A proteinaceous toxin, named Latroeggtoxin-V, was mined from the identified transcriptome. In this study, the gene of Latroeggtoxin-V was cloned and heterologously expressed, and the anticancer activity of the recombinant Latroeggtoxin-V (rLatroeggtoxin-V) was characterized. Activity assay found that rLatroeggtoxin-V could selectively act on breast cancer line MDA-MB-231 cells, not only arresting their cell cycle, inhibiting their proliferation and migration, but also inducing their apoptosis. Bioinformatics analysis suggested that Latroeggtoxin-V belongs to the ATPase inhibitor protein family and the further activity assay showed that the rLatroeggtoxin-V inhibited the activity of the Na⁺/K⁺-ATPase in MDA-MB-231 cells in a concentration-dependent manner, suggesting that the anticancer activity of Latroeggtoxin-V is based on its affecting the ion transport and receptor functions of Na⁺/K⁺-ATPase. The present work not only laid the foundation for the utilization of Latroeggtoxin-V in the anticancer drug development and the related fields, but also provided a new paradigm for exploration of the proteinaceous toxins under the direction of transcriptomics and bioinformatics.


Assuntos
Viúva Negra/genética , Neoplasias da Mama/patologia , Óvulo/química , Transcriptoma , Sequência de Aminoácidos , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Sequência de Bases , Neoplasias da Mama/enzimologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Clonagem Molecular , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Homologia de Sequência de Aminoácidos , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores
19.
J Pharmacol Sci ; 138(3): 167-175, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30322800

RESUMO

Na+, K+-ATPase is a highly expressed membrane protein. Dysfunction of Na+, K+-ATPase has been implicated in the pathophysiology of several neurodegenerative and psychiatric disorders, however, the underlying mechanism of neuronal cell death resulting from Na+, K+-ATPase dysfunction is poorly understood. Here, we investigated the mechanism of neurotoxicity due to Na+, K+-ATPase inhibition using rat organotypic hippocampal slice cultures. Treatment with ouabain, a Na+, K+-ATPase inhibitor, increased the ratio of propidium iodide-positive cells among NeuN-positive cells in the hippocampal CA1 region, which was prevented by MK-801 and d-AP5, specific blockers of the N-methyl-d-aspartate (NMDA) receptor. EGTA, a Ca2+-chelating agent, also protected neurons from ouabain-induced injury. We observed that astrocytes expressed the glutamate aspartate transporter (GLAST), and ouabain changed the immunoreactive area of GFAP-positive astrocytes as well as GLAST. We also observed that ouabain increased the number of Iba1-positive microglial cells in a time-dependent manner. Furthermore, lithium carbonate, a mood-stabilizing drug, protected hippocampal neurons and reduced disturbances of astrocytes and microglia after ouabain treatment. Notably, lithium carbonate improved ouabain-induced decreases in GLAST intensity in astrocytes. These results suggest that glial cell abnormalities resulting in excessive extracellular concentrations of glutamate contribute to neurotoxicity due to Na+, K+-ATPase dysfunction in the hippocampal CA1 region.


Assuntos
Região CA1 Hipocampal/efeitos dos fármacos , Região CA1 Hipocampal/patologia , Morte Celular/efeitos dos fármacos , Transportador 1 de Aminoácido Excitatório/metabolismo , Microglia/efeitos dos fármacos , Microglia/patologia , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , Animais , Astrócitos/metabolismo , Contagem de Células , Células Cultivadas , Maleato de Dizocilpina/farmacologia , Ácido Egtázico/farmacologia , Técnicas In Vitro , Carbonato de Lítio/farmacologia , Ouabaína/antagonistas & inibidores , Ouabaína/farmacologia , Ratos , Valina/análogos & derivados , Valina/farmacologia
20.
Cell ; 175(5): 1213-1227.e18, 2018 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-30318147

RESUMO

Neurons use two main schemes to encode information: rate coding (frequency of firing) and temporal coding (timing or pattern of firing). While the importance of rate coding is well established, it remains controversial whether temporal codes alone are sufficient for controlling behavior. Moreover, the molecular mechanisms underlying the generation of specific temporal codes are enigmatic. Here, we show in Drosophila clock neurons that distinct temporal spike patterns, dissociated from changes in firing rate, encode time-dependent arousal and regulate sleep. From a large-scale genetic screen, we identify the molecular pathways mediating the circadian-dependent changes in ionic flux and spike morphology that rhythmically modulate spike timing. Remarkably, the daytime spiking pattern alone is sufficient to drive plasticity in downstream arousal neurons, leading to increased firing of these cells. These findings demonstrate a causal role for temporal coding in behavior and define a form of synaptic plasticity triggered solely by temporal spike patterns.


Assuntos
Plasticidade Neuronal , Sono/fisiologia , Potenciais de Ação , Animais , Relógios Circadianos/fisiologia , Drosophila , Proteínas de Drosophila/antagonistas & inibidores , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Modelos Neurológicos , Neurônios/metabolismo , Optogenética , Canais de Potássio/genética , Canais de Potássio/metabolismo , Canais de Potássio Cálcio-Ativados/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Transdução de Sinais , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , ATPase Trocadora de Sódio-Potássio/genética , ATPase Trocadora de Sódio-Potássio/metabolismo , Transmissão Sináptica
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