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1.
Cells ; 11(17)2022 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-36078160

RESUMO

Beta-amyloid (Aß) has a dual role, both as an important factor in the pathology of Alzheimer's disease and as a regulator in brain physiology. The inhibitory effect of Aß42 oligomers on Na,K-ATPase contributes to neuronal dysfunction in Alzheimer's disease. Still, the physiological role of the monomeric form of Aß42 interaction with Na,K-ATPase remains unclear. We report that Na,K-ATPase serves as a receptor for Aß42 monomer, triggering Src kinase activation. The co-localization of Aß42 with α1- and ß1-subunits of Na,K-ATPase, and Na,K-ATPase with Src kinase in SH-SY5Y neuroblastoma cells, was observed. Treatment of cells with 100 nM Aß42 causes Src kinase activation, but does not alter Na,K-ATPase transport activity. The interaction of Aß42 with α1ß1 Na,K-ATPase isozyme leads to activation of Src kinase associated with the enzyme. Notably, prevention of Na,K-ATPase:Src kinase interaction by a specific inhibitor pNaKtide disrupts the Aß-induced Src kinase activation. Stimulatory effect of Aß42 on Src kinase was lost under hypoxic conditions, which was similar to the effect of specific Na,K-ATPase ligands, the cardiotonic steroids. Our findings identify Na,K-ATPase as a Aß42 receptor, thus opening a prospect on exploring the physiological and pathological Src kinase activation caused by Aß42 in the nervous system.


Assuntos
Peptídeos beta-Amiloides , ATPase Trocadora de Sódio-Potássio , Quinases da Família src , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Humanos , Neuroblastoma , Sódio/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Quinases da Família src/metabolismo
2.
Int J Mol Sci ; 24(1)2022 Dec 22.
Artigo em Inglês | MEDLINE | ID: mdl-36613623

RESUMO

The Alzheimer's disease (AD)-associated breakdown of the blood-brain barrier (BBB) promotes the accumulation of beta-amyloid peptide (Aß) in the brain as the BBB cells provide Aß transport from the brain parenchyma to the blood, and vice versa. The breakdown of the BBB during AD may be caused by the emergence of blood-borne Aß pathogenic forms, such as structurally and chemically modified Aß species; their effect on the BBB cells has not yet been studied. Here, we report that the effects of Aß42, Aß42, containing isomerized Asp7 residue (iso-Aß42) or phosphorylated Ser8 residue (p-Aß42) on the mitochondrial potential and respiration are closely related to the redox status changes in the mouse brain endothelial cells bEnd.3. Aß42 and iso-Aß42 cause a significant increase in nitric oxide, reactive oxygen species, glutathione, cytosolic calcium and the mitochondrial potential after 4 h of incubation. P-Aß42 either does not affect or its effect develops after 24 h of incubation. Aß42 and iso-Aß42 activate mitochondrial respiration compared to p-Aß42. The isomerized form promotes a greater cytotoxicity and mitochondrial dysfunction, causing maximum oxidative stress. Thus, Aß42, p-Aß42 and iso-Aß42 isoforms differently affect the BBBs' cell redox parameters, significantly modulating the functioning of the mitochondria. The changes in the level of modified Aß forms can contribute to the BBBs' breakdown during AD.


Assuntos
Doença de Alzheimer , Peptídeos beta-Amiloides , Animais , Camundongos , Peptídeos beta-Amiloides/metabolismo , Barreira Hematoencefálica/metabolismo , Células Endoteliais/metabolismo , Doença de Alzheimer/metabolismo , Oxirredução , Endotélio/metabolismo , Fragmentos de Peptídeos/metabolismo
3.
Int J Mol Sci ; 22(24)2021 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-34948068

RESUMO

Cardiotonic steroids (CTSs) are specific inhibitors of Na,K-ATPase (NKA). They induce diverse physiological effects and were investigated as potential drugs in heart diseases, hypertension, neuroinflammation, antiviral and cancer therapy. Here, we compared the inhibition mode and binding of CTSs, such as ouabain, digoxin and marinobufagenin to NKA from pig and rat kidneys, containing CTSs-sensitive (α1S) and -resistant (α1R) α1-subunit, respectively. Marinobufagenin in contrast to ouabain and digoxin interacted with α1S-NKA reversibly, and its binding constant was reduced due to the decrease in the deepening in the CTSs-binding site and a lower number of contacts between the site and the inhibitor. The formation of a hydrogen bond between Arg111 and Asp122 in α1R-NKA induced the reduction in CTSs' steroid core deepening that led to the reversible inhibition of α1R-NKA by ouabain and digoxin and the absence of marinobufagenin's effect on α1R-NKA activity. Our results elucidate that the difference in signaling, and cytotoxic effects of CTSs may be due to the distinction in the deepening of CTSs into the binding side that, in turn, is a result of a bent-in inhibitor steroid core (marinobufagenin in α1S-NKA) or the change of the width of CTSs-binding cavity (all CTSs in α1R-NKA).


Assuntos
Bufanolídeos/farmacologia , Digoxina/farmacologia , Rim/enzimologia , Ouabaína/farmacologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Sítios de Ligação , Glicosídeos Cardíacos/farmacologia , Ligação de Hidrogênio , Rim/efeitos dos fármacos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Ratos , ATPase Trocadora de Sódio-Potássio/química , Suínos
4.
Genes Dis ; 8(3): 259-271, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33997173

RESUMO

With an exception of few reports, the plasma concentration of ouabain and marinobufagenin, mostly studied cardiotonic steroids (CTS) assessed by immunoassay techniques, is less than 1 nM. During the last 3 decades, the implication of these endogenous CTS in the pathogenesis of hypertension and other volume-expanded disorders is widely disputed. The threshold for inhibition by CTS of human and rodent α1-Na,K-ATPase is ∼1 and 1000 nM, respectively, that rules out the functioning of endogenous CTS (ECTS) as natriuretic hormones and regulators of cell adhesion, cell-to-cell communication, gene transcription and translation, which are mediated by dissipation of the transmembrane gradients of monovalent cations. In several types of cells ouabain and marinobufagenin at concentrations corresponding to its plasma level activate Na,K-ATPase, decrease the [Na+]i/[K+]i-ratio and increase cell proliferation. Possible physiological significance and mechanism of non-canonical Na+ i/K+ i-dependent and Na+ i/K+ i-independent cell responses to CTS are discussed.

5.
Int J Mol Sci ; 21(21)2020 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-33121152

RESUMO

Stimulus-dependent elevation of intracellular Ca2+ affects gene expression via well-documented calmodulin-mediated signaling pathways. Recently, we found that the addition of extra- and intracellular Ca2+ chelators increased, rather than decreased, the number of genes expressed, and that this is affected by the elevation of [Na+]i/[K+]i-ratio. This assumes the existence of a novel Na+i/K+i-mediated Ca2+i-independent mechanism of excitation-transcription coupling. To identify upstream Na+i/K+i-sensitive genes, we examined the kinetics of transcriptomic changes in human umbilical vein endothelial cells (HUVEC) subjected to Na,K-ATPase inhibition by ouabain or K+-free medium. According to our data, microRNAs, transcription factors, and proteins involved in immune response and inflammation might be considered as key components of Na+i/K+i-mediated excitation-transcription coupling. Special attention was focused on the FOS gene and the possible mechanism of transcription regulation via G-quadruplexes, non-canonical secondary structures of nucleic acids, whose stability depends on [Na+]i/[K+]i-ratio. Verification of the [Na+]i/[K+]i-sensitive transcription regulation mechanism should be continued in forthcoming studies.


Assuntos
Células Endoteliais/metabolismo , Perfilação da Expressão Gênica/métodos , Ouabaína/farmacologia , Proteínas Proto-Oncogênicas c-fos/genética , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Quadruplex G , Regulação da Expressão Gênica/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana , Humanos , Conformação Molecular , Proteínas Proto-Oncogênicas c-fos/química , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , Transcrição Gênica
6.
Molecules ; 22(4)2017 Apr 14.
Artigo em Inglês | MEDLINE | ID: mdl-28420099

RESUMO

Na⁺,K⁺-ATPase is the only known receptor of cardiotonic steroids (CTS) whose interaction with catalytic α-subunits leads to inhibition of this enzyme. As predicted, CTS affect numerous cellular functions related to the maintenance of the transmembrane gradient of monovalent cations, such as electrical membrane potential, cell volume, transepithelial movement of salt and osmotically-obliged water, symport of Na⁺ with inorganic phosphate, glucose, amino acids, nucleotides, etc. During the last two decades, it was shown that side-by-side with these canonical Na⁺i/K⁺i-dependent cellular responses, long-term exposure to CTS affects transcription, translation, tight junction, cell adhesion and exhibits tissue-specific impact on cell survival and death. It was also shown that CTS trigger diverse signaling cascades via conformational transitions of the Na⁺,K⁺-ATPase α-subunit that, in turn, results in the activation of membrane-associated non-receptor tyrosine kinase Src, phosphatidylinositol 3-kinase and the inositol 1,4,5-triphosphate receptor. These findings allowed researchers to propose that endogenous CTS might be considered as a novel class of steroid hormones. We focus our review on the analysis of the relative impact Na⁺i,K⁺i-mediated and -independent pathways in cellular responses evoked by CTS.


Assuntos
Glicosídeos Cardíacos/farmacologia , Transdução de Sinais/efeitos dos fármacos , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Glicosídeos Cardíacos/química , Adesão Celular/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Bombas de Íon/metabolismo , Extratos Vegetais/química , Extratos Vegetais/farmacologia , ATPase Trocadora de Sódio-Potássio/química , Relação Estrutura-Atividade
7.
Sci Rep ; 7: 45403, 2017 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-28345607

RESUMO

Recent studies demonstrated that in addition to Na+,K+-ATPase inhibition cardiotonic steroids (CTSs) affect diverse intracellular signaling pathways. This study examines the relative impact of [Na+]i/[K+]i-mediated and -independent signaling in transcriptomic changes triggered by the endogenous CTSs ouabain and marinobufagenin (MBG) in human umbilical vein endothelial cells (HUVEC). We noted that prolongation of incubation increased the apparent affinity for ouabain estimated by the loss of [K+]i and gain of [Na+]i. Six hour exposure of HUVEC to 100 and 3,000 nM ouabain resulted in elevation of the [Na+]i/[K+]i ratio by ~15 and 80-fold and differential expression of 258 and 2185 transcripts, respectively. Neither [Na+]i/[K+]i ratio nor transcriptome were affected by 6-h incubation with 30 nM ouabain. The 96-h incubation with 3 nM ouabain or 30 nM MBG elevated the [Na+]i/[K+]i ratio by ~14 and 3-fold and led to differential expression of 880 and 484 transcripts, respectively. These parameters were not changed after 96-h incubation with 1 nM ouabain or 10 nM MBG. Thus, our results demonstrate that elevation of the [Na+]i/[K+]i ratio is an obligatory step for transcriptomic changes evoked by CTS in HUVEC. The molecular origin of upstream [Na+]i/[K+]i sensors involved in transcription regulation should be identified in forthcoming studies.


Assuntos
Glicosídeos Cardíacos/farmacologia , Cardiotônicos/farmacologia , Íons/metabolismo , Potássio/metabolismo , Sódio/metabolismo , Transcriptoma/efeitos dos fármacos , Bufanolídeos/farmacologia , Linhagem Celular , Regulação da Expressão Gênica/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana , Humanos , Ouabaína/farmacologia , Transdução de Sinais/efeitos dos fármacos , ATPase Trocadora de Sódio-Potássio/metabolismo , Transcrição Gênica/efeitos dos fármacos
8.
Apoptosis ; 20(9): 1200-10, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26067145

RESUMO

In rodents, ubiquitous α1-Na(+), K(+)-ATPase is inhibited by ouabain and other cardiotonic steroids (CTS) at ~10(3)-fold higher concentrations than those effective in other mammals. To examine the specific roles of the CTS-sensitive α1S- and CTS-resistant α1R-Na(+), K(+)-ATPase isoforms, we compared the effects of ouabain on intracellular Na(+) and K(+) content, cell survival, and mitogen-activated protein kinases (MAPK) in human and rat vascular smooth muscle cells (HASMC and RASMC), human and rat endothelial cells (HUVEC and RAEC), and human and rat brain astrocytes. 6-h exposure of HASMC and HUVEC to 3 µM ouabain dramatically increased the intracellular [Na(+)]/[K(+)] ratio to the same extend as in RASMC and RAEC treated with 3000 µM ouabain. In 24, 3 µM ouabain triggered the death of all types of human cells used in this study. Unlike human cells, we did not detect any effect of 3000-5000 µM ouabain on the survival of rat cells, or smooth muscle cells from mouse aorta (MASMC). Unlike in the wild-type α1(R/R) mouse, ouabain triggered death of MASMC from α1(S/S) mouse expressing human α1-Na(+), K(+)-ATPase. Furthermore, transfection of HUVEC with rat α1R-Na(+), K(+)-ATPase protected them from the ouabain-induced death. In HUVEC, ouabain led to phosphorylation of p38 MAPK, whereas in RAEC it stimulated phosphorylation of ERK1/2. Overall, our results, demonstrate that the drastic differences in cytotoxic action of ouabain on human and rodent cells are caused by unique features of α1S/α1R-Na(+), K(+)-ATPase, rather than by any downstream CTS-sensitive/resistant components of the cell death machinery.


Assuntos
Cardiotônicos/toxicidade , Morte Celular/efeitos dos fármacos , Ouabaína/toxicidade , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Animais Recém-Nascidos , Astrócitos/metabolismo , Biomarcadores/metabolismo , Encéfalo/citologia , Linhagem Celular , Células Epiteliais/metabolismo , Humanos , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Músculo Liso/citologia , Potássio/metabolismo , Estrutura Terciária de Proteína , Ratos Sprague-Dawley , Sódio/metabolismo
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