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1.
Adv Exp Med Biol ; 1131: 131-161, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31646509

RESUMO

Calcium (Ca2+) is a fundamental regulator of cell fate and intracellular Ca2+ homeostasis is crucial for proper function of the nerve cells. Given the complexity of neurons, a constellation of mechanisms finely tunes the intracellular Ca2+ signaling. We are focusing on the sarco/endoplasmic reticulum (SR/ER) calcium (Ca2+)-ATPase (SERCA) pump, an integral ER protein. SERCA's well established role is to preserve low cytosolic Ca2+ levels ([Ca2+]cyt), by pumping free Ca2+ ions into the ER lumen, utilizing ATP hydrolysis. The SERCA pumps are encoded by three distinct genes, SERCA1-3, resulting in 12 known protein isoforms, with tissue-dependent expression patterns. Despite the well-established structure and function of the SERCA pumps, their role in the central nervous system is not clear yet. Interestingly, SERCA-mediated Ca2+ dyshomeostasis has been associated with neuropathological conditions, such as bipolar disorder, schizophrenia, Parkinson's disease and Alzheimer's disease. We summarize here current evidence suggesting a role for SERCA in the neurobiology of neuropsychiatric and neurodegenerative disorders, thus highlighting the importance of this pump in brain physiology and pathophysiology.


Assuntos
Encéfalo , Retículo Endoplasmático , Doenças do Sistema Nervoso , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático , Encéfalo/enzimologia , Encéfalo/patologia , Retículo Endoplasmático/enzimologia , Regulação Enzimológica da Expressão Gênica , Homeostase , Humanos , Doenças do Sistema Nervoso/enzimologia , Doenças do Sistema Nervoso/fisiopatologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo
2.
Adv Exp Med Biol ; 1131: 337-370, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31646517

RESUMO

The sarcoplasmic/endoplasmic reticulum (SR/ER) is the main intracellular calcium (Ca2+) pool in muscle and non-muscle eukaryotic cells, respectively. The reticulum accumulates Ca2+ against its electrochemical gradient by the action of sarco/endoplasmic reticulum calcium ATPases (SERCA pumps), and the capacity of this Ca2+ store is increased by the presence of Ca2+ binding proteins in the lumen of the reticulum. A diversity of physical and chemical signals, activate the main Ca2+ release channels, i.e. ryanodine receptors (RyRs) and inositol (1, 4, 5) trisphosphate receptors (IP3Rs), to produce transient elevations of the cytoplasmic calcium concentration ([Ca2+]i) while the reticulum is being depleted of Ca2+. This picture is incomplete because it implies that the elements involved in the Ca2+ release process are acting alone and independently of each other. However, it appears that the Ca2+ released by RyRs and IP3Rs is trapped in luminal Ca2+ binding proteins (Ca2+ lattice), which are associated with these release channels, and the activation of these channels appears to facilitate that the trapped Ca2+ ions become available for release. This situation makes the initial stage of the Ca2+ release process a highly efficient one; accordingly, there is a large increase in the [Ca2+]i with minimal reductions in the bulk of the free luminal SR/ER [Ca2+] ([Ca2+]SR/ER). Additionally, it has been shown that active SERCA pumps are required for attaining this highly efficient Ca2+ release process. All these data indicate that Ca2+ release by the SR/ER is a highly regulated event and not just Ca2+ coming down its electrochemical gradient via the open release channels. One obvious advantage of this sophisticated Ca2+ release process is to avoid depletion of the ER Ca2+ store and accordingly, to prevent the activation of ER stress during each Ca2+ release event.


Assuntos
Cálcio , Retículo Endoplasmático , Retículo Sarcoplasmático , Animais , Cálcio/metabolismo , Sinalização do Cálcio , Retículo Endoplasmático/metabolismo , Humanos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo
3.
Chem Commun (Camb) ; 55(57): 8231-8234, 2019 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-31241075

RESUMO

Withangulatin A (WA) has been reported to exhibit potent antitumor activity. However, its possible mechanism and direct proteomic targets remain unknown. Herein we report the subcellular localization of WA by designing and synthesizing its fluorescent analogues with coumarin moieties. Furthermore, sarco/endoplasmic reticulum calcium-ATPase (SERCA)2 was identified as the potential target of WA for its antitumor activity by chemical proteomics.


Assuntos
Corantes Fluorescentes/química , Pregnenos/análise , Proteômica/métodos , Linhagem Celular Tumoral , Cumarínicos/química , Humanos , Microscopia de Fluorescência , Pregnenos/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/análise , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo
4.
Nat Commun ; 10(1): 2299, 2019 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-31127110

RESUMO

Ca2+ coordinates diverse cellular processes, yet how function-specific signals arise is enigmatic. We describe a cell-wide network of distinct cytoplasmic nanocourses with the nucleus at its centre, demarcated by sarcoplasmic reticulum (SR) junctions (≤400 nm across) that restrict Ca2+ diffusion and by nanocourse-specific Ca2+-pumps that facilitate signal segregation. Ryanodine receptor subtype 1 (RyR1) supports relaxation of arterial myocytes by unloading Ca2+ into peripheral nanocourses delimited by plasmalemma-SR junctions, fed by sarco/endoplasmic reticulum Ca2+ ATPase 2b (SERCA2b). Conversely, stimulus-specified increases in Ca2+ flux through RyR2/3 clusters selects for rapid propagation of Ca2+ signals throughout deeper extraperinuclear nanocourses and thus myocyte contraction. Nuclear envelope invaginations incorporating SERCA1 in their outer nuclear membranes demarcate further diverse networks of cytoplasmic nanocourses that receive Ca2+ signals through discrete RyR1 clusters, impacting gene expression through epigenetic marks segregated by their associated invaginations. Critically, this circuit is not hardwired and remodels for different outputs during cell proliferation.


Assuntos
Sinalização do Cálcio/fisiologia , Citosol/metabolismo , Animais , Membrana Celular/metabolismo , Proliferação de Células/fisiologia , Células Cultivadas , Masculino , Células Musculares/fisiologia , Contração Muscular/fisiologia , Músculo Esquelético/citologia , Músculo Esquelético/fisiologia , Membrana Nuclear/metabolismo , Cultura Primária de Células , Ratos , Ratos Sprague-Dawley , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo
5.
Fitoterapia ; 137: 104150, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30995564

RESUMO

Schefflera kwangsiensis Merr. ex H.L. Li (Araliaceae) is a widely used traditional Chinese medicine for pain management in the clinic. In the present study, we isolated a previously undescribed lupane saponin, designated as schekwanglupaside C (Sch C) from the ethanolic extract of S. kwangsiensis. The structure of Sch C was determined by comprehensive spectroscopic and spectrometric analyses and chemical degradation. In primary cultured cortical neurons, Sch C altered the pattern of spontaneous Ca2+ oscillation (SCO) with a slight increase in the frequency of SCO right after addition and a gradual decrease in the frequency and amplitude of SCO, that dynamic change mimicked by an activator of sarcoplasmic reticulum Ca2+-ATPase (SERCA). The IC50 values for Sch C suppression of the frequency and amplitude of SCO were 1.75 and 2.51 µM, respectively. Furthermore, we demonstrated that Sch C is a potent SERCA activator (EC50 = 1.20 µM). Given the pivotal role of SERCA in the progression of neuropathic pain and neurodegenerative diseases, Sch C represents a new drug lead compound to develop the treatment of neuropathic pain and Alzheimer's disease.


Assuntos
Araliaceae/química , Neurônios/efeitos dos fármacos , Saponinas/farmacologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Triterpenos/farmacologia , Animais , Cálcio/metabolismo , Células Cultivadas , China , Feminino , Masculino , Camundongos Endogâmicos C57BL , Estrutura Molecular , Compostos Fitoquímicos/isolamento & purificação , Compostos Fitoquímicos/farmacologia , Componentes Aéreos da Planta/química , Saponinas/isolamento & purificação , Retículo Sarcoplasmático/enzimologia , Triterpenos/isolamento & purificação
6.
J Physiol Biochem ; 75(1): 109-115, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30756238

RESUMO

The purpose of this study was to examine the effects of interferon-γ (IFN-γ) on calcium movement in rat ventricular myocytes. L-type Ca2+ currents (ICa,L) were recorded with the whole-cell configuration of the patch-clamp techniques. IFN-γ induces current density reduction at the test potential of 0 mV by 47.6 ± 7.4%. Heparin, a selective inhibitor of inositol-1,4,5-triphosphate (IP3)-induced Ca2+ release, applied via a patch pipette, induced an ICa,L amplitude decrease of about 46 ± 5.6%. The addition of IFN-γ to heparin-treated cells has no effect on ICa,L. Ryanodine induced an ICa,L current amplitude decrease of 35.1 ± 6.2%. The addition of IFN-γ to ryanodine-treated cells caused an additional ICa,L inhibiting of 17.6 ± 4.8%. Both cyclopiazonic acid (CPA), a specific SERCA inhibitor, and a combination of CPA and ryanodine caused a significant reduction of the ICa,L amplitudes. Subsequent addition of IFN-γ inhibited ICa,L for an additional 16.3 ± 4.4%. The employment of chelerythrine in this study prevented IFN-γ-induced L-type Ca2+ channel inhibition in only 10 min from the start of perfusion. Proposed mechanisms of regulation involved IFN-γ-induced IP3-sensitive Ca2+ release probably by a Ca2+-dependent translocation of PKC from the cytoplasm to the cell membrane as the obligatory first step of the IFN-γ-induced PKC-dependent L-type Ca2+ channel inhibition.


Assuntos
Canais de Cálcio Tipo L/metabolismo , Cálcio/metabolismo , Interferon gama/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Transdução de Sinais/efeitos dos fármacos , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Benzofenantridinas/farmacologia , Ventrículos do Coração/citologia , Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/metabolismo , Heparina/farmacologia , Indóis/farmacologia , Inositol 1,4,5-Trifosfato/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Técnicas de Patch-Clamp , Cultura Primária de Células , Proteína Quinase C/metabolismo , Transporte Proteico , Ratos , Ratos Wistar , Rianodina/farmacologia , Retículo Sarcoplasmático/efeitos dos fármacos , Retículo Sarcoplasmático/metabolismo
7.
J Agric Food Chem ; 67(11): 3242-3248, 2019 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-30807139

RESUMO

The research was performed to investigate the difference of activity, expression, and S-nitrosylation of calcium transfer proteins between pale, soft, and exudative (PSE) and red, firm, and non-exudative (RFN) pork. Seven PSE and seven RFN pork longissimus thoracis (LT) muscles were chosen according to pH and L* at 1 h post-mortem and identified by drip loss at 24 h. The nitric oxide synthase (NOS) activity and neuronal nitric oxide synthase (nNOS) expression showed a significant difference between two groups ( p < 0.05). PSE meat had a considerably higher sarcoplasmic calcium concentration compared to RFN meat at 1 h post-mortem aging ( p < 0.05). In PSE meat, the expression of ryanodine receptor 1 (RyR1) and sarcoplasmic reticulum calcium ATPase 1 (SERCA1) was lower than that in RFN meat, while the relative S-nitrosylation level of RyR1 and SERCA1 was higher ( p < 0.05). In addition, a lower activity of SERCA was detected in PSE meat compared to RFN meat ( p < 0.05). Those results indicate that S-nitrosylation of RyR1 and SERCA1 can putatively play a crucial part in regulating calcium homeostasis. A high level of RyR1 and SERCA1 S-nitrosylation can induce the imbalance of calcium in cytoplasm, leading to accelerated pH decline and the development of PSE meat.


Assuntos
Carne/análise , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Suínos/genética , Animais , Cálcio/metabolismo , Cor , Manipulação de Alimentos , Músculo Esquelético/química , Músculo Esquelético/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Suínos/metabolismo
8.
J Chem Theory Comput ; 15(4): 2692-2705, 2019 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-30807147

RESUMO

Sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) is a transmembrane pump that plays an important role in transporting calcium into the sarcoplasmic reticulum (SR). While calcium (Ca2+) binds SERCA with micromolar affinity, magnesium (Mg2+) and potassium (K+) also compete with Ca2+ binding. However, the molecular bases for these competing ions' influence on the SERCA function and the selectivity of the pump for Ca2+ are not well-established. We therefore used in silico methods to resolve molecular determinants of cation binding in the canonical site I and II Ca2+ binding sites via (1) triplicate molecular dynamics (MD) simulations of Mg2+, Ca2+, and K+-bound SERCA, (2) mean spherical approximation (MSA) theory to score the affinity and selectivity of cation binding to the MD-resolved structures, and (3) state models of SERCA turnover informed from MSA-derived affinity data. Our key findings are that (a) coordination at sites I and II is optimized for Ca2+ and to a lesser extent for Mg2+ and K+, as determined by MD-derived cation-amino acid oxygen and bound water configurations, (b) the impaired coordination and high desolvation cost for Mg2+ precludes favorable Mg2+ binding relative to Ca2+, while K+ has limited capacity to bind site I, and (c) Mg2+ most likely acts as inhibitor and K+ as intermediate in SERCA's reaction cycle, based on a best-fit state model of SERCA turnover. These findings provide a quantitative basis for SERCA function that leverages molecular-scale thermodynamic data and rationalizes enzyme activity across broad ranges of K+, Ca2+, and Mg2+ concentrations.


Assuntos
ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Termodinâmica , Animais , Sítios de Ligação , Cálcio/metabolismo , Cátions/metabolismo , Magnésio/metabolismo , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Potássio/metabolismo , Ligação Proteica , Coelhos , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/química
9.
Can J Physiol Pharmacol ; 97(5): 413-421, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30730760

RESUMO

Statins are determined to have various pleiotropic effects apart from their lipid-lowering properties. Herein, we investigated the direct effects of atorvastatin on gastric smooth muscle tone. Atorvastatin effectively relaxed isolated rat gastric fundus strips precontracted with acetylcholine, potassium chloride, and serotonin. Incubation of the strips with nitric oxide synthase inhibitor, l-NOARG (10-4 M, 20 min), l-type voltage-operated Ca2+ channel (VOCC) blocker, nifedipine (10-6 M, 30 min), KATP channel blocker, glibenclamide (10-5 M, 30 min), or precursor of cholesterol, mevalonate (10-2 M, 45 min) did not change the relaxations to atorvastatin. However, pretreatment of fundus strips with atorvastatin (3×10-5-3×10-4 M, 30 min) inhibited the contractions to calcium chloride (10-4-10-1 M), acetylcholine (10-4 M), and caffeine (20 mM) in the calcium-free medium. Moreover, atorvastatin reduced the contractions induced by sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) inhibitor, cyclopiazonic acid (10-7-3×10-5 M). The current study demonstrated that atorvastatin produces an acute relaxant effect on gastric fundus strips, which appears to be mediated by several Ca2+-signalling mechanisms such as the blockade of l-type VOCC-independent Ca2+ entry, decrease in smooth muscle Ca2+ sensitivity, inhibition of IP3- and ryanodine-sensitive intracellular stores to mediate Ca2+ release, as well as the activation of SERCA. This acute relaxing effect seems unlikely to be related with nitric oxide, KATP channels, and the mevalonate pathway.


Assuntos
Atorvastatina/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Relaxamento Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Músculo Liso/fisiologia , Animais , Fundo Gástrico/efeitos dos fármacos , Fundo Gástrico/fisiologia , Masculino , Músculo Liso/citologia , Ratos , Ratos Wistar , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo
10.
Methods Mol Biol ; 1929: 539-550, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30710295

RESUMO

Calumenin is a secretory pathway protein regulating different endoplasmic reticulum (ER) proteins such as the sarco-endoplasmic reticulum calcium ATPase (SERCA) pumps. Combined with its diverse cellular distribution, its calcium-binding ability, and its interaction with proteins involved in calcium signaling, it is easy to speculate on future description of important roles of calumenin in calcium homeostasis in many cell types, as it was initially observed in muscle cells. In this chapter, we describe basic techniques to modulate calumenin expression and detect its impact on ER calcium content using classic transfection and Western blot techniques, as well as ER calcium measurement using microplate reader.


Assuntos
Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Sinalização do Cálcio , Linhagem Celular , Inativação Gênica , Humanos , Ligação Proteica , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Regulação para Cima
11.
Nitric Oxide ; 86: 12-20, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30772501

RESUMO

PURPOSE: This study investigated the intracellular mechanisms involved in the vasodilatation induced by the classic NO donor SNP and the non-classic NO donor cis-[Ru(bpy)2(py)(NO2)](PF6) (or RuBPY) in mesenteric resistance arteries obtained from renal hypertensive (2K-1C) and normotensive (2K) rats. METHODS: On the basis of fluorimetric assays in cultured vascular smooth muscle cells (VSMCs) isolated from 2K-1C and 2K rats, we measured NO release from SNP and RuBPY, cytosolic Ca2+ concentration ([Ca2+]c), and reactive oxygen species (ROS) with the selective probes DAF-2DA, Fluo-3AM and the more selective probe for peroxynitrite (7-CBA), respectively. We determined isometric tension in mesenteric arteries to assess SNP- and RuBPY-induced relaxation. RESULTS: SNP and RuBPY released NO in comparable amounts in cultured aortic VSMCs from hypertensive 2K-1C and normotensive 2K rats. The NO0 scavenger hydroxocobalamin blunted NO release. Sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) inhibition with thapsigargin reduced [Ca2+]c in normotensive 2K rat VSMCs only. ROS amounts were greater in hypertensive 2K-1C than in normotensive 2K rat VSMCs, but neither SNP nor RuBPY altered ROS concentrations in any of the groups. SNP and RuBPY induced similar relaxation in hypertensive 2K-1C and normotensive 2K rat mesenteric resistance arteries. The SNP and RuBPY-induced relaxation involves sGC and PKG activation. On the other hand, SNP but not RuBPY activates K+ channels. Interestingly, SERCA inhibition reduces SNP induced relaxation only in normotensive 2K rat mesenteric arteries whereas RuBPY-induced relaxation does not involve SERCA activation in both normotensive and hypertensive arteries. CONCLUSION: Our results indicate that SNP and RuBPY-induced mesenteric resistance artery relaxation involves NO/sGC/cGMP/PKG pathway activation. K+ channels and SERCA activation is required to SNP but not for RuBPY-induced relaxation. Moreover, SERCA seems to be impaired in hypertensive 2K-1C rat mesenteric resistance arteries although it does not impact SNP- or RuBPY-induced relaxation.


Assuntos
Complexos de Coordenação/farmacologia , Hipertensão Renal/fisiopatologia , Doadores de Óxido Nítrico/farmacologia , Nitroprussiato/farmacologia , Vasodilatação/efeitos dos fármacos , Animais , Masculino , Artérias Mesentéricas/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Óxido Nítrico/metabolismo , Canais de Potássio/metabolismo , Ratos Wistar , Rutênio/química , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Guanilil Ciclase Solúvel/metabolismo
12.
Mol Genet Genomic Med ; 7(3): e552, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30688039

RESUMO

BACKGROUND: Pathogenic mutations causing aberrant splicing are often difficult to detect. Standard variant analysis of next-generation sequence (NGS) data focuses on canonical splice sites. Noncanonical splice sites are more difficult to ascertain. METHODS: We developed a bioinformatics pipeline that screens existing NGS data for potentially aberrant novel essential splice sites (PANESS) and performed a pilot study on a family with a myotonic disorder. Further analyses were performed via qRT-PCR, immunoblotting, and immunohistochemistry. RNAi knockdown studies were performed in Drosophila to model the gene deficiency. RESULTS: The PANESS pipeline identified a homozygous ATP2A1 variant (NC_000016.9:g.28905928G>A; NM_004320.4:c.1287G>A:p.(Glu429=)) that was predicted to cause the omission of exon 11. Aberrant splicing of ATP2A1 was confirmed via qRT-PCR, and abnormal expression of the protein product sarcoplasmic/endoplasmic reticulum Ca++ ATPase 1 (SERCA1) was demonstrated in quadriceps femoris tissue from the proband. Ubiquitous knockdown of SERCA led to lethality in Drosophila, as did knockdown targeting differentiating or fusing myoblasts. CONCLUSIONS: This study confirms the potential of novel in silico algorithms to detect cryptic mutations in existing NGS data; expands the phenotypic spectrum of ATP2A1 mutations beyond classic Brody myopathy; and suggests that genetic testing of ATP2A1 should be considered in patients with clinical myotonia.


Assuntos
Biologia Computacional/métodos , Testes Genéticos/métodos , Miotonia Congênita/genética , Sítios de Splice de RNA/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Sequenciamento Completo do Exoma/métodos , Algoritmos , Animais , Células Cultivadas , Drosophila melanogaster , Humanos , Masculino , Músculo Esquelético/metabolismo , Mutação , Miotonia Congênita/patologia , Fenótipo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Adulto Jovem
13.
Mol Nutr Food Res ; 63(7): e1800813, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30632684

RESUMO

SCOPE: The mechanisms and involvement of uncoupling protein 1 (UCP1) in the protection from obesity and insulin resistance induced by intake of a high-fat diet rich in omega-3 (n-3) fatty acids are investigated. METHODS AND RESULTS: C57BL/6J mice are fed either a low-fat (control group) or one of two isocaloric high-fat diets containing either lard (HFD) or fish oil (HFN3) as fat source and evaluated for body weight, adiposity, energy expenditure, glucose homeostasis, and inguinal white and interscapular brown adipose tissue (iWAT and iBAT, respectively) gene expression, lipidome, and mitochondrial bioenergetics. HFN3 intake protected from obesity, glucose and insulin intolerances, and hyperinsulinemia. This is associated with increased energy expenditure, iWAT UCP1 expression, and incorporation of n-3 eicosapentaenoic and docosahexaenoic fatty acids in iWAT and iBAT triacylglycerol. Importantly, HFN3 is equally effective in reducing body weight gain, adiposity, and glucose intolerance and increasing energy expenditure in wild-type and UCP1-deficient mice without recruiting other thermogenic processes in iWAT and iBAT, such as mitochondrial uncoupling and SERCA-mediated calcium and creatine-driven substrate cyclings. CONCLUSION: Intake of a high-fat diet rich in omega-3 fatty acids protects both wild-type and UCP1-deficient mice from obesity and insulin resistance by increasing energy expenditure through unknown mechanisms.


Assuntos
Metabolismo Energético/efeitos dos fármacos , Óleos de Peixe/farmacologia , Intolerância à Glucose/dietoterapia , Obesidade/prevenção & controle , Proteína Desacopladora 1/genética , Tecido Adiposo Marrom/efeitos dos fármacos , Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Branco/efeitos dos fármacos , Tecido Adiposo Branco/metabolismo , Animais , Dieta Hiperlipídica/efeitos adversos , Metabolismo Energético/genética , Ácidos Graxos Ômega-3/análise , Ácidos Graxos Ômega-3/farmacologia , Óleos de Peixe/química , Intolerância à Glucose/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/etiologia , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Termogênese/efeitos dos fármacos , Termogênese/genética , Proteína Desacopladora 1/metabolismo
14.
J Mol Biol ; 430(24): 5050-5065, 2018 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-30539761

RESUMO

The calcium pump of the sarcoplasmic reticulum (SERCA) is an ATP-driven active transporter of Ca2+ ions that functions via an "alternating-access" cycle mechanism. In each cycle, SERCA transports two Ca2+ ions toward the lumen of the sarcoplasmic reticulum and two to three protons to the cytoplasm. How the latter conformational transition is coupled to cytoplasmic release of protons remains poorly understood. The present computational study shows how the mechanism of proton countertransport is coupled to the alternating access gating process in SERCA. Molecular dynamics simulation trajectories are generated starting from a series of configurations taken along the E2 to E1 transition pathway determined by the string method with swarms-of-trajectories. Simulations of different protonation configurations at the binding sites reveal how deprotonation events affect the opening of the cytoplasmic gate. The results show that there is a strong coupling between the chronological order of deprotonation, the entry of water molecules into the TM region, and the opening of the cytoplasmic gate. Deprotonation of E309 and E771 is sequential with E309 being the first to lose the proton. The deprotonation promotes the opening of the cytoplasmic gate but leads to a productive gating transition only if it occurs after the transmembrane domain has reached an intermediate conformation. Deprotonation of E309 and E771 is unproductive when it occurs too early because it causes the re-opening of the luminal gate.


Assuntos
Cálcio/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/química , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Retículo Sarcoplasmático/metabolismo , Sítios de Ligação , Citoplasma/metabolismo , Modelos Moleculares , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica , Prótons
15.
Cell Physiol Biochem ; 50(1): 28-40, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30278458

RESUMO

BACKGROUND/AIMS: Total saponins of Aralia elata (Miq) Seem (AS) from the Chinese traditional herb Long ya Aralia chinensis L. reportedly provide cardioprotective effects, but the exact mechanisms require further study. Previous studies have showed that myocardial ischemia/ reperfusion injury (MIRI) was related to calcium homeostasis imbalance and endoplasmic reticulum stress (ERS). Thus, this study aimed to demonstrate protective effects of AS on MIRI. METHODS: After administrating AS for 5 days, the left anterior descending artery coronary artery of Sprague-Dawley (SD) rats was ligated for 30 min. After 48 h of reperfusion, haemodynamics, Evans blue/ 2,3,5-triphenyltetrazolium chloride (TTC) staining, hematoxylin-eosin (HE) staining, masson staining and the levels of lactate dehydrogenase (LDH) and creatine kinase (CK), superoxide dismutase (SOD), malondialdehyde (MDA) were detected to assess MIRI. ATPase activity and Western Blot were used to study the mechanisms. RESULTS: Compared with IR group, AS treatment groups could significantly reduce myocardial infarct size; improve myocardial pathologic progress; decrease content of LDH, CK, and MDA; increase content of SOD; and restore the activities of Ca2+-Mg2+-ATPase, Na+-K+-ATPase, sarcoplasmic reticulum Ca2+-ATPases (SERCA), and calcineurin (CaN). AS treatment groups also significantly up-regulated the expression of GRP78, C/EBP homologous protein (CHOP), and Bax, and down-regulated the expression of Bcl-2, all similar to the effects of ERS. CONCLUSION: These findings illustrated that AS could prevent myocardial ischemia/reperfusion injury and reduce calcium homeostasis imbalance and ERS-related apoptosis.


Assuntos
Apoptose/efeitos dos fármacos , Aralia/química , Cálcio/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Saponinas/farmacologia , Animais , Aralia/metabolismo , Creatina Quinase/metabolismo , Regulação para Baixo , Proteínas de Choque Térmico/metabolismo , Masculino , Malondialdeído/metabolismo , Infarto do Miocárdio/patologia , Infarto do Miocárdio/prevenção & controle , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Ratos Sprague-Dawley , Saponinas/química , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/antagonistas & inibidores , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Superóxido Dismutase/metabolismo , Fator de Transcrição CHOP/metabolismo , Proteína X Associada a bcl-2/metabolismo
16.
Life Sci ; 210: 29-39, 2018 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-30107170

RESUMO

AIM: Epidermal growth factor receptor (EGFR) related reactive oxygen species (ROS) generation results in myocardial damage. We aimed to investigate the role of gefitinib (EGFR-tyrosine kinase inhibitor) in diabetic cardiomyopathy (DbCM). METHOD: DbCM was induced by injecting streptozotocin (55 mg/kg for 5 consecutive days) to C57/BL6 mice intraperitoneally. Diabetic C57/BL6 mice (fasting blood glucose level ≥ 250 mg/dl) were allocated into four study groups and treated with two doses of gefitinib (30 mg/kg and 350 mg/kg per day) as well as ramipril (3 mg/kg/day) for four weeks. KEY FINDINGS: We observed a significant correlation between persistent hyperglycaemia with cardiac remodeling and alterations in myocardial architecture. Gefitinib significantly prevented lipid peroxidation (MDA), damage of antioxidant enzymes like superoxide dismutase (SOD), Catalase, glutathione (GSH) and thioredoxin reductase (TrxR). Gefitinib also prevented hypertrophy of myocardium evidenced by reduced heart weight to body weight ratio and TGF­ß related collagen deposition. Gefitinib maintained cardiac biomarkers like lactate dehydrogenase (LDH), Creatine Kinase­MB, brain natriuretic peptide (BNP) and cardiac Troponin­I (cTroponinI) indicating reduced myocardial damage. Decreased sarcoplasmic endoplasmic reticulum Ca2 + ATPase2a (SERCA2a) and sodium­calcium exchanger-1 (NCX1) protein depletion after gefitinib administration indicated improved Ca2+ homeostasis during myocardial contractility. Histopathology and transmission electron microscopy clearly showed almost normal myofibrils and mitochondrial arrangements in gefitinib treated mice. SIGNIFICANCE: Our findings suggest that gefitinib protects myocardial damage in DbCM via balancing oxidant-antioxidant system, decreased collagen deposition as well as improved CKMB, BNP, cTroponinI and SERCA2a/NCX-1. Thereby, it indicated that gefitinib may be a potential therapeutic drug for treating DbCM.


Assuntos
Diabetes Mellitus Experimental/complicações , Cardiomiopatias Diabéticas/tratamento farmacológico , Receptores ErbB/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Quinazolinas/farmacologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Animais , Células Cultivadas , Cardiomiopatias Diabéticas/etiologia , Cardiomiopatias Diabéticas/patologia , Gefitinibe , Camundongos , Camundongos Endogâmicos C57BL , Miocárdio/metabolismo , Miocárdio/patologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Trocador de Sódio e Cálcio/genética
17.
Development ; 145(17)2018 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-30093550

RESUMO

The cytoplasm of striated myofibers contains a large number of membrane organelles, including sarcoplasmic reticulum (SR), T-tubules and the nuclear membrane. These organelles maintain a characteristic juxtaposition that appears to be essential for efficient inter-membranous exchange of RNA, proteins and ions. We found that the membrane-associated Muscle-specific α2/δ (Ma2/d) subunit of the Ca2+ channel complex localizes to the SR and T-tubules, and accumulates at the myonuclear surfaces. Furthermore, Ma2/d mutant larval muscles exhibit nuclear positioning defects, disruption of the nuclear-SR juxtapositioning, as well as impaired larval locomotion. Ma2/d localization at the nuclear membrane depends on the proper function of the nesprin ortholog Msp300 and the BAR domain protein Amphiphysin (Amph). Importantly, live imaging of muscle contraction in intact Drosophila larvae indicated altered distribution of Sarco/Endoplamic Reticulum Ca2+-ATPase (SERCA) around the myonuclei of Ma2/d mutant larvae. Co-immunoprecipitation analysis supports association between Ma2/d and Amph, and indirectly with Msp300. We therefore suggest that Ma2/d, in association with Msp300 and Amph, mediates interactions between the SR and the nuclear membrane.


Assuntos
Transporte Biológico/fisiologia , Canais de Cálcio/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas Musculares/metabolismo , Miofibrilas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Membrana Nuclear/metabolismo , Retículo Sarcoplasmático/metabolismo , Animais , Animais Geneticamente Modificados , Cálcio/metabolismo , Drosophila , Contração Muscular/fisiologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo
18.
Int J Mol Sci ; 19(8)2018 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-30044420

RESUMO

Spontaneous beating of the heart pacemaker, the sinoatrial node, is generated by sinoatrial node cells (SANC) and caused by gradual change of the membrane potential called diastolic depolarization (DD). Submembrane local Ca2+ releases (LCR) from sarcoplasmic reticulum (SR) occur during late DD and activate an inward Na⁺/Ca2+ exchange current, which accelerates the DD rate leading to earlier occurrence of an action potential. A comparison of intrinsic SR Ca2+ cycling revealed that, at similar physiological Ca2+ concentrations, LCRs are large and rhythmic in permeabilized SANC, but small and random in permeabilized ventricular myocytes (VM). Permeabilized SANC spontaneously released more Ca2+ from SR than VM, despite comparable SR Ca2+ content in both cell types. In this review we discuss specific patterns of expression and distribution of SR Ca2+ cycling proteins (SR Ca2+ ATPase (SERCA2), phospholamban (PLB) and ryanodine receptors (RyR)) in SANC and ventricular myocytes. We link ability of SANC to generate larger and rhythmic LCRs with increased abundance of SERCA2, reduced abundance of the SERCA inhibitor PLB. In addition, an increase in intracellular [Ca2+] increases phosphorylation of both PLB and RyR exclusively in SANC. The differences in SR Ca2+ cycling protein expression between SANC and VM provide insights into diverse regulation of intrinsic SR Ca2+ cycling that drives automaticity of SANC.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Cálcio/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Nó Sinoatrial/fisiologia , Animais , Potenciais da Membrana/fisiologia , Miócitos Cardíacos/metabolismo , Fosforilação , Coelhos , Retículo Sarcoplasmático/fisiologia , Nó Sinoatrial/citologia , Sódio/metabolismo
19.
Cell Physiol Biochem ; 48(1): 75-86, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30001530

RESUMO

BACKGROUND/AIMS: Nuclear localization leucine-rich-repeat protein 1 (NLRP1) is a cytoplasmic protein, involved in autoimmune diseases, mammalian reproduction, neuronal cell death, and stroke. However, the role of NLRP1 in cardiac hypertrophy remains unclear. We used in vivo and in vitro models to investigate the effects of NLRP1 on cardiac hypertrophy. METHODS: We used NLRP1-deficient mice and cultured neonatal rat cardiomyocytes with gain and loss of NLRP1 function. Cardiac hypertrophy was estimated by echocardiographic and hemodynamic measurements, and by pathological and molecular analysis. RESULTS: Eight weeks after aortic banding (AB), NLRP1 deficiency significantly inhibited aortic banding-induced cardiac hypertrophy, inflammation, and fibrosis. Activation of MAPK, NF-κB, and TGF-ß/Smad pathways was reduced in NLRP1-knockout (KO) mice compared with that in wild-type (WT) mice. Consistent with these results, in vitro studies, performed using cultured neonatal mouse cardiomyocytes, confirmed that NLRP1 deficiency protects against cardiomyocyte hypertrophy induced by isoproterenol (PE); this protective activity was associated with the arrest of MAPK and NF-κB signaling. CONCLUSIONS: Our data illustrates that NLRP1 plays a crucial role in the development of cardiac hypertrophy via positive regulation of the MAPK, NF-κB, and TGF-ß/Smad signaling pathways.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Cardiomegalia/patologia , Pressão , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Proteínas Reguladoras de Apoptose/genética , Fator Natriurético Atrial/metabolismo , Cardiomegalia/induzido quimicamente , Cardiomegalia/prevenção & controle , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Miocárdio/metabolismo , Miocárdio/patologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , NF-kappa B/metabolismo , Peptídeo Natriurético Encefálico/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Ratos , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Transdução de Sinais
20.
Cell Physiol Biochem ; 47(3): 1230-1243, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29913456

RESUMO

BACKGROUND/AIMS: Dietary polyphenols from green tea have been shown to possess cardio-protective activities in different experimental models of heart diseases and age-related ventricular dysfunction. The present study was aimed at evaluating whether long term in vivo administration of green tea extracts (GTE), can exert positive effects on the normal heart, with focus on the underlying mechanisms. METHODS: The study population consisted of 20 male adult Wistar rats. Ten animals were given 40 mL/day tap water solution of GTE (concentration 0.3%) for 4 weeks (GTE group). The same volume of water was administered to the 10 remaining control rats (CTRL). Then, in vivo and ex vivo measurements of cardiac function were performed in the same animal, at the organ (hemodynamics) and cellular (cardiomyocyte mechanical properties and intracellular calcium dynamics) levels. On cardiomyocytes and myocardial tissue samples collected from the same in vivo studied animals, we evaluated: (1) the intracellular content of ATP, (2) the endogenous mitochondrial respiration, (3) the expression levels of the Sarcoplasmic Reticulum Ca2+-dependent ATPase 2a (SERCA2), the Phospholamban (PLB) and the phosphorylated form of PLB, the L-type Ca2+ channel, the Na+-Ca2+ exchanger, and the ryanodine receptor 2. RESULTS: GTE cardiomyocytes exhibited a hyperdynamic contractility compared with CTRL (the rate of shortening and re-lengthening, the fraction of shortening, the amplitude of calcium transient, and the rate of cytosolic calcium removal were significantly increased). A faster isovolumic relaxation was also observed at the organ level. Consistent with functional data, we measured a significant increase in the intracellular ATP content supported by enhanced endogenous mitochondrial respiration in GTE cardiomyocytes, as well as higher values of the ratios phosphorylated-PLB/PLB and SERCA2/PLB. CONCLUSIONS: Long-term in vivo administration of GTE improves cell mechanical properties and intracellular calcium dynamics in normal cardiomyocytes, by increasing energy availability and removing the inhibitory effect of PLB on SERCA2.


Assuntos
Trifosfato de Adenosina/biossíntese , Sinalização do Cálcio/efeitos dos fármacos , Proteínas de Ligação ao Cálcio/metabolismo , Metabolismo Energético/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Polifenóis/farmacologia , Chá/química , Administração Oral , Animais , Masculino , Miócitos Cardíacos/citologia , Fosforilação/efeitos dos fármacos , Polifenóis/química , Ratos , Ratos Wistar , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo
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