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1.
Int J Mol Sci ; 25(3)2024 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-38338921

RESUMO

Bipolar disorder (BD) is a severe and common chronic mental illness characterized by recurrent mood swings between depression and mania. The biological basis of the disease is poorly understood, and its treatment is unsatisfactory. Na+, K+-ATPase is a major plasma membrane transporter and signal transducer. The catalytic α subunit of this enzyme is the binding site for cardiac steroids. Three α isoforms of the Na+, K+-ATPase are present in the brain. Previous studies have supported the involvement of the Na+, K+-ATPase and endogenous cardiac steroids (ECS) in the etiology of BD. Decreased brain ECS has been found to elicit anti-manic and anti-depressive-like behaviors in mice and rats. However, the identity of the specific α isoform involved in these behavioral effects is unknown. Here, we demonstrated that decreasing ECS through intracerebroventricular (i.c.v.) administration of anti-ouabain antibodies (anti-Ou-Ab) decreased the activity of α1+/- mice in forced swimming tests but did not change the activity in wild type (wt) mice. This treatment also affected exploratory and anxiety behaviors in α1+/- but not wt mice, as measured in open field tests. The i.c.v. administration of anti-Ou-Ab decreased brain ECS and increased brain Na+, K+-ATPase activity in wt and α1+/- mice. The serum ECS was lower in α1+/- than wt mice. In addition, a study in human participants demonstrated that serum ECS significantly decreased after treatment. These results suggest that the Na+, K+-ATPase α1 isoform is involved in depressive- and manic-like behaviors and support that the Na+, K+-ATPase/ECS system participates in the etiology of BD.


Assuntos
Depressão , ATPase Trocadora de Sódio-Potássio , Humanos , Camundongos , Ratos , Animais , ATPase Trocadora de Sódio-Potássio/genética , ATPase Trocadora de Sódio-Potássio/metabolismo , Ouabaína/metabolismo , Isoformas de Proteínas/metabolismo , Esteroides
2.
Int J Mol Sci ; 23(22)2022 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-36430373

RESUMO

Bipolar disorder (BD) is a severe and common chronic mental illness. The biological basis of the disease is poorly understood and its treatment is unsatisfactory. Our previous studies supported the notion that alterations in Na+, K+-ATPase activity were involved in the etiology of BD. As various chemical elements inhibit Na+, K+-ATPase, we determined the concentration of 26 elements in the serum of BD patients before and after treatment and in postmortem brain samples from BD patients, and compared them with matched controls. The only element that was reduced significantly in the serum following treatment was vanadium (V). Furthermore, the concentration of V was significantly lower in the pre-frontal cortex of BD patients compared with that of the controls. Intracerebroventricular administration of V in mice elicited anxiolytic and depressive activities, concomitantly inhibited brain Na+, K+-ATPase activity, and increased extracellular signal-regulated kinase phosphorylation. A hypothesis associating V with BD was set forth decades ago but eventually faded out. Our results are in accord with the hypothesis and advocate for a thorough examination of the possible involvement of chemical elements, V in particular, in BD.


Assuntos
Transtorno Bipolar , Animais , Camundongos , Transtorno Bipolar/tratamento farmacológico , Vanádio/farmacologia , Encéfalo , Lobo Frontal , Adenosina Trifosfatases
3.
Int J Mol Sci ; 23(7)2022 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-35409366

RESUMO

Bufalin and other cardiac steroids (CS) have been used for centuries for the treatment of congestive heart failure, arrhythmias, and other maladies. However, toxicity and the small therapeutic window of this family of steroids limit their use. Therefore, attempts to synthesize a potent, but less toxic, CS are of major importance. In the present study, two novel bufalin derivatives were synthesized and some of their pharmacological properties were characterized. The reaction of bufalin with Ishikawa's reagent resulted in the production of two novel bufalin derivatives: bufalin 2,3-ene and bufalin 3,4-ene. The compounds were purified with TLC and HPLC and their structure was verified with UV, NMR, and MS analyses. The biological activities of these compounds were evaluated by testing their ability to inhibit the Na+, K+-ATPase activity of the brain microsomal fraction to induce cytotoxic activity against the NCI-60 human tumor cell line panel and non-cancer human cells, and to increase the force of contraction of quail embryonic heart muscle cells in culture. The two steroids exhibited biological activities similar to those of other CS in the tested experimental systems, but with reduced cytotoxicity, advocating their development as drugs for the treatment of heart failure and arrhythmias.


Assuntos
Bufanolídeos , Ouabaína , Arritmias Cardíacas/tratamento farmacológico , Bufanolídeos/metabolismo , Bufanolídeos/farmacologia , Humanos , Microssomos/metabolismo , Ouabaína/farmacologia , ATPase Trocadora de Sódio-Potássio/metabolismo
4.
Int J Mol Sci ; 21(16)2020 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-32824628

RESUMO

Bipolar disorder is a chronic multifactorial psychiatric illness that affects the mood, cognition, and functioning of about 1-2% of the world's population. Its biological basis is unknown, and its treatment is unsatisfactory. The α1, α2, and α3 isoforms of the Na+, K+-ATPase, an essential membrane transporter, are vital for neuronal and glial function. The enzyme and its regulators, endogenous cardiac steroids like ouabain and marinobufagenin, are implicated in neuropsychiatric disorders, bipolar disorder in particular. Here, we address the hypothesis that the α isoforms of the Na+, K+-ATPase and its regulators are altered in the prefrontal cortex of bipolar disease patients. The α isoforms were determined by Western blot and ouabain and marinobufagenin by specific and sensitive immunoassays. We found that the α2 and α3 isoforms were significantly higher and marinobufagenin levels were significantly lower in the prefrontal cortex of the bipolar disease patients compared with those in the control. A positive correlation was found between the levels of the three α isoforms in all samples and between the α1 isoform and ouabain levels in the controls. These results are in accordance with the notion that the Na+, K+-ATPase-endogenous cardiac steroids system is involved in bipolar disease and suggest that it may be used as a target for drug development.


Assuntos
Transtorno Bipolar/metabolismo , Bufanolídeos/metabolismo , Ouabaína/metabolismo , Córtex Pré-Frontal/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Adulto , Transtorno Bipolar/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ATPase Trocadora de Sódio-Potássio/genética
5.
PLoS One ; 14(6): e0218041, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31173612

RESUMO

There is strong evidence that neuronal hyper-excitability underlies migraine, and may or may not be preceded by cortical spreading depression. However, the mechanisms for cortical spreading depression and/or migraine are not established. Previous studies reported that cerebrospinal fluid (CSF) [Na+] is higher during migraine, and that higher extracellular [Na+] leads to hyper-excitability. We raise the hypothesis that altered choroid plexus Na+, K+-ATPase activity can cause both migraine phenomena: inhibition raises CSF [K+] and initiates cortical spreading depression, while activation raises CSF [Na+] and causes migraine. In this study, we examined levels of specific Na+, K+-ATPase inhibitors, endogenous ouabain-like compounds (EOLC), in CSF from migraineurs and controls. CSF EOLC levels were significantly lower during ictal migraine (0.4 nM +/- 0.09) than from either controls (1.8 nM +/- 0.4) or interictal migraineurs (3.1 nM +/- 1.9). Blood plasma EOLC levels were higher in migraineurs than controls, but did not differ between ictal and interictal states. In a Sprague-Dawley rat model of nitroglycerin-triggered central sensitization, we changed the concentrations of EOLC and CSF sodium, and measured aversive mechanical threshold (von Frey hairs), trigeminal nucleus caudalis activation (cFos), and CSF [Na+] (ultra-high field 23Na MRI). Animals were sensitized by three independent treatments: intraperitoneal nitroglycerin, immunodepleting EOLC from cerebral ventricles, or cerebroventricular infusion of higher CSF [Na+]. Conversely, nitroglycerin-triggered sensitization was prevented by either vascular or cerebroventricular delivery of the specific Na+, K+-ATPase inhibitor, ouabain. These results affirm our hypothesis that higher CSF [Na+] is linked to human migraine and to a rodent migraine model, and demonstrate that EOLC regulates them both. Our data suggest that altered choroid plexus Na+, K+-ATPase activity is a common source of these changes, and may be the initiating mechanism in migraine.


Assuntos
Líquido Cefalorraquidiano/metabolismo , Íons/metabolismo , Transtornos de Enxaqueca/etiologia , Transtornos de Enxaqueca/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Sódio/metabolismo , Adolescente , Adulto , Idoso , Animais , Plexo Corióideo/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Ouabaína/metabolismo , Ratos , Ratos Sprague-Dawley , Adulto Jovem
6.
J Psychiatr Res ; 115: 21-28, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31082653

RESUMO

Na+, K+-ATPase is an essential membrane transporter. In the brain, the α3 isoform of Na+, K+-ATPase is vital for neuronal function. The enzyme and its regulators, endogenous cardiac steroids (ECS), were implicated in neuropsychiatric disorders. GABAergic neurotransmission was also studied extensively in diseases such as schizophrenia and bipolar disorder (BD). Post mortem brain samples from subjects with depression, schizophrenia or BD and non-psychiatric controls were provided by the Stanley Medical Research Institute. ECS levels were determined by ELISA. Expression levels of the three Na+, K+-ATPase-α isoforms, α1, α2 and α3, were determined by Western blot analysis. The α3 levels in GABAergic neurons in different regions of the brain were quantified by fluorescence immunohistochemistry. The results show that Na+, K+ -ATPase α3 isoform levels were lower in GABAergic neurons in the frontal cortex in BD and schizophrenia as compared with the controls (n = 15 subjects per group). A study on a 'mini-cohort' (n = 3 subjects per group) showed that the α3 isoform levels were also lower in GABAergic neurons in the hippocampus, but not amygdala, of bipolar and schizophrenic subjects. In the temporal cortex, higher Na+, K+ -ATPase α3 protein levels were found in the three psychiatric groups. No significant differences in ECS levels were found in this brain area. This is the first report on the distribution of α3 in specific neurons in the human brain in association with mental illness. These results strengthen the hypothesis for the involvement of Na+, K+ -ATPase in neuropsychiatric diseases.


Assuntos
Transtorno Bipolar/enzimologia , Transtorno Depressivo/enzimologia , Neurônios GABAérgicos/enzimologia , Interneurônios/enzimologia , Córtex Pré-Frontal/enzimologia , Esquizofrenia/enzimologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Bancos de Tecidos , Adulto , Tonsila do Cerebelo/enzimologia , Hipocampo/enzimologia , Humanos , Córtex Pré-Frontal/patologia , Isoformas de Proteínas , Lobo Temporal/enzimologia
7.
J Cardiovasc Pharmacol Ther ; 24(1): 78-89, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30033751

RESUMO

Cardiac steroids (CSs), such as ouabain and digoxin, increase the force of contraction of heart muscle and are used for the treatment of congestive heart failure (CHF). However, their small therapeutic window limits their use. It is well established that Na+, K+-ATPase inhibition mediates CS-induced increase in heart contractility. Recently, the involvement of intracellular signal transduction was implicated in this effect. The aim of the present study was to test the hypothesis that combined treatment with ouabain and Akt inhibitor (MK-2206) augments ouabain-induced inotropy in mammalian models. We demonstrate that the combined treatment led to an ouabain-induced increase in contractility at concentrations at which ouabain alone was ineffective. This was shown in 3 experimental systems: neonatal primary rat cardiomyocytes, a Langendorff preparation, and an in vivo myocardial infarction induced by left anterior descending coronary artery (LAD) ligation. Furthermore, cell viability experiments revealed that this treatment protected primary cardiomyocytes from MK-2206 toxicity and in vivo reduced the size of scar tissue 10 days post-LAD ligation. We propose that Akt activity imposes a constant inhibitory force on muscle contraction, which is attenuated by low concentrations of MK-2206, resulting in potentiation of the ouabain effect. This demonstration of the increase in the CS effect advocates the development of the combined treatment in CHF.


Assuntos
Cardiotônicos/farmacologia , Compostos Heterocíclicos com 3 Anéis/farmacologia , Contração Miocárdica/efeitos dos fármacos , Infarto do Miocárdio/tratamento farmacológico , Miócitos Cardíacos/efeitos dos fármacos , Ouabaína/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Função Ventricular Esquerda/efeitos dos fármacos , Animais , Células Cultivadas , Modelos Animais de Doenças , Sinergismo Farmacológico , Quimioterapia Combinada , Preparação de Coração Isolado , Masculino , Infarto do Miocárdio/enzimologia , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Miócitos Cardíacos/enzimologia , Miócitos Cardíacos/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos Wistar , Transdução de Sinais
8.
Int J Mol Sci ; 19(8)2018 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-30087257

RESUMO

Bipolar disorder (BD) is a severe and common chronic mental illness characterized by recurrent mood swings between depression and mania. The biological basis of the disease is poorly understood and its treatment is unsatisfactory. Although in past decades the "monoamine hypothesis" has dominated our understanding of both the pathophysiology of depressive disorders and the action of pharmacological treatments, recent studies focus on the involvement of additional neurotransmitters/neuromodulators systems and cellular processes in BD. Here, evidence for the participation of Na⁺, K⁺-ATPase and its endogenous regulators, the endogenous cardiac steroids (ECS), in the etiology of BD is reviewed. Proof for the involvement of brain Na⁺, K⁺-ATPase and ECS in behavior is summarized and it is hypothesized that ECS-Na⁺, K⁺-ATPase-induced activation of intracellular signaling participates in the mechanisms underlying BD. We propose that the activation of ERK, AKT, and NFκB, resulting from ECS-Na⁺, K⁺-ATPase interaction, modifies neuronal activity and neurotransmission which, in turn, participate in the regulation of behavior and BD. These observations suggest Na⁺, K⁺-ATPase-mediated signaling is a potential target for drug development for the treatment of BD.


Assuntos
Transtorno Bipolar/metabolismo , Transdução de Sinais , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Transtorno Bipolar/etiologia , Transtorno Bipolar/patologia , Humanos , Sistema de Sinalização das MAP Quinases , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Esteroides/metabolismo
9.
J Pharmacol Exp Ther ; 357(2): 345-56, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26941172

RESUMO

Interaction of cardiac steroids (CS) with the Na(+), K(+)-ATPase elicits, in addition to inhibition of the enzyme's activity, the activation of intracellular signaling such as extracellular signal-regulated (ERK) and protein kinase B (Akt). We hypothesized that the activities of these pathways are involved in CS-induced increase in heart contractility. This hypothesis was tested using in vivo and ex vivo wild type (WT) and sarcoplasmic reticulum Ca(2+) atpase1a-deficient zebrafish (accordion, acc mutant) experimental model. Heart contractility was measured in vivo and in primary cardiomyocytes in WT zebrafish larvae and acc mutant. Ca(2+) transients were determined ex vivo in adult zebrafish hearts. CS dose dependently augmented the force of contraction of larvae heart muscle and cardiomyocytes and increased Ca(2+) transients in WT but not in acc mutant. CS in vivo increased the phosphorylation rate of ERK and Akt in the adult zebrafish heart of the two strains. Pretreatment of WT zebrafish larvae or cardiomyocytes with specific MAPK inhibitors completely abolished the CS-induced increase in contractility. On the contrary, pretreatment with Akt inhibitor significantly enhanced the CS-induced increase in heart contractility both in vivo and ex vivo without affecting CS-induced Ca(2+) transients. Furthermore, pretreatment of the acc mutant larvae or cardiomyocytes with Akt inhibitor restored the CS-induced increase in heart contractility also without affecting Ca(2+) transients. These results support the notion that the activity of MAPK pathway is obligatory for CS-induced increases in heart muscle contractility. Akt activity, on the other hand, plays a negative role, via Ca(2+) independent mechanisms, in CS action. These findings point to novel potential pharmacological intervention to increase CS efficacy.


Assuntos
Cardiotônicos/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Contração Miocárdica/efeitos dos fármacos , Proteína Oncogênica v-akt/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Esteroides/farmacologia , Animais , Sinalização do Cálcio/efeitos dos fármacos , Larva , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Mutação , Miócitos Cardíacos/efeitos dos fármacos , Proteína Oncogênica v-akt/antagonistas & inibidores , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/deficiência , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética
10.
Int J Cardiol ; 209: 296-306, 2016 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-26913371

RESUMO

BACKGROUND: The developmental origin of the c-kit expressing progenitor cell pool in the adult heart has remained elusive. Recently, it has been discovered that the injured heart is enriched with c-kit(+) cells, which also express the hematopoietic marker CD45. METHODS AND RESULTS: In this study, we characterize the phenotype and transcriptome of the c-kit+/CD45+/CD11b+/Flk-1+/Sca-1±(B-type) cell population, originating from the left atrial appendage. These cells are defined as cardiac macrophage progenitors. We also demonstrate that the CD45+ progenitor cell population activates heart development, neural crest and pluripotency-associated pathways in vitro, in conjunction with CD45 down-regulation, and acquire a c-kit+/CD45-/CD11b-/Flk-1-/Sca-1+ (A-type) phenotype through cell fusion and asymmetric division. This putative spontaneous reprogramming evolves into a highly proliferative, partially myogenic phenotype (C-type). CONCLUSIONS: Our data suggests that A-type cells and cardiac macrophage precursor cells (B-type) have a common lineage origin, possibly resolving some current conundrums in the field of cardiac regeneration.


Assuntos
Apêndice Atrial/fisiologia , Células-Tronco Hematopoéticas/fisiologia , Antígenos Comuns de Leucócito/fisiologia , Macrófagos/fisiologia , Fenótipo , Proteínas Proto-Oncogênicas c-kit/fisiologia , Animais , Apêndice Atrial/citologia , Células Cultivadas , Técnicas de Reprogramação Celular/métodos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos
11.
Can J Physiol Pharmacol ; 90(10): 1386-93, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22966876

RESUMO

Natriuretic peptides and digitalis-like compounds serve as regulators of homeostasis, including control of volume expansion and blood pressure. The aim of the present study was to explore possible interactions between atrial natriuretic peptide (ANP) and ouabain in the heart. ANP (1 nmol/L) had no effect in papillary muscle preparations from guinea pigs. Ouabain (1 µmol/L) induced positive inotropic effect. The addition of ANP prior to ouabain resulted in a significant decrease in the ouabain-induced positive inotropic effect, manifested as an attenuated increase in twitch maximal upward force slope and resting muscular tension. In addition, ANP caused an increase in Na⁺-K⁺-ATPase activity in heart microsomal preparations. The effect of ouabain on Na⁺-K⁺-ATPase activity was shown in a biphasic manner. Ouabain (0.01-1 nmol/L) had a small but significant increase on pump activity, but higher doses of ouabain inhibited activity. ANP attenuated ouabain-induced Na⁺-K⁺-ATPase activity. Furthermore, ouabain (50 nmol/L) or ANP (10 nmol/L) alone induced Akt activation in cardiomyocytes. However, ANP blocked ouabain-induced Akt activation. These results point to the existence of interactions between ANP and ouabain on Na⁺-K⁺-ATPase signaling and function in the heart, which may be mediated by regulation of Na⁺-K⁺-ATPase activity and (or) signal transduction mechanisms.


Assuntos
Fator Natriurético Atrial/metabolismo , Cardiotônicos/farmacologia , Contração Miocárdica/efeitos dos fármacos , Ouabaína/farmacologia , Músculos Papilares/efeitos dos fármacos , Animais , Cardiotônicos/antagonistas & inibidores , Células Cultivadas , Cobaias , Técnicas In Vitro , Masculino , Camundongos , Camundongos Transgênicos , Microssomos/efeitos dos fármacos , Microssomos/enzimologia , Microssomos/metabolismo , Tono Muscular/efeitos dos fármacos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Ouabaína/antagonistas & inibidores , Músculos Papilares/enzimologia , Músculos Papilares/metabolismo , Proteínas Proto-Oncogênicas c-akt/agonistas , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , ATPase Trocadora de Sódio-Potássio/química , ATPase Trocadora de Sódio-Potássio/genética , ATPase Trocadora de Sódio-Potássio/metabolismo , Regulação para Cima/efeitos dos fármacos
13.
J Biol Chem ; 287(9): 6518-29, 2012 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-22228762

RESUMO

Cardiac steroids (CS), an important class of naturally occurring compounds, are synthesized in plants and animals. The only established receptor for CS is the ubiquitous Na(+),K(+)-ATPase, a major plasma membrane transporter. The binding of CS to Na(+),K(+)-ATPase causes the inhibition of Na(+) and K(+) transport and elicits cell-specific activation of several intracellular signaling mechanisms. It is well documented that the interaction of CS with Na(+),K(+)-ATPase is responsible for numerous changes in basic cellular physiological properties, such as electrical plasma membrane potential, cell volume, intracellular [Ca(2+)] and pH, endocytosed membrane traffic, and the transport of other solutes. In the present study we show that CS induces the formation of dark structures adjacent to the nucleus in human NT2 and ACHN cells. These structures, which are not surrounded by membranes, are clusters of glycogen and a distorted microtubule network. Formation of these clusters results from a relocation of glycogen and microtubules in the cells, two processes that are independent of one another. The molecular mechanisms underlying the formation of the clusters are mediated by the Na(+),K(+)-ATPase, ERK1/2 signaling pathway, and an additional unknown factor. Similar glycogen clusters are induced by hypoxia, suggesting that the CS-induced structural change, described in this study, may be part of a new type of cellular stress response.


Assuntos
Bufanolídeos/farmacologia , Glicogênio/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , Adenocarcinoma , Cardiotônicos/farmacologia , Hipóxia Celular/fisiologia , Digoxigenina/farmacologia , Humanos , Neoplasias Renais , Sistema de Sinalização das MAP Quinases/fisiologia , Microscopia Eletrônica , Microtúbulos/ultraestrutura , Células-Tronco Neurais/citologia , Nocodazol/farmacologia , Ouabaína/análogos & derivados , Ouabaína/farmacologia , Potássio/farmacologia , RNA Interferente Pequeno/genética , ATPase Trocadora de Sódio-Potássio/genética , ATPase Trocadora de Sódio-Potássio/metabolismo , Estresse Fisiológico/fisiologia , Moduladores de Tubulina/farmacologia , Células Tumorais Cultivadas
14.
Am J Physiol Cell Physiol ; 302(2): C442-52, 2012 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-22031604

RESUMO

The endogenous cardiac steroid-like compounds, endogenous ouabain (EO) in particular, are present in the human circulation and are considered putative ligands of the inhibitory binding site of the plasma membrane Na(+)-K(+)-ATPase. A vast amount of data shows that, when added to cell cultures, these steroids promote the growth of cardiac, vascular, and epithelial cells. However, the involvement of the endogenous compounds in the regulation of cell viability and proliferation has never been addressed experimentally. In this study, we show that EO is present in mammalian sera and cerebral spinal fluid, as well as in commercial bovine and horse sera. The lowering of serum EO concentration by the addition of specific anti-ouabain antibodies caused a decrease in the viability of several cultured cell lines. Among these, neuronal NT2 cells were mostly affected, whereas no reduction in viability was seen in rat neuroendocrine PC12 and monkey kidney COS-7 cells. The anti-ouabain antibody-induced reduction in NT2 cell viability was significantly attenuated by the addition of ouabain and was not observed in cells growing in serum-free media. Furthermore, the addition to the medium of low concentrations (nM) of the cardenolide ouabain, but not of the bufadienolide bufalin, increased NT2 and PC12 cell viability and proliferation. In addition, at these concentrations both ouabain and bufalin caused the activation of ERK1/2 in the NT2 cells. The specific ERK1/2 inhibitor U0126 inhibited both the ouabain-induced activation of the enzyme and the increase in cell viability. Furthermore, anti-ouabain antibodies attenuated serum-stimulated ERK1/2 activity in NT2 but not in PC12 cells. Cumulatively, our results suggest that EO plays a significant role in the regulation of cell viability. In addition, our findings support the notion that activation of the ERK1/2 signaling pathway is obligatory but not sufficient for the induction of cell viability by EO.


Assuntos
Sobrevivência Celular/fisiologia , Inibidores Enzimáticos/sangue , Inibidores Enzimáticos/líquido cefalorraquidiano , Ouabaína/sangue , Ouabaína/líquido cefalorraquidiano , Animais , Anticorpos/metabolismo , Bufanolídeos/metabolismo , Bufanolídeos/farmacologia , Butadienos/metabolismo , Butadienos/farmacologia , Células COS , Bovinos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Chlorocebus aethiops , Relação Dose-Resposta a Droga , Ativação Enzimática , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Cavalos , Humanos , Nitrilas/metabolismo , Nitrilas/farmacologia , Ouabaína/farmacologia , Células PC12 , Ratos
15.
Am J Physiol Heart Circ Physiol ; 297(6): H2026-34, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19837951

RESUMO

Endogenous ouabain (EO)-like compounds are synthesized in and released from the adrenal gland. Although EO has been implicated in several pathological states such as hypertension and heart and kidney failure, its physiological roles in normal animal have not been elucidated. To address this issue, we studied the effects of reduction in plasma EO resulting from antiouabain antibody administration. Normal rats were treated for 28 days with antiouabain antibodies or rabbit IgG as control. Infusions were delivered through a jugular vein cannula by osmotic pumps, and blood pressure was monitored by tail-cuff plethysmography. The animals were housed in metabolic cages to measure water and food consumption and urine excretion. After 28 days, the thoracic aorta was isolated and used to study phenylephrine-induced contraction and atrial natriuretic peptide (ANP)-induced vasorelaxation. The adrenal gland cortex was enlarged in the antiouabain antibody-treated rats. Moreover, on the second day of treatment, there was a significant transient reduction in natriuresis in the antiouabain antibody-treated rats, suggesting that EO is a natriuretic hormone. Reduction in natriuresis was also observed when EO levels were reduced by active immunization resulting from sequential injection of ouabain-albumin. Furthermore, following 28 days of treatment, the response to phenylephrine was significantly lowered and that to ANP was significantly increased in aortic rings from antiouabain antibody-treated rats. These findings show for the first time that circulatory ouabain plausibly originating in the adrenal has physiological roles controlling vasculature tone and sodium homeostasis in normal rats.


Assuntos
Córtex Suprarrenal/metabolismo , Aorta Torácica/metabolismo , Cardenolídeos/sangue , Rim/metabolismo , Natriurese , Saponinas/sangue , Vasoconstrição , Vasodilatação , Aldosterona/sangue , Animais , Anticorpos/administração & dosagem , Aorta Torácica/efeitos dos fármacos , Fator Natriurético Atrial/metabolismo , Pressão Sanguínea , Cardenolídeos/imunologia , Corticosterona/sangue , Relação Dose-Resposta a Droga , Homeostase , Infusões Intravenosas , Masculino , Fenilefrina/farmacologia , Coelhos , Ratos , Ratos Wistar , Saponinas/imunologia , Sódio/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Fatores de Tempo , Vasoconstrição/efeitos dos fármacos , Vasoconstritores/farmacologia , Aumento de Peso
16.
Biol Psychiatry ; 65(11): 985-91, 2009 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-19058785

RESUMO

BACKGROUND: The sodium- and potassium-activated adenosine triphosphatase (Na+, K+-ATPase) is a major plasma membrane transporter for sodium and potassium. We recently suggested that bipolar disorders (BD) may be associated with alterations in brain Na+, K+-ATPase. We further conjectured that the differences in Na+, K+-ATPase in BD patients could result partially from genetic variations in Na+, K+-ATPase alpha isoforms. METHODS: To test our hypothesis, we undertook a comprehensive study of 13 tagged single nucleotide polymorphisms (SNPs) across the three genes of the brain alpha isoforms of Na+, K+- ATPase (ATP1A1, ATP1A2, and ATP1A3, which encode the three alpha isoforms, alpha1, alpha2, and alpha3, respectively) identified using HapMap data and the Haploview algorithm. Altogether, 126 subjects diagnosed with BD from 118 families were genotyped (parents and affected siblings). Both individual SNPs and haplotypes were tested for association using family-based association tests as provided in the UNPHASED and PBAT set of programs. RESULTS: Significant nominal association with BD was observed for six single SNPs (alpha1: rs11805078; alpha2: rs2070704, rs1016732, rs2854248, and rs2295623; alpha3: rs919390) in the three genes of Na+, K+-ATPase alpha isoforms. Haplotype analysis of the alpha2 isoform (ATP1A2 gene) showed a significant association with two loci haplotypes with BD (rs2295623: rs2070704; global p value = .0198, following a permutation test). CONCLUSIONS: This study demonstrates for the first time that genetic variations in Na+, K+-ATPase are associated with BD, suggesting a role of this enzyme in the etiology of this disease.


Assuntos
Transtorno Bipolar/genética , Predisposição Genética para Doença , Polimorfismo de Nucleotídeo Único/genética , Isoformas de Proteínas/genética , ATPase Trocadora de Sódio-Potássio/genética , Análise Mutacional de DNA , Saúde da Família , Feminino , Frequência do Gene , Genótipo , Humanos , Masculino , ATPase Trocadora de Sódio-Potássio/classificação
17.
Am J Physiol Cell Physiol ; 293(3): C885-96, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17553933

RESUMO

Plasma membrane Na(+)-K(+)-ATPase, which drives potassium into and sodium out of the cell, has important roles in numerous physiological processes. Cardiac steroids (CS), such as ouabain and bufalin, specifically interact with the pump and affect ionic homeostasis, signal transduction, and endocytosed membrane traffic. CS-like compounds are present in mammalian tissues, synthesized in the adrenal gland, and considered to be new family of steroid hormones. In this study, the mechanism of Na(+)-K(+)-ATPase involvement in the regulation of endocytosis is explored. We show that the effects of various CS on changes in endosomal pH are mediated by the pump and correspond to their effects on endosomal membrane traffic. In addition, it was found that CS-induced changes in endocytosed membrane traffic were dependent on alterations in [Na(+)] and [H(+)] in the endosome. Furthermore, we show that various CS differentially regulate endosomal pH and membrane traffic. The results suggest that these differences are due to specific binding characteristics. Based on our observations, we propose that Na(+)-K(+)-ATPase is a key player in the regulation of endosomal pH and endocytosed membrane traffic. Furthermore, our results raise the possibility that CS-like hormones regulate differentially intracellular membrane traffic.


Assuntos
Cardiotônicos/farmacologia , Endocitose/fisiologia , Ouabaína/farmacologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Células-Tronco/enzimologia , Ácidos/metabolismo , Bufanolídeos/farmacologia , Cardiotônicos/metabolismo , Linhagem Celular , Membrana Celular/enzimologia , Digoxina/metabolismo , Digoxina/farmacologia , Endocitose/efeitos dos fármacos , Endossomos/enzimologia , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Humanos , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Neurônios/citologia , Ouabaína/metabolismo , Potássio/metabolismo , Transporte Proteico/fisiologia , Sódio/metabolismo , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , Células-Tronco/citologia , Transferrina/metabolismo , Trítio
18.
Life Sci ; 80(23): 2093-2107, 2007 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-17499813

RESUMO

Digitalis-like compounds (DLC) are a family of steroid hormones synthesized in and released from the adrenal gland. DLC, the structure of which resembles that of plant cardiac glycosides, bind to and inhibit the activity of the ubiquitous cell surface enzyme Na(+), K(+)-ATPase. However, there is a large body of evidence suggesting that the regulation of ion transport by Na(+), K(+)-ATPase is not the only physiological role of DLC. The binding of DLC to Na(+), K(+)-ATPase induces the activation of various signal transduction cascades that activate changes in intracellular Ca(++) homeostasis, and in specific gene expression. These, in turn, stimulate endocytosis and affect cell growth and proliferation. At the systemic level, DLC were shown to be involved in the regulation of major physiological parameters including water and salt homeostasis, cardiac contractility and rhythm, systemic blood pressure and behavior. Furthermore, the DLC system has been implicated in several pathological conditions, including cardiac arrhythmias, hypertension, cancer and depressive disorders. This review evaluates the evidence for the different aspects of DLC action and delineates open questions in the field.


Assuntos
Adenosina Trifosfatases/metabolismo , Digitalis/metabolismo , Potássio/química , Sódio/metabolismo , Esteroides/metabolismo , Animais , Transporte Biológico , Endocitose , Humanos , Íons , Modelos Biológicos , Natriuréticos/metabolismo , Ouabaína/farmacologia , Extratos Vegetais/farmacologia , ATPase Trocadora de Sódio-Potássio/química
19.
Biol Psychiatry ; 60(5): 491-9, 2006 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-16712803

RESUMO

BACKGROUND: Sodium and potassium-activated adenosine triphosphatase (Na(+), K(+)-ATPase) and endogenous digitalis-like compounds (DLC) in the brain have been implicated in the pathogenesis of mood disorders. This hypothesis was examined by the determination of Na(+), K(+)-ATPase/DLC system in parietal cortex of patients with different mood disorders and two animal models of depression. METHODS: Na(+), K(+)-ATPase concentrations in human brain synaptosomal fractions, from patients with mood disorders, schizophrenia, and normal individuals, were determined by (3)H-ouabain binding assay. Alpha isoforms were quantified by Western blotting. Brain DLC were measured using sensitive enzyme linked immunosorbant assay (ELISA). The effects of ouabain and ouabain-antibodies on behavior were determined in two animal models of depression. RESULTS: (3)H-ouabain binding in bipolar patients was significantly lower than in major depressed and schizophrenic patients. Na(+), K(+)-ATPase alpha isoforms in synaptosomal fractions were not different among the groups. DLC levels in the parietal cortex of bipolar patients were significantly higher than in normal individuals and depressed patients. Injection of lipopolysaccharide (intraperitoneally) to rats elicited depression-like symptoms, which were significantly attenuated by pre-injection of ouabain-antibodies. Injection of ouabain and ouabain-antibodies (intracerebroventricular) reduced depression-like symptoms in the forced swimming test in rats. CONCLUSIONS: The results support the possibility that Na(+), K(+)-ATPase and endogenous DLC participate in the pathogenesis of depressive disorders.


Assuntos
Bufanolídeos/metabolismo , Cardenolídeos/metabolismo , Transtorno Depressivo/enzimologia , Lobo Parietal/enzimologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Sinaptossomos/enzimologia , Adulto , Animais , Comportamento Animal/fisiologia , Transtorno Bipolar/enzimologia , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Transtornos do Humor/enzimologia , Ouabaína/metabolismo , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Valores de Referência , Esquizofrenia/enzimologia
20.
Mol Biol Cell ; 15(3): 1044-54, 2004 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-14718569

RESUMO

Cardiac steroids (CSs) are specific inhibitors of Na+, K(+)-ATPase activity. Although the presence of CS-like compounds in animal tissues has been established, their physiological role is not evident. In the present study, treatment of human NT2 cells with physiological concentrations (nanomolar) of CSs caused the accumulation of large vesicles adjacent to the nucleus. Experiments using N-(3-triethylammonium propyl)-4-(dibutilamino)styryl-pyrodinum dibromide, transferrin, low-density lipoprotein, and selected anti-transferrin receptor and Rab protein antibodies revealed that CSs induced changes in endocytosis-dependent membrane traffic. Our data indicate that the CS-induced accumulation of cytoplasmic membrane components is a result of inhibited recycling within the late endocytic pathway. Furthermore, our results support the notion that the CS-induced changes in membrane traffic is mediated by the Na+, K(+)-ATPase. These phenomena were apparent in NT2 cells at nanomolar concentrations of CSs and were observed also in other human cell lines, pointing to the generality of this phenomenon. Based on these observations, we propose that the endogenous CS-like compounds are physiological regulators of recycling of endocytosed membrane proteins and cargo.


Assuntos
Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Endocitose/efeitos dos fármacos , Esteroides/farmacologia , Transferrina/metabolismo , Bufanolídeos/farmacologia , Membrana Celular/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Células Cultivadas , Endocitose/fisiologia , Inibidores Enzimáticos/farmacologia , Humanos , Lipoproteínas LDL , Microscopia de Fluorescência , Miocárdio/metabolismo , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , ATPase Trocadora de Sódio-Potássio/metabolismo
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