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MAIN CONCLUSION: Four cultivars of Paeonia lactiflora pollen have a different viability after cryopreservation, and that the difference of pollen viability is related to calcium ions and cell wall deposition. Cryopreservation is a vital technique for preserving germplasm resources, offering extensive application prospects. Understanding the factors influencing pollen viability after cryopreservation is crucial for the permanent preservation and exchange of pollen resources. This study investigated pollen from four Paeonia lactiflora cultivars with varying viability after cryopreservation, aiming to determine whether calcium ions (Ca2+) and cell wall deposition affect these viability changes. The results showed that Ca2+-ATPase activity and cytoplasmic Ca2+ of all four cultivars exhibited an increasing trend after cryopreservation; the calmodulin (CaM) content varied with cultivars. Correlation analysis showed that fresh pollen viability was significantly negatively correlated with cytoplasmic Ca2+ content and positively correlated with Ca2+-ATPase activity, while pollen viability after cryopreservation exhibited a significantly negative correlation with cytoplasmic Ca2+ content and a positive correlation with CaM content. The pollen cell wall of the cultivar 'Zi Feng Chao Yang' (ZFCY), which showed increased viability after cryopreservation, contained significantly higher levels of low-temperature tolerance-related phospholipids and proteins compared to other cultivars. Additionally, all cultivars maintained a clear Ca2+ gradient at the tips of pollen tubes after cryopreservation, without significant callose accumulation. These findings suggest that differences in Ca2+ signaling and cell wall components deposition influence changes in pollen viability after cryopreservation, and the Ca2+ gradient and callose at the tip of pollen tubes are not responsible for preventing pollen tube growth.
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Calcio , Pared Celular , Criopreservación , Paeonia , Polen , Pared Celular/metabolismo , Criopreservación/métodos , Calcio/metabolismo , Polen/fisiología , Paeonia/fisiología , Paeonia/metabolismo , Calmodulina/metabolismo , Supervivencia CelularRESUMEN
The molecular mechanisms leading to saliva secretion are largely established, but factors that underlie secretory hypofunction, specifically related to the autoimmune disease Sjögren's syndrome (SS) are not fully understood. A major conundrum is the lack of association between the severity of salivary gland immune cell infiltration and glandular hypofunction. SS-like disease was induced by treatment with DMXAA, a small molecule agonist of murine STING. We have previously shown that the extent of salivary secretion is correlated with the magnitude of intracellular Ca2+ signals (Takano et al., 2021). Contrary to our expectations, despite a significant reduction in fluid secretion, neural stimulation resulted in enhanced Ca2+ signals with altered spatiotemporal characteristics in vivo. Muscarinic stimulation resulted in reduced activation of the Ca2+-activated Cl- channel, TMEM16a, although there were no changes in channel abundance or absolute sensitivity to Ca2+. Super-resolution microscopy revealed a disruption in the colocalization of Inositol 1,4,5-trisphosphate receptor Ca2+ release channels with TMEM16a, and channel activation was reduced when intracellular Ca2+ buffering was increased. These data indicate altered local peripheral coupling between the channels. Appropriate Ca2+ signaling is also pivotal for mitochondrial morphology and bioenergetics. Disrupted mitochondrial morphology and reduced oxygen consumption rate were observed in DMXAA-treated animals. In summary, early in SS disease, dysregulated Ca2+ signals lead to decreased fluid secretion and disrupted mitochondrial function contributing to salivary gland hypofunction.
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Anoctamina-1 , Señalización del Calcio , Modelos Animales de Enfermedad , Mitocondrias , Síndrome de Sjögren , Animales , Síndrome de Sjögren/metabolismo , Ratones , Mitocondrias/metabolismo , Anoctamina-1/metabolismo , Calcio/metabolismo , Glándulas Salivales/metabolismo , Glándulas Salivales/patología , Femenino , Ratones Endogámicos C57BLRESUMEN
The clinical management of severe COVID-19 cases is not yet well resolved. Therefore, it is important to identify and characterize cell signaling pathways involved in virus pathogenesis that can be targeted therapeutically. Envelope (E) protein is a structural protein of the virus, which is known to be highly expressed in the infected host cell and is a key virulence factor; however, its role is poorly characterized. The E protein is a single-pass transmembrane protein that can assemble into a pentamer forming a viroporin, perturbing Ca2+ homeostasis. Because it is structurally similar to regulins such as, for example, phospholamban, that regulate the sarco/endoplasmic reticulum calcium ATPases (SERCA), we investigated whether the SARS-CoV-2 E protein affects the SERCA system as an exoregulin. Using FRET experiments we demonstrate that E protein can form oligomers with regulins, and thus can alter the monomer/multimer regulin ratio and consequently influence their interactions with SERCAs. We also confirm that a direct interaction between E protein and SERCA2b results in a decrease in SERCA-mediated ER Ca2+ reload. Structural modeling of the complexes indicates an overlapping interaction site for E protein and endogenous regulins. Our results reveal novel links in the host-virus interaction network that play an important role in viral pathogenesis and may provide a new therapeutic target for managing severe inflammatory responses induced by SARS-CoV-2.
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COVID-19 , Señalización del Calcio , Proteínas de la Envoltura de Coronavirus , SARS-CoV-2 , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Humanos , SARS-CoV-2/metabolismo , COVID-19/virología , COVID-19/metabolismo , Proteínas de la Envoltura de Coronavirus/metabolismo , Proteínas de Unión al Calcio/metabolismo , Calcio/metabolismo , Unión ProteicaRESUMEN
BACKGROUND: Mast cells are key effector cells that elicit immunoglobulin E (IgE)-mediated allergic inflammations. Allergen cross-linking of IgE bound to the high-affinity IgE receptor, FcεRI, on mast cells triggers signaling cascades that activate signal proteins and evoke extracellular Ca2+ influx, which are crucial for cytokine production. The ß2-adrenergic receptor (Adrb2) on mast cells negatively regulates FcεRI signaling, as demonstrated by the inhibition of IgE/antigen (Ag)-induced activation by Adrb2 agonists. OBJECTIVE: Although ß2-adrenergic-related reagents are known to influence mast cell functions, the specific intrinsic role of Adrb2 in these cells is not fully understood, potentially because of off-target effects. In this study, the additional roles of Adrb2 in mast cells were investigated, specifically the involvement of Adrb2 in FcεRI signaling, using Adrb2-/- mice. METHODS: Adrb2-/- mice were used to investigate the roles of Adrb2 in mast cells by examining bone marrow-derived mast cells (BMMCs) for surface expression of mast cell markers, granule numbers, and gene expression of mast cell proteases. Cytokine production, Ca2+ influx, and nuclear factor of activated T cells (NFAT) nuclear translocation were measured in Adrb2-/- and Adrb2+/+ BMMCs upon IgE/Ag stimulation. RESULTS: Adrb2-/- did not affect the generation of BMMCs, their surface expression of mast cell markers, granule numbers, or gene expression of mast cell proteases, indicating that the absence of Adrb2 had no adverse effect on mast cell development. However, Adrb2-/- BMMCs exhibited reduced tumor necrosis factor α (TNFα) production and diminished Ca2⺠influx upon IgE/Ag stimulation, which correlated with decreased NFAT translocation. Restoration of Adrb2 in Adrb2-/- BMMCs rescued cytokine production. Notably, FcεRI-mediated phosphorylation of the phospholipase PLCγ1 and mitogen-activated protein kinases (MAPKs) remained unchanged in the absence of Adrb2. CONCLUSION: These results suggest that Adrb2 has a novel ligand-independent function, increasing Ca2+ entry in mast cells when stimulated with IgE/Ag.
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Increasing evidence suggests that vitamin D is one of the causes of accelerated development of Insulin Resistance (IR) and islet cell secret dysfunction. Numerous studies have shown that vitamin D can reduce inflammation, activate the transcription of the insulin receptors and related genes, and increase insulin-mediated glucose transport, thereby reducing IR. This article reviews the molecular mechanisms related to vitamin D deficiency and pancreatic ß-cell dysfunction in patients with Type 2 Diabetes (T2D).
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Nuclear Ca²âº signaling is crucial for symbiotic interactions between legumes and beneficial microbes, such as rhizobia and arbuscular mycorrhizal fungi. Key to generating repetitive nuclear Ca²âº oscillations are the ion channels DMI1 and CNGC15. Despite over 20 years of research on symbiotic nuclear Ca²âº spiking, important questions remain, including the exact function of the DMI1 channel. This review highlights recent developments that have filled knowledge gaps regarding the regulation of CNGC15 and its interplay with DMI1. We also explore new insights into the evolutionary conservation of DMI1-induced symbiotic nuclear Ca²âº oscillations and the roles of CNGC15 and DMI1 beyond symbiosis, such as in nitrate signaling, and discuss new questions this raises. As we delve deeper into the regulatory mechanisms and evolutionary history of these ion channels, we move closer to fully understanding the roles of nuclear Ca²âº signaling in plant life.
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Toxoplasma gondii, the causative parasite of toxoplasmosis, is an apicomplexan parasite that infects warm-blooded mammals. The ability of the calcium-binding proteins (CBPs) to transport large amounts of Ca2+ appears to be critical for the biological activity of T. gondii. However, the functions of some members of the CBP family have not yet been deciphered. Here, we characterized a putative CBP of T. gondii, TgpCaBP (TGME49_229480), which is composed of four EF-hand motifs with Ca2+-binding capability. TgpCaBP was localized in the cytosol and ER of T. gondii, and parasites lacking the TgpCaBP gene exhibited diminished abilities in cell invasion, intracellular growth, egress, and motility. These phenomena were due to the abnormalities in intracellular Ca2+ efflux and ER Ca2+ storage, and the reduction in motility was associated with a decrease in the discharge of secretory proteins. Therefore, we propose that TgpCaBP is a Ca2+ transporter and signaling molecule involved in Ca2+ regulation and parasitization in the hosts.IMPORTANCECa2+ signaling is essential in the development of T. gondii. In this study, we identified a calcium-binding protein in T. gondii, named TgpCaBP, which actively regulates intracellular Ca2+ levels in the parasite. Deletion of the gene coding for TgpCaBP caused serious deficits in the parasite's ability to maintain a stable intracellular calcium environment, which also impaired the secretory protein discharged from the parasite, and its capacity of gliding motility, cell invasion, intracellular growth, and egress from host cells. In summary, we have identified a novel calcium-binding protein, TgpCaBP, in the zoonotic parasite T. gondii, which is a potential therapeutic target for toxoplasmosis.
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Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia. Excessive stimulation of the IP3 signalling pathway has been linked to AF through abnormal calcium handling. However, little is known about the mechanisms involved in this process. We expressed the fluorescence resonance energy transfer (FRET) based cytosolic cAMP sensor EPAC-SH187 in neonatal rat atrial myocytes (NRAMs) and neonatal rat ventricular myocytes (NRVMs). In NRAMs, addition of the α-1 agonist phenylephrine (PE, 3 µM) resulted in a FRET change 21.20 ± 7.43 % and addition of membrane permeant IP3 derivative, 2,3,6-tri-O-Butyryl-myo-IP3(1,4,5)-hexakis(acetoxymethyl)ester (IP3-AM, 20 µM) resulted in a peak of 20.31 ± 6.74 %. These FRET changes imply an increase in cAMP. Prior application of IP3 receptor (IP3R) inhibitors 2-Aminoethyl diphenylborinate (2-APB, 2.5µM) or Xestospongin-C (0.3 µM) significantly inhibited the change in FRET in NRAMs in response to PE. Xestospongin-C (0.3 µM) significantly inhibited the change in FRET in NRAMs in response to IP3-AM. The FRET change in response to PE in NRVMs were not inhibited by 2-APB or Xestospongin-C. Finally, the localisation of cAMP signals was tested by expressing the FRET-based cAMP sensor, AKAP79-CUTie, which targets the intracellular surface of the plasmalemma. We found in NRAMs that PE led to FRET change corresponding to an increase in cAMP that was inhibited by 2-APB and Xestospongin C. These data support further investigation of the pro-arrhythmic nature and components of IP3 induced cAMP signalling to identify potential pharmacological targets.
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Pathogen-responsive immune-related genes (resistance genes [R-genes]) and hormones are crucial mediators of systemic acquired resistance (SAR). However, their integrated functions in regulating SAR signaling components in local and distal leaves remain largely unknown. To characterize SAR in the Xanthomonas campestris pv. campestris (Xcc)-Brassica napus pathosystem, the responses of R-genes, (leaf and phloem) hormone levels, H2O2 levels, and Ca2+ signaling-related genes were assessed in local and distal leaves of plants exposed to four Xcc-treatments: Non-inoculation (control), only secondary Xcc-inoculation in distal leaves (C-Xcc), only primary Xcc-inoculation in local leaves (Xcc), and both primary and secondary Xcc-inoculation (X-Xcc). The primary Xcc-inoculation provoked disease symptoms as evidenced by enlarged destructive necrosis in the local leaves of Xcc and X-Xcc plants 7 days post-inoculation. Comparing visual symptoms in distal leaves 5 days post-secondary inoculation, yellowish necrotic lesions were clearly observed in non Xcc-primed plants (C-Xcc), whereas no visual symptom was developed in Xcc-primed plants (X-Xcc), demonstrating SAR. Pathogen resistance in X-Xcc plants was characterized by distinct upregulations in expression of the PAMP-triggered immunity (PTI)-related kinase-encoding gene, BIK1, the (CC-NB-LRR-type) R-gene, ZAR1, and its signaling-related gene, NDR1, with a concurrent enhancement of the kinase-encoding gene, MAPK6, and a depression of the (TIR-NB-LRR-type) R-gene, TAO1, and its signaling-related gene, SGT1, in distal leaves. Further, in X-Xcc plants, higher salicylic acid (SA) and jasmonic acid (JA) levels, both in phloem and distal leaves, were accompanied by enhanced expressions of the SA-signaling gene, NPR3, the JA-signaling genes, LOX2 and PDF1.2, and the Ca2+-signaling genes, CAS and CBP60g. However, in distal leaves of C-Xcc plants, an increase in SA level resulted in an antagonistic depression of JA, which enhanced only SA-dependent signaling, EDS1 and NPR1. These results demonstrate that primary Xcc-inoculation in local leaves induces resistance to subsequent pathogen attack by upregulating BIK1-ZAR1-mediated synergistic interactions with SA and JA signaling as a crucial component of SAR.
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Ciclopentanos , Oxilipinas , Enfermedades de las Plantas , Ácido Salicílico , Transducción de Señal , Ácido Salicílico/metabolismo , Enfermedades de las Plantas/microbiología , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/inmunología , Ciclopentanos/metabolismo , Oxilipinas/metabolismo , Transducción de Señal/genética , Xanthomonas campestris/fisiología , Xanthomonas campestris/patogenicidad , Hojas de la Planta/microbiología , Hojas de la Planta/genética , Brassica napus/microbiología , Brassica napus/genética , Brassica napus/fisiología , Regulación de la Expresión Génica de las Plantas , Resistencia a la Enfermedad/genética , Peróxido de Hidrógeno/metabolismo , Inmunidad de la Planta/genéticaRESUMEN
The impacts of L-glutamate (L-Glu) treatment on chilling injury (CI), Ca2+ signaling, mitochondrial ultrastructure, and metabolisms of reactive oxygen species (ROS), γ-aminobutyric acid (GABA), energy of prune fruit under chilling stress were studied. The results found that the optimal concentration of L-Glu to suppress CI occurrence and maintain quality in prune fruit was 0.1 g L-1, which also enhanced the PdGLRs expression, cytoplasmic Ca2+ concentration, the contents of CaM, and CML under cold stress. Moreover, L-Glu treatment could reduce ROS accumulation and increase GABA content, and energy level, contributing to maintaining the integrity of the mitochondrial structure in cold-stored prune fruit. More importantly, PdGLRs expression and CaM/CML content positively correlated with antioxidant enzyme activities, GABA shunt, and energy status in prune fruit. These results indicated that the enhanced cold resistance of L-Glu-treated prunes might be attributed to the activated Ca2+ signaling, thus improving the antioxidant capacity, GABA, and energy levels.
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Frío , Metabolismo Energético , Frutas , Ácido Glutámico , Especies Reactivas de Oxígeno , Ácido gamma-Aminobutírico , Especies Reactivas de Oxígeno/metabolismo , Frutas/química , Frutas/metabolismo , Frutas/efectos de los fármacos , Ácido Glutámico/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Ácido gamma-Aminobutírico/análisis , Metabolismo Energético/efectos de los fármacos , Homeostasis/efectos de los fármacosRESUMEN
The conjunctiva has immune-responsive properties to protect the eye from infections. Its innate immune system reacts against external pathogens, such as fungi. The complement factor C5a is an important contributor to the initial immune response. It is known that activation of transient-receptor-potential-vanilloid 1 (TRPV1) and TRP-melastatin 8 (TRPM8) channels is involved in different immune reactions and inflammation in the human body. The aim of this study was to determine if C5a and mucor racemosus e voluminae cellulae (MR) modulate Ca2+-signaling through changes in TRPs activity in human conjunctival epithelial cells (HCjECs). Furthermore, crosstalk was examined between C5a and MR in mediating calcium regulation. Intracellular Ca2+-concentration ([Ca2+]i) was measured by fluorescence calcium imaging, and whole-cell currents were recorded using the planar-patch-clamp technique. MR was used as a purified extract. Application of C5a (0.05-50 ng/mL) increased both [Ca2+]i and whole-cell currents, which were suppressed by either the TRPV1-blocker AMG 9810 or the TRPM8-blocker AMTB (both 20 µM). The N-terminal peptide C5L2p (20-50 ng/mL) blocked rises in [Ca2+]i induced by C5a. Moreover, the MR-induced rise in Ca2+-influx was suppressed by AMG 9810 and AMTB, as well as 0.05 ng/mL C5a. In conclusion, crosstalk between C5a and MR controls human conjunctival cell function through modulating interactions between TRPV1 and TRPM8 channel activity.
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Calcio , Complemento C5a , Conjuntiva , Células Epiteliales , Humanos , Células Epiteliales/metabolismo , Células Epiteliales/microbiología , Conjuntiva/metabolismo , Conjuntiva/microbiología , Calcio/metabolismo , Complemento C5a/metabolismo , Canales de Potencial de Receptor Transitorio/metabolismo , Señalización del Calcio , Canales Catiónicos TRPV/metabolismoRESUMEN
Since the initial identification of oncogenic Wnt in mice and Drosophila, the Wnt signaling pathway has been subjected to thorough and extensive investigation. Persistent activation of Wnt signaling exerts diverse cancer characteristics, encompassing tumor initiation, tumor growth, cell senescence, cell death, differentiation, and metastasis. Here we review the principal signaling mechanisms and the regulatory influence of pathway-intrinsic and extrinsic kinases on cancer progression. Additionally, we underscore the divergences and intricate interplays of the canonical and non-canonical Wnt signaling pathways and their critical influence in cancer pathophysiology, exhibiting both growth-promoting and growth-suppressing roles across diverse cancer types.
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ORAI1 is an intrinsic component of store-operated calcium entry (SOCE) that strictly regulates Ca2+ influx in most non-excitable cells. ORAI1 is overexpressed in a wide variety of cancers, and its signal transduction has been associated with chemotherapy resistance. There is extensive proteomic interaction of ORAI1 with other channels and effectors, resulting in various altered phenotypes. However, the transcription regulation of ORAI1 is not well understood. We have found a putative G-quadruplex (G4) motif, ORAI1-Pu, in the upstream promoter region of the gene, having regulatory functions. High-resolution 3-D NMR structure elucidation suggests that ORAI1-Pu is a stable parallel-stranded G4, having a long 8-nt loop imparting dynamics without affecting the structural stability. The protruded loop further houses an E-box motif that provides a docking site for transcription factors like Zeb1. The G4 structure was also endogenously observed using Chromatin Immunoprecipitation (ChIP) with anti-G4 antibody (BG4) in the MDA-MB-231 cell line overexpressing ORAI1. Ligand-mediated stabilization suggested that the stabilized G4 represses transcription in cancer cell line MDA-MB-231. Downregulation of transcription further led to decreased Ca2+ entry by the SOCE pathway, as observed by live-cell Fura-2 Ca2+ imaging.
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Calcio , G-Cuádruplex , Proteína ORAI1 , Regiones Promotoras Genéticas , Neoplasias de la Mama Triple Negativas , Humanos , Proteína ORAI1/metabolismo , Proteína ORAI1/genética , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patología , Neoplasias de la Mama Triple Negativas/genética , Calcio/metabolismo , Línea Celular Tumoral , Elementos E-Box/genética , Femenino , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/metabolismo , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/genética , Regulación Neoplásica de la Expresión GénicaRESUMEN
The gut microbiota and their metabolites are closely linked to obesity-related diseases, such as type 2 diabetes, but their causal relationship and underlying mechanisms remain largely elusive. Here, we found that dysbiosis-induced tyramine (TA) suppresses high-fat diet (HFD)-mediated insulin resistance in both Drosophila and mice. In Drosophila, HFD increases cytosolic Ca2+ signaling in enterocytes, which, in turn, suppresses intestinal lipid levels. 16 S rRNA sequencing and metabolomics revealed that HFD leads to increased prevalence of tyrosine decarboxylase (Tdc)-expressing bacteria and resulting tyramine production. Tyramine acts on the tyramine receptor, TyrR1, to promote cytosolic Ca2+ signaling and activation of the CRTC-CREB complex to transcriptionally suppress dietary lipid digestion and lipogenesis in enterocytes, while promoting mitochondrial biogenesis. Furthermore, the tyramine-induced cytosolic Ca2+ signaling is sufficient to suppress HFD-induced obesity and insulin resistance in Drosophila. In mice, tyramine intake also improves glucose tolerance and insulin sensitivity under HFD. These results indicate that dysbiosis-induced tyramine suppresses insulin resistance in both flies and mice under HFD, suggesting a potential therapeutic strategy for related metabolic disorders, such as diabetes.
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Señalización del Calcio , Dieta Alta en Grasa , Microbioma Gastrointestinal , Resistencia a la Insulina , Tiramina , Animales , Tiramina/metabolismo , Tiramina/farmacología , Microbioma Gastrointestinal/efectos de los fármacos , Dieta Alta en Grasa/efectos adversos , Ratones , Señalización del Calcio/efectos de los fármacos , Obesidad/metabolismo , Obesidad/microbiología , Obesidad/etiología , Masculino , Drosophila/metabolismo , Disbiosis/metabolismo , Disbiosis/microbiología , Ratones Endogámicos C57BL , Drosophila melanogaster/microbiología , Drosophila melanogaster/metabolismo , Enterocitos/metabolismo , Enterocitos/efectos de los fármacosRESUMEN
BACKGROUND: Vascular smooth muscle cell (VSMC) proliferation is involved in many types of arterial diseases, including neointima hyperplasia, in which Ca2+ has been recognized as a key player. However, the physiological role of Ca2+ release via inositol 1,4,5-trisphosphate receptors (IP3Rs) from endoplasmic reticulum in regulating VSMC proliferation has not been well determined. METHODS AND RESULTS: Both in vitro cell culture models and in vivo mouse models were generated to investigate the role of IP3Rs in regulating VSMC proliferation. Expression of all 3 IP3R subtypes was increased in cultured VSMCs upon platelet-derived growth factor-BB and FBS stimulation as well as in the left carotid artery undergoing intimal thickening after vascular occlusion. Genetic ablation of all 3 IP3R subtypes abolished endoplasmic reticulum Ca2+ release in cultured VSMCs, significantly reduced cell proliferation induced by platelet-derived growth factor-BB and FBS stimulation, and also decreased cell migration of VSMCs. Furthermore, smooth muscle-specific deletion of all IP3R subtypes in adult mice dramatically attenuated neointima formation induced by left carotid artery ligation, accompanied by significant decreases in cell proliferation and matrix metalloproteinase-9 expression in injured vessels. Mechanistically, IP3R-mediated Ca2+ release may activate cAMP response element-binding protein, a key player in controlling VSMC proliferation, via Ca2+/calmodulin-dependent protein kinase II and Akt. Loss of IP3Rs suppressed cAMP response element-binding protein phosphorylation at Ser133 in both cultured VSMCs and injured vessels, whereas application of Ca2+ permeable ionophore, ionomycin, can reverse cAMP response element-binding protein phosphorylation in IP3R triple knockout VSMCs. CONCLUSIONS: Our results demonstrated an essential role of IP3R-mediated Ca2+ release from endoplasmic reticulum in regulating cAMP response element-binding protein activation, VSMC proliferation, and neointima formation in mouse arteries.
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Proliferación Celular , Receptores de Inositol 1,4,5-Trifosfato , Músculo Liso Vascular , Miocitos del Músculo Liso , Neointima , Animales , Masculino , Ratones , Becaplermina/farmacología , Becaplermina/metabolismo , Calcio/metabolismo , Señalización del Calcio , Traumatismos de las Arterias Carótidas/patología , Traumatismos de las Arterias Carótidas/metabolismo , Traumatismos de las Arterias Carótidas/genética , Movimiento Celular , Células Cultivadas , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Modelos Animales de Enfermedad , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/patología , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/genética , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Neointima/patología , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
Pyruvate kinase M2 (PKM2) is a key glycolytic enzyme interacting with the inositol 1,4,5-trisphosphate receptor (IP3R). This interaction suppresses IP3R-mediated cytosolic [Ca2+] rises. As PKM2 exists in monomeric, dimeric and tetrameric forms displaying different properties including catalytic activity, we investigated the molecular determinants of PKM2 enabling its interaction with IP3Rs. Treatment of HeLa cells with TEPP-46, a compound stabilizing the tetrameric form of PKM2, increased both its catalytic activity and the suppression of IP3R-mediated Ca2+ signals. Consistently, in PKM2 knock-out HeLa cells, PKM2C424L, a tetrameric, highly active PKM2 mutant, but not inactive PKM2K270M or the less active PKM2K305Q, suppressed IP3R-mediated Ca2+ release. Surprisingly, however, in vitro assays did not reveal a direct interaction between purified PKM2 and either the purified Fragment 5 of IP3R1 (a.a. 1932-2216) or the therein located D5SD peptide (a.a. 2078-2098 of IP3R1), the presumed interaction sites of PKM2 on the IP3R. Moreover, on-nucleus patch clamp of heterologously expressed IP3R1 in DT40 cells devoid of endogenous IP3Rs did not reveal any functional effect of purified wild-type PKM2, mutant PKM2 or PKM1 proteins. These results indicate that an additional factor mediates the regulation of the IP3R by PKM2 in cellulo. Immunoprecipitation of GRP75 using HeLa cell lysates co-precipitated IP3R1, IP3R3 and PKM2. Moreover, the D5SD peptide not only disrupted PKM2:IP3R, but also PKM2:GRP75 and GRP75:IP3R interactions. Our data therefore support a model in which catalytically active, tetrameric PKM2 suppresses Ca2+ signaling via the IP3R through a multiprotein complex involving GRP75.
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Receptores de Inositol 1,4,5-Trifosfato , Proteínas de la Membrana , Humanos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/genética , Células HeLa , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Piruvato Quinasa/metabolismo , Piruvato Quinasa/genética , Señalización del Calcio , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP70 de Choque Térmico/genética , Calcio/metabolismo , Unión Proteica , Multimerización de ProteínaRESUMEN
Lysosomal Ca2+ signaling is emerging as a crucial regulator of endothelial Ca2+ dynamics. Ca2+ release from the acidic vesicles in response to extracellular stimulation is usually promoted via Two Pore Channels (TPCs) and is amplified by endoplasmic reticulum (ER)-embedded inositol-1,3,4-trisphosphate (InsP3) receptors and ryanodine receptors. Emerging evidence suggests that sub-cellular Ca2+ signals in vascular endothelial cells can also be generated by the Transient Receptor Potential Mucolipin 1 channel (TRPML1) channel, which controls vesicle trafficking, autophagy and gene expression. Herein, we adopted a multidisciplinary approach, including live cell imaging, pharmacological manipulation, and gene targeting, revealing that TRPML1 protein is expressed and triggers global Ca2+ signals in the human brain microvascular endothelial cell line, hCMEC/D3. The direct stimulation of TRPML1 with both the synthetic agonist, ML-SA1, and the endogenous ligand phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) induced a significant increase in [Ca2+]i, that was reduced by pharmacological blockade and genetic silencing of TRPML1. In addition, TRPML1-mediated lysosomal Ca2+ release was sustained both by lysosomal Ca2+ release and ER Ca2+- release through inositol-1,4,5-trisphophate receptors and store-operated Ca2+ entry. Notably, interfering with TRPML1-mediated lysosomal Ca2+ mobilization led to a decrease in the free ER Ca2+ concentration. Imaging of DAF-FM fluorescence revealed that TRPML1 stimulation could also induce a significant Ca2+-dependent increase in nitric oxide concentration. Finally, the pharmacological and genetic blockade of TRPML1 impaired ATP-induced intracellular Ca2+ release and NO production. These findings, therefore, shed novel light on the mechanisms whereby the lysosomal Ca2+ store can shape endothelial Ca2+ signaling and Ca2+-dependent functions in vascular endothelial cells.
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OBJECTIVE: To explore the mechanism by which soybean isoflavone (SI) reduces calcium overload induced by cerebral ischemia-reperfusion (I/R). METHODS: Forty-eight SD rats were randomized into 4 groups to receive sham operation, cerebral middle artery occlusion for 2 h followed by 24 h of reperfusion (I/R model group), or injection of adeno-associated virus carrying Frizzled-2 siRNA or empty viral vector into the lateral cerebral ventricle after modeling.Western blotting was used to examine Frizzled-2 knockdown efficiency and changes in protein expressions in the Wnt/Ca2+ signaling pathway.Calcium levels and pathological changes in the ischemic penumbra (IP) were measured using calcium chromogenic assay and HE staining, respectively.Another 72 SD randomly allocated for sham operation, I/R modeling, or soy isoflavones pretreatment before modeling were examined for regional cerebral blood flow using a Doppler flowmeter, and the cerebral infarct volume was assessed using TTC staining.Pathologies in the IP area were evaluated using HE and Nissl staining, and ROS level, Ca2+ level, cell apoptosis, and intracellular calcium concentration were analyzed using immunofluorescence assay or flow cytometry; the protein expressions of Wnt5a, Frizzled-2, and P-CaMK â ¡ in the IP were detected with Western blotting and immunohistochemistry. RESULTS: In rats with cerebral I/R, Frizzled-2 knockdown significantly lowered calcium concentration (P < 0.001) and the expression levels of Wnt5a, Frizzled-2, and P-CaMK â ¡ in the IP area.In soy isoflavones-pretreated rats, calcium concentration, ROS and MDA levels, cell apoptosis rate, cerebral infarct volume, and expression levels of Wnt/Ca2+ signaling pathway-related proteins were all significantly lower while SOD level was higher than those in rats in I/R model group. CONCLUSION: Soy isoflavones can mitigate calcium overload in rats with cerebral I/R by inhibiting the Wnt/Ca2+ signaling pathway.
Asunto(s)
Isquemia Encefálica , Calcio , Glycine max , Isoflavonas , Ratas Sprague-Dawley , Daño por Reperfusión , Vía de Señalización Wnt , Animales , Isoflavonas/farmacología , Isoflavonas/uso terapéutico , Ratas , Daño por Reperfusión/metabolismo , Daño por Reperfusión/prevención & control , Vía de Señalización Wnt/efectos de los fármacos , Isquemia Encefálica/metabolismo , Calcio/metabolismo , Glycine max/química , Apoptosis/efectos de los fármacos , Masculino , Proteína Wnt-5a/metabolismo , ARN Interferente Pequeño/genéticaRESUMEN
Early in the development of Type 2 diabetes (T2D), metabolic stress brought on by insulin resistance and nutrient overload causes ß-cell hyperstimulation. Herein we summarize recent studies that have explored the premise that an increase in the intracellular Ca2+ concentration ([Ca2+]i), brought on by persistent metabolic stimulation of ß-cells, causes ß-cell dysfunction and failure by adversely affecting ß-cell function, structure, and identity. This mini-review builds on several recent reviews that also describe how excess [Ca2+]i impairs ß-cell function.
Asunto(s)
Señalización del Calcio , Diabetes Mellitus Tipo 2 , Células Secretoras de Insulina , Estrés Fisiológico , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patología , Humanos , Señalización del Calcio/fisiología , Animales , Estrés Fisiológico/fisiología , Diabetes Mellitus Tipo 2/metabolismo , Calcio/metabolismo , Resistencia a la Insulina/fisiologíaRESUMEN
Intracellular Ca2+ can be conveniently monitored by sensitive Ca2+ fluorescent dyes in live cells. The Gαq involved lipid signaling pathways and, thus, can be studied by intracellular Ca2+ imaging. Here we describe the protocols to measure intracellular Ca2+ for studying PEG2-EP1 activity in esophageal smooth muscle cells. The ratiometric Fura-2 imaging provides quantitative data, and the Fluo-4 confocal microscopic imaging has high-spatial resolution.