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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.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 36(8): 794-797, 2019 Aug 10.
Artigo em Chinês | MEDLINE | ID: mdl-31400130

RESUMO

OBJECTIVE: To explore the molecular basis for a pedigree affected with Darier-White disease. METHODS: Genomic DNA was isolated from 3 patients and 1 unaffected member from the pedigree, as well as 80 healthy controls. Targeted sequence capture and next-generation sequencing were used to screen mutations of skin disease-related genes. Candidate mutations were verified by Sanger sequencing, and co-segregation analysis was carried out to confirm the pathogenicity of mutation. Conservation analysis and protein structure and function were also predicted with Bioinformatic tools. RESULTS: A heterozygous mutation c.2246G>T (p.G749V) was identified in exon 15 of ATP2A2 gene in all 3 patients from the pedigree, but not in the unaffected member or 80 healthy controls. The corresponding amino acid was highly conserved, and mutation of which can lead to structural and functional changes of the protein. CONCLUSION: The c.2246G>T missense mutation of the ATP2A2 gene probably underlies the Darier-White disease in this pedigree by causing damages to the structure and function of sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (SERCA2).


Assuntos
Doença de Darier/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Heterozigoto , Humanos , Mutação de Sentido Incorreto , Linhagem
4.
Adv Exp Med Biol ; 1124: 313-328, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31183833

RESUMO

Veins exhibit spontaneous contractile activity, a phenomenon generally termed vasomotion. This is mediated by spontaneous rhythmical contractions of mural cells (i.e. smooth muscle cells (SMCs) or pericytes) in the wall of the vessel. Vasomotion occurs through interconnected oscillators within and between mural cells, entraining their cycles. Pharmacological studies indicate that a key oscillator underlying vasomotion is the rhythmical calcium ion (Ca2+) release-refill cycle of Ca2+ stores. This occurs through opening of inositol 1,4,5-trisphosphate receptor (IP3R)- and/or ryanodine receptor (RyR)-operated Ca2+ release channels in the sarcoplasmic/endoplasmic (SR/ER) reticulum and refilling by the SR/ER reticulum Ca2+ATPase (SERCA). Released Ca2+ from stores near the plasma membrane diffuse through the cytosol to open Ca2+-activated chloride (Cl-) channels, this generating inward current through an efflux of Cl-. The resultant depolarisation leads to the opening of voltage-dependent Ca2+ channels and possibly increased production of IP3, which through Ca2+-induced Ca2+ release (CICR) of IP3Rs and/or RyRs and IP3R-mediated Ca2+ release provide a means by which store oscillators entrain their activity. Intercellular entrainment normally involves current flow through gap junctions that interconnect mural cells and in many cases this is aided by additional connectivity through the endothelium. Once entrainment has occurred the substantial Ca2+ entry that results from the near-synchronous depolarisations leads to rhythmical contractions of the mural cells, this often leading to vessel constriction. The basis for venous/venular vasomotion has yet to be fully delineated but could improve both venous drainage and capillary/venular absorption of blood plasma-associated fluids.


Assuntos
Sinalização do Cálcio , Contração Muscular , Miócitos de Músculo Liso/fisiologia , Veias/fisiologia , Cálcio/fisiologia , Membrana Celular , Retículo Endoplasmático/fisiologia , Humanos , Canal de Liberação de Cálcio do Receptor de Rianodina/fisiologia , Retículo Sarcoplasmático/fisiologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/fisiologia
5.
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
6.
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
7.
BMC Cancer ; 19(1): 381, 2019 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-31023247

RESUMO

BACKGROUND: Salinomycin is a monocarboxylic polyether antibiotic and is a potential chemotherapy drug. Our previous studies showed that salinomycin inhibited cell growth and targeted CSCs in prostate cancer. However, the precise target of salinomycin action is unclear. METHODS: In this work, we analyzed and identified differentially expressed genes (DEGs) after treatment with or without salinomycin using a gene expression microarray in vitro (PC-3 cells) and in vivo (NOD/SCID mice xenograft model generated from implanted PC-3 cells). Western blotting and immunohistochemical staining were used to analyze the expression of ATP2A3 and endoplasmic reticulum (ER) stress biomarkers. Flow cytometry was used to analyze the cell cycle, apoptosis and intracellular Ca2+ concentration. RESULTS: A significantly upregulated gene, ATPase sarcoplasmatic/endoplasmatic reticulum Ca2+ transporting 3 (ATP2A3), was successfully identified. In subsequent studies, we found that ATP2A3 overexpression could trigger ER stress and exert anti-cancer effects in PC-3 and DU145 cells. ATP2A3 was slightly expressed, but the ER stress biomarkers showed strong staining in prostate cancer tissues. We also found that salinomycin could trigger ER stress, which might be related to ATP2A3-mediated Ca2+ release in PC-3 cells. Furthermore, we found that salinomycin-triggered ER stress could promote apoptosis and thus exert anti-cancer effects in prostate cancer cells. CONCLUSION: This study demonstrates that ATP2A3 might be one of the potential targets for salinomycin, which can inhibit Ca2+ release and trigger ER stress to exert anti-cancer effects.


Assuntos
Estresse do Retículo Endoplasmático/efeitos dos fármacos , Neoplasias da Próstata/tratamento farmacológico , Piranos/administração & dosagem , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Animais , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Camundongos , Células PC-3 , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Transdução de Sinais/efeitos dos fármacos , Ativação Transcricional/genética , Ensaios Antitumorais Modelo de Xenoenxerto
8.
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
9.
Science ; 363(6430): 948-955, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30819957

RESUMO

We investigated the roles of components of neuronal synapses for development of the Drosophila air sac primordium (ASP). The ASP, an epithelial tube, extends specialized signaling filopodia called cytonemes that take up signals such as Dpp (Decapentaplegic, a homolog of the vertebrate bone morphogenetic protein) from the wing imaginal disc. Dpp signaling in the ASP was compromised if disc cells lacked Synaptobrevin and Synaptotagmin-1 (which function in vesicle transport at neuronal synapses), the glutamate transporter, and a voltage-gated calcium channel, or if ASP cells lacked Synaptotagmin-4 or the glutamate receptor GluRII. Transient elevations of intracellular calcium in ASP cytonemes correlate with signaling activity. Calcium transients in ASP cells depend on GluRII, are activated by l-glutamate and by stimulation of an optogenetic ion channel expressed in the wing disc, and are inhibited by EGTA and by the GluR inhibitor NASPM (1-naphthylacetyl spermine trihydrochloride). Activation of GluRII is essential but not sufficient for signaling. Cytoneme-mediated signaling is glutamatergic.


Assuntos
Sinalização do Cálcio , Proteínas de Drosophila/fisiologia , Glutamatos/fisiologia , Discos Imaginais/fisiologia , Receptores Ionotrópicos de Glutamato/fisiologia , Sinapses/fisiologia , Animais , Animais Geneticamente Modificados , Canais de Cálcio/fisiologia , Drosophila melanogaster/fisiologia , Imagem Óptica , Pseudópodes/fisiologia , Proteínas R-SNARE/fisiologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/fisiologia , Sinaptotagmina I/fisiologia , Técnicas de Cultura de Tecidos
10.
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
11.
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
12.
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
13.
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
14.
Molecules ; 24(3)2019 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-30717322

RESUMO

Arsenic trioxide (ATO) has been verified as a breakthrough with respect to the management of acute promyelocytic leukemia (APL) in recent decades but associated with some serious adverse phenomena, particularly cardiac functional abnormalities. Salvianolic acid A (Sal A) is a major effective component in treating ATO-induced cardiotoxicity. Therefore, the objective of our study was to assess whether Sal A had protective effects by the regulation of calcium homeostasis and endoplasmic reticulum (ER) stress. For the in vivo study, BALB/c mice were treated with ATO and/or Sal A via daily tail vein injections for two weeks. For the in vitro study, we detected the effects of ATO and/or Sal A in real time using adult rat ventricular myocytes (ARVMs) and an IonOptix MyoCam system. Our results showed that Sal A pretreatment alleviated cardiac dysfunction and Ca2+ overload induced by ATO in vivo and vitro. Moreover, Sal A increased sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA) activity and expression, alleviated [Ca2+]ER depletion, and decreased ER stress-related protein expression. Sal A protects the heart from ATO-induced injury and its administration correlates with the modulation of SERCA, the recovery of Ca2+ homeostasis, and the down-regulation of ER stress-mediated apoptosis.


Assuntos
Trióxido de Arsênio/efeitos adversos , Ácidos Cafeicos/administração & dosagem , Cardiotoxicidade/tratamento farmacológico , Lactatos/administração & dosagem , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Animais , Apoptose/efeitos dos fármacos , Trióxido de Arsênio/administração & dosagem , Cálcio/metabolismo , Cardiotoxicidade/etiologia , Modelos Animais de Doenças , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Homeostase/efeitos dos fármacos , Humanos , Leucemia Promielocítica Aguda/complicações , Leucemia Promielocítica Aguda/tratamento farmacológico , Camundongos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/enzimologia , Ratos , Retículo Sarcoplasmático/efeitos dos fármacos
15.
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
16.
J Vet Intern Med ; 33(2): 933-941, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30720217

RESUMO

BACKGROUND: Sarcolipin (SLN), myoregulin (MRLN), and dwarf open reading frame (DWORF) are transmembrane regulators of the sarcoplasmic reticulum calcium transporting ATPase (SERCA) that we hypothesized played a role in recurrent exertional rhabdomyolysis (RER). OBJECTIVES: Compare coding sequences of SLN, MRLN, DWORF across species and between RER and control horses. Compare expression of muscle Ca2+ regulatory genes between RER and control horses. ANIMALS: Twenty Thoroughbreds (TB), 5 Standardbreds (STD), 6 Quarter Horses (QH) with RER and 39 breed-matched controls. METHODS: Sanger sequencing of SERCA regulatory genes with comparison of amino acid (AA) sequences among control, RER horses, human, mouse, and rabbit reference genomes. In RER and control gluteal muscle, quantitative real-time polymerase chain reaction of SERCA regulatory peptides, the calcium release channel (RYR1), and its accessory proteins calsequestrin (CASQ1), and calstabin (FKBP1A). RESULTS: The SLN gene was the highest expressed horse SERCA regulatory gene with a uniquely truncated AA sequence (29 versus 31) versus other species. Coding sequences of SLN, MRLN, and DWORF were identical in RER and control horses. A sex-by-phenotype effect occurred with lower CASQ1 expression in RER males versus control males (P < .001) and RER females (P = .05) and higher FKBP1A (P = .01) expression in RER males versus control males. CONCLUSIONS AND CLINICAL IMPORTANCE: The SLN gene encodes a uniquely truncated peptide in the horse versus other species. Variants in the coding sequence of SLN, MLRN, or DWORF were not associated with RER. Males with RER have differential gene expression that could reflect adaptations to stabilize RYR1.


Assuntos
Doenças dos Cavalos/genética , Rabdomiólise/veterinária , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Sequência de Aminoácidos , Animais , Estudos de Casos e Controles , Feminino , Expressão Gênica , Cavalos , Humanos , Masculino , Camundongos , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Músculo Esquelético , Coelhos , Rabdomiólise/genética
17.
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
18.
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
19.
Mol Carcinog ; 58(6): 887-897, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30657210

RESUMO

The knowledge about the role of calcium-regulated pathways in cancer cell growth and differentiation could be useful for the development of new therapeutic approaches to diminish its mortality. The ATP2A genes encode for SERCA pumps, which modulate cytosolic Ca2+ concentration, regulating various cellular processes including cell growth. ATP2A3 gene transcriptional down-regulation has been reported in gastric and colon cancer, but there is still a lack of understanding about the epigenetic processes regulating its transcription. In this work, we report that butyrate, trichostatin A, and 5-azacytidine treatments increase SERCA3 expression, increased apoptosis, and decreased cell viability of the KATO-III gastric carcinoma cell line. We analyzed the methylation profile of the ATP2A3 gene promoter CpG island, finding clones with methylated status through -280 to -135 promoter region, harboring Sp1 and AP-2 binding sites, which could have a role in transcriptional repression. Post-translational modifications of histones show a major role in the ATP2A3 transcriptional regulation, and our results show histones marks linked to transcriptional repression associated with the -262 to -135 region, this repressive context changed to transcriptional permissive through SERCA3 re-expressing conditions. These results suggest that the nucleotide sequence from -280 to -135 position is an ATP2A3 epigenetic regulatory CpG region in KATO-III cells. Analyses of online-databases show a decreased SERCA3 expression in gastric and colon tumors, as well as overall survival results, showed that high SERCA3 expression could serve as a favorable prognostic marker for colon and gastric cancer patients.


Assuntos
Neoplasias do Colo/genética , Epigênese Genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Neoplasias Gástricas/genética , Sítios de Ligação , Linhagem Celular Tumoral , Ilhas de CpG , Metilação de DNA , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Histonas/metabolismo , Humanos , Regiões Promotoras Genéticas , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/química , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Fator de Transcrição Sp1/metabolismo , Análise de Sobrevida
20.
Thromb Haemost ; 119(3): 384-396, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30650444

RESUMO

In obesity, platelets are described as hyperactive, mainly based on increased platelet size and presence of pro-thrombotic plasmatic molecules. We explored platelet functions, including calcium signalling in obesity, and the effect of weight loss. We included 40 obese patients (women with body mass index [BMI] of ≥ 35 kg/m2) who were to undergo gastric bypass surgery and 40 healthy lean subjects (women with BMI of < 25 kg/m2) as a control group. Approximately 1 year after surgery, the obese patients lost weight (75% had a BMI < 35 kg/m2). They were explored a second time with the same healthy control for the same platelet experiments. Compared with controls, obese patients' platelets displayed reduced sensitivity to thrombin (aggregation EC50 increased by 1.9 ± 0.3-fold, p = 0.005) and a lower Ca2+ response (70 ± 7% decrease, p < 10-4). In 17 pairs of patients, we performed additional experiments: in obese patients' platelets, thrombin-induced αIIbß3 activation was significantly lower (p = 0.003) and sarco-endoplasmic reticulum Ca2+ATPase (SERCA3) expression was decreased (48 ± 6% decrease, p < 10-4). These differences were abolished after weight loss. Interestingly, pharmacological inhibition of SERCA3 activity in control group's platelets mimicked similar alterations than in obese patients' platelets and was associated with defective adenosine diphosphate (ADP) secretion. Addition of ADP to agonist restored platelet functions in obese patients and in SERCA3-inhibited control platelets (five experiments) confirming the direct involvement of the SERCA3-dependent ADP secretion pathway. This is the first study demonstrating that platelets from obese patients are hypo-reactive, due to a deficiency of SERCA3-dependent ADP secretion. Weight loss restores SERCA3 activity and subsequent calcium signalling, αIIbß3 activation, platelet aggregation and ADP secretion.


Assuntos
Difosfato de Adenosina/sangue , Plaquetas/metabolismo , Derivação Gástrica , Obesidade/cirurgia , Ativação Plaquetária , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/sangue , Perda de Peso , Adulto , Sinalização do Cálcio , Feminino , Humanos , Obesidade/sangue , Obesidade/diagnóstico , Obesidade/fisiopatologia , Paris , Agregação Plaquetária , Testes de Função Plaquetária , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Via Secretória , Fatores de Tempo , Resultado do Tratamento
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