RESUMO
Transient receptor potential canonical 4 (TRPC4) channel is a nonselective calcium-permeable cation channels. In intestinal smooth muscle cells, TRPC4 currents contribute more than 80% to muscarinic cationic current (mIcat). With its inward-rectifying current-voltage relationship and high calcium permeability, TRPC4 channels permit calcium influx once the channel is opened by muscarinic receptor stimulation. Polyamines are known to inhibit nonselective cation channels that mediate the generation of mIcat. Moreover, it is reported that TRPC4 channels are blocked by the intracellular spermine through electrostatic interaction with glutamate residues (E728, E729). Here, we investigated the correlation between the magnitude of channel inactivation by spermine and the magnitude of channel conductance. We also found additional spermine binding sites in TRPC4. We evaluated channel activity with electrophysiological recordings and revalidated structural significance based on Cryo-EM structure, which was resolved recently. We found that there is no correlation between magnitude of inhibitory action of spermine and magnitude of maximum current of the channel. In intracellular region, TRPC4 attracts spermine at channel periphery by reducing access resistance, and acidic residues contribute to blocking action of intracellular spermine; channel periphery, E649; cytosolic space, D629, D649, and E687.
Assuntos
Aminoácidos , Sítios de Ligação , Cálcio , Citosol , Ácido Glutâmico , Miócitos de Músculo Liso , Permeabilidade , Poliaminas , Receptores Muscarínicos , Espermina , Canais de Potencial de Receptor TransitórioRESUMO
Campylobacter is a worldwide foodborne pathogen, associated with human gastroenteritis. The efficient translocation of Campylobacter and its ability to secrete toxins into host cells are the 2 key features of Campylobacter pathophysiology which trigger inflammation in intestinal cells and contribute to the development of gastrointestinal symptoms, particularly diarrhoea, in humans. The purpose of conducting this literature review is to summarise the current understanding of: i) the human immune responses involved in the elimination of Campylobacter infection and ii) the resistance potential in Campylobacter against these immune responses. This review has highlighted that the intestinal epithelial cells are the preliminary cells which sense Campylobacter cells by means of their cell-surface and cytosolic receptors, activate various receptors-dependent signalling pathways, and recruit the innate immune cells to the site of inflammation. The innate immune system, adaptive immune system, and networking between these systems play a crucial role in bacterial clearance. Different cellular constituents of Campylobacter, mainly cell membrane lipooligosaccharides, capsule, and toxins, provide protection to Campylobacter against the human immune system mediated killing. This review has also identified gaps in knowledge, which are related to the activation of following during Campylobacter infection: i) cathelicidins, bactericidal permeability-increasing proteins, chemokines, and inflammasomes in intestinal epithelial cells; ii) siglec-7 receptors in dendritic cell; iii) acute phase proteins in serum; and iv) T-cell subsets in lymphoid nodules. This review evaluates the existing literature to improve the understanding of human immunity against Campylobacter infection and identify some of the knowledge gaps for future research.
Assuntos
Humanos , Proteínas de Fase Aguda , Células Apresentadoras de Antígenos , Infecções por Campylobacter , Campylobacter , Catelicidinas , Membrana Celular , Quimiocinas , Citosol , Células Dendríticas , Células Epiteliais , Gastroenterite , Síndrome de Guillain-Barré , Homicídio , Sistema Imunitário , Inflamassomos , Inflamação , Subpopulações de Linfócitos T , Receptores Toll-LikeRESUMO
BACKGROUND: Mesenchymal stem cells (MSCs) have strong self-renewal ability and multiple differentiation potential. Some studies confirmed that spreading shape and area of single MSCs influence cell differentiation, but few studies focused on the effect of the circularity of cell shape on the osteogenic differentiation of MSCs with a confined area during osteogenic process.METHODS: In the present study, MSCs were seeded on a micropatterned island with a spreading area lower than that of a freely spreading area. The patterns had circularities of 1.0 or 0.4, respectively, and areas of 314, 628, or 1256 µm² . After the cells were grown on a micropatterned surface for 1 or 3 days, cell apoptosis and F-actin were stained and analyzed. In addition, the expression of β-catenin and three osteogenic differentiation markers were immunofluorescently stained and analyzed, respectively.RESULTS: Of these MSCs, the ones with star-like shapes and large areas promoted the expression of osteogenic differentiation markers and the survival of cells. The expression of F-actin and its cytosolic distribution or orientation also correlated with the spreading shape and area. When actin polymerization was inhibited by cytochalasin D, the shape-regulated differentiation and apoptosis of MSCs with the confined spreading area were abolished.CONCLUSION: This study demonstrated that a spreading shape of low circularity and a larger spreading area are beneficial to the survival and osteogenic differentiation of individual MSCs, which may be regulated through the cytosolic expression and distribution of F-actin.
Assuntos
Actinas , Antígenos de Diferenciação , Apoptose , Diferenciação Celular , Forma Celular , Citocalasina D , Citosol , Células-Tronco Mesenquimais , Osteogênese , Polimerização , PolímerosRESUMO
OBJECTIVE@#Reactive oxygen species (ROS) are involved in a variety of biological phenomena and serve both deleterious and beneficial roles. ROS quantification and assessment of reaction networks are desirable but difficult because of their short half-life and high reactivity. Here, we describe a pro-oxidative model in a single human lung carcinoma SPC-A-1 cell that was created by application of extracellular H2O2 stimuli.@*METHODS@#Modified microfluidics and imaging techniques were used to determine O2 •- levels and construct an O2 •- reaction network. To elucidate the consequences of increased O2 •- input, the mitochondria were given a central role in the oxidative stress mode, by manipulating mitochondria-interrelated cytosolic Ca2+ levels, mitochondrial Ca2+ uptake, auto-amplification of intracellular ROS and the intrinsic apoptotic pathway.@*RESULTS AND CONCLUSIONS@#Results from a modified microchip demonstrated that 1 mmol/L H2O2 induced a rapid increase in cellular O2 •- levels (>27 vs. >406 amol in 20 min), leading to increased cellular oxidizing power (evaluated by ROS levels) and decreased reducing power (evaluated by glutathione (GSH) levels). In addition, we examined the dynamics of cytosolic Ca2+ and mitochondrial Ca2+ by confocal laser scanning microscopy and confirmed that Ca2+ stores in the endoplasmic reticulum were the primary source of H2O2-induced cytosolic Ca2+ bursts. It is clear that mitochondria have pivotal roles in determining how exogenous oxidative stress affects cell fate. The stress response involves the transfer of Ca2+ signals between organelles, ROS auto-amplification, mitochondrial dysfunction, and a caspase-dependent apoptotic pathway.
Assuntos
Humanos , Apoptose , Cálcio/metabolismo , Sinalização do Cálcio , Caspases/metabolismo , Linhagem Celular Tumoral , Linhagem da Célula , Citosol/metabolismo , Glutationa/metabolismo , Peróxido de Hidrogênio/química , Mitocôndrias/metabolismo , Oxirredução , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Superóxidos/químicaRESUMO
Depending on the intracellular buffering of calcium by chelation, zinc has the following two apparent effects on neuronal excitability: enhancement or reduction. Zinc increased tonic activity in the depolarized state when neurons were intracellularly dialyzed with EGTA but attenuated the neuronal activity when BAPTA was used as an intracellular calcium buffer. This suggests that neuronal excitability can be modulated by zinc, depending on the internal calcium buffering capacity. In this study, we elucidated the mechanisms of zinc-mediated alterations in neuronal excitability and determined the effect of calcium-related channels on zinc-mediated alterations in excitability. The zinc-induced augmentation of firing activity was mediated via the inhibition of small-conductance calcium-activated potassium (SK) channels with not only the contribution of voltage-gated L-type calcium channels (VGCCs) and ryanodine receptors (RyRs), but also through the activation of VGCCs via melastatin-like transient receptor potential channels. We suggest that zinc modulates the dopaminergic neuronal activity by regulating not only SK channels as calcium sensors, but also VGCCs or RyRs as calcium sources. Our results suggest that the cytosolic calcium-buffering capacity can tightly regulate zinc-induced neuronal firing patterns and that local calcium-signaling domains can determine the physiological and pathological state of synaptic activity in the dopaminergic system.
Assuntos
Animais , Ratos , Cálcio , Canais de Cálcio Tipo L , Citosol , Neurônios Dopaminérgicos , Ácido Egtázico , Eletrofisiologia , Incêndios , Neurônios , Potássio , Canal de Liberação de Cálcio do Receptor de Rianodina , Canais de Potencial de Receptor Transitório , ZincoRESUMO
Melatonin is a neurotransmitter that modulates various physiological phenomena including regulation and maintenance of the circadian rhythm. Nicotinic acetylcholine receptors (nAChRs) play an important role in oral functions including orofacial muscle contraction, salivary secretion, and tooth development. However, knowledge regarding physiological crosstalk between melatonin and nAChRs is limited. In the present study, the melatonin-mediated modulation of nAChR functions using bovine adrenal chromaffin cells, a representative model for the study of nAChRs, was investigated. Melatonin inhibited the nicotinic agonist dimethylphenylpiperazinium (DMPP) iodide-induced cytosolic free Ca²⁺ concentration ([Ca²⁺](i)) increase and norepinephrine secretion in a concentration-dependent manner. The inhibitory effect of melatonin on the DMPP-induced [Ca²⁺](i) increase was observed when the melatonin treatment was performed simultaneously with DMPP. The results indicate that melatonin inhibits nAChR functions in both peripheral and central nervous systems.
Assuntos
Sinalização do Cálcio , Sistema Nervoso Central , Células Cromafins , Ritmo Circadiano , Citosol , Iodeto de Dimetilfenilpiperazina , Melatonina , Contração Muscular , Neurotransmissores , Agonistas Nicotínicos , Norepinefrina , Fenômenos Fisiológicos , Receptores Nicotínicos , DenteRESUMO
G protein-coupled receptors (GPCRs) are membrane receptors whose agonist-induced dynamic conformational changes trigger heterotrimeric G protein activation, followed by GRK-mediated phosphorylation and arrestin-mediated desensitization. Cytosolic regions of GPCRs have been studied extensively because they are direct contact sites with G proteins, GRKs, and arrestins. Among various cytosolic regions, the role of helix 8 is least understood, although a few studies have suggested that it is involved in G protein activation, receptor localization, and/or internalization. In the present study, we investigated the role of helix 8 in dopamine receptor signaling focusing on dopamine D1 receptor (D1R) and dopamine D2 receptor (D2R). D1R couples exclusively to Gs, whereas D2R couples exclusively to Gi. Bioinformatic analysis implied that the sequences of helix 8 may affect GPCR-G protein coupling selectivity; therefore, we evaluated if swapping helix 8 between D1R and D2R changed G protein selectivity. Our results suggest that helix 8 is not involved in D1R-Gs or D2R-Gi coupling selectivity. Instead, we observed that D1R with D2R helix 8 or D1R with an increased number of hydrophobic residues in helix 8 relative to wild-type showed diminished β-arrestin-mediated desensitization, resulting in increased Gs signaling.
Assuntos
Arrestina , Arrestinas , Biologia Computacional , Citosol , Dopamina , Características da Família , Proteínas de Ligação ao GTP , Membranas , Fosforilação , Receptores de Dopamina D1 , Receptores de Dopamina D2 , Receptores DopaminérgicosRESUMO
In vitro prediction of hepatotoxicity can enhance the performance of non-clinical animal testing for identifying chemical hazards. In this study, we assessed high-content analysis (HCA) using multi-parameter cell-based assays as an in vitro hepatotoxicity testing model using various hepatotoxicants and human hepatocytes such as HepG2 cells and human primary hepatocytes (hPHs). Both hepatocyte types were exposed separately to multiple doses of ten hepatotoxicants associated with liver injury whose mechanisms of action have been described. HCA data were obtained using fluorescence probes for nuclear size (Hoechst), mitochondrial membrane potential (TMRM), cytosolic free calcium (Fluo-4AM), and lipid peroxidation (BODIPY). Cellular alterations were observed in response to all hepatotoxicants tested. The most sensitive parameter was TMRM, with high sensitivity at a low dose, next was BODIPY, followed by Fluo-4AM. HCA data from HepG2 cells and hPHs were generally concordant, although some inconsistencies were noted. Both hepatocyte types showed mild or severe mitochondrial impairment and lipid peroxidation in response to several hepatotoxicants. The results demonstrate that the application of HCA to in vitro hepatotoxicity testing enables more efficient hazard identification, and further, they suggest that certain parameters could serve as sensitive endpoints for predicting the hepatotoxic potential of chemical compounds.
Assuntos
Animais , Humanos , Cálcio , Citosol , Fluorescência , Células Hep G2 , Hepatócitos , Técnicas In Vitro , Peroxidação de Lipídeos , Fígado , Potencial da Membrana MitocondrialRESUMO
BACKGROUND/OBJECTIVES: Although aged black garlic has various biological activities such as anti-allergy, anti-inflammation and neuroprotection, effect of aged black garlic on chemically contact dermatitis is unclarified. MATERIALS/METHODS: To evaluate anti-dermatitic activity of aged black garlic extract, we investigated effects of a fraction of aged black garlic extract (BG10) on both in vivo and in vitro. RESULTS: BG10 almost inhibited formation of nitric monoxide and interleukin-6 (IL-6; IC50, 7.07 µg/mL) at 25 µg/mL, and dose-dependently reduced production of tumor necrosis factor-α (TNF-α; IC50, 52.07 µg/mL) and prostaglandin E2 (IC50, 38.46 µg/mL) in lipopolysaccharide-stimulated RAW264.7 cells. In addition, BG10 significantly inhibited the expression of inducible nitric oxide synthase, cyclooxygenase-2 and nuclear NF-κB, and improved that of cytosolic levels of NF-κB and IκBα in the cells. Consistent with in vitro studies, BG10 (0.5 mg/mL) not only reduced ear edema but also suppressed the formation of IL-6 and TNF-α induced by 12-O-tetradecanoylphorbol-13-acetate in ear tissues of mice. CONCLUSIONS: These findings suggest BG10 has anti-dermatitic activity through inhibiting activation of macrophages. Therefore, such effects of BG10 may provide information for the application of aged black garlic for prevention and therapy of contact dermatitis.
Assuntos
Animais , Camundongos , Ciclo-Oxigenase 2 , Citocinas , Citosol , Dermatite , Dermatite de Contato , Dinoprostona , Orelha , Edema , Alho , Técnicas In Vitro , Concentração Inibidora 50 , Interleucina-6 , Macrófagos , Necrose , Neuroproteção , NF-kappa B , Óxido Nítrico Sintase Tipo IIRESUMO
A better understanding of the underlying mechanisms by which signals from the fetus initiate human parturition is required. Our recent findings support the core hypothesis that oxidative stress (OS) and cellular senescence of the fetal membranes (amnion and chorion) trigger human parturition. Fetal membrane cell senescence at term is a natural physiological response to OS that occurs as a result of increased metabolic demands by the maturing fetus. Fetal membrane senescence is affected by the activation of the p38 mitogen activated kinase-mediated pathway. Similarly, various risk factors of preterm labor and premature rupture of the membranes also cause OS-induced senescence. Data suggest that fetal cell senescence causes inflammatory senescence-associated secretory phenotype (SASP) release. Besides SASP, high mobility group box 1 and cell-free fetal telomere fragments translocate from the nucleus to the cytosol in senescent cells, where they represent damage-associated molecular pattern markers (DAMPs). In fetal membranes, both SASPs and DAMPs augment fetal cell senescence and an associated ‘sterile’ inflammatory reaction. In senescent cells, DAMPs are encapsulated in extracellular vesicles, specifically exosomes, which are 30–150 nm particles, and propagated to distant sites. Exosomes traffic from the fetus to the maternal side and cause labor-associated inflammatory changes in maternal uterine tissues. Thus, fetal membrane senescence and the inflammation generated from this process functions as a paracrine signaling system during parturition. A better understanding of the premature activation of these signals can provide insights into the mechanisms by which fetal signals initiate preterm parturition.
Assuntos
Feminino , Humanos , Gravidez , Envelhecimento , Senescência Celular , Citosol , Exossomos , Vesículas Extracelulares , Membranas Extraembrionárias , Feto , Inflamação , Membranas , Trabalho de Parto Prematuro , Estresse Oxidativo , Comunicação Parácrina , Parto , Fenótipo , Nascimento Prematuro , Fatores de Risco , Ruptura , TelômeroRESUMO
In 1923, Dr. Warburg had observed that tumors acidified the Ringer solution when 13 mM glucose was added, which was identified as being due to lactate. When glucose is the only source of nutrient, it can serve for both biosynthesis and energy production. However, a series of studies revealed that the cancer cell consumes glucose for biosynthesis through fermentation, not for energy supply, under physiological conditions. Recently, a new observation was made that there is a metabolic symbiosis in which glycolytic and oxidative tumor cells mutually regulate their energy metabolism. Hypoxic cancer cells use glucose for glycolytic metabolism and release lactate which is used by oxygenated cancer cells. This study challenged the Warburg effect, because Warburg claimed that fermentation by irreversible damaging of mitochondria is a fundamental cause of cancer. However, recent studies revealed that mitochondria in cancer cell show active function of oxidative phosphorylation although TCA cycle is stalled. It was also shown that blocking cytosolic NADH production by aldehyde dehydrogenase inhibition, combined with oxidative phosphorylation inhibition, resulted in up to 80% decrease of ATP production, which resulted in a significant regression of tumor growth in the NSCLC model. This suggests a new theory that NADH production in the cytosol plays a key role of ATP production through the mitochondrial electron transport chain in cancer cells, while NADH production is mostly occupied inside mitochondria in normal cells.
Assuntos
Trifosfato de Adenosina , Aldeído Desidrogenase , Citosol , Transporte de Elétrons , Metabolismo Energético , Fermentação , Glucose , Ácido Láctico , Metabolismo , Mitocôndrias , NAD , Fosforilação Oxidativa , Oxigênio , SimbioseRESUMO
Nucleotide-binding domain 1 (Nod1) is a cytosolic receptor that is responsible for the recognition of a bacterial peptidoglycan motif containing meso-diaminophimelic acid. In this study, we sought to identify the role of Nod1 in host defense in vivo against pulmonary infection by multidrug resistant Acinetobacter baumannii. Wildtype (WT) and Nod1-deficient mice were intranasally infected with 3×107 CFU of A. baumannii and sacrificed at 1 and 3 days post-infection (dpi). Bacterial CFUs, cytokines production, histopathology, and mouse β-defensins (mBD) in the lungs of infected mice were evaluated. The production of cytokines in response to A. baumannii was also measured in WT and Nod1-deficient macrophages. The bacterial clearance in the lungs was not affected by Nod1 deficiency. Levels of IL-6, TNF-α, and IL-1β in the lung homogenates were comparable at days 1 and 3 between WT and Nod1-deficient mice, except the TNF-α level at day 3, which was higher in Nod1-deficient mice. There was no significant difference in lung pathology and expression of mBDs (mBD1, 2, 3, and 4) between WT and Nod1-deficient mice infected with A. baumannii. The production of IL-6, TNF-α, and NO by macrophages in response to A. baumannii was also comparable in WT and Nod1-deficient mice. Our results indicated that Nod1 does not play an important role in host immune responses against A. baumannii infection.
Assuntos
Animais , Camundongos , Acinetobacter baumannii , Acinetobacter , Citocinas , Citosol , Interleucina-6 , Pulmão , Macrófagos , Patologia , PeptidoglicanoRESUMO
The nucleotide-binding oligomerization domain-like receptors (NOD-like receptors, NLRs) are intracellular sensors. Most of them positively affect inflammatory responses, particularly the inflammasome forming NLRs. On the other hand, several studies on gene-deficient mice have revealed that several NLRs negatively influence innate immune responses. Some recent studies have identified a novel sub-group of non-inflammasome forming NLRs that negatively influence different pathways related to inflammation and carcinogenesis. Cytosolic pattern recognition receptor NRLC3 is a negative regulator of innate immune response. In this review we will discuss finding related with NLRC3 and its mechanism by which it alter cancer pathogenesis. Recently, it has been found that mice deficient in Nlrc3 are hyper-susceptible to colitis and colitis-associated colon carcinogenesis. Oncogenic inhibitory effect of NLRC3 is more dominant in epithelial compartment than hematopoietic compartment. It down regulates mTOR signaling and reduce cell proliferation. NLRC3 interact with PI3Ks and suppress activation of PI3K dependent kinase AKT. Understanding the role of NLRC3 in cancer may facilitate the recognition of new therapeutic strategies.
Assuntos
Animais , Camundongos , Carcinogênese , Proliferação de Células , Colite , Colo , Neoplasias do Colo , Citosol , Mãos , Imunidade Inata , Inflamassomos , Inflamação , FosfotransferasesRESUMO
PURPOSE: The dried body of Scolopendra subspinipes mutilans has long been used as a traditional Korean medicinal food, but little is known about its mechanisms of action. In this study, we investigated the anti-inflammatory activities of Scolopendra subspinipes mutilans and possible mechanisms in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. METHODS: Cytotoxicity of Scolopendra subspinipes mutilans extract (SSME) was measured by MTT assay, anti-inflammatory activities were analyzed by nitric oxide (NO) production, the expression of inducible NO synthase (iNOS) and the mRNA level of pro-inflammatory cytokines such as interleukin-1β (IL-1β) and interleukin-6 (IL-6). Nuclear translocation of nuclear factor-kappa B (NF-κB) p65 subunit and degradation of inhibitory kappa B (IκB) were examined by western blot. RESULTS: SSME inhibited LPS-induced NO production and iNOS expression without cytotoxicity. Up-regulation of LPS-induced pro-inflammatory cytokines, IL-1β and IL-6 was dose dependently attenuated by SSME. Exposure of pyrrolidine dithiocarbamate, an NF-κB specific inhibitor, accelerated the inhibitory effects of SSME on NO production and iNOS expression in LPS-stimulated cells. Moreover, translocation of NF-κB from the cytosol to the nucleus and degradation of IκB were decreased by treatment with SSME in LPS-induced cells. CONCLUSION: These results suggest that the SSME might have the inhibitory effects on inflammation, partly through inhibition of the NF-κB signaling pathway.
Assuntos
Western Blotting , Citocinas , Citosol , Inflamação , Interleucina-6 , Óxido Nítrico , Óxido Nítrico Sintase , RNA Mensageiro , Regulação para CimaRESUMO
α-Iso-cubebene (ICB) is a dibenzocyclooctadiene lignin contained in Schisandra chinensis (SC), a well-known medicinal herb that ameliorates cardiovascular symptoms, but the mechanism responsible for this activity has not been determined. To determine the role played by ICB on the regulation of vascular tone, we investigated the inhibitory effects of ICB on vascular contractile responses by adrenergic α-receptor agonists. In addition, we investigated the role on myosin light chain (MLC) phosphorylation and cytosolic calcium concentration in vascular smooth muscle cells (VSMC). In aortic rings isolated from C57BL/6J mice, ICB significantly attenuated the contraction induced by phenylephrine (PE) and norepinephrine (NE), whereas ICB had no effects on KCl (60 mM)-induced contraction. In vasculatures precontracted with PE, ICB caused marked relaxation of aortic rings with or without endothelium, suggesting a direct effect on VSMC. In cultured rat VSMC, PE or NE increased MLC phosphorylation and increased cytosolic calcium levels. Both of these effects were significantly suppressed by ICB. In conclusion, our results showed that ICB regulated vascular tone by inhibiting MLC phosphorylation and calcium flux into VSMC, and suggest that ICB has anti-hypertensive properties and therapeutic potential for cardiovascular disorders related to vascular hypertension.
Assuntos
Animais , Camundongos , Ratos , Aorta Torácica , Cálcio , Citosol , Endotélio , Hipertensão , Lignina , Músculo Liso Vascular , Cadeias Leves de Miosina , Miosinas , Norepinefrina , Fenilefrina , Fosforilação , Plantas Medicinais , Relaxamento , SchisandraRESUMO
Intracellular Ca²⁺ mobilization is closely linked with the initiation of salivary secretion in parotid acinar cells. Reactive oxygen species (ROS) are known to be related to a variety of oxidative stress-induced cellular disorders and believed to be involved in salivary impairments. In this study, we investigated the underlying mechanism of hydrogen peroxide (H₂O₂) on cytosolic Ca²⁺ accumulation in mouse parotid acinar cells. Intracellular Ca²⁺ levels were slowly elevated when 1 mM H₂O₂ was perfused in the presence of normal extracellular Ca²⁺. In a Ca²⁺-free medium, 1 mM H₂O₂ still enhanced the intracellular Ca²⁺ level. Ca²⁺ entry tested using manganese quenching technique was not affected by perfusion of 1 mM H₂O₂. On the other hand, 10 mM H₂O₂ induced more rapid Ca²⁺ accumulation and facilitated Ca²⁺ entry from extracellular fluid. Ca²⁺ refill into intracellular Ca²⁺ store and inositol 1,4,5-trisphosphate (1 µM)-induced Ca²⁺ release from Ca²⁺ store was not affected by 1 mM H₂O₂ in permeabilized cells. Ca²⁺ efflux through plasma membrane Ca²⁺-ATPase (PMCA) was markedly blocked by 1 mM H₂O₂ in thapsigargin-treated intact acinar cells. Antioxidants, either catalase or dithiothreitol, completely protected H₂O₂-induced Ca²⁺ accumulation through PMCA inactivation. From the above results, we suggest that excessive production of H₂O₂ under pathological conditions may lead to cytosolic Ca²⁺ accumulation and that the primary mechanism of H₂O₂-induced Ca²⁺ accumulation is likely to inhibit Ca²⁺ efflux through PMCA rather than mobilize Ca²⁺ ions from extracellular medium or intracellular stores in mouse parotid acinar cells.
Assuntos
Animais , Camundongos , Células Acinares , Antioxidantes , Cálcio , Catalase , Membrana Celular , Citosol , Ditiotreitol , Líquido Extracelular , Mãos , Peróxido de Hidrogênio , Hidrogênio , Inositol 1,4,5-Trifosfato , Íons , Manganês , Perfusão , ATPases Transportadoras de Cálcio da Membrana Plasmática , Plasma , Espécies Reativas de OxigênioRESUMO
NADPH-oxidase (NOX) mediated superoxide originally found on leukocytes, but now recognized in several types of cells in the brain. It has been shown to play an important role in the progression of stroke and related cerebrovascular disease. NOX is a multisubunit complex consisting of 2 membrane-associated and 4 cytosolic subunits. NOX activation occurs when cytosolic subunits translocate to the membrane, leading to transport electrons to oxygen, thus producing superoxide. Superoxide produced by NOX is thought to function in long-term potentiation and intercellular signaling, but excessive production is damaging and has been implicated to play an important role in the progression of ischemic brain. Thus, inhibition of NOX activity may prove to be a promising treatment for ischemic brain as well as an adjunctive agent to prevent its secondary complications. There is mounting evidence that NOX inhibition in the ischemic brain is neuroprotective, and targeting NOX in circulating immune cells will also improve outcome. This review will focus on therapeutic effects of NOX assembly inhibitors in brain ischemia and stroke. However, the lack of specificity and toxicities of existing inhibitors are clear hurdles that will need to be overcome before this class of compounds could be translated clinically.
Assuntos
Encéfalo , Isquemia Encefálica , Transtornos Cerebrovasculares , Citosol , Leucócitos , Potenciação de Longa Duração , Membranas , NADPH Oxidases , Oxigênio , Sensibilidade e Especificidade , Acidente Vascular Cerebral , Superóxidos , Usos TerapêuticosRESUMO
Free fatty acids (FFAs) are important substrates for mitochondrial oxidative metabolism and ATP synthesis but also cause serious stress to various tissues, contributing to the development of metabolic diseases. CD36 is a major mediator of cellular FFA uptake. Inside the cell, saturated FFAs are able to induce the production of cytosolic and mitochondrial reactive oxygen species (ROS), which can be prevented by co-exposure to unsaturated FFAs. There are close connections between oxidative stress and organellar Ca²⁺ homeostasis. Highly oxidative conditions induced by palmitate trigger aberrant endoplasmic reticulum (ER) Ca²⁺ release and thereby deplete ER Ca²⁺ stores. The resulting ER Ca²⁺ deficiency impairs chaperones of the protein folding machinery, leading to the accumulation of misfolded proteins. This ER stress may further aggravate oxidative stress by augmenting ER ROS production. Secondary to ER Ca²⁺ release, cytosolic and mitochondrial matrix Ca²⁺ concentrations can also be altered. In addition, plasmalemmal ion channels operated by ER Ca²⁺ depletion mediate persistent Ca²⁺ influx, further impairing cytosolic and mitochondrial Ca²⁺ homeostasis. Mitochondrial Ca²⁺ overload causes superoxide production and functional impairment, culminating in apoptosis. This vicious cycle of lipotoxicity occurs in multiple tissues, resulting in β-cell failure and insulin resistance in target tissues, and further aggravates diabetic complications.
Assuntos
Trifosfato de Adenosina , Apoptose , Cálcio , Citosol , Complicações do Diabetes , Retículo Endoplasmático , Ácidos Graxos não Esterificados , Homeostase , Resistência à Insulina , Canais Iônicos , Doenças Metabólicas , Metabolismo , Estresse Oxidativo , Dobramento de Proteína , Espécies Reativas de Oxigênio , SuperóxidosRESUMO
OBJECTIVE: Retinal dehydrogenase 1 (RALDH1) is a cytosolic enzyme which acts both as a source of retinoic acid (RA) and as a detoxification enzyme. RALDH1 has key functions in the midbrain dopaminergic system, which influences motivation, cognition, and social behavior. Since dopamine has been increasingly linked to autism spectrum disorder (ASD), we asked whether RALDH1 could contribute to the autistic phenotype. Therefore, we investigated for the first time the levels of RALDH1 in autistic patients. To further assess the detoxification function of RALDH1, we also explored 4-hydroxynonenal protein adducts (4-HNE PAs) and reduced glutathione (GSH) levels. Moreover, considering the effect of testosterone on RALDH1 expression, we measured the second to fourth digit ratio (2D:4D ratio) for both hands, which reflects exposure to prenatal testosterone. METHODS: Male patients with ASD (n=18; age, 62.9±4.3 months) and healthy controls (n=13; age, 78.1±4.9 months) were examined. Erythrocyte RALDH1, serum 4-HNE PAs and erythrocyte GSH levels were measured using colorimetric assays, and digit lengths were measured using digital calipers. RESULTS: We found significantly lower (−42.9%) RALDH1 levels in autistic patients as compared to controls (p=0.032). However, there was no difference in 4-HNE PAs levels (p=0.368), GSH levels (p=0.586), or 2D:4D ratios (p=0.246 in the left hand, p=0.584 in the right hand) between healthy controls and autistic subjects. CONCLUSION: We concluded that a subset of autistic patients had a low RALDH1 level. These results suggest that low RALDH1 levels could contribute to the autistic phenotype by reflecting a dopaminergic dysfunction.
Assuntos
Humanos , Masculino , Transtorno do Espectro Autista , Transtorno Autístico , Cognição , Citosol , Dopamina , Eritrócitos , Glutationa , Mãos , Mesencéfalo , Motivação , Fenótipo , Retinal Desidrogenase , Retinaldeído , Comportamento Social , Testosterona , TretinoínaRESUMO
The main task of skeletal muscle is contraction and relaxation for body movement and posture maintenance. During contraction and relaxation, Ca²⁺ in the cytosol has a critical role in activating and deactivating a series of contractile proteins. In skeletal muscle, the cytosolic Ca²⁺ level is mainly determined by Ca²⁺ movements between the cytosol and the sarcoplasmic reticulum. The importance of Ca²⁺ entry from extracellular spaces to the cytosol has gained significant attention over the past decade. Store-operated Ca²⁺ entry with a low amplitude and relatively slow kinetics is a main extracellular Ca²⁺ entryway into skeletal muscle. Herein, recent studies on extracellular Ca²⁺ entry into skeletal muscle are reviewed along with descriptions of the proteins that are related to extracellular Ca²⁺ entry and their influences on skeletal muscle function and disease.