RESUMEN
Cell-to-cell transmission of α-synuclein (α-syn) pathology underlies the spread of neurodegeneration in Parkinson's disease. α-Syn secretion is an important factor in the transmission of α-syn pathology. However, it is unclear how α-syn secretion is therapeutically modulated. Here, we investigated effects of monoamine oxidase (MAO)-B inhibitor selegiline on α-syn secretion. Treatment with selegiline promoted α-syn secretion in mouse primary cortical neuron cultures, and this increase was kept under glial cell-eliminated condition by Ara-C. Selegiline-induced α-syn secretion was blocked by cytosolic Ca2+ chelator BAPTA-AM in primary neurons. Selegiline-induced α-syn secretion was retained in MAOA siRNA knockdown, whereas it was abrogated by ATG5 knockdown in SH-SY5Y cells. Selegiline increased LC3-II generation with a reduction in intracellular p62/SQSTM1 levels in primary neurons. The increase in LC3-II generation was blocked by co-treatment with BAPTA-AM in primary neurons. Additionally, fractionation experiments showed that selegiline-induced α-syn secretion occurred in non-extracellular vesicle fractions of primary neurons and SH-SY5Y cells. Collectively, these findings show that selegiline promotes neuronal autophagy involving secretion of non-exosomal α-syn via a change of cytosolic Ca2+ levels.
Asunto(s)
Autofagia , Neuronas , Selegilina , alfa-Sinucleína , Selegilina/farmacología , Animales , Autofagia/efectos de los fármacos , alfa-Sinucleína/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Ratones , Monoaminooxidasa/metabolismo , Humanos , Calcio/metabolismo , Células Cultivadas , Inhibidores de la Monoaminooxidasa/farmacología , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Ratones Endogámicos C57BL , Línea Celular Tumoral , Proteína 5 Relacionada con la Autofagia/metabolismo , Proteína 5 Relacionada con la Autofagia/genéticaRESUMEN
Extracellular secretion is an essential mechanism for α-synuclein (α-syn) proteostasis. Although it has been reported that neuronal activity affects α-syn secretion, the underlying mechanisms remain unclear. Here, we investigated the autophagic processes that regulate the physiological release of α-syn in mouse primary cortical neurons and SH-SY5Y cells. Stimulating neuronal activity with glutamate or depolarization with high KCl enhanced α-syn secretion. This glutamate-induced α-syn secretion was blocked by a mixture of NMDA receptor antagonist AP5 and AMPA receptor antagonist NBQX, as well as by cytosolic Ca2+ chelator BAPTA-AM. Additionally, mTOR inhibitor rapamycin increased α-syn and p62/SQSTM1 (p62) secretion, and this effect of rapamycin was reduced in primary cortical neurons deficient in the autophagy regulator beclin 1 (derived from BECN1+/- mice). Glutamate-induced α-syn and p62 secretion was suppressed by the knockdown of ATG5, which is required for autophagosome formation. Glutamate increased LC3-II generation and decreased intracellular p62 levels, and the increase in LC3-II levels was blocked by BAPTA-AM. Moreover, glutamate promoted co-localization of α-syn with LC3-positive puncta, but not with LAMP1-positive structures in the neuronal somas. Glutamate-induced α-syn and p62 secretion were also reduced by the knockdown of RAB8A, which is required for autophagosome fusion with the plasma membrane. Collectively, these findings suggest that stimulating neuronal activity mediates autophagic α-syn secretion in a cytosolic Ca2+-dependent manner, and autophagosomes may participate in autophagic secretion by functioning as α-syn carriers.
Asunto(s)
Autofagia , Neuronas , Proteína Sequestosoma-1 , alfa-Sinucleína , Animales , alfa-Sinucleína/metabolismo , alfa-Sinucleína/genética , Neuronas/metabolismo , Ratones , Humanos , Proteína Sequestosoma-1/metabolismo , Proteína Sequestosoma-1/genética , Proteína 5 Relacionada con la Autofagia/metabolismo , Proteína 5 Relacionada con la Autofagia/genética , Ácido Glutámico/metabolismo , Beclina-1/metabolismo , Beclina-1/genética , Calcio/metabolismo , Línea Celular Tumoral , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Sirolimus/farmacologíaRESUMEN
Antisense oligonucleotide (ASO) therapy is a novel therapeutic approach in which ASO specifically binds target mRNA, resulting in mRNA degradation; however, cellular uptake of ASOs remains critically low, warranting improvement. Transient receptor potential canonical (TRPC) channels regulate Ca2+ influx and are activated upon stimulation by phospholipase C-generated diacylglycerol. Herein, we report that a novel TRPC3/C6/C7 activator, L687, can induce cellular ASO uptake. L687-induced ASO uptake was enhanced in a dose- and incubation-time-dependent manner. L687 enhanced the knockdown activity of various ASOs both in vitro and in vivo. Notably, suppression of TRPC3/C6 by specific siRNAs reduced ASO uptake in A549 cells. Application of BAPTA-AM, a Ca2+ chelator, and SKF96365, a TRPC3/C6 inhibitor, suppressed Ca2+ influx via TRPC3/C6, resulting in reduced ASO uptake, thereby suggesting that Ca2+ influx via TRPC3/C6 is critical for L687-mediated increased ASO uptake. L687 also induced dextran uptake, indicating that L687 increased endocytosis. Adding ASO to L687 resulted in endosome accumulation; however, the endosomal membrane disruptor UNC7938 facilitated endosomal escape and enhanced knockdown activity. We discovered a new function for TRPC activators regarding ASO trafficking in target cells. Our findings provide an opportunity to formulate an innovative drug delivery system for the therapeutic development of ASO.
Asunto(s)
Calcio , Oligonucleótidos Antisentido , Canales Catiónicos TRPC , Humanos , Oligonucleótidos Antisentido/farmacología , Oligonucleótidos Antisentido/metabolismo , Canales Catiónicos TRPC/metabolismo , Canales Catiónicos TRPC/genética , Canales Catiónicos TRPC/antagonistas & inhibidores , Calcio/metabolismo , Células A549 , Animales , Ratones , Imidazoles/farmacología , Canal Catiónico TRPC6/metabolismo , Canal Catiónico TRPC6/genética , Canal Catiónico TRPC6/antagonistas & inhibidores , Ácido Egtácico/farmacología , Ácido Egtácico/análogos & derivados , Endosomas/metabolismo , Endosomas/efectos de los fármacos , Línea Celular TumoralRESUMEN
Intracellular Ca2+ signals play a vital role in a broad range of cell biological and physiological processes in all eukaryotic cell types. Dysregulation of Ca2+ signaling has been implicated in numerous human diseases. Over the past four decades, the understanding of how cells use Ca2+ as a messenger has flourished, largely because of the development of reporters that enable visualization of Ca2+ signals in different cellular compartments, and tools that can modulate cellular Ca2+ signaling. One such tool that is frequently used is BAPTA; a fast, high-affinity Ca2+-chelating molecule. By making use of a cell-permeable acetoxymethyl ester (AM) variant, BAPTA can be readily loaded into the cytosol of cells (referred to as BAPTAi), where it is trapped and able to buffer changes in cytosolic Ca2+. Due to the ease of loading of the AM version of BAPTA, this reagent has been used in hundreds of studies to probe the role of Ca2+ signaling in specific processes. As such, for decades, researchers have almost universally attributed changes in biological processes caused by BAPTAi to the involvement of Ca2+ signaling. However, BAPTAi has often been used without any form of control, and in many cases has neither been shown to be retained in cells for the duration of experiments nor to buffer any Ca2+ signals. Moreover, increasing evidence points to off-target cellular effects of BAPTA that are clearly not related to Ca2+ chelation. Here, we briefly introduce Ca2+ signaling and the history of Ca2+ chelators and fluorescent Ca2+ indicators. We highlight Ca2+-independent effects of BAPTAi on a broad range of molecular targets and describe some of BAPTAi's impacts on cell functions that occur independently of its Ca2+-chelating properties. Finally, we propose strategies for determining whether Ca2+ chelation, the binding of other metal ions, or off-target interactions with cell components are responsible for BAPTAi's effect on a particular process and suggest some future research directions.
Asunto(s)
Quelantes , Humanos , Ácido Egtácico/farmacología , Quelantes/farmacología , CitosolRESUMEN
Selective calcium chelator 1,2-Bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA) is a common tool to investigate calcium signaling. However, BAPTA expresses various effects on intracellular calcium signaling, which are not related to its ability to bind Ca2+. In patch clamp experiments, we investigated calcium chelation independent effects of BAPTA on endogenous calcium-activated chloride channels ANO6 (TMEM16F) in HEK293T cells. We have found that application of BAPTA to intracellular solution led to two distinct effects on channels properties. On the one hand, application of BAPTA acutely reduced amplitude of endogenous ANO6 channels induced by 10 µM Ca2+ in single channel recordings. On the other hand, BAPTA application by itself induced ANO6 channel activity in the absence of the intracellular calcium elevation. Open channel probability was enhanced by increasing the intracellular BAPTA concentration from 0.1 to 1 and 10 mM. Another calcium chelator EGTA did not demonstrate chelation independent effects on the ANO6 activity in the same conditions. Due to off-target effects BAPTA should be used with caution when studying calcium-activated ANO6 channels.
Asunto(s)
Canales de Calcio , Calcio , Humanos , Ácido Egtácico/farmacología , Calcio/metabolismo , Células HEK293 , Quelantes del Calcio/farmacologíaRESUMEN
Acetylcholine can excite neurons by suppressing M-type (KCNQ) potassium channels. This effect is mediated by M1 muscarinic receptors coupled to the Gq protein. Although PIP2 depletion and PKC activation have been strongly suggested to contribute to muscarinic inhibition of M currents (IM), direct evidence is lacking. We investigated the mechanism involved in muscarinic inhibition of IM with Ca2+ measurement and electrophysiological studies in both neuronal (rat sympathetic neurons) and heterologous (HEK cells expressing KCNQ2/KCNQ3) preparations. We found that muscarinic inhibition of IM was not blocked either by PIP2 or by calphostin C, a PKC inhibitor. We then examined whether muscarinic inhibition of IM uses multiple signaling pathways by blocking both PIP2 depletion and PKC activation. This maneuver, however, did not block muscarinic inhibition of IM. Additionally, muscarinic inhibition of IM was not prevented either by sequestering of G-protein ßγ subunits from Gα-transducin or anti-Gßγ antibody or by preventing intracellular trafficking of channel proteins with blebbistatin, a class-II myosin inhibitor. Finally, we re-examined the role of Ca2+ signals in muscarinic inhibition of IM. Ca2+ measurements showed that muscarinic stimulation increased intracellular Ca2+ and was comparable to the Ca2+ mobilizing effect of bradykinin. Accordingly, 20-mM of BAPTA significantly suppressed muscarinic inhibition of IM. In contrast, muscarinic inhibition of IM was completely insensitive to 20-mM EGTA. Taken together, these data suggest a role of Ca2+ signaling in muscarinic modulation of IM. The differential effects of EGTA and BAPTA imply that Ca2+ microdomains or spatially local Ca2+ signals contribute to inhibition of IM.
Asunto(s)
Neuronas , Transducción de Señal , Ratas , Animales , Ácido Egtácico/metabolismo , Ácido Egtácico/farmacología , Neuronas/metabolismo , Colinérgicos/metabolismo , Colinérgicos/farmacologíaRESUMEN
This study was designed to determine the inhibitory effect of astragaloside â £(AS-â £), a principal bioactive component extracted from the Chinese medicinal Astragali Radix, on the inflammatory response of vascular endothelial cells induced by angiotensin â ¡(Ang â ¡), the most major pathogenic factor for cardiovascular diseases, and to clarify the role of calcium(Ca~(2+))/phosphatidylinosi-tol-3-kinase(PI3K)/protein kinase B(Akt)/endothelial nitric oxide synthase(eNOS)/nitric oxide(NO) pathway in the process. To be specific, human umbilical vein endothelial cells(HUVECs) were cultured in the presence of AS-â £ with or without the specific inhibitor of NO synthase(NG-monomethyl-L-arginine, L-NMMA), inhibitor of PI3K/Akt signaling pathway(LY294002), or Ca~(2+)-chelating agent(ethylene glycol tetraacetic acid, EGTA) prior to Ang â ¡ stimulation. The inhibitory effect of AS-â £ on Ang â ¡-induced inflammatory response and the involved mechanism was determined with enzyme-linked immunosorbent assay(ELISA), cell-based ELISA assay, Western blot, and monocyte adhesion assay which determined the fluorescently labeled human monocytic cell line(THP-1) adhered to Ang â ¡-stimulated endothelial cells. AS-â £ increased the production of NO by HUVECs in a dose-and time-dependent manner(P<0.05) and raised the level of phosphorylated eNOS(P<0.05). The above AS-â £-induced changes were abolished by pretreatment with L-NMMA, LY294002, or EGTA. Compared with the control group, Ang â ¡ obviously enhanced the production and release of cytokines(tumor necrosis factor-α, interleukin-6), chemokines(monocyte chemoattractant protein-1) and adhesion molecules(intercellular adhesion molecule-1, vascular cellular adhesion molecule-1), and the number of monocytes adhered to HUVECs(P<0.05), which were accompanied by the enhanced levels of phosphorylated inhibitor of nuclear factor-κBα protein and activities of nuclear factor-κB(NF-κB)(P<0.05). This study also demonstrated that Ang â ¡-induced inflammatory response was inhibited by pretreatment with AS-â £(P<0.05). In addition, the inhibitory effect of AS-â £ was abrogated by pretreatment with L-NMMA, LY294002, or EGTA(P<0.05). This study provides a direct link between AS-â £ and Ca~(2+)/PI3K/Akt/eNOS/NO pathway in AS-â £-mediated anti-inflammatory actions in endothelial cells exposed to Ang â ¡. The results indicate that AS-â £ attenuates endothelial cell-mediated inflammatory response induced by Ang â ¡ via the activation of Ca~(2+)/PI3K/Akt/eNOS/NO signaling pathway.
Asunto(s)
Angiotensina II , Proteínas Proto-Oncogénicas c-akt , Humanos , Angiotensina II/metabolismo , Angiotensina II/farmacología , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , omega-N-Metilarginina/metabolismo , omega-N-Metilarginina/farmacología , Ácido Egtácico/metabolismo , Ácido Egtácico/farmacología , Células Endoteliales de la Vena Umbilical Humana , FN-kappa B/genética , FN-kappa B/metabolismo , Óxido Nítrico/metabolismo , Células CultivadasRESUMEN
To explore whether there is an interaction between melatonin (MT) and calcium (Ca2+) in regulating heat tolerance of plants, we analyzed the response of endogenous MT and Ca2+ to heat stress, and examined the effect of MT and Ca2+ on the reactive oxygen (ROS) accumulation, antioxidant system, and transcripts of heat shock factor (HSF) and heat shock proteins (HSPs) of cucumber seedlings under high temperature stress. Seedlings were foliar sprayed with 100 µmol·L-1 MT, 10 mmol·L-1 CaCl2, 3 mmol·L-1 ethylene glycol tetraacetic acid (EGTA, Ca2+ chelating agent) +100 µmol·L-1 MT, 0.05 mmol·L-1 chlorpromazine (calmodulin antagonist, CPZ) +100 µmol·L-1 MT, 100 µmol·L-1 p-chlorophenylalanine (p-CPA, inhibitor of MT) +10 mmol·L-1 CaCl2 or deionized water (H2O), respectively. The results showed that both endogenous MT and Ca2+ in cucumber seedlings were induced by high temperature stress. The seedlings treated with exogenous MT showed significant increases in the mRNA expression of calmodulin (CaM), calcium-dependent protein kinase (CDPK5), calcineurin B-like protein (CBL3) and CBL interacting protein kinase (CIPK2) compared with the control at normal temperature. The mRNA levels of tryptophane decarboxylase (TDC), 5-hydroxytryptamine-N-acetyltransferase (SNAT) and N-acetyl-5-hydroxytryptamine methyltransferase (ASMT), key genes of MT biosynthesis and endogenous MT content were also induced by Ca2+ in cucumber seedlings. Exogenous MT and CaCl2 alleviated the heat-induced oxidative damage through increasing antioxidant ability, reducing the accumulation of reactive oxygen species (ROS), and upregulating the mRNA abundances of HSF7, HSP70.1 and HSP70.11, as evidenced by mild thermal damage symptoms, lower heat injury index and electrolyte leakage under heat stress. The positive effect of MT-induced antioxidant capacity and mRNA expression of HSPs was removed by adding EGTA and CPZ in stressed seedlings. Similarly, the mitigating role of Ca2+ in the peroxidation damage to high temperature stress was reversed by p-CPA. These results suggested that both MT and Ca2+ could induce heat tolerance of cucumber seedlings, which had crosstalk in the process of heat stress signal transduction.
Asunto(s)
Cucumis sativus , Melatonina , Cucumis sativus/genética , Melatonina/farmacología , Calcio , Antioxidantes/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Calmodulina/genética , Calmodulina/metabolismo , Calmodulina/farmacología , Ácido Egtácico/farmacología , Cloruro de Calcio/metabolismo , Cloruro de Calcio/farmacología , Temperatura , Estrés Fisiológico , Plantones/fisiología , ARN Mensajero/metabolismo , ARN Mensajero/farmacologíaRESUMEN
BACKGROUND: A novel N-methyl-D-aspartate receptor (NMDAR) allosteric modulator, rapastinel (RAP, formerly GLYX-13), elicits long-lasting antidepressant-like effects by enhancing long-term potentiation (LTP) of synaptic transmission. RAP elicits these effects by binding to a unique site in the extracellular region of the NMDAR complex, transiently enhancing NMDAR-gated current in pyramidal neurons of both hippocampus and medial prefrontal cortex. METHODS: We compared efficacy of RAP in modulating Schaffer collateral-evoked NMDAR-currents as a function of kinetics of the Ca2+ chelator in the intracellular solution, using whole-cell patch-clamp recordings. The intracellular solution contained either the slow Ca2+ chelator EGTA [3,12-bis(carboxymethyl)-6,9-dioxa-3,12-diazatetradecane-1,14-dioic acid, 0.5 mmol/l] or the 40-500-fold kinetically faster, more selective Ca2+ chelator BAPTA {2,2',2â³,2â´-[ethane-1,2-diylbis(oxy-2,1-phenylenenitrilo)] tetraacetic acid, 5 mmol/l}. NMDAR-gated currents were pharmacologically isolated by bath application of the 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid receptor antagonist 6-nitro-2,3-dioxo-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (10 µmol/l) plus the GABA receptor blocker bicuculline (20 µmol/l). RESULTS: When the slow Ca2+ chelator EGTA was in the intracellular solution, RAP elicited significant enhancement of NMDAR-gated current at a 1 µmol/l concentration, and significantly reduced current at 10 µmol/l. In contrast, when recording with the 40-500-fold kinetically faster, more selective Ca2+ chelator BAPTA, NMDAR current increased in magnitude by 84% as BAPTA washed into the cell, and the enhancement of NMDAR current by 1 µmol/l RAP was completely blocked. Interestingly, the reduction in NMDAR current from 10 µmol/l RAP was not affected by the presence of BAPTA in the recording pipette, indicating that this effect is mediated by a different mechanism. CONCLUSION: Extracellular binding of RAP to the NMDAR produces a novel, long-range reduction in affinity of the Ca2+ inactivation site on the NMDAR C-terminus accessible to the intracellular space. This action underlies enhancement in NMDAR-gated conductance elicited by RAP.
Asunto(s)
Calcio , Receptores de N-Metil-D-Aspartato , Quelantes/farmacología , Ácido Egtácico/farmacología , Hipocampo/fisiología , OligopéptidosRESUMEN
BACKGROUND: Mechanical stimuli induce the release of adenosine triphosphate into the extracellular environment by human periodontal ligament cells (hPDLCs). Extracellular adenosine triphosphate (eATP) plays the role in both inflammation and osteogenic differentiation. eATP involves in immunosuppressive action by increasing immunosuppressive molecules IDO and IFNγ expression on immune cells. However, the role of eATP on the immunomodulation of hPDLCs remains unclear. This study aimed to examine the effects of eATP on the IDO and IFNγ expression of hPDLCs and the participation of purinergic P2 receptors in this phenomenon. METHODS: hPDLCs were treated with eATP. The mRNA and protein expression of indoleamine-pyrrole 2,3-dioxygenase (IDO) and interferon-gamma (IFNγ) were determined. The role of the purinergic P2 receptor was determined using calcium chelator (EGTA) and PKC inhibitor (PKCi). Chemical inhibitors (KN62 and BBG), small interfering RNA (siRNA), and P2 X7 receptor agonist (BzATP) were used to confirm the involvement of P2 X7 receptors on IDO and IFNγ induction by hPDLCs. RESULTS: eATP significantly enhanced mRNA expression of IDO and IFNγ. Moreover, eATP increased kynurenine which is the active metabolite of tryptophan breakdown catalyzed by the IDO enzyme and significantly induced IFNγ protein expression. EGTA and PKCi reduced eATP-induced IDO and IFNγ expressions by hPDLCs, confirming the role of calcium signaling. Chemical P2 X7 inhibitors (KN62 and BBG) and siRNA targeting the P2 X7 receptor significantly inhibited the eATP-induced IDO and IFNγ production. Correspondingly, BzATP markedly increased IDO and IFNγ expression. CONCLUSION: eATP induced immunosuppressive function of hPDLCs by promoting IDO and IFNγ production via P2 X7 receptor signaling. eATP may become a promising target for periodontal regeneration by modulating immune response and further triggering tissue healing.
Asunto(s)
Indolamina-Pirrol 2,3,-Dioxigenasa , Interferón gamma , Ligamento Periodontal , Receptores Purinérgicos P2X7 , Adenosina Trifosfato/farmacología , Células Cultivadas , Ácido Egtácico/farmacología , Humanos , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Interferón gamma/metabolismo , Osteogénesis , ARN Mensajero , ARN Interferente Pequeño , Receptores Purinérgicos P2X7/metabolismoRESUMEN
KEY MESSAGE: After cryopreservation, the Ca2+ content increased, which affected the intracellular ROS content, then participated in the occurrence of programmed cell death in pollen. Programmed cell death (PCD) is one of the reasons for the decline in pollen viability after cryopreservation. However, the role of calcium ions (Ca2+) in PCD during pollen cryopreservation has not been revealed in the existing studies. In this study, Paeonia lactiflora 'Fen Yu Nu' pollen was used as the research material for investigating the effects of Ca2+ changes on PCD indices and reactive oxygen species (ROS) during pollen cryopreservation. The results showed that after cryopreservation, with the decrease of pollen viability, the Ca2+ content significantly increased. The regulation of Ca2+ content had a significant effect on PCD indices, which showed that the Ca2+ carrier A23187 accelerated the decrease of mitochondrial membrane potential level and increased the activity of caspase-3-like and caspase-9-like proteases and the apoptosis rate. The expression levels of partial pro-PCD genes were upregulated, the anti-PCD gene BI-1 was downregulated, and the addition of Ca2+-chelating agent EGTA had the opposite effect. The addition of the Ca2+ carrier A23187 after cryopreservation significantly increased the ROS content of pollen, the addition of the Ca2+-chelating agent EGTA had the opposite effect, and Ca2+ regulators also had significant effects on the contents of ROS production and clearance-related substances. Ca2+ affected intracellular ROS content by acting on the ROS production and clearance system during the cryopreservation of pollen and is thus involved in the occurrence of PCD.
Asunto(s)
Apoptosis , Polen , Calcimicina/metabolismo , Calcimicina/farmacología , Quelantes/farmacología , Criopreservación/métodos , Ácido Egtácico/metabolismo , Ácido Egtácico/farmacología , Polen/genética , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Capsaicin and zinc have recently been highlighted as potential treatments for glucose metabolism disorders; however, the effect of these two natural compounds on signalling pathways involved in glucose metabolism is still uncertain. In this study, we assessed the capsaicin- or zinc- induced activation of signalling molecules including calcium/calmodulin-dependent protein kinase 2 (CAMKK2), cAMP-response element-binding protein (CREB), and target of rapamycin kinase complex 1 (TORC1). Moreover, the expression status of genes associated with the control of glucose metabolism was measured in treated cells. The activation of cell signalling proteins was then evaluated in capsaicin- or zinc treated cells in the presence or absence of cell-permeant calcium chelator (BAPTA-AM) and the CAMKK inhibitor (STO-609). Finally, capsaicin- and zinc-induced glucose uptake was measured in the cells pre-treated with or without BAPTA-AM. Our results indicate that calcium flux induced by capsaicin or zinc led to activation of calcium signalling molecules and promoting glucose uptake in skeletal muscle cells. Pharmacological inhibition of CAMKK diminished activation of signalling molecules. Moreover, we observed an increase in intracellular cAMP levels in the cells after treatment with capsaicin and zinc. Our data show that capsaicin and zinc mediate glucose uptake in C2C12 skeletal muscle cells through the activation of calcium signalling.
Asunto(s)
Señalización del Calcio/efectos de los fármacos , Calcio/metabolismo , Capsaicina/farmacología , Glucosa/metabolismo , Fibras Musculares Esqueléticas/efectos de los fármacos , Zinc/farmacología , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Bencimidazoles/farmacología , Quinasa de la Proteína Quinasa Dependiente de Calcio-Calmodulina/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Metabolismo de los Hidratos de Carbono/efectos de los fármacos , Línea Celular , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Fibras Musculares Esqueléticas/metabolismo , Naftalimidas/farmacología , Fosforilación/efectos de los fármacosRESUMEN
Calcium homeostasis plays a vital role in protecting against Alzheimer's disease (AD). In this study, amyloid-ß (Aß)-induced C. elegans models of AD were used to elucidate the mechanisms underlying calcium homeostasis in AD. Calcium acetate increased the intracellular calcium content, exacerbated Aß 1-42 aggregation, which is closely associated with oxidative stress, aggravated neuronal degeneration and dysfunction, and shortened the lifespan of the C. elegans models. Ethylene glycol tetraacetic acid (EGTA) and nimodipine were used to decrease the intracellular calcium content. Both EGTA and nimodipine showed remarkable inhibitory effects on Aß 1-42 aggregations by increasing oxidative stress resistance. Moreover, both compounds significantly delayed the onset of Aß-induced paralysis, rescued memory deficits, ameliorated behavioral dysfunction, decreased the vulnerability of two major (GABAergic and dopaminergic) neurons and synapses, and extended the lifespan of the C. elegans AD models. Furthermore, RNA sequencing of nimodipine-treated worms revealed numerous downstream differentially expressed genes related to calcium signaling. Nimodipine-induced inhibition of selective voltage-gated calcium channels was shown to activate other calcium channels of the plasma membrane (clhm-1) and endoplasmic reticulum (unc-68), in addition to sodium-calcium exchanger channels (ncx-1). These channels collaborated to activate downstream events to resist oxidative stress through glutathione S-transferase activity mediated by HPGD and skn-1, as verified by RNA interference. These results may be applied for the treatment of Alzheimer's disease.
Asunto(s)
Enfermedad de Alzheimer , Proteínas de Caenorhabditis elegans , Animales , Enfermedad de Alzheimer/metabolismo , Caenorhabditis elegans , Calcio/metabolismo , Nimodipina/farmacología , Nimodipina/uso terapéutico , Ácido Egtácico/metabolismo , Ácido Egtácico/farmacología , Estrés Oxidativo , Péptidos beta-Amiloides/metabolismo , Canales de Calcio/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Proteínas de Caenorhabditis elegans/metabolismoRESUMEN
Rationale: Disruption of intracellular calcium (Ca2+) homeostasis is implicated in inflammatory responses. Here we investigated endoplasmic reticulum (ER) Ca2+ efflux through the Inositol 1,4,5-trisphosphate receptor (IP3R) as a potential mechanism of inflammatory pathophysiology in a ventilator-induced lung injury (VILI) mouse model. Methods: C57BL/6 mice were exposed to mechanical ventilation using high tidal volume (HTV). Mice were pretreated with the IP3R agonist carbachol, IP3R inhibitor 2-aminoethoxydiphenyl borate (2-APB) or the Ca2+ chelator BAPTA-AM. Lung tissues and bronchoalveolar lavage fluid (BALF) were collected to measure Ca2+ concentrations, inflammatory responses and mRNA/protein expression associated with ER stress, NLRP3 inflammasome activation and inflammation. Analyses were conducted in concert with cultured murine lung cell lines. Results: Lungs from mice subjected to HTV displayed upregulated IP3R expression in ER and mitochondrial-associated-membranes (MAMs), with enhanced formation of MAMs. Moreover, HTV disrupted Ca2+ homeostasis, with increased flux from the ER to the cytoplasm and mitochondria. Administration of carbachol aggravated HTV-induced lung injury and inflammation while pretreatment with 2-APB or BAPTA-AM largely prevented these effects. HTV activated the IRE1α and PERK arms of the ER stress signaling response and induced mitochondrial dysfunction-NLRP3 inflammasome activation in an IP3R-dependent manner. Similarly, disruption of IP3R/Ca2+ in MLE12 and RAW264.7 cells using carbachol lead to inflammatory responses, and stimulated ER stress and mitochondrial dysfunction. Conclusion: Increase in IP3R-mediated Ca2+ release is involved in the inflammatory pathophysiology of VILI via ER stress and mitochondrial dysfunction. Antagonizing IP3R/Ca2+ and/or maintaining Ca2+ homeostasis in lung tissue represents a prospective treatment approach for VILI.
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Compuestos de Boro/farmacología , Quelantes del Calcio/farmacología , Señalización del Calcio/efectos de los fármacos , Calcio/metabolismo , Ácido Egtácico/análogos & derivados , Retículo Endoplásmico/efectos de los fármacos , Receptores de Inositol 1,4,5-Trifosfato/antagonistas & inhibidores , Pulmón/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Lesión Pulmonar Inducida por Ventilación Mecánica/prevención & control , Animales , Apoptosis/efectos de los fármacos , Carbacol/toxicidad , Modelos Animales de Enfermedad , Ácido Egtácico/farmacología , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/patología , Estrés del Retículo Endoplásmico/efectos de los fármacos , Inflamasomas/metabolismo , Mediadores de Inflamación/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Pulmón/metabolismo , Pulmón/patología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Macrófagos/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Mitocondrias/patología , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Células RAW 264.7 , Lesión Pulmonar Inducida por Ventilación Mecánica/metabolismo , Lesión Pulmonar Inducida por Ventilación Mecánica/patologíaRESUMEN
Osteoarthritis (OA) is a common joint disease that is characterized by cartilage degradation. Iron deposition in the joints is common during the pathogenic progression of OA and recent studies have indicated that iron overload is an important contributor to OA progression. Calcium chelators have been reported to inhibit iron influx via modulating transferrin receptor protein 1 internalization, and they have been identified as a potential approach to the treatment of iron overloadinduced diseases. The aim of the present study was to investigate the effect of calcium chelators on the progression of iron overloadinduced OA. Primary chondrocytes were treated with various concentrations of ferric ammonium citrate (FAC) to mimic iron overload in vitro, followed by cotreatment with the calcium chelator BAPTA acetoxymethyl ester (BAPTAAM). Subsequently, intracellular iron levels, cell viability, reactive oxygen species (ROS) levels, mitochondrial function and morphological changes, as well as MMP levels, were detected using commercial kits. It was demonstrated that FAC treatment significantly promoted chondrocyte apoptosis and the expression of MMPs, and these effects were reversed by cotreatment with BAPTAAM. Moreover, BAPTAAM suppressed iron influx into chondrocytes and inhibited iron overloadinduced ROS production and mitochondrial dysfunction. These results indicated that calcium chelators may be of value in the treatment of iron metabolismrelated diseases and iron overloadinduced OA progression.
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Quelantes del Calcio/farmacología , Enfermedades de los Cartílagos/tratamiento farmacológico , Condrocitos/efectos de los fármacos , Ácido Egtácico/análogos & derivados , Sobrecarga de Hierro/complicaciones , Enfermedades Mitocondriales/tratamiento farmacológico , Sustancias Protectoras/farmacología , Animales , Apoptosis/efectos de los fármacos , Enfermedades de los Cartílagos/metabolismo , Cartílago Articular/efectos de los fármacos , Cartílago Articular/metabolismo , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Condrocitos/metabolismo , Ácido Egtácico/farmacología , Compuestos Férricos/farmacología , Sobrecarga de Hierro/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Enfermedades Mitocondriales/inducido químicamente , Enfermedades Mitocondriales/metabolismo , Osteoartritis/tratamiento farmacológico , Osteoartritis/metabolismo , Compuestos de Amonio Cuaternario/farmacología , Especies Reactivas de Oxígeno/metabolismoRESUMEN
BACKGROUND: Phagocytosis is an essential process that maintains cellular homeostasis. In the epidermis, the phagocytosis of melanosomes into keratinocytes is important to protect their DNA against damage from ultraviolet B (UVB) radiation. Furthermore, it is considered that UVB activates the phagocytosis by keratinocytes but the detailed mechanism involved is not fully understood. OBJECTIVE: To clarify the mechanism of UVB-enhanced phagocytosis in keratinocytes, we investigated the relationship between the phagocytic ability of keratinocytes and the cell cycle stage of keratinocytes. METHODS: The phagocytic ability of keratinocytes was evaluated using the incorporation of fluorescent beads after exposure to UVB or oxidative stress. S-phase was evaluated by BrdU incorporation and immunostaining of cyclin D1. Intracellular calcium levels of keratinocytes were measured using the probe Fluo-4AM. RESULTS: The phagocytosis of fluorescent beads into keratinocytes was enhanced by UVB and also by oxidative stress. We found that keratinocytes exposed to UVB or oxidative stress were at S-phase of the cell cycle. Furthermore, keratinocytes synchronized to S-phase showed a higher phagocytic ability according to the increased intracellular ROS level. The UVB-enhanced phagocytosis and entrance into S-phase of keratinocytes was abolished by ascorbic acid, a typical antioxidant. Keratinocytes synchronized to S-phase and exposed to UVB or oxidative stress had increased levels of intracellular calcium and their enhanced phagocytic abilities were diminished by the calcium ion chelator BAPTA-AM. CONCLUSION: Taken together, intracellular oxidative stress induced by intracellular calcium influx mediates the UVB-enhanced phagocytic ability of keratinocytes accumulating at S-phase of the cell cycle.
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Calcio/metabolismo , Queratinocitos/efectos de la radiación , Fagocitosis/efectos de la radiación , Puntos de Control de la Fase S del Ciclo Celular/efectos de la radiación , Rayos Ultravioleta/efectos adversos , Antioxidantes/farmacología , Ácido Ascórbico/farmacología , Línea Celular , Quelantes/farmacología , Daño del ADN/efectos de los fármacos , Daño del ADN/efectos de la radiación , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Humanos , Queratinocitos/citología , Queratinocitos/metabolismo , Melaninas/biosíntesis , Melanosomas/metabolismo , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/genética , Estrés Oxidativo/efectos de la radiación , Fagocitosis/efectos de los fármacos , Fagocitosis/genética , Especies Reactivas de Oxígeno/metabolismoRESUMEN
In 15% of cases, autosomal dominant polycystic kidney disease arises from defects in polycystin-2 (PC2). PC2 is a member of the polycystin transient receptor potential subfamily of cation-conducting channels and is expressed in the endoplasmic reticulum and primary cilium of renal epithelial cells. PC2 opposes a procystogenic influence of the cilium, and it has been proposed that this beneficial effect is mediated in part by a flow of Ca2+ through PC2 channels into the primary cilium. However, previous efforts to determine the permeability of PC2 channels to Ca2+ have yielded widely varying results. Here, we report the mean macroscopic Ca2+ influx through native PC2 channels in the primary cilia of mIMCD-3 cells, which are derived from the murine inner medullary collecting duct. Under conditions designed to isolate inward Ca2+ currents, a small inward Ca2+ current was detected in cilia with active PC2 channels but not in cilia lacking those channels. The current was activated by the addition of 10 µM internal Ca2+, which is known to activate ciliary PC2 channels. It was blocked by 10 µM isosakuranetin, which blocks the same channels. On average, the current amplitude was -1.8 pA at -190 mV; its conductance from -50 to -200 mV averaged 20 pS. Thus, native PC2 channels of renal primary cilia are able to conduct a small but detectable Ca2+ influx under the conditions tested. The possible consequences of this influx are discussed.NEW & NOTEWORTHY In autosomal dominant polycystic kidney disease, it is proposed that Ca2+ entering the primary cilium through polycystin-2 (PC2) channels may limit the formation of cysts. Recent studies predict that any macroscopic Ca2+ influx through these channels should be small. We report that the native PC2 channels in primary cilia of cultured renal epithelial cells can allow a small macroscopic calcium influx. This may allow a significant accumulation of Ca2+ in the cilium in vivo.
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Canales de Calcio/fisiología , Calcio/metabolismo , Cilios/fisiología , Fenómenos Electrofisiológicos , Canales Catiónicos TRPP/metabolismo , Animales , Línea Celular , Quelantes/farmacología , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Células Epiteliales , Túbulos Renales Colectores/citología , RatonesRESUMEN
AIM: Despite animal evidence of a role of calcium in the pathogenesis of spinal cord injury, several studies conducted in the past found calcium blockade ineffective. However, those studies involved oral or parenteral administration of Ca++ antagonists. We hypothesized that Ca++ blockade might be effective with local/immediate application (LIA) at the time of neural injury. METHODS: In this study, we assessed the effects of LIA of BAPTA (1,2-bis (o-aminophenoxy) ethane-N, N, N', N'-tetraacetic acid), a cell-permeable highly selective Ca++ chelator, after spinal cord transection (SCT) in mice over 4 weeks. Effects of BAPTA were assessed behaviorally and with immunohistochemistry. Concurrently, BAPTA was submitted for the first time to multimodality assessment in an in vitro model of neural damage as a possible spinal neuroprotectant. RESULTS: We demonstrate that BAPTA alleviates neuronal apoptosis caused by physical damage by inhibition of neuronal apoptosis and reactive oxygen species (ROS) generation. This translates to enhanced preservation of electrophysiological function and superior behavioral recovery. CONCLUSION: This study shows for the first time that local/immediate application of Ca++ chelator BAPTA is strongly neuroprotective after severe spinal cord injury.
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Quelantes del Calcio/uso terapéutico , Ácido Egtácico/análogos & derivados , Fármacos Neuroprotectores/uso terapéutico , Recuperación de la Función/efectos de los fármacos , Traumatismos de la Médula Espinal/tratamiento farmacológico , Vértebras Torácicas/lesiones , Animales , Quelantes del Calcio/farmacología , Células Cultivadas , Ácido Egtácico/farmacología , Ácido Egtácico/uso terapéutico , Femenino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Neuronas/efectos de los fármacos , Neuronas/fisiología , Neuroprotección/efectos de los fármacos , Neuroprotección/fisiología , Fármacos Neuroprotectores/farmacología , Especies Reactivas de Oxígeno/metabolismo , Recuperación de la Función/fisiología , Traumatismos de la Médula Espinal/metabolismo , Traumatismos de la Médula Espinal/patologíaRESUMEN
We present the first direct nuclear magnetic resonance (NMR) evidence of enhanced entry of Ca2+ ions into human erythrocytes (red blood cells; RBCs), when these cells are mechanically distorted. For this we loaded the RBCs with the fluorinated Ca2+ chelator, 1,2-bis(2-amino-5-fluorophenoxy)ethane-N,N,N',N'-tetraacetic acid (5FBAPTA), and recorded 19F NMR spectra. The RBCs were suspended in gelatin gel in a special stretching/compression apparatus. The 5FBAPTA was loaded into the cells as the tetraacetoxymethyl ester; and 13C NMR spectroscopy with [1,6-13C]D-glucose as substrate showed active glycolysis albeit at a reduced rate in cell suspensions and gels. The enhancement of Ca2+ influx is concluded to be via the mechanosensitive cation channel Piezo1. The increased rate of influx brought about by the activator of Piezo1, 2-[5-[[(2,6-dichlorophenyl)methyl]thio]-1,3,4-thiadiazol-2-yl]-pyrazine (Yoda1) supported this conclusion; while the specificity of the cation-sensing by 5FBAPTA was confirmed by using the Ca2+ ionophore, A23187.
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Calcio/metabolismo , Eritrocitos/metabolismo , Mecanotransducción Celular/fisiología , Transporte Biológico , Canales de Calcio/metabolismo , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Eritrocitos/patología , Femenino , Flúor , Imagen por Resonancia Magnética con Fluor-19/métodos , Glucosa , Glucólisis , Humanos , Canales Iónicos/metabolismo , Espectroscopía de Resonancia Magnética/métodos , MasculinoRESUMEN
BACKGROUND & AIMS: In upper airway cells, T helper 2 cytokines that signal through interleukin-4 (IL-4) receptor-α have been shown to stimulate eotaxin-3 secretion via a nongastric proton pump (ngH+,K+ATPase). To seek novel targets for eosinophilic esophagitis (EoE) treatments, we evaluated ngH+,K+ATPase expression in EoE squamous cells, and explored molecular pathways involved in eotaxin-3 secretion by IL-4 receptor-α signaling. METHODS: ngH+,K+ATPase expression in EoE cells was evaluated by quantitative real-time polymerase chain reaction and Western blotting. IL-4-stimulated eotaxin-3 secretion was measured by enzyme-linked immunosorbent assay after treatment with omeprazole, SCH 28080 (potassium-competitive acid blocker), ethylene glycol-bis(ß-aminoethyl)-N,N,N',N'-tetraacetoxymethyl ester (calcium chelator), 2-aminoethoxydiphenyl borate (inhibitor of endoplasmic reticulum calcium release), verapamil, and diltiazem (L-type calcium channel inhibitors). Intracellular calcium transients were measured by Fluo-4 fluorescence. Key experiments were confirmed in EoE primary cells and in RNA sequencing datasets from mucosal biopsies of patients with EoE and controls. RESULTS: EoE cells expressed ngH+,K+ATPase messenger RNA and protein. Omeprazole and SCH 28080 decreased IL-4-stimulated eotaxin-3 secretion. IL-4 increased intracellular calcium transients, and IL-4-stimulated eotaxin-3 secretion was blocked by ethylene glycol-bis(ß-aminoethyl)-N,N,N',N'-tetraacetoxymethyl ester, 2-aminoethoxydiphenyl borate, verapamil, and diltiazem. The combination of omeprazole and verapamil suppressed IL-4-stimulated eotaxin-3 secretion more than either agent alone. EoE biopsies expressed higher ngH+,K+ATPase and exhibited more calcium signaling than controls. CONCLUSIONS: EoE cells express a nongastric proton pump that mediates T helper 2 cytokine-stimulated eotaxin-3 secretion. IL-4 induces calcium release from the endoplasmic reticulum and calcium entry via L-type calcium channels, increasing intracellular calcium that contributes to eotaxin-3 secretion by EoE cells. L-type calcium channel inhibitors block T helper 2 cytokine-stimulated eotaxin-3 secretion, suggesting a potential role for these agents in EoE treatment.