Cytoplasmic Calcium Concentration Research Articles

Phosphorylation of CAX transporters controls Ca2+ homeostasis

Plants rely on a sophisticated innate immune system to recognize and defend against invading pathogens. The first line of plant innate immunity called pattern-triggered immunity (PTI) is initiated upon detection of pathogen-associated molecular patterns (PAMPs) by plasma membrane-localized pattern recognition receptors (PRRs) (Jones and Dangl, 2006). A well-characterized PRR is the Arabidopsis receptor-like kinase (RLK) FLS2, which complexes with the co-receptor BAK1 to detect the bacterial PAMP flagellin (or its major epitope flg22) (Zipfel, 2014). Downstream of PAMP-triggered PRR activation, the receptor-like cytoplasmic kinases (RLCKs), such as BIK1 and RIPK, associate with and are activated by PRRs to trigger diverse downstream immune signaling events, including reactive oxygen species (ROS) burst, phosphatidic acid (PA) production, increase of cytoplasmic calcium ([Ca2+]cyt) concentration, and activation of mitogen-activated protein kinase (MAPK) cascades, which collectively lead to the establishment of plant PTI (Bigeard et al., 2015).

Sex-dependent intra-islet structural rearrangements affecting alpha-to-beta cell interactions lead to adaptive enhancements of Ca2+ dynamics in prediabetic beta cells

Aims/hypothesisPrediabetic pancreatic beta cells can adapt their function to maintain normoglycaemia for a limited period of time, after which diabetes mellitus will manifest upon beta cell exhaustion. Understanding sex-specific beta cell compensatory mechanisms and their failure in prediabetes (impaired glucose tolerance) is crucial for early disease diagnosis and individualised treatment. Our aims were as follows: (1) to determine the key time points of the progression from beta cells’ functional adaptations to their failure in vivo; and (2) to mechanistically explain in vivo sex-specific beta cell compensatory mechanisms and their failure in prediabetes.MethodsIslets from male and female transgenic Ins1CreERT2-GCaMP3 mice were transplanted into the anterior chamber of the eye of 10- to 12-week-old sex-matched C57BL/6J mice. Recipient mice were fed either a control diet (CD) or western diet (WD) for a maximum of 4 months. Metabolic variables were evaluated monthly. Beta cell cytoplasmic free calcium concentration ([Ca2+]i) dynamics were monitored in vivo longitudinally by image fluorescence of the GCaMP3 reporter islets. Global islet beta cell [Ca2+]i dynamics in line with single beta cell [Ca2+]i analysis were used for beta cell coordination studies. The glucagon receptor antagonist L-168,049 (4 mmol/l) was applied topically to the transplanted eyes to evaluate in vivo the effect of glucagon on beta cell [Ca2+]idynamics. Human islets from non-diabetic women and men were cultured for 24 h in either a control medium or high-fat/high-glucose medium in the presence or absence of the glucagon receptor antagonist L-168,049. [Ca2+]i dynamics of human islets were evaluated in vitro after 1 h exposure to Fura-10.ResultsMice fed a WD for 1 month displayed increased beta cell [Ca2+]i dynamics linked to enhanced insulin secretion as a functional compensatory mechanism in prediabetes. Recruitment of inactive beta cells in WD-fed mice explained the improved beta cell function adaptation observed in vivo; this occurred in a sex-specific manner. Mechanistically, this was attributable to an intra-islet structural rearrangement involving alpha cells. These sex-dependent cytoarchitecture reorganisations, observed in both mice and humans, induced enhanced paracrine input from adjacent alpha cells, adjusting the glucose setpoint and amplifying the insulin secretion pathway. When WD feeding was prolonged, female mice maintained the adaptive mechanism due to their intrinsically high proportion of alpha cells. In males, [Ca2+]i dynamics progressively declined subsequent to glucose stimulation while insulin secretion continue to increase, suggesting uncoordinated beta cell function as an early sign of diabetes.Conclusions/interpretationWe identified increased coordination of [Ca2+]i dynamics as a beta cell functional adaptation mechanisms in prediabetes. Importantly, we uncovered the mechanisms by which sex-dependent beta cell [Ca2+]i dynamics coordination is orchestrated by an intra-islet structure reorganisation increasing the paracrine input from alpha cells on beta cell function. Moreover, we identified reduced [Ca2+]i dynamics coordination in response to glucose as an early sign of diabetes preceding beta cell secretory dysfunction, with males being more vulnerable. Alterations in coordination capacity of [Ca2+]i dynamics may thus serve as an early marker for beta cell failure in prediabetes.Graphical

Host transcriptome response to heat stress and Eimeria maxima infection in meat-type chickens.

Eimeria (E.) maxima parasite infects chickens' midgut disrupting the jejunal and ileal mucosa causing high morbidity and mortality. Heat stress (HS) is a seasonal stressor that impacts biological functions leading to poor performance. This study elucidates how HS, E. maxima infection, and their combination affect the ileum transcriptome. Two-hundred and forty 2-week-old males Ross708 chickens were randomly allocated into four treatment groups: thermoneutral-control (TNc), thermoneutral-infected (TNi), heat-stress control (HSc), and heat stress-infected (HSi), with 6 replicates each of 10 birds. Infected groups received 200x103 sporulated E. maxima oocysts/bird, and heat-treated groups were raised at 35°C. At 6-day post-treatment, ileums of five randomly selected chickens per group were sampled, RNA was extracted and sequenced. A total of 413, 3377, 1908, and 2304 DEGs were identified when applying the comparisons: TNc vs HSc, TNc vs TNi, HSi vs HSc, and TNi vs HSi, respectively, at cutoff ≥1.2-fold change (FDR: q<0.05). HSc vs TNc showed upregulation of lipid metabolic pathways and degradation/metabolism of multiple amino acids; and downregulation of most immune-related and protein synthesis pathways. TNc vs TNi displayed upregulation of most of immune-associated pathways and eukaryotic mRNA maturation pathways; and downregulation of fatty acid metabolism and multiple amino acid metabolism pathways including tryptophan. Comparing HSi versus HSc and TNi revealed that combining the two stressors restored the expression of some cellular functions, e.g., oxidative phosphorylation and protein synthesis; and downregulate immune response pathways associated with E. maxima infection. During E. maxima infection under HS the calcium signaling pathway was downregulated, including genes responsible for increasing the cytoplasmic calcium concentration; and tryptophan metabolism was upregulated, including genes that contribute to catabolizing tryptophan through serotonin and indole pathways; which might result in reducing the cytoplasmic pool of nutrients and calcium available for the parasite to scavenge and consequently might affect the parasite's reproductive ability.

Tacrolimus impairs airway mucociliary clearance of rats

ObjectivesTacrolimus (TAC) is the most widely used immunosuppressive agent after lung transplantation. Considering that the ciliary beat frequency (CBF) mainly depends on the cytoplasmic calcium concentration and that TAC can affect this due to its binding with the intracellular immunophilin FKBP12, we hypothesized that TAC could also impair the airway mucociliary clearance of rats. MethodsSixty rats were divided into two groups (n = 30 each): Control = water; TAC = tacrolimus. After 7, 15 or 30 days of treatment, ten animals from each group were euthanized and the following parameters were studied: mucus transportability, CBF, mucociliary transport velocity (MCTV), and neutral and acid mucus production. ResultsThere was a significant decrease in CBF (Control vs TAC: 7 days, p = 0.008; 15 days, p = 0.007; 30 days, p = 0.001) and MCTV (Control vs TAC: 7 days, p = 0.004; 15 days, p < 0.001; 30 days, p < 0.001) in all immunosuppressed animals. TAC therapy also caused an increase in acid mucus production at all treatment times (Control vs TAC: 7 days, p = 0.001; 15 days, p = 0.043; 30 days, p = 0.001). ConclusionsTAC impairs airway mucociliary clearance of rats.

Septin6 as a new approach for AD treatment

The number of Alzheimer’s disease (AD) patients is increasing and new therapeutic approaches need to be proposed urgently. In recent years, some researchers have focused on the relationship between calcium homeostasis and AD; however, selective regulation of abnormal calcium signaling pathways and related targets of action remain unclear, presenting a challenge. Gerard Griffioen’s team in Belgium has proposed a self-reinforcing amplification between cytoplasmic calcium concentration [Ca2+]cyto and AD pathology in previous studies, discovering a new kind of small-molecule scaffold protein. The protein, ReS19-T, can stabilize the structure of septin filaments and significantly improve the core pathology of AD by inhibiting the pathological activation of store-operated calcium entry (SOCE) and restoring calcium homeostasis, thereby suggesting a new avenue for therapeutic intervention. However, there is still a way to go before clinical application. There are some questions. SEP2/6/7 hexamer plays a role in maintaining immune function, so could ReS19-T affect this function and impact immune responses? Moreover, both Stim1 and Orai (affected by TRPC) contribute to SOCE. The TRPC-specific inhibitor SKF-96365 is highly selective, and its relationship with AD remains to be investigated. Future studies might use SKF-96365 to validate the therapeutic effect of Res19-T. In conclusion, Septin6 as a new approach to AD treatment expects more relevant research to emerge.

Identification of Bioactive Phytocytokines Using Transcriptomic Data and Plant Bioassays.

Plant genomes contain thousands of short open reading frames that encode putative peptides. Some of these peptides play important signaling roles in response to environmental stress. Here we describe a pipeline used to identify the CTNIP/SMALL PHYTOCYTOKINES REGULATING DEFENSE AND WATER LOSS (SCREW) family of phytocytokines, based upon their transcriptional upregulation during biotic stress. Moreover, we describe approaches to assay their activity in planta by measuring increases in cytoplasmic calcium concentration, reactive oxygen species production, and mitogen-activated protein kinase phosphorylation.

Mitochondrial Oxidative Stress Is the General Reason for Apoptosis Induced by Different-Valence Heavy Metals in Cells and Mitochondria.

This review analyzes the causes and consequences of apoptosis resulting from oxidative stress that occurs in mitochondria and cells exposed to the toxic effects of different-valence heavy metals (Ag+, Tl+, Hg2+, Cd2+, Pb2+, Al3+, Ga3+, In3+, As3+, Sb3+, Cr6+, and U6+). The problems of the relationship between the integration of these toxic metals into molecular mechanisms with the subsequent development of pathophysiological processes and the appearance of diseases caused by the accumulation of these metals in the body are also addressed in this review. Such apoptosis is characterized by a reduction in cell viability, the activation of caspase-3 and caspase-9, the expression of pro-apoptotic genes (Bax and Bcl-2), and the activation of protein kinases (ERK, JNK, p53, and p38) by mitogens. Moreover, the oxidative stress manifests as the mitochondrial permeability transition pore (MPTP) opening, mitochondrial swelling, an increase in the production of reactive oxygen species (ROS) and H2O2, lipid peroxidation, cytochrome c release, a decline in the inner mitochondrial membrane potential (ΔΨmito), a decrease in ATP synthesis, and reduced glutathione and oxygen consumption as well as cytoplasm and matrix calcium overload due to Ca2+ release from the endoplasmic reticulum (ER). The apoptosis and respiratory dysfunction induced by these metals are discussed regarding their interaction with cellular and mitochondrial thiol groups and Fe2+ metabolism disturbance. Similarities and differences in the toxic effects of Tl+ from those of other heavy metals under review are discussed. Similarities may be due to the increase in the cytoplasmic calcium concentration induced by Tl+ and these metals. One difference discussed is the failure to decrease Tl+ toxicity through metallothionein-dependent mechanisms. Another difference could be the decrease in reduced glutathione in the matrix due to the reversible oxidation of Tl+ to Tl3+ near the centers of ROS generation in the respiratory chain. The latter may explain why thallium toxicity to humans turned out to be higher than the toxicity of mercury, lead, cadmium, copper, and zinc.

Possible role of high calcium concentrations in rat neocortical neurons in inducing hyper excitatory behavior during emergence from sevoflurane: a proposed pathophysiology.

Sevoflurane has been shown to increase the incidence of emergence delirium in children; however, the mechanism remains unclear. Sevoflurane increases cytoplasmic calcium concentration which in turn may play a role in emergence delirium. This study aimed to investigate the level of intracellular calcium in rats experiencing hyperexcitatory behavior after exposure to sevoflurane, as well as the role of magnesium in preventing this phenomenon. After ethical approval, 2-5-week-old Sprague-Dawley rats (n = 34) were insufflated with sevoflurane in a modified anesthesia chamber. One group received magnesium sulphate intraperitoneally. After termination of sevoflurane exposure, the occurrence of hyperexcitation was observed. Brain tissue samples from the rats were studied for intracellular calcium levels under a two-channel laser scanning confocal microscope and were quantitatively calculated using ratiometric calculation. The presence of inflammation or oxidative stress reaction was assessed using nuclear factor κB and malondialdehyde. The incidence of hyperexcitatory behavior post sevoflurane exposure was 9 in 16 rats in the observation group and none in the magnesium group. Tests for inflammation and oxidative stress were within normal limits in both groups. The rats showing hyperexcitation had a higher level of cytosol calcium concentration compared to the other groups. To conclude, the calcium concentration of neocortical neurons in Sprague-Dawley rats with hyperexcitatory behavior is increased after exposure to sevoflurane. Administration of magnesium sulphate can prevent the occurrence of hyperexcitation in experimental animals.

Synergistic Interaction of 5-HT1B and 5-HT2B Receptors in Cytoplasmic Ca2+ Regulation in Human Umbilical Vein Endothelial Cells: Possible Involvement in Pathologies.

The aim of this work was to explore the involvement of 5-HT1B and 5-HT2B receptors (5-HT1BR and 5-HT2BR) in the regulation of free cytoplasmic calcium concentration ([Ca2+]i) in human umbilical vein endothelial cells (HUVEC). We have shown by quantitative PCR analysis, that 5-HT1BR and 5-HT2BR mRNAs levels are almost equal in HUVEC. Immunofluorescent staining demonstrated, that 5-HT1BR and 5-HT2BR are expressed both in plasma membrane and inside the cells. Intracellular 5-HT1BR are localized mainly in the nuclear region, whereas 5-HT2BR receptors are almost evenly distributed in HUVEC. 5-HT, 5-HT1BR agonist CGS12066B, or 5-HT2BR agonist BW723C86 added to HUVEC caused a slight increase in [Ca2+]i, which was much lower than that of histamine, ATP, or SFLLRN, an agonist of protease-activated receptors (PAR1). However, activation of 5-HT1BR with CGS12066B followed by activation of 5-HT2BR with BW723C86 manifested a synergism of response, since several-fold higher rise in [Ca2+]i occurred. CGS12066B caused more than a 5-fold increase in [Ca2+]i rise in HUVEC in response to 5-HT. This 5-HT induced [Ca2+]i rise was abolished by 5-HT2BR antagonist RS127445, indicating that extracellular 5-HT acts through 5-HT2BR. Synergistic [Ca2+]i rise in response to activation of 5-HT1BR and 5-HT2BR persisted in a calcium-free medium. It was suppressed by the phospholipase C inhibitor U73122 and was not inhibited by the ryanodine and NAADP receptors antagonists dantrolene and NED-19. [Ca2+]i measurements in single cells demonstrated that activation of 5-HT2BR alone by BW723C86 caused single asynchronous [Ca2+]i oscillations in 19.8 ± 4.2% (n = 3) of HUVEC that occur with a long delay (66.1 ± 4.3 s, n = 71). On the contrary, histamine causes a simultaneous and almost immediate increase in [Ca2+]i in all the cells. Pre-activation of 5-HT1BR by CGS12066B led to a 3-4 fold increase in the number of HUVEC responding to BW723C86, to synchronization of their responses with a delay shortening, and to the bursts of [Ca2+]i oscillations in addition to single oscillations. In conclusion, to get a full rise of [Ca2+]i in HUVEC in response to 5-HT, simultaneous activation of 5-HT1BR and 5-HT2BR is required. 5-HT causes an increase in [Ca2+]i via 5-HT2BR while 5-HT1BR could be activated by the membrane-permeable agonist CGS12066B. We hypothesized that CGS12066B acts via intracellular 5-HT1BR inaccessible to extracellular 5-HT. Intracellular 5-HT1BR might be activated by 5-HT which could be accumulated in EC under certain pathological conditions.

Abstract P2136: TpTe Interval Behavior And Mortality In Wistar Rats Submitted To Cardiac Ischemia And Reperfusion Treated With The Heparin Fragment Trisulfated Disaccharide

Introduction: The Tp-Te ekg interval reflects ventricular transmural dispersion, being used for prediction of ventricular arrhythmia and sudden death. The heparin fragment called Trisulfated Disaccharide (TD), which has no effect on blood coagulation and acts on the sodium-calcium exchanger accelerating the output of calcium from the cell, decreases cytoplasmic calcium concentrations in situations of calcium overload due to ischemia and reperfusion (I-R). Objective: To evaluate the behavior of the Tp-Te interval and mortality due to cardiac arest in Wistar rats submitted to surgicaly induced I-R under treatment with TD at reperfusion. Materials and Method: Wistar rats (n=8) with average weight of 300g were anesthetized and under assisted breathing were electrocardiographically monitored and submitted to surgical cardiac ischemia by ligation of the anterior descending artery for 10 min, with reperfusion thereafter. The Tp-Te interval was analyzed before ischemia, at 10 min after ischemia induction and at 10 min after reperfusion, The incidence of arrhythmias was determined throughout the recording period. In the study group intravenous (0.2 mg/kg) TD was injected concomitantly with the reperfusion. In the control group (n=8), the same procedure was performed, without the TD infusion. Results: During ischemia there was a similar alteration of the Tp-Te interval in both groups when compared to the pre-ischemic phase, with an increase of about 90% in magnitude, as well as the presence of ventricular arrhythmias, without animal mortality. During reperfusion Tp-Te interval returned to normal in the treated group and there was no mortality. In the control group, the magnitude of the Tp-Te increase was maintained. The presence of ventricular arrhythmias was similar, but total atrioventricular block and/or sustained arrhythmia occurred in 50% of the animals, followed by animals death. Conclusion: Treatment with TD at the time of reperfusion normalized the Tp-Te interval 10 min after TD infusion as well as reduced mortality in wistar rats submitted to I-R, and may be useful in the protection of organs submitted to the same I-R conditions (transplants, surgical and hemodynamic procedures), where calcium overload is determinant in morbidity and mortality.

Isotherm kinetics of PIP2 bound gelsolin inactivation

Actin monomers (G-actin) and filaments (F-actin) have dynamical rearrangement thus manage cellular motility, division and transport processes. The gelsolin (GSN) regulates the remodeling of cytoskeleton. After the activation of GSN by calcium ions, it can sever actin filaments then capped at its barbed end. In the cytoplasm, GSN manages the cellular motions and morphology. Phosphatidylinositol 4,5-bisphosphate (PIP2) is involved in signal transduction and the regulation of the actin cytoskeleton by regulation of actin-binding proteins. GSN can bind to PIP2 and thus can be localized in the near of the plasma membrane and released from the end of F-actin. We test here with isoperibol calorimetry the enthalpy change, within the interplay between GSN and F-actin under nano-, micro- and millimolar calcium concentrations and express the importance of PIP2 binding for the inactivation of GSN. As we have demonstrated here that PIP2 binding stabilizes the structure of gelsolin and reduces its actin monomer binding activity under nanomolar calcium as the typical cytoplasmic calcium concentration of resting cells. The gelsolin shows partial activity under micromolar and total activity with strong responses under millimolar calcium. If gelsolin-capped filaments point at the plasma membrane helps the binding between gelsolin and PIP2, and hence, filament uncapping in case of resting cells. We presume that the low free calcium concentration keeps on the structure of gelsolin which is able to bind actin within the cooperativity of actin bound calcium. Gelsolin can help to manage monomer pool far from the membrane and it can be linked to a basic sensory mechanism which drives the direction of filament growth in the near of the membrane.

Combined FRET-FLIM and NAD(P)H FLIM to Analyze B Cell Receptor Signaling Induced Metabolic Activity of Germinal Center B Cells In Vivo.

Affinity maturation of B cell clones within germinal centers constitutes an important mechanism for immune memory. During this process, B cell receptor signaling capacity is tested in multiple rounds of positive selection. Antigen stimulation and co-stimulatory signals mobilize calcium to switch on gene expression leading to proliferation and survival and to differentiation into memory B cells and plasma cells. Additionally, all these processes require adaption of B cell metabolism, and calcium signaling and metabolic pathways are closely interlinked. Mitochondrial adaption, ROS production, and NADPH oxidase activation are involved in cell fate decisions, but it remains elusive to what extent, especially because the analysis of these dynamic processes in germinal centers has to take place in vivo. Here, we introduce a quantitative intravital imaging method for combined measurement of cytoplasmic calcium concentration and enzymatic fingerprinting in germinal center B cells as a possible tool in order to further examine the relationship of calcium signaling and immunometabolism.

Effect of Buffers with Multiple Binding Sites on Calcium Waves

The existence and properties of intracellular waves of increased free cytoplasmic calcium concentration (calcium waves) are strongly affected by the binding and unbinding of calcium ions to a multitude of different buffers in the cell. These buffers can be mobile or immobile and, in general, have multiple binding sites that are not independent. Previous theoretical studies have focused on the case when each buffer molecule binds a single calcium ion. In this study, we analyze how calcium waves are affected by calcium buffers with two non-independent binding sites, and show that the interactions between the calcium binding sites can result in the emergence of new behaviors. In particular, for certain combinations of kinetic parameters, the profiles of buffer molecules with one calcium ion bound can be non-monotone.

Activation of Piezo1 Increases Na,K-ATPase-Mediated Ion Transport in Mouse Lens.

Lens ion homeostasis depends on Na,K-ATPase and NKCC1. TRPV4 and TRPV1 channels, which are mechanosensitive, play important roles in mechanisms that regulate the activity of these transporters. Here, we examined another mechanosensitive channel, piezo1, which is also expressed in the lens. The purpose of the study was to examine piezo1 function. Recognizing that activation of TRPV4 and TRPV1 causes changes in lens ion transport mechanisms, we carried out studies to determine whether piezo1 activation changes either Na,K-ATPase-mediated or NKCC1-mediated ion transport. We also examined channel function of piezo1 by measuring calcium entry. Rb uptake was measured as an index of inwardly directed potassium transport by intact mouse lenses. Intracellular calcium concentration was measured in Fura-2 loaded cells by a ratiometric imaging technique. Piezo1 immunolocalization was most evident in the lens epithelium. Potassium (Rb) uptake was increased in intact lenses as well as in cultured lens epithelium exposed to Yoda1, a piezo1 agonist. The majority of Rb uptake is Na,K-ATPase-dependent, although there also is a significant NKCC-dependent component. In the presence of ouabain, an Na,K-ATPase inhibitor, Yoda1 did not increase Rb uptake. In contrast, Yoda1 increased Rb uptake to a similar degree in the presence or absence of 1 µM bumetanide, an NKCC inhibitor. The Rb uptake response to Yoda1 was inhibited by the selective piezo1 antagonist GsMTx4, and also by the nonselective antagonists ruthenium red and gadolinium. In parallel studies, Yoda1 was observed to increase cytoplasmic calcium concentration in cells loaded with Fura-2. The calcium response to Yoda1 was abolished by gadolinium or ruthenium red. The calcium and Rb uptake responses to Yoda1 were absent in calcium-free bathing solution, consistent with calcium entry when piezo1 is activated. Taken together, these findings point to stimulation of Na,K-ATPase, but not NKCC, when piezo1 is activated. Na,K-ATPase is the principal mechanism responsible for ion and water homeostasis in the lens. The functional role of lens piezo1 is a topic for further study.

Involvement of calcium ions in amyloid-β-induced lamin fragmentation

Amyloid-β (Aβ) peptide, the main pathogenic peptide in Alzheimer's disease, has been shown to induce an increase in cytoplasmic calcium concentration (CCC). In the current study, we explored the cytotoxic signal transduction pathway in 42-amino-acid Aβ (Aβ42)-treated HeLa cells in relation to the increase in CCC. The increase in CCC was prominent in cells treated twice with oligomeric Aβ42. We previously showed that double treatment also promoted Aβ-induced lamin fragmentation (AILF), which appears to be mediated by cathepsin L. Apoptotic caspase activation was a downstream event of AILF. The Ca2+ chelator BAPTA-AM suppressed cell death, cathepsin L activation, AILF, and caspase activation in Aβ-treated cells. These results indicate that Aβ42 induces an increase in CCC, which is an event upstream of the cytotoxic processes. The products of AILF are different from those produced by other cell death-inducing agents, such as staurosporine, which induces caspase-6-mediated lamin fragmentation (CMLF). CMLF was unaffected by BAPTA-AM and was not detected in cells treated with Aβ42, indicating that Aβ42 peptide induced a specific cytotoxic pathway involving AILF via increased CCC. We confirmed that the same processes (except caspase activation) operated in Aβ42-treated neuroblastoma SH-SY5Y cells.

Dynamic Regulation of Mitochondrial [Ca2+] in Hippocampal Neurons.

While neuronal mitochondria have been studied extensively in their role in health and disease, the rules that govern calcium regulation in mitochondria remain somewhat vague. In the present study using cultured rat hippocampal neurons transfected with the mtRCaMP mitochondrial calcium sensor, we investigated the effects of cytosolic calcium surges on the dynamics of mitochondrial calcium ([Ca2+]m). Cytosolic calcium ([Ca2+]c) was measured using the high affinity sensor Fluo-2. We recorded two types of calcium events: local and global ones. Local events were limited to a small, 2–5 µm section of the dendrite, presumably caused by local synaptic activity, while global events were associated with network bursts and extended throughout the imaged dendrite. In both cases, cytosolic surges were followed by a delayed rise in [Ca2+]m. In global events, the rise lasted longer and was observed in all mitochondrial clusters. At the end of the descending part of the global event, [Ca2+]m was still high. Global events were accompanied by short and rather high [Ca2+]m surges which we called spikelets, and were present until the complete decay of the cytosolic event. In the case of local events, selective short-term responses were limited to the part of the mitochondrial cluster that was located directly in the center of [Ca2+]c activity, and faded quickly, while responses in the neighboring regions were rarely observed. Caffeine (which recruits ryanodine receptors to supply calcium to the mitochondria), and carbonyl cyanide m-chlorophenyl hydrazine (CCCP, a mitochondrial uncoupler) could affect [Ca2+]m in both global and local events. We constructed a computational model to simulate the fundamental role of mitochondria in restricting calcium signals within a narrow range under synapses, preventing diffusion into adjacent regions of the dendrite. Our results indicate that local cytoplasmic and mitochondrial calcium concentrations are highly correlated. This reflects a key role of signaling pathways that connect the postsynaptic membrane to local mitochondrial clusters.

Chlorpyrifos exposure induces calcium-dependent necrosis in carp (Cyprinus carpio) lymphocytes via the inhibition of T cell receptor gamma (TCR γ)

The insecticide chlorpyrifos plays an important role in agricultural production and is widely used because of its excellent insecticidal ability. However, the mechanism by which chlorpyrifos causes lymphocyte death remains unclear. In this study, transcriptomic techniques were used to analyze the head kidney tissues of carp (Cyprinus carpio) treated with chlorpyrifos. Subsequently, we screened out differentially expressed genes (DEGs) and performed the corresponding processing in the head kidney lymphocyte. Then, the intracellular calcium content and necrosis were detected by fluorescence staining, real-time fluorescence quantitative PCR, and flow cytometry. Our results showed that the expression of T cell receptor gamma (TCR γ) was significantly decreased, and TCR γ was inhibited after chlorpyrifos treatment. Also, TCR γ significantly increased the abundance of calcium channel messenger RNA (mRNA). To verify this result, we established the TCR γ overexpression group and found that the reverse results were observed in TCR γ of in the overexpression group. The results of cytoplasmic calcium concentration detection, calcium staining, and flow cytometry confirmed the conclusion of increased calcium in the cytoplasm. The function of TCR γ significantly enhanced the mRNA expression levels of necrosis-related genes, and this conclusion was evidenced by the result of necrotic flow detection. Our results showed that chlorpyrifos could inhibit TCR γ in carp lymphocytes and induce calcium-dependent necrosis.

IN VITRO ЭКСПЕРИМЕНТАЛЬНОЕ ИЗУЧЕНИЕ ТКАНЕВОЙ И КЛЕТОЧНОЙ ТОКСИЧНОСТИ НОВЫХ ЛЕКАРСТВЕННЫХ ВЕЩЕСТВ

Abstract. We studied the neurotoxic/neurotropic effects of a new derivative of dimethylacetamide and benzoic acid LHT-5-01 in a mixed neuroglial culture to determine both the effect of various concentrations of the compound on cell survival and their interaction with the glutamate noncainate receptor. To do this, a mixed neuroglial culture of the hippocampus of linear newborn (P 1-3) Sprague Dawley rats was resuspended in a neurobasal medium containing 0.5 mmol glutamine, 2% Supplement B27 and 15 μg/ml gentamicin. The cytoplasmic concentration of calcium in the ionized form [Ca2+]i was determined using a Fura-2 fluorescent probe (USA). The viability of culture cells—neurons and glial cells—was determined by the immunocytochemical method. Experimental studies were performed on cultures aged from 7 to 9 days, to which the test compound of dimethylacetamide LHT-5-01 was added at various concentrations in the laboratory of intracellular signaling of the Institute of Cell Biophysics of the Russian Academy of Sciences - a separate division of the Federal State Budgetary Scientific Institution Federal Research Center "Pushchino Scientific Center for Biological Research of the Russian Academy of Sciences" (Pushchino) in strict accordance with the literary descriptions. Incubation of the culture with 10 μM of the LHT-5-01 compound completely suppresses the first phase of receptor activity, but does not prevent the formation of the second phase of NMDA receptor excitation. The established activity can be considered in the aspect of the prevention of glutamate-mediated excitotoxicity of the test compound.

Oxidative stress impairs the meat quality of broiler by damaging mitochondrial function, affecting calcium metabolism and leading to ferroptosis.

ObjectiveThis work was conducted to investigate the effects of oxidative stress on meat quality, mitochondrial function, calcium metabolism and ferroptosis of broilers.MethodsIn this study, a total of 144 one-day-old male Ross 308 chicks were divided into 3 groups (control group, saline group, and hydrogen peroxide [H2O2] group) with 6 replicates of 8 broilers each. The study lasted for 42 d. The broilers in the saline and H2O2 groups were intraperitoneally injected with 0.75% saline and 10.0% H2O2 on the 16th and 37th day of the experimental period respectively, the injection volumes were 1.0 mL/kg of broiler body weight. On the 42nd day of the experimental period, two chicks were randomly selected from each cage, a total of thirty-six chicks were stunned by electric shock and slaughtered to collect breast muscle samples.ResultsThe H2O2 exposure reduced pH value, increased drip loss and shear force of breast meat (p<0.05), impaired the ultrastructure and function of mitochondria. The H2O2 exposure damaged the antioxidant system in mitochondria, excessive reactive oxygen species carbonylation modified calcium channels on mitochondria, which impaired the activities of key enzymes on calcium channel, resulted in the increased calcium concentration in cytoplasm and mitochondria (p<0.05). In addition, the H2O2 exposure increased the iron content and lipid peroxidation (p<0.05), which induced ferroptosis.ConclusionOxidative stress could impair meat quality by causing mitochondrial dysfunction, resulting in calcium metabolism disorder and ferroptosis.

Arginine promotes myogenic differentiation and myotube formation through the elevation of cytoplasmic calcium concentration.

This study aimed to explore the mechanism underlying arginine-promoted myogenesis of myoblasts. C2C12 cells were cultured with a medium containing 0.1, 0.4, 0.8, or 1.2 mmol/L arginine, respectively. Cell proliferation, viability, differentiation indexes, cytoplasmic Ca2+ concentration, and relative mRNA expression levels of myogenic regulatory factors (MRF) and key Ca2+ channels were measured in the absence or presence of 2 chemical inhibitors, dantrolene (DAN, 10 μmol/L) and nisoldipine (NIS, 10 μmol/L), respectively. Results demonstrated that arginine promoted myogenic differentiation and myotube formation. Compared with the control (0.4 mmol/L arginine), 1.2 mmol/L arginine upregulated the relative mRNA expression levels of myogenin (MyoG) and Myomaker at d 2 during myogenic induction (P < 0.05). Cytoplasmic Ca2+ concentrations were significantly elevated by arginine supplementation at d 2 and 4 (P < 0.05). Relative mRNA expression levels of Ca2+ channels including the type 1 ryanodine receptor (RyR1) and voltage-gated Ca2+ channel (Cav1.1) were upregulated by 1.2 mmol/L arginine during 2-d myogenic induction (P < 0.01). However, arginine-promoted myogenic potential of myoblasts was remarkably compromised by DAN and NIS, respectively (P < 0.05). These findings evidenced that the supplementation of arginine promoted myogenic differentiation and myotube formation through increasing cytoplasmic Ca2+ concentration from both extracellular and sarcoplasmic reticulum Ca2+.