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1.
Int J Mol Sci ; 22(19)2021 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-34639150

RESUMO

This review presents the latest data on the importance of selenium nanoparticles in human health, their use in medicine, and the main known methods of their production by various methods. In recent years, a multifaceted study of nanoscale complexes in medicine, including selenium nanoparticles, has become very important in view of a number of positive features that make it possible to create new drugs based on them or significantly improve the properties of existing drugs. It is known that selenium is an essential trace element that is part of key antioxidant enzymes. In mammals, there are 25 selenoproteins, in which selenium is a key component of the active site. The important role of selenium in human health has been repeatedly proven by several hundred works in the past few decades; in recent years, the study of selenium nanocomplexes has become the focus of researchers. A large amount of accumulated data requires generalization and systematization in order to improve understanding of the key mechanisms and prospects for the use of selenium nanoparticles in medicine, which is the purpose of this review.

2.
Int J Mol Sci ; 22(15)2021 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-34360859

RESUMO

The aim of the study was to investigate the mechanisms of Ca2+ oscillation generation upon activation of connexin-43 and regulation of the lipolysis/lipogenesis balance in white adipocytes through vesicular ATP release. With fluorescence microscopy it was revealed that a decrease in the concentration of extracellular calcium ([Ca2+]ex) results in two types of Ca2+ responses in white adipocytes: Ca2+ oscillations and transient Ca2+ signals. It was found that activation of the connexin half-channels is involved in the generation of Ca2+ oscillations, since the blockers of the connexin hemichannels-carbenoxolone, octanol, proadifen and Gap26-as well as Cx43 gene knockdown led to complete suppression of these signals. The activation of Cx43 in response to the reduction of [Ca2+]ex was confirmed by TIRF microscopy. It was shown that in response to the activation of Cx43, ATP-containing vesicles were released from the adipocytes. This process was suppressed by knockdown of the Cx43 gene and by bafilomycin A1, an inhibitor of vacuolar ATPase. At the level of intracellular signaling, the generation of Ca2+ oscillations in white adipocytes in response to a decrease in [Ca2+]ex occurred due to the mobilization of the Ca2+ ions from the thapsigargin-sensitive Ca2+ pool of IP3R as a result of activation of the purinergic P2Y1 receptors and phosphoinositide signaling pathway. After activation of Cx43 and generation of the Ca2+ oscillations, changes in the expression levels of key genes and their encoding proteins involved in the regulation of lipolysis were observed in white adipocytes. This effect was accompanied by a decrease in the number of adipocytes containing lipid droplets, while inhibition or knockdown of Cx43 led to inhibition of lipolysis and accumulation of lipid droplets. In this study, we investigated the mechanism of Ca2+ oscillation generation in white adipocytes in response to a decrease in the concentration of Ca2+ ions in the external environment and established an interplay between periodic Ca2+ modes and the regulation of the lipolysis/lipogenesis balance.


Assuntos
Adipócitos Brancos/metabolismo , Cálcio/metabolismo , Conexina 43/fisiologia , Lipogênese , Lipólise , Adipócitos Brancos/citologia , Animais , Sinalização do Cálcio , Células Cultivadas , Camundongos
3.
Int J Mol Sci ; 22(16)2021 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-34445509

RESUMO

Ischemia-like conditions reflect almost the entire spectrum of events that occur during cerebral ischemia, including the induction of oxidative stress, Ca2+ overload, glutamate excitotoxicity, and activation of necrosis and apoptosis in brain cells. Mechanisms for the protective effects of the antioxidant enzyme peroxiredoxin-6 (Prx-6) on hippocampal cells during oxygen-glucose deprivation/reoxygenation (OGD/R) were investigated. Using the methods of fluorescence microscopy, inhibitory analysis, vitality tests and PCR, it was shown that 24-h incubation of mixed hippocampal cell cultures with Prx-6 does not affect the generation of a reversible phase of a OGD-induced rise in Ca2+ ions in cytosol ([Ca2+]i), but inhibits a global increase in [Ca2+]i in astrocytes completely and in neurons by 70%. In addition, after 40 min of OGD, cell necrosis is suppressed, especially in the astrocyte population. This effect is associated with the complex action of Prx-6 on neuroglial networks. As an antioxidant, Prx-6 has a more pronounced and astrocyte-directed effect, compared to the exogenous antioxidant vitamin E (Vit E). Prx-6 inhibits ROS production in mitochondria by increasing the antioxidant capacity of cells and altering the expression of genes encoding redox status proteins. Due to the close bond between [Ca2+]i and intracellular ROS, this effect of Prx-6 is one of its protective mechanisms. Moreover, Prx-6 effectively suppresses not only necrosis, but also apoptosis during OGD and reoxygenation. Incubation with Prx-6 leads to activation of the basic expression of genes encoding protective kinases-PI3K, CaMKII, PKC, anti-apoptotic proteins-Stat3 and Bcl-2, while inhibiting the expression of signaling kinases and factors involved in apoptosis activation-Ikk, Src, NF-κb, Caspase-3, p53, Fas, etc. This effect on the basic expression of the genome leads to the cell preconditions, which is expressed in the inhibition of caspase-3 during OGD/reoxygenation. A significant effect of Prx-6 is directed on suppression of the level of pro-inflammatory cytokine IL-1ß and factor TNFα, as well as genes encoding NMDA- and kainate receptor subunits, which was established for the first time for this antioxidant enzyme. The protective effect of Prx-6 is due to its antioxidant properties, since mutant Prx-6 (mutPrx-6, Prx6-C47S) leads to polar opposite effects, contributing to oxidative stress, activation of apoptosis and cell death through receptor action on TLR4.


Assuntos
Astrócitos/citologia , Hipocampo/citologia , Peroxirredoxina VI/metabolismo , Traumatismo por Reperfusão/metabolismo , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Cálcio/metabolismo , Células Cultivadas , Citosol/metabolismo , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Interleucina-1beta/metabolismo , Microscopia de Fluorescência , Peroxirredoxina VI/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
4.
Int J Mol Sci ; 22(15)2021 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-34360564

RESUMO

In recent decades, studies on the functional features of Se nanoparticles (SeNP) have gained great popularity due to their high biocompatibility, stability, and pronounced selectivity. A large number of works prove the anticarcinogenic effect of SeNP. In this work, the molecular mechanisms regulating the cytotoxic effects of SeNP, obtained by laser ablation, were studied by the example of four human cancer cell lines: A-172 (glioblastoma), Caco-2, (colorectal adenocarcinoma), DU-145 (prostate carcinoma), MCF-7 (breast adenocarcinoma). It was found that SeNP had different concentration-dependent effects on cancer cells of the four studied human lines. SeNP at concentrations of less than 1 µg/mL had no cytotoxic effect on the studied cancer cells, with the exception of the A-172 cell line, for which 0.5 µg/mL SeNP was the minimum concentration affecting its metabolic activity. It was shown that SeNP concentration-dependently caused cancer cell apoptosis, but not necrosis. In addition, it was found that SeNP enhanced the expression of pro-apoptotic genes in almost all cancer cell lines, with the exception of Caco-2 and activated various pathways of adaptive and pro-apoptotic signaling pathways of UPR. Different effects of SeNP on the expression of ER-resident selenoproteins and selenium-containing glutathione peroxidases and thioredoxin reductases, depending on the cell line, were established. In addition, SeNP triggered Ca2+ signals in all investigated cancer cell lines. Different sensitivity of cancer cell lines to SeNP can determine the induction of the process of apoptosis in them through regulation of the Ca2+ signaling system, mechanisms of ER stress, and activation of various expression patterns of genes encoding pro-apoptotic proteins.


Assuntos
Antineoplásicos/administração & dosagem , Apoptose , Citotoxinas/administração & dosagem , Nanopartículas/administração & dosagem , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Selênio/química , Antineoplásicos/química , Citotoxinas/química , Humanos , Nanopartículas/química , Transdução de Sinais , Células Tumorais Cultivadas
5.
Int J Mol Sci ; 22(12)2021 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-34205571

RESUMO

Studies of recent decades have repeatedly demonstrated the cytotoxic effect of selenium-containing compounds on cancer cells of various origins. Particular attention in these studies is paid to methylseleninic acid, a widespread selenium-containing compound of organic nature, for several reasons: it has a selective cytotoxic effect on cancer cells, it is cytotoxic in small doses, it is able to generate methylselenol, excluding the action of the enzyme ß-lyase. All these qualities make methylseleninic acid an attractive substrate for the production of anticancer drugs on its basis with a well-pronounced selective effect. However, the studies available to date indicate that there is no strictly specific molecular mechanism of its cytotoxic effect in relation to different cancer cell lines and cancer models. This review contains generalized information on the dose- and time-dependent regulation of the toxic effect of methylseleninic acid on the proliferative properties of a number of cancer cell lines. In addition, special attention in this review is paid to the influence of this selenium-containing compound on the regulation of endoplasmic reticulum stress and on the expression of seven selenoproteins, which are localized in the endoplasmic reticulum.


Assuntos
Carcinogênese/efeitos dos fármacos , Citotoxinas/uso terapêutico , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Compostos Organosselênicos/toxicidade , Animais , Humanos , Compostos Organosselênicos/uso terapêutico , Selenoproteínas/metabolismo
6.
Int J Mol Sci ; 22(11)2021 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-34073140

RESUMO

Transcription factors Satb1 and Satb2 are involved in the processes of cortex development and maturation of neurons. Alterations in the expression of their target genes can lead to neurodegenerative processes. Molecular and cellular mechanisms of regulation of neurotransmission by these transcription factors remain poorly understood. In this study, we have shown that transcription factors Satb1 and Satb2 participate in the regulation of genes encoding the NMDA-, AMPA-, and KA- receptor subunits and the inhibitory GABA(A) receptor. Deletion of gene for either Satb1 or Satb2 homologous factors induces the expression of genes encoding the NMDA receptor subunits, thereby leading to higher amplitudes of Ca2+-signals in neurons derived from the Satb1-deficient (Satb1fl/+ * NexCre/+) and Satb1-null mice (Satb1fl/fl * NexCre/+) in response to the selective agonist reducing the EC50 for the NMDA receptor. Simultaneously, there is an increase in the expression of the Gria2 gene, encoding the AMPA receptor subunit, thus decreasing the Ca2+-signals of neurons in response to the treatment with a selective agonist (5-Fluorowillardiine (FW)). The Satb1 deletion increases the sensitivity of the KA receptor to the agonist (domoic acid), in the cortical neurons of the Satb1-deficient mice but decreases it in the Satb1-null mice. At the same time, the Satb2 deletion decreases Ca2+-signals and the sensitivity of the KA receptor to the agonist in neurons from the Satb1-null and the Satb1-deficient mice. The Satb1 deletion affects the development of the inhibitory system of neurotransmission resulting in the suppression of the neuron maturation process and switching the GABAergic responses from excitatory to inhibitory, while the Satb2 deletion has a similar effect only in the Satb1-null mice. We show that the Satb1 and Satb2 transcription factors are involved in the regulation of the transmission of excitatory signals and inhibition of the neuronal network in the cortical cell culture.


Assuntos
Sinalização do Cálcio , Proteínas de Ligação à Região de Interação com a Matriz/fisiologia , Neuroglia , Receptores de Glutamato/metabolismo , Transmissão Sináptica , Fatores de Transcrição/fisiologia , Animais , Células Cultivadas , Feminino , Masculino , Camundongos , Camundongos Knockout , Neuroglia/citologia , Neuroglia/metabolismo , Receptores de GABA/metabolismo
7.
Int J Mol Sci ; 22(10)2021 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-34065973

RESUMO

Various types of cells demonstrate ubiquitous rhythmicity registered as simple and complex Ca2+-oscillations, spikes, waves, and triggering phenomena mediated by G-protein and tyrosine kinase coupled receptors. Phospholipase C/IP3-receptors (PLC/IP3R) and endothelial NO-synthase/Ryanodine receptors (NOS/RyR)-dependent Ca2+ signaling systems, organized as multivariate positive feedback generators (PLC-G and NOS-G), underlie this rhythmicity. Loss of rhythmicity at obesity may indicate deregulation of these signaling systems. To issue the impact of cell size, receptors' interplay, and obesity on the regulation of PLC-G and NOS-G, we applied fluorescent microscopy, immunochemical staining, and inhibitory analysis using cultured adipocytes of epididumal white adipose tissue of mice. Acetylcholine, norepinephrine, atrial natriuretic peptide, bradykinin, cholecystokinin, angiotensin II, and insulin evoked complex [Ca2+]i responses in adipocytes, implicating NOS-G or PLC-G. At low sub-threshold concentrations, acetylcholine and norepinephrine or acetylcholine and peptide hormones (in paired combinations) recruited NOS-G, based on G proteins subunits interplay and signaling amplification. Rhythmicity was cell size- dependent and disappeared in hypertrophied cells filled with lipids. Contrary to control cells, adipocytes of obese hyperglycemic and hypertensive mice, growing on glucose, did not accumulate lipids and demonstrated hormonal resistance being non responsive to any hormone applied. Preincubation of preadipocytes with palmitoyl-L-carnitine (100 nM) provided accumulation of lipids, increased expression and clustering of IP3R and RyR proteins, and partially restored hormonal sensitivity and rhythmicity (5-15% vs. 30-80% in control cells), while adipocytes of diabetic mice were not responsive at all. Here, we presented a detailed kinetic model of NOS-G and discussed its control. Collectively, we may suggest that universal mechanisms underlie loss of rhythmicity, Ca2+-signaling systems deregulation, and development of general hormonal resistance to obesity.


Assuntos
Adipócitos Brancos/metabolismo , Sinalização do Cálcio , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Obesidade/metabolismo , Adipócitos Brancos/citologia , Adipócitos Brancos/efeitos dos fármacos , Animais , Sinalização do Cálcio/efeitos dos fármacos , Tamanho Celular , Células Cultivadas , Diabetes Mellitus Tipo 2/etiologia , Dieta Hiperlipídica/efeitos adversos , Epididimo , Proteínas de Ligação ao GTP/metabolismo , Masculino , Camundongos , Óxido Nítrico Sintase Tipo III/metabolismo , Obesidade/induzido quimicamente , Palmitoilcarnitina/farmacologia , Periodicidade , Cultura Primária de Células , Fosfolipases Tipo C/metabolismo
8.
J Neurosci ; 40(49): 9364-9371, 2020 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-33122390

RESUMO

Mechanosensitivity is a well-known feature of astrocytes, however, its underlying mechanisms and functional significance remain unclear. There is evidence that astrocytes are acutely sensitive to decreases in cerebral perfusion pressure and may function as intracranial baroreceptors, tuned to monitor brain blood flow. This study investigated the mechanosensory signaling in brainstem astrocytes, as these cells reside alongside the cardiovascular control circuits and mediate increases in blood pressure and heart rate induced by falls in brain perfusion. It was found that mechanical stimulation-evoked Ca2+ responses in astrocytes of the rat brainstem were blocked by (1) antagonists of connexin channels, connexin 43 (Cx43) blocking peptide Gap26, or Cx43 gene knock-down; (2) antagonists of TRPV4 channels; (3) antagonist of P2Y1 receptors for ATP; and (4) inhibitors of phospholipase C or IP3 receptors. Proximity ligation assay demonstrated interaction between TRPV4 and Cx43 channels in astrocytes. Dye loading experiments showed that mechanical stimulation increased open probability of carboxyfluorescein-permeable membrane channels. These data suggest that mechanosensory Ca2+ responses in astrocytes are mediated by interaction between TRPV4 and Cx43 channels, leading to Cx43-mediated release of ATP which propagates/amplifies Ca2+ signals via P2Y1 receptors and Ca2+ recruitment from the intracellular stores. In astrocyte-specific Cx43 knock-out mice the magnitude of heart rate responses to acute increases in intracranial pressure was not affected by Cx43 deficiency. However, these animals displayed lower heart rates at different levels of cerebral perfusion, supporting the hypothesis of connexin hemichannel-mediated release of signaling molecules by astrocytes having an excitatory action on the CNS sympathetic control circuits.SIGNIFICANCE STATEMENT There is evidence suggesting that astrocytes may function as intracranial baroreceptors that play an important role in the control of systemic and cerebral circulation. To function as intracranial baroreceptors, astrocytes must possess a specialized membrane mechanism that makes them exquisitely sensitive to mechanical stimuli. This study shows that opening of connexin 43 (Cx43) hemichannels leading to the release of ATP is the key central event underlying mechanosensory Ca2+ responses in astrocytes. This astroglial mechanism plays an important role in the autonomic control of heart rate. These data add to the growing body of evidence suggesting that astrocytes function as versatile surveyors of the CNS metabolic milieu, tuned to detect conditions of potential metabolic threat, such as hypoxia, hypercapnia, and reduced perfusion.


Assuntos
Astrócitos/fisiologia , Mecanotransdução Celular/fisiologia , Trifosfato de Adenosina/metabolismo , Animais , Pressão Sanguínea/efeitos dos fármacos , Tronco Encefálico/citologia , Tronco Encefálico/efeitos dos fármacos , Tronco Encefálico/fisiologia , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Circulação Cerebrovascular/fisiologia , Conexina 43/antagonistas & inibidores , Conexina 43/genética , Feminino , Frequência Cardíaca/fisiologia , Masculino , Mecanotransdução Celular/efeitos dos fármacos , Camundongos , Camundongos Knockout , Peptídeos/antagonistas & inibidores , Peptídeos/genética , Estimulação Física , Ratos , Receptores Purinérgicos P2Y1/efeitos dos fármacos , Canais de Cátion TRPV/antagonistas & inibidores , Canais de Cátion TRPV/genética
9.
Int J Neurosci ; : 1-12, 2020 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-32727246

RESUMO

OBJECTIVE: This study aimed to investigate the connection between the mutation of the Sip1 transcription factor and impaired Ca2+-signaling, which reflects changes in neurotransmission in the cerebral cortex in vitro. METHODS: We used mixed neuroglial cortical cell cultures derived from Sip1 mutant mice. The cells were loaded with a fluorescent ratiometric calcium-sensitive probe Fura-2 AM and epileptiform activity was modeled by excluding magnesium ions from the external media or adding a GABA(A) receptor antagonist, bicuculline. Intracellular calcium dynamics were recorded using fluorescence microscopy. To identify the level of gene expression, the Real-Time PCR method was used. RESULTS: It was found that cortical neurons isolated from homozygous (Sip1fl/fl) mice with the Sip1 mutation demonstrate suppressed Ca2+ signals in models of epileptiform activity in vitro. Wild-type cortical neurons are characterized by synchronous high-frequency and high-amplitude Ca2+ oscillations occurring in all neurons of the network in response to Mg2+-free medium and bicuculline. But cortical Sip1fl/fl neurons only single Ca2+ pulses or attenuated Ca2+ oscillations are recorded and only in single neurons, while most of the cell network does not respond to these stimuli. This signal deficiency of Sip1fl/fl neurons correlates with a suppressed expression level of the genes encoding the subunits of NMDA, AMPA, and KA receptors; protein kinases PKA, JNK, CaMKII; and also the transcription factor Hif1α. These negative effects were partially abolished when Sip1fl/fl neurons are grown in media with anti-inflammatory cytokine IL-10. IL-10 increases the expression of the above-mentioned genes but not to the level of expression in wild-type. At the same time, the amplitudes of Ca2+ signals increase in response to the selective agonists of NMDA, AMPA and KA receptors, and the proportion of neurons responding with Ca2+ oscillations to a Mg2+-free medium and bicuculline increases. CONCLUSION: IL-10 restores neurotransmission in neuronal networks with the Sip1 mutation by regulating the expression of genes encoding signaling proteins.

10.
Mol Cell Biochem ; 456(1-2): 191-204, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30756222

RESUMO

Intracellular Са2+ controls its own level by regulation of Ca2+ transport across the plasma and organellar membranes, often acting via calmodulin (CaM). Drugs antagonizing CaM action induce an increase in cytosolic Ca2+ concentration in different cells. We have found persistent Са2+ oscillations in cultured white adipocytes in response to calmidazolium (CMZ). They appeared at [CMZ] > 1 µM as repetitive sharp spikes mainly superimposed on a transient or elevated baseline. Similar oscillations were observed when we used trifluoperazine. Oscillations evoked by 5 µM CMZ resulted from the release of stored Ca2+ and were supported by Са2+ entry. Inhibition of store-operated channels by YM-58483 or 2-APB did not change the responses. Phospholipase A2 inhibited by AACOCF3 was responsible for initial Ca2+ mobilization, but not for subsequent oscillations, whereas inhibition of iPLA2 by BEL had no effect. Phospholipase C was partially involved in both stages as revealed with U73122. Intracellular Са2+ stores engaged by CMZ were entirely dependent on thapsigargin. The oscillations existed in the presence of inhibitors of ryanodine or inositol 1,4,5-trisphosphate receptors, or antagonists of Ca2+ transport by lysosome-like acidic stores. Carbenoxolone or octanol, blockers of hemichannels (connexons), when applied for two hours, prevented oscillations but did not affect the initial Са2+ release. Incubation with La3+ for 2 or 24 h inhibited all responses to CMZ, retaining the thapsigargin-induced Ca2+ rise. These results suggest that Ca2+-CaM regulation suppresses La3+-sensitive channels in non-acidic organelles, of which arachidonate-activated channels initiate Ca2+ oscillations, and connexons are intimately implicated in their generation mechanism.


Assuntos
Adipócitos Brancos/metabolismo , Ácido Araquidônico/metabolismo , Canais de Cálcio/metabolismo , Sinalização do Cálcio , Cálcio/metabolismo , Calmodulina/metabolismo , Adipócitos Brancos/citologia , Animais , Imidazóis/farmacologia , Camundongos
11.
Arch Biochem Biophys ; 654: 126-135, 2018 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-30056076

RESUMO

The Sip1 mutation plays the main role in pathogenesis of the Mowat-Wilson syndrome, which is characterized by the pronounced epileptic symptoms. Cortical neurons of homozygous mice with Sip1 mutation are resistant to AMPA receptor activators. Disturbances of the excitatory signaling components are also observed on such a phenomenon of neuroplasticity as hypoxic preconditioning. In this work, the mechanisms of loss of the AMPA receptor's ability to precondition by episodes of short-term hypoxia were investigated on cortical neurons derived from the Sip1 homozygous mice. The preconditioning effect was estimated by the level of suppression of the AMPA receptors activity with hypoxia episodes. Using fluorescence microscopy, we have shown that cortical neurons from the Sip1fl/fl mice are characterized by the absence of hypoxic preconditioning effect, whereas the amplitude of Ca2+-responses to the application of the AMPA receptor agonist, 5-Fluorowillardiine, in neurons from the Sip1 mice brainstem is suppressed by brief episodes of hypoxia. The mechanism responsible for this process is hypoxia-induced desensitization of the AMPA receptors, which is absent in the cortex neurons possessing the Sip1 mutation. However, the appearance of preconditioning in these neurons can be induced by phosphoinositide-3-kinase activation with a selective activator or an anti-inflammatory cytokine interleukin-10.


Assuntos
Córtex Cerebral/fisiopatologia , Hipóxia/fisiopatologia , Interleucina-10/fisiologia , Mutação , Proteínas do Tecido Nervoso/fisiologia , Neurônios/fisiologia , Receptores de AMPA/fisiologia , Alanina/análogos & derivados , Alanina/farmacologia , Animais , Cálcio/metabolismo , Sinalização do Cálcio , Ativação Enzimática , Agonistas de Aminoácidos Excitatórios/farmacologia , Camundongos , Microscopia de Fluorescência , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Pirimidinas/farmacologia , Receptores de AMPA/agonistas
12.
Nat Commun ; 9(1): 370, 2018 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-29371650

RESUMO

Astrocytes are implicated in modulation of neuronal excitability and synaptic function, but it remains unknown if these glial cells can directly control activities of motor circuits to influence complex behaviors in vivo. This study focused on the vital respiratory rhythm-generating circuits of the preBötzinger complex (preBötC) and determined how compromised function of local astrocytes affects breathing in conscious experimental animals (rats). Vesicular release mechanisms in astrocytes were disrupted by virally driven expression of either the dominant-negative SNARE protein or light chain of tetanus toxin. We show that blockade of vesicular release in preBötC astrocytes reduces the resting breathing rate and frequency of periodic sighs, decreases rhythm variability, impairs respiratory responses to hypoxia and hypercapnia, and dramatically reduces the exercise capacity. These findings indicate that astrocytes modulate the activity of CNS circuits generating the respiratory rhythm, critically contribute to adaptive respiratory responses in conditions of increased metabolic demand and determine the exercise capacity.


Assuntos
Astrócitos/fisiologia , Tronco Encefálico/fisiologia , Periodicidade , Condicionamento Físico Animal/fisiologia , Respiração , Potenciais de Ação/fisiologia , Adenoviridae/genética , Adenoviridae/metabolismo , Animais , Animais Recém-Nascidos , Astrócitos/citologia , Tronco Encefálico/citologia , Cálcio/metabolismo , Feminino , Regulação da Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Hipercapnia/metabolismo , Hipercapnia/fisiopatologia , Hipóxia/metabolismo , Hipóxia/fisiopatologia , Masculino , Bulbo/citologia , Bulbo/fisiologia , Cultura Primária de Células , Ratos , Ratos Sprague-Dawley , Proteínas SNARE/antagonistas & inibidores , Proteínas SNARE/genética , Proteínas SNARE/metabolismo
13.
Neurosci Lett ; 650: 180-186, 2017 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-28455101

RESUMO

Smad-interacting protein-1 (Sip1) [Zinc finger homeobox (Zfhx1b), Zeb2] is a transcription factor implicated in the genesis of Mowat-Wilson syndrome (MWS) in humans. MWS is a rare genetic autosomal dominant disease caused by a mutation in the Sip1 gene (aka Zeb2 or Zfhx1b) mapped to 2q22.3 locus. MWS affects 1 in every 50-100 newborns worldwide. It is characterized by mental retardation, small stature, typical facial abnormalities as well as disturbances in the development of the cardio-vascular and renal systems as well as some other organs. Sip1 mutations cause abnormal neurogenesis in the brain during development as well as susceptibility to epileptic seizures. In the current study we investigated the role of the Sip1 gene in the activity of NMDA-, AMPA- and KA- receptors. We showed that a particular Sip1 mutation in the mouse causes changes in the activity of both NMDA- and AMPA- receptors in the neocortical neurons in vitro. We demonstrate that neocortical neurons that have only one copy of Sip1 (heterozygous, Sip1fI/wt), are more sensitive to both NMDA- and AMPA- receptors agonists as compared to wild type neurons (Sip1wt/wt). This is reflected in higher amplitudes of agonist induced Ca2+ signals as well as a lower half maximal effective concentration (ЕC50). In contrast, neurons from homozygous Sip1 mice (Sip1fI/fI), demonstrate higher resistance to these respective receptor agonists. This is reflected in lower amplitudes of Ca2+-responses and so a higher concentration of receptor activators is required for activation.


Assuntos
Sinalização do Cálcio/fisiologia , Córtex Cerebral/fisiologia , Proteínas do Tecido Nervoso/genética , Neurônios/fisiologia , Receptores de AMPA/metabolismo , Receptores de Ácido Caínico/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Potenciais de Ação/fisiologia , Animais , Variações do Número de Cópias de DNA/genética , Camundongos , Camundongos Transgênicos , Mutação
14.
Arch Biochem Biophys ; 615: 35-43, 2017 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-28063948

RESUMO

In the present work we compared the protective effect of anti-inflammatory cytokine IL-10 with the action of a PI3-kinase selective activator 740 Y-P, selective agonists of alpha-2 adrenoreceptor, guanfacine and UK-14,304, and compounds having antioxidant effect: recombinant human peroxiredoxin 6 and B27, in hippocampal cell culture during OGD (ischemia-like conditions). It has been shown that the response of cells to OGD in the control includes two phases. The first phase was accompanied by an increase in the frequency of spontaneous synchronous Ca2+-oscillations (SSCO) in neurons and Ca2+-pulse in astrocytes. Spontaneous Ca2+ events in astrocytes during ischemia in control experiments disappeared. The second phase started after a few minutes of OGD and looked like a sharp/avalanche, global synchronic (within 20 s) increase in [Ca2+]i in many cells. Within 1 h after OGD, a mass death of cells, primarily astrocytes, was observed. To study the protective action of the compounds, cells were incubated in the presence of the neuroprotective agents for 10-40 min or 24 h before ischemia. All the neuroprotective agents delayed a global [Ca2+]i increase during OGD or completely inhibited this process and increased cell survival.


Assuntos
Antioxidantes/metabolismo , Hipocampo/citologia , Interleucina-10/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Receptores Adrenérgicos alfa 2/metabolismo , Agonistas Adrenérgicos/metabolismo , Animais , Astrócitos/citologia , Encéfalo/metabolismo , Cálcio/metabolismo , Morte Celular , Sobrevivência Celular , Células Cultivadas , Guanfacina/química , Isquemia/metabolismo , Oscilometria , Ratos
15.
Arch Biochem Biophys ; 593: 38-49, 2016 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-26850364

RESUMO

Angiotensin II (Ang II) is an important mammalian neurohormone involved in reninangiotensin system. Ang II is produced both constitutively and locally by RAS systems, including white fat adipocytes. The influence of Ang II on adipocytes is complex, affecting different systems of signal transduction from early Са(2+) responses to cell proliferation and differentiation, triglyceride accumulation, expression of adipokine-encoding genes and adipokine secretion. It is known that white fat adipocytes express all RAS components and Ang II receptors (АТ1 and АТ2). The current work was carried out with the primary white adipocytes culture, and Са(2+) signaling pathways activated by Ang II were investigated using fluorescent microscopy. Са(2+)-oscillations and transient responses of differentiated adipocytes to Ang II were registered in cells with both small and multiple lipid inclusions. Using inhibitory analysis and selective antagonists, we now show that Ang II initiates periodic Са(2+)-oscillations and transient responses by activating АТ1 and АТ2 receptors and involving branched signaling cascades: 1) Ang II → Gq → PLC → IP3 → IP3Rs → Ca(2+) 2) Gßγ → PI3Kγ → PKB 3) PKB → eNOS → NO → PKG 4) CD38 → cADPR → RyRs → Ca(2+) In these cascades, AT1 receptors play the leading role. The results of the present work open a perspective of using Ang II for correction of signal resistance of adipocytes often observed during obesity and type 2 diabetes.


Assuntos
Adipócitos Brancos/metabolismo , Angiotensina II/metabolismo , Sinalização do Cálcio , Adipócitos Brancos/citologia , Adipócitos Brancos/efeitos dos fármacos , Angiotensina II/farmacologia , Animais , Cálcio/metabolismo , Diferenciação Celular , Gotículas Lipídicas/ultraestrutura , Camundongos , Cultura Primária de Células , Receptor Tipo 1 de Angiotensina/metabolismo , Receptor Tipo 2 de Angiotensina/metabolismo
16.
Neurosci Lett ; 571: 55-60, 2014 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-24796809

RESUMO

It is suggested that anti-inflammatory cytokine interleukin-10 (IL-10) mediates the delayed protective effects through activation of Jak-Stat3, PI3K-Akt and NF-κB signaling pathways. However, our previous experiments have demonstrated that IL-10 is capable to exert the rapid neuroprotective action through modulation of hypoxia-induced intracellular Ca(2+) ([Ca(2+)]i) response. The first purpose of the present study was to evaluate the neuroprotective effects of IL-10 using three models of the ischemic insults in rats: permanent middle cerebral artery occlusion, ischemia in acute hippocampal slices in vitro and ischemia in cultured hippocampal cells in vitro. The second purpose of the study was to elucidate a role of [Ca(2+)]i changes in the mechanisms underlying IL-10 elicited protection of neurons and astrocytes from ischemia-induced death in cultures of primary hippocampal cells. The data presented here shown that anti-inflammatory cytokine IL-10 is capable to induce a resistance of the brain cells to ischemia-evoked damages in in vivo and in vitro models of the ischemic insults in rats. This protective effect in cultured hippocampal cells is developed rapidly after application of IL-10 and strongly associated with the IL-10 elicited elimination of [Ca(2+)]i response to ischemia. Thus, our results provide the evidence that anti-inflammatory cytokine IL-10, in addition to an activation of the canonical signaling pathways, is capable to exert the rapid neuroprotective effects through transcription-independent modulation of ischemia-induced intracellular Ca(2+) responses in the brain cells.


Assuntos
Isquemia Encefálica/patologia , Cálcio/metabolismo , Interleucina-10/metabolismo , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Astrócitos/patologia , Infarto Encefálico/etiologia , Infarto Encefálico/metabolismo , Infarto Encefálico/patologia , Isquemia Encefálica/etiologia , Isquemia Encefálica/metabolismo , Morte Celular , Hipóxia Celular , Células Cultivadas , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Hipocampo/patologia , Infarto da Artéria Cerebral Média/complicações , Infarto da Artéria Cerebral Média/metabolismo , Infarto da Artéria Cerebral Média/patologia , Interleucina-10/farmacologia , Masculino , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Cultura Primária de Células , Ratos Sprague-Dawley , Ratos Wistar
17.
Exp Neurol ; 250: 1-7, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24041985

RESUMO

We have previously developed a rat hippocampal neuronal cell model for the registration of the preconditioning effect and posthypoxic hyperexcitability (Turovskaya et al., 2011). Repeated episodes of short-term hypoxia are reported to suppress the amplitude of Ca(2+) response to NMDA in majority of neurons, reflecting the effect of preconditioning in the culture. In addition, exposure to hypoxia causes posthypoxic hyperexcitability: this is characterized by the onset of spontaneous synchronous Ca(2+) transients in a population of neurons in a neural network during the period of reoxygenation after each hypoxic episode. The nature of this phenomenon is unknown, although it has been observed that there always exists a minority of neurons in which there is no effect of hypoxic preconditioning. In this small population of neurons, the amplitude of Ca(2+) response to NMDA is not suppressed, but rather increases after each episode of hypoxia. Here we report the type of these neurons and their role in the generation of posthypoxic hyperexcitability. We compared the effect of short-term hypoxia on the amplitude of the Ca(2+) response to NMDA and the Ca(2+) transient generation in two populations of neurons - inhibitory GABAergic and excitatory glutamatergic. We have demonstrated that the neurons in which the preconditioning effect was not observed are GABAergic. Moreover at the instant moment of the posthypoxic synchronous Ca(2+)-transient generation (during reoxygenation) there is a global increase of [Ca(2+)]i and subsequent apoptosis in some GABAergic neurons. Anti-inflammatory cytokine interleukin-10 prevents the development of posthypoxic hyperexcitability, inhibiting the spontaneous synchronous Ca(2+) transients. At the same time, interleukin-10 protects GABAergic neurons from death, by restoring the effect of hypoxic preconditioning in them. Activation of one of the signaling pathways initiated by interleukin-10 appears to be necessary for the development of hypoxic preconditioning in GABAergic neurons. Overall our results indicate that short-term episodes of hypoxia can damage GABAergic neurons and weaken the inhibitory action of GABAergic neurons in a neural network. Activation of PI3K-dependent survival signaling pathways in neurons of this type is a possible strategy to protect these cells against hypoxia.


Assuntos
Apoptose/fisiologia , Neurônios GABAérgicos/patologia , Hipocampo/patologia , Hipóxia/metabolismo , Interleucina-10/metabolismo , Precondicionamento Isquêmico , Animais , Apoptose/efeitos dos fármacos , Cálcio/metabolismo , Hipóxia Celular/efeitos dos fármacos , Hipóxia Celular/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Aminoácidos Excitatórios/farmacologia , Neurônios GABAérgicos/metabolismo , Hipocampo/irrigação sanguínea , Hipocampo/metabolismo , Imuno-Histoquímica , N-Metilaspartato/farmacologia , Ratos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia
18.
PLoS One ; 8(5): e63483, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23696827

RESUMO

PURPOSE: This study investigated possible mechanisms of autoregulation of Ca(2+) signalling pathways in adipocytes responsible for Ca(2+) and NO oscillations and switching phenomena promoted by acetylcholine (ACh), norepinephrine (NE) and atrial natriuretic peptide (ANP). METHODS: Fluorescent microscopy was used to detect changes in Ca(2+) and NO in cultures of rodent white adipocytes. Agonists and inhibitors were applied to characterize the involvement of various enzymes and Ca(2+)-channels in Ca(2+) signalling pathways. RESULTS: ACh activating M3-muscarinic receptors and Gßγ protein dependent phosphatidylinositol 3 kinase induces Ca(2+) and NO oscillations in adipocytes. At low concentrations of ACh which are insufficient to induce oscillations, NE or α1, α2-adrenergic agonists act by amplifying the effect of ACh to promote Ca(2+) oscillations or switching phenomena. SNAP, 8-Br-cAMP, NAD and ANP may also produce similar set of dynamic regimes. These regimes arise from activation of the ryanodine receptor (RyR) with the implication of a long positive feedback loop (PFL): Ca(2+)→NO→cGMP→cADPR→Ca(2+), which determines periodic or steady operation of a short PFL based on Ca(2+)-induced Ca(2+) release via RyR by generating cADPR, a coagonist of Ca(2+) at the RyR. Interplay between these two loops may be responsible for the observed effects. Several other PFLs, based on activation of endothelial nitric oxide synthase or of protein kinase B by Ca(2+)-dependent kinases, may reinforce functioning of main PFL and enhance reliability. All observed regimes are independent of operation of the phospholipase C/Ca(2+)-signalling axis, which may be switched off due to negative feedback arising from phosphorylation of the inositol-3-phosphate receptor by protein kinase G. CONCLUSIONS: This study presents a kinetic model of Ca(2+)-signalling system operating in adipocytes and integrating signals from various agonists, which describes it as multivariable multi feedback network with a family of nested positive feedback.


Assuntos
Acetilcolina/farmacologia , Adipócitos/metabolismo , Fator Natriurético Atrial/metabolismo , Cálcio/metabolismo , ADP-Ribose Cíclica/metabolismo , GMP Cíclico/metabolismo , Óxidos de Nitrogênio/metabolismo , Norepinefrina/metabolismo , Adipócitos/efeitos dos fármacos , Animais , Camundongos , Transdução de Sinais/efeitos dos fármacos
19.
Neurosci Lett ; 516(1): 151-5, 2012 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-22498075

RESUMO

The goal of this study was to evaluate an effect of interleukin-10 (IL-10) on the Ca(2+) response induced by repeated NMDA receptor activation with brief hypoxia in cultured hippocampal neurons. We focused on the importance of internal Ca(2+) stores in the modulation of this Ca(2+) response by IL-10. To test this, we compared roles of InsP(3)- and ryanodine-sensitive internal stores in the effects of IL-10. Measurements of intracellular cytosolic calcium concentration ([Ca(2+)](i)) in cultured hippocampal neurons were made by imaging Fura-2AM loaded hippocampal cells. Repeated episodes of NMDA receptor activation with brief hypoxia induced the spontaneous (s) [Ca(2+)](i) increases about 3 min after each hypoxic episode. The amplitude of the s[Ca(2+)](i) increases was progressively enhanced from the first hypoxic episode to the third one. IL-10 (1 ng/ml) abolished these s[Ca(2+)](i) increases. Exposure of cultured hippocampal neurons with thapsigargin (1 µM) or an inhibitor of phospholipase C (U73122, 1 µM) for 10 min also abolished the s[Ca(2+)](i) increases. On the other hand, antagonist of ryanodine receptors (ryanodine, 1 µM) did not affect this Ca(2+) response. These studies appear to provide the first evidence that Ca(2+) release from internal stores is affected by anti-inflammatory cytokine IL-10 in brain neurons. It is suggested that these data increase our understanding of the neuroprotective mechanisms of IL-10 in the early phase of hypoxia.


Assuntos
Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Hipocampo/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Interleucina-10/metabolismo , Neurônios/metabolismo , Animais , Animais Recém-Nascidos , Hipóxia Celular , Células Cultivadas , Hipocampo/citologia , Ratos , Ratos Wistar , Receptores de N-Metil-D-Aspartato
20.
Neurosci Lett ; 496(1): 11-4, 2011 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-21458539

RESUMO

The aim of this study was to evaluate the intracellular cytosolic calcium concentration ([Ca(2+)](i)) changes induced by activation of ionotropic glutamate receptors in cultured hippocampal neurons after repeated brief episodes of hypoxia. To investigate what kinds of ionotropic glutamate receptors are involved we used specific agonists for AMPA- and NMDA-type glutamate receptors. Measurements of [Ca(2+)](i) in cultured hippocampal neurons were made by imaging Fura-2AM loaded hippocampal cells. In the rat hippocampal slice method, field potential measurements in CA1 pyramidal neurons were used. The main result of our study is that brief hypoxic episodes progressively depress the [Ca(2+)](i) increases induced by agonists of AMPA and NMDA glutamate receptors in cultured hippocampal neurons. An effectiveness of this depression is increased from the first hypoxic episode to the third one. Hypoxic preconditioning effect is observed during 10-20 min after termination of hypoxic episode and depends on [Ca(2+)](i) response amplitudes to agonists before hypoxia. In contrast to AMPA receptor activation, NMDA receptor activation before hypoxia induce the spontaneous [Ca(2+)](i) increase about 3 min after each hypoxic episode. These spontaneous [Ca(2+)](i) increases may be an indicator of the development of posthypoxic hyperexcitability in hippocampal neurons. Our results suggest that brief hypoxia-induced depression of the glutamate receptor-mediated [Ca(2+)](i) responses contributes to the development of rapid hypoxic preconditioning in hippocampal CA1 neurons.


Assuntos
Cálcio/metabolismo , Hipóxia Celular/fisiologia , Hipocampo/citologia , Neurônios/metabolismo , Receptores Ionotrópicos de Glutamato/metabolismo , Animais , Hipóxia Celular/efeitos dos fármacos , Agonistas de Aminoácidos Excitatórios/farmacologia , Técnicas In Vitro , Líquido Intracelular/efeitos dos fármacos , Líquido Intracelular/metabolismo , Masculino , N-Metilaspartato/farmacologia , Neurônios/citologia , Neurônios/efeitos dos fármacos , Ratos , Ratos Wistar , Fatores de Tempo , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico/farmacologia
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