Cytosolic Calcium Concentration Research Articles

Purinergic agonists increase [Ca2+]i in rat conjunctival goblet cells through ryanodine receptor type 3.

ATP and benzoylbenzoyl-ATP (BzATP) increase free cytosolic Ca2+ concentration ([Ca2+]i) in conjunctival goblet cells (CGCs) resulting in mucin secretion. The purpose of this study was to investigate the source of the Ca2+i mobilized by ATP and BzATP. First-passage cultured rat CGCs were incubated with Fura-2/AM, and [Ca2+]i was measured under several conditions with ATP and BzATP stimulation. The following conditions were used: 1) preincubation with the Ca2+ chelator EGTA, 2) preincubation with the SERCA inhibitor thapsigargin (10-6 M), which depletes ER Ca2+ stores, 3) preincubation with phospholipase C (PLC) or protein kinase A (PKA) inhibitor, or 4) preincubation with the voltage-gated calcium channel antagonist nifedipine (10-5 M) and the ryanodine receptor (RyR) antagonist dantrolene (10-5 M). Immunofluorescence microscopy (IF) and quantitative reverse transcription polymerase chain reaction (RT-qPCR) were used to investigate RyR presence in rat and human CGCs. ATP-stimulated peak [Ca2+]i was significantly lower after chelating Ca2+i with 2 mM EGTA in Ca2+-free buffer. The peak [Ca2+]i increase in CGCs preincubated with thapsigargin, the PKA inhibitor H89, nifedipine, and dantrolene, but not the PLC inhibitor, was reduced for ATP at 10-5 M and BzATP at 10-4 M. Incubating CGCs with dantrolene alone decreased [Ca2+]i and induced CGC cell death at a high concentration. RyR3 was detected in rat and human CGCs with IF and RT-qPCR. We conclude that ATP- and BzATP-induced Ca2+i increases originate from the ER and that RyR3 may be an essential regulator of CGC [Ca2+]i. This study contributes to the understanding of diseases arising from defective Ca2+ signaling in nonexcitable cells.NEW & NOTEWORTHY ATP and benzoylbenzoyl-ATP (BzATP) induce mucin secretion through an increase in free cytosolic calcium concentration ([Ca2+]i) in conjunctival goblet cells (CGCs). The mechanisms through which ATP and BzATP increase [Ca2+]i in CGCs are unclear. Ryanodine receptors (RyRs) are fundamental in [Ca2+]i regulation in excitable cells. Herein, we find that ATP and BzATP increase [Ca2+]i through the activation of protein kinase A, voltage-gated calcium channels, and RyRs, and that RyRs are crucial for nonexcitable CGCs' Ca2+i homeostasis.

A Comprehensive Fuzzy Model for Understanding Neuronal Calcium Distribution in Presence of VGCC, Na+/Ca2+ Exchanger, Buffer, and ER Fluxes.

Free Calcium ions in the cytosol are essential for many physiological and physical functions. The free calcium ions are commonly regarded as a second messenger, are an essential part of brain communication. Numerous physiological activities, such as calcium buffering and calcium ion channel flow, etc. influence the cytosolic calcium concentration. In light of the above, the primary goal of this study is to develop a model of calcium distribution in neuron cells when a Voltage-Gated Calcium Channel and Sodium Calcium Exchanger are present. As we know, decreased buffer levels and increased calcium activity in the Voltage-Gated Calcium Channel and Sodium Calcium Exchanger lead to Alzheimer's disease. Due to these changes, the calcium diffusion in that location becomes disrupted and impacted by Alzheimer's disease. The model has been constructed by considering key factors like buffers and ER fluxes when Voltage-Gated Calcium Channels and Sodium Calcium Exchangers are present. Based on the physiological conditions of the parameters, appropriate boundary conditions have been constructed in the fuzzy environment. This model is considered a fuzzy boundary value problem with the source term and initial boundary conditions are modeled by triangular fuzzy functions. In this, paper we observed the approximate solution of the mathematical model which was investigated by the fuzzy undetermined coefficient method. The solution has been performed through MATLAB and numerical results have been computed using simulation. The observation made that the proper operation of the Voltage-Gated Calcium Channel and Sodium Calcium Exchanger is critical for maintaining the delicate equilibrium of calcium ions, which regulates vital cellular activities. Dysregulation of Voltage-Gated Calcium Channel and Sodium Calcium Exchanger activity has been linked to neurodegenerative illnesses like Alzheimer's disease.

In Cardiac Myocytes the SGLT2 Inhibitor Ertugliflozin Attenuates Cytosolic Ca2+ Peaks After Adrenergic Stimulation

A novel class of antidiabetic drugs, called gliflozins, inhibit sodium-glucose cotransporter 2 (SGLT2i) to lower blood glucose levels in patients with Type 2 diabetes (T2DM). Gliflozins reduce mortality and heart failure (HF) hospitalizations in HF patients with or without T2DM, but the molecular mechanisms are unclear. We hypothesize that modulating effects on cardiac calcium homeostasis are involved in SGLT2i-induced benefits during HF, and that the SGLT2i ertugliflozin (ERTU) will alter cytosolic calcium concentration ([Ca2+]cyt) in contracting cardiomyocytes after adrenergic stimulation. Cardiomyocytes were isolated from 0–2-day old C57BL/6J mice, treated for 72 hours with either ERTU (100 nM, and 1 μM), the NHE-1 inhibitor cariporide (CARI, 10 μM), ERTU and CARI, or vehicle (Ctrl). After 72 hours, cells were either A) loaded with Fura-2, baseline cytosolic calcium levels ([Ca2+]cyt) recorded, cells stimulated with phenylephrine (PE, 100 μM), and [Ca2+]cyt recorded for additional 30 minutes, B) lysed and protein isolated for analysis by immunoblotting, or C) fixed for immunohistochemical assessment. Results: 100 nM and 1 uM ERTU reduced [Ca2+]cyt peaks after PE, but 100 nM ERTU to a lesser degree. In contrast, 72-hour-long NHE1 inhibition with CARI increased [Ca2+]cyt at baseline when compared to vehicle and ERTU, and further enhanced [Ca2+]cyt peaks after PE. Immunoblotting revealed increased protein levels of S16 phosphorylated phospholamban, decreased total phospholamban, and upregulated sarcomeric α-actinin post-ERTU. Immunohistochemical assessment of sarcomeric α-actinin localization revealed improved sarcomeric Z-line organization after ERTU administration. Sustained exposure to the SGLT2i ERTU reduced, but the NHE-1 inhibitor CARI increased [Ca2+]cyt in contracting cardiac myocytes. These data indicate that ERTU’s beneficial effect on [Ca2+]cyt homeostasis is independent of NHE1 inhibition. Since disturbed calcium handling and calcium overload are hallmarks of HF, reduced [Ca2+]cyt levels after sympathetic stimulation may contribute to the cardioprotective effect of SGLT2i. Further studies are needed to unravel the molecular mechanisms behind ERTU’s influence on cardiac calcium homeostasis. This study was funded by Merck & Co., Inc. (Kenilworth, NJ). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Elevated intracellular Ca2+ functions downstream of mitodysfunction to induce Wallerian-like degeneration and necroptosis in organophosphorus-induced delayed neuropathy

Neurotoxic organophosphorus compounds can induce a type of delayed neuropathy in humans and sensitive animals, known as organophosphorus-induced delayed neuropathy (OPIDN). OPIDN is characterized by axonal degeneration akin to Wallerian-like degeneration, which is thought to be caused by increased intra-axonal Ca2+ concentrations. This study was designed to investigate that deregulated cytosolic Ca2+ may function downstream of mitodysfunction in activating Wallerian-like degeneration and necroptosis in OPIDN. Adult hens were administrated a single dosage of 750 mg/kg tri-ortho-cresyl phosphate (TOCP), and then sacrificed at 1 day, 5 day, 10 day and 21 day post-exposure, respectively. Sciatic nerves and spinal cords were examined for pathological changes and proteins expression related to Wallerian-like degeneration and necroptosis. In vitro experiments using differentiated neuro-2a (N2a) cells were conducted to investigate the relationship among mitochondrial dysfunction, Ca2+ influx, axonal degeneration, and necroptosis. The cells were co-administered with the Ca2+-chelator BAPTA-AM, the TRPA1 channel inhibitor HC030031, the RIPK1 inhibitor Necrostatin-1, and the mitochondrial-targeted antioxidant MitoQ along with TOCP. Results demonstrated an increase in cytosolic calcium concentration and key proteins associated with Wallerian degeneration and necroptosis in both in vivo and in vitro models after TOCP exposure. Moreover, co-administration with BATPA-AM or HC030031 significantly attenuated the loss of NMNAT2 and STMN2 in N2a cells, as well as the upregulation of SARM1, RIPK1 and p-MLKL. In contrast, Necrostatin-1 treatment only inhibited the TOCP-induced elevation of p-MLKL. Notably, pharmacological protection of mitochondrial function with MitoQ effectively alleviated the increase in intracellular Ca2+ following TOCP and mitigated axonal degeneration and necroptosis in N2a cells, supporting mitochondrial dysfunction as an upstream event of the intracellular Ca2+ imbalance and neuronal damage in OPIDN. These findings suggest that mitochondrial dysfunction post-TOCP intoxication leads to an elevated intracellular Ca2+ concentration, which plays a pivotal role in the initiation and development of OPIDN through inducing SARM1-mediated axonal degeneration and activating the necroptotic signaling pathway.

Piezo1 stretch-activated channel activity differs between murine bone marrow-derived and cardiac tissue-resident macrophages.

Macrophages (MΦ) play pivotal roles in tissue homeostasis and repair. Their mechanical environment has been identified as a key modulator of various cell functions, and MΦ mechanosensitivity is likely to be critical - in particular in a rhythmically contracting organ such as the heart. Cultured MΦ, differentiated in vitro from bone marrow (MΦBM), form a popular research model. This study explores the activity of mechanosensitive ion channels (MSC) in murine MΦBM and compares it to MSC activity in MΦ enzymatically isolated from cardiac tissue (tissue-resident MΦ; MΦTR). We show that MΦBM and MΦTR have stretch-induced currents, indicating the presence of functional MSC in their plasma membrane. The current profiles in MΦBM and in MΦTR show characteristics of cation non-selective MSC such as Piezo1 or transient receptor potential channels. While Piezo1 ion channel activity is detectable in the plasma membrane of MΦBM using the patch-clamp technique, or by measuring cytosolic calcium concentration upon perfusion with the Piezo1 channel agonist Yoda1, no Piezo1 channel activity was observed in MΦTR. The selective transient receptor potential vanilloid 4 (TRPV4) channel agonist GSK1016790A induces calcium entry in MΦTR and in MΦBM. In MΦ isolated from left-ventricular scar tissue 28days after cryoablation, stretch-induced current characteristics are not significantly different compared to non-injured control tissue, even though scarred ventricular tissue is expected to be mechanically remodelled and to contain an altered composition of pre-existing cardiac and circulation-recruited MΦ. Our data suggest that the in vitro differentiation protocols used to obtain MΦBM generate cells that differ from MΦ recruited from the circulation during tissue repair in vivo. Further investigations are needed to explore MSC identity in lineage-traced MΦ in scar tissue, and to compare mechanosensitivity of circulating monocytes with that of MΦBM. KEY POINTS: Bone marrow-derived (MΦBM) and tissue resident (MΦTR) macrophages have stretch-induced currents, indicating expression of functional mechanosensitive channels (MSC) in their plasma membrane. Stretch-activated current profiles show characteristics of cation non-selective MSC; and mRNA coding for MSC, including Piezo1 and TRPV4, is expressed in murine MΦBM and in MΦTR. Calcium entry upon pharmacological activation of TRPV4 confirms functionality of the channel in MΦTR and in MΦBM. Piezo1 ion channel activity is detected in the plasma membrane of MΦBM but not in MΦTR, suggesting that MΦBM may not be a good model to study the mechanotransduction of MΦTR. Stretch-induced currents, Piezo1mRNA expression and response to pharmacological activation are not significantly changed in cardiac MΦ 28days after cryoinjury compared to sham operated mice.

Adenosine receptors are the on-and-off switch of astrocytic cannabinoid type 1 (CB1) receptor effect upon synaptic plasticity in the medial prefrontal cortex.

The medial prefrontal cortex (mPFC) is involved in cognitive functions such as working memory. Astrocytic cannabinoid type 1 receptor (CB1R) induces cytosolic calcium (Ca2+) concentration changes with an impact on neuronal function. mPFC astrocytes also express adenosine A1 and A2A receptors (A1R, A2AR), being unknown the crosstalk between CB1R and adenosine receptors in these cells. We show here that a further level of regulation of astrocyte Ca2+ signaling occurs through CB1R-A2AR or CB1R-A1R heteromers that ultimately impact mPFC synaptic plasticity. CB1R-mediated Ca2+ transients increased and decreased when A1R and A2AR were activated, respectively, unveiling adenosine receptors as modulators of astrocytic CB1R. CB1R activation leads to an enhancement of long-term potentiation (LTP) in the mPFC, under the control of A1R but not of A2AR. Notably, in IP3R2KO mice, that do not show astrocytic Ca2+ level elevations, CB1R activation decreases LTP, which is not modified by A1R or A2AR. The present work suggests that CB1R has a homeostatic role on mPFC LTP, under the control of A1R, probably due to physical crosstalk between these receptors in astrocytes that ultimately alters CB1R Ca2+ signaling.

Phenanthroline relaxes uterine contractions induced by diverse contractile agents by decreasing cytosolic calcium concentration

Uterine contractions during labor and preterm labor are influenced by a complex interplay of factors, including hormones and inflammatory mediators. This complexity may contribute to the limited efficacy of current tocolytics for preterm labor, a significant challenge in obstetrics with 15 million cases annually and approximately 1 million resulting deaths worldwide. We have previously shown that the myometrium expresses bitter taste receptors (TAS2Rs) and that their activation leads to uterine relaxation. Here, we investigated whether the selective TAS2R5 agonist phenanthroline can induce relaxation across a spectrum of human uterine contractions and whether the underlying mechanism involves changes in intracellular Ca2+ signaling. We performed experiments using samples from pregnant women undergoing scheduled cesarean delivery, assessing responses to various inflammatory mediators and oxytocin with and without phenanthroline. Our results showed that phenanthroline concentration-dependently inhibited contractions induced by PGF2α, U46619, 5-HT, endothelin-1 and oxytocin. Furthermore, in hTERT-infected human myometrial cells exposed to uterotonics, phenanthroline effectively suppressed the increase in intracellular Ca2+ concentration induced by PGF2α, U46619, oxytocin, and endothelin-1. These results suggest that the selective TAS2R5 agonist may not only significantly reduce uterine contractions but also decrease intracellular Ca2+ levels. This study highlights the potential development of TAS2R5 agonists as a new class of uterine relaxants, providing a novel avenue for improving the management of preterm labor.

Gap Junction Channel Regulation: A Tale of Two Gates-Voltage Sensitivity of the Chemical Gate and Chemical Sensitivity of the Fast Voltage Gate.

Gap junction channels are regulated by gates sensitive to cytosolic acidification and trans-junctional voltage (Vj). We propose that the chemical gate is a calmodulin (CaM) lobe. The fast-Vj gate is made primarily by the connexin's NH2-terminus domain (NT). The chemical gate closes the channel slowly and completely, while the fast-Vj gate closes the channel rapidly but incompletely. The chemical gate closes with increased cytosolic calcium concentration [Ca2+]i and with Vj gradients at Vj's negative side. In contrast, the fast-Vj gate closes at the positive or negative side of Vj depending on the connexin (Cx) type. Cxs with positively charged NT close at Vj's negative side, while those with negatively charged NT close at Vj's positive side. Cytosolic acidification alters in opposite ways the sensitivity of the fast-Vj gate: it increases the Vj sensitivity of negative gaters and decreases that of positive gaters. While the fast-Vj gate closes and opens instantaneously, the chemical gate often shows fluctuations, likely to reflect the shifting of the gate (CaM's N-lobe) in and out of the channel's pore.

The C5aR1 complement receptor: A novel immunomodulator of insulin action in skeletal muscle.

The complement system constitutes an integral component of the innate immune system and plays a critical role in adaptive immunity. Activation of this system engenders the production of complement peptide fragments, including C5a, which engage G-protein coupled receptors predominantly expressed in immune-associated cells, such as neutrophils, initiating pro-inflammatory responses. Intriguingly, our investigation has unveiled the presence of C5a receptor 1 (C5aR1) expression within skeletal muscle, a key metabolic tissue and primary target of insulin. Herein, we demonstrate that C5aR1 activation by C5a in differentiated human skeletal muscle cells elicits acute suppression of insulin signalling. This suppression manifests as impaired insulin-dependent association between IRS1 and the p85 subunit of PI3-kinase, a 50% reduction in Akt phosphorylation, and a 60% decline in insulin-stimulated glucose uptake. This impairment in insulin signalling is associated with a three-fold elevation in intramyocellular diacylglycerol (DAG) levels and a two-fold increase in cytosolic calcium content, which promote PKC-mediated IRS1 inhibition via enhanced phosphorylation at IRS1 Ser1101. Significantly, our findings demonstrate that structurally diverse C5aR1 antagonists, along with genetic deletion or stable silencing of C5aR1 by 80% using short-hairpin RNA, effectively attenuate repression of insulin signalling by C5a in LHCN-M2 human skeletal myotubes. These results underscore the potential of heightened C5aR1 activation, characteristic of obesity and chronic inflammatory conditions, to detrimentally impact insulin function within skeletal muscle cells. Additionally, the study suggests that agents targeting the C5a-C5aR axis, originally devised for mitigating complement-dependent inflammatory conditions, may offer therapeutic avenues to ameliorate immune-driven insulin resistance in key peripheral metabolic tissues, including skeletal muscle.

Electrochemical cellular biosensor combined with fluorescence microscopy: An investigation of subtle changes in response of cells to a drug

AbstractThis study presents the combination of electrochemical cell based biosensor and fluorescence microscopy on a single platform to track biomolecular processes contributing to the morphology changes of the cells. Initial experiments demonstrate that using interdigitated electrodes where the gold microfingers were comparable in width and spacing to a single cell have the optimal sensitivity in the final electrochemical cell based‐sensing device. This was determined by measuring the cell index, based on the impedance analysis of bare and cell‐covered microelectrodes. The fabrication of the electrodes on a glass substrate enabled the capture of high‐resolution fluorescence microscopy images of single cells and related intracellular calcium release inside the HeLa cells via a window incorporated into the gold microelectrode design. As an illustration of the enhanced capability of the combined approach over traditional impedance cellular assay, the opto‐electric assay was utilized as a functional readout for G protein couple receptor activation. The simultaneous examinations of cells stimulated with histamine demonstrated an association between time courses of changes in cytosolic calcium concentration and reductions in cell‐cell adhesions.

Regulatory effect of bacterial melanin on the isoforms of new superoxide-producing associates from rat tissues in rotenone-induced Parkinson’s disease

According to recent research, selective neuronal vulnerability in Parkinson’s disease (PD) results from several phenotypic traits, including calcium-dependent, feed-forward control of mitochondrial respiration leading to elevated reactive oxygen species and cytosolic calcium concentration, an extensive axonal arbor, and a reactive neurotransmitter. Therefore, antioxidant therapy is a promising direction in the treatment of PD. In vitro studies have indicated the survival-promoting activity of bacterial melanin (BM) on midbrain dopaminergic neuron cultures. It has been established that BM has a number of protective and anti-inflammatory properties, so there is a high probability of a protective effect of BM in the early stages of PD. In this study, PD was induced through the unilateral intracerebral administration of rotenone followed by bacterial melanin. Tissues (brain, lungs, and small intestine) from the observed groups underwent isolation and purification to extract isoforms of new thermostable superoxide (О2−)-producing associates between NADPH-containing lipoprotein (NLP) and NADPH oxidase-Nox (NLP-Nox). The optical absorption spectral characteristics, specific amounts, stationary concentration of the produced О2−, and the content of NADPH in the observed associates were determined. The optical absorption spectra of the NLP-Nox isoforms in the visible and UV regions in the experimental groups did not differ from those of the control group. However, compared with the control group, the specific content of the total fractions of NLP-Nox isoforms associated with PD groups was higher, especially in the small intestine. These findings suggest that the described changes may represent a novel mechanism for rotenone-induced PD. Furthermore, bacterial melanin demonstrated antioxidant properties and regulated membrane formation in the brain, lung, and small intestine. This regulation occurred by inhibiting the release of new membrane-bound formations (NLP-Nox associates) from these membranes while simultaneously regulating the steady-state concentration of the formed О2−.

Analyzing fuzzy boundary value problems: a study on the influence of mitochondria and ER fluxes on calcium ions in neuron cells.

Cytosolic-free calcium ions play an important role in various physical and physiological processes. A vital component of neural signaling is the free calcium ion concentration often known as the second messenger. There are many parameters that effect the cytosolic free calcium concentration like buffer, voltage-gated ion channels, Endoplasmic reticulum, Mitochondria, etc. Mitochondria are small organelles located within thenervous system that are involved in processes within cells such as calcium homeostasis management, energy generation, response to stress, and cell demise pathways. In this work, a mathematical model with fuzzy boundary values has been developed to study the effect of Mitochondria and ER fluxes on free Calcium ions. The intended findings are displayed utilizingthe physiological understanding that amyloid beta plaques and tangles of neurofibrillary fibers have been identified as the two main causes of AD. The key conclusion of the work is the investigation of [Formula: see text] for healthy cells and cells affected by Alzheimer's disease, which may aid in the study of such processes for computational scientists and medical practitioners. Also, it has been shown that when a unique solution is found for a specific precise problem, it also successfully deals with any underlying ambiguity within the problem by utilizinga technique based on the principles of linear transformation. Furthermore, the comparison between the analytical approach and the generalized hukuhara derivative approach is shown here, which illustrates the benefits of the analytical approach. The simulation is carried out in MATLAB.

CaMKK2 and CHK1 phosphorylate human STN1 in response to replication stress to protect stalled forks from aberrant resection

Keeping replication fork stable is essential for safeguarding genome integrity; hence, its protection is highly regulated. The CTC1-STN1-TEN1 (CST) complex protects stalled forks from aberrant MRE11-mediated nascent strand DNA degradation (NSD). However, the activation mechanism for CST at forks is unknown. Here, we report that STN1 is phosphorylated in its intrinsic disordered region. Loss of STN1 phosphorylation reduces the replication stress-induced STN1 localization to stalled forks, elevates NSD, increases MRE11 access to stalled forks, and decreases RAD51 localization at forks, leading to increased genome instability under perturbed DNA replication condition. STN1 is phosphorylated by both the ATR-CHK1 and the calcium-sensing kinase CaMKK2 in response to hydroxyurea/aphidicolin treatment or elevated cytosolic calcium concentration. Cancer-associated STN1 variants impair STN1 phosphorylation, conferring inability of fork protection. Collectively, our study uncovers that CaMKK2 and ATR-CHK1 target STN1 to enable its fork protective function, and suggests an important role of STN1 phosphorylation in cancer development.

The WRKY46–MYC2 module plays a critical role in E-2-hexenal-induced anti-herbivore responses by promoting flavonoid accumulation

Volatile organic compounds (VOCs) play key roles in plant–plant communication, especially in response to pest attack. E-2-hexenal is an important component of VOCs, but it is unclear whether it can induce endogenous plant resistance to insects. Here, we show that E-2-hexenal activates early signaling events in Arabidopsis (Arabidopsis thaliana) mesophyll cells, including an H2O2 burst at the plasma membrane, the directed flow of calcium ions, and an increase in cytosolic calcium concentration. Treatment of wild-type Arabidopsis plants with E-2-hexenal increases their resistance when challenged with the diamondback moth Plutella xylostella L., and this phenomenon is largely lost in the wrky46 mutant. Mechanistically, E-2-hexenal induces the expression of WRKY46 and MYC2, and the physical interaction of their encoded proteins was verified by yeast two-hybrid, firefly luciferase complementation imaging, and in vitro pull-down assays. The WRKY46–MYC2 complex directly binds to the promoter of RBOHD to promote its expression, as demonstrated by luciferase reporter, yeast one-hybrid, chromatin immunoprecipitation, and electrophoretic mobility shift assays. This module also positively regulates the expression of E-2-hexenal-induced naringenin biosynthesis genes (TT4 and CHIL) and the accumulation of total flavonoids, thereby modulating plant tolerance to insects. Together, our results highlight an important role for the WRKY46–MYC2 module in the E-2-hexenal-induced defense response of Arabidopsis, providing new insights into the mechanisms by which VOCs trigger plant defense responses.

Studying the role of random translocation of GLUT4 in cardiomyocytes on calcium oscillations

Defective excitation-contraction coupling (ECC) is one of the causes of contractile dysfunction linked to diabetic cardiomyopathy (DCM). This defect in ECC is mainly caused by the dysregulation of the calcium homeostasis in normal cardiomyocytes due to prolonged elevated blood glucose levels. Different types of channels inside the cardiomyocytes help maintain calcium homeostasis in the cell. Free cytosolic calcium concentration changes are one of several variables that affect complicated physiological cardiac processes and significantly impact proper heart functioning. Glucose concentration in the cardiac cell also plays a vital role in altering the calcium homeostasis in the DCM condition. In the current study, we develop a mathematical model that explicitly represents many of the known signalling components mediating translocation of the insulin-responsive glucose transporter type 4 (GLUT4) to gain insight into the complexities of metabolic insulin signalling pathways in cardiac cells. We examined the effect of the random movement of GLUT4 responsible for the entry of glucose in the cardiomyocytes. In non-paced cardiac cells calcium remains in the steady state in the absence of external pacing and avoid oscillation for healthy cardiomyocyte functioning. Intracellular calcium transients were observed during action potential. We investigated the circumstances under which the system transits from a stable state to an oscillatory state. We studied the effect of random translocation of GLUT4 on calcium oscillation. By altering system parameters, we induced insulin resistance in cardiomyocytes to mimic diabetic conditions and proposed some potential restoration strategies to restore normal calcium dynamics. Early bifurcation was observed with the introduction of randomness in the system.

Cerium Oxide Nanoparticles Protect Cortical Astrocytes from Oxygen-Glucose Deprivation through Activation of the Ca2+ Signaling System.

Most of the works aimed at studying the cytoprotective properties of nanocerium are usually focused on the mechanisms of regulation of the redox status in cells while the complex effects of nanocerium on calcium homeostasis, the expression of pro-apoptotic and protective proteins are generally overlooked. There is a problem of a strong dependence of the effects of cerium oxide nanoparticles on their size, method of preparation and origin, which significantly limits their use in medicine. In this study, using the methods of molecular biology, immunocytochemistry, fluorescence microscopy and inhibitory analysis, the cytoprotective effect of cerium oxide nanoparticles obtained by laser ablation on cultured astrocytes of the cerebral cortex under oxygen-glucose deprivation (OGD) and reoxygenation (ischemia-like conditions) are shown. The concentration effects of cerium oxide nanoparticles on ROS production by astrocytes in an acute experiment and the effects of cell pre-incubation with nanocerium on ROS production under OGD conditions were studied. The dose dependence for nanocerium protection of cortical astrocytes from a global increase in calcium ions during oxygen-glucose deprivation and cell death were demonstrated. The concentration range of cerium oxide nanoparticles at which they have a pro-oxidant effect on cells has been identified. The effect of nanocerium concentrations on astrocyte preconditioning, accompanied by increased expression of protective proteins and limited ROS production induced by oxygen-glucose deprivation, has been investigated. In particular, a correlation was found between an increase in the concentration of cytosolic calcium under the action of nanocerium and the suppression of cell death. As a result, the positive and negative effects of nanocerium under oxygen-glucose deprivation and reoxygenation in astrocytes were revealed at the molecular level. Nanocerium was found to act as a "double-edged sword" and to have a strictly defined concentration therapeutic "window".

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.

Calcineurin-Dependent Homeostatic Response of C. elegans Muscle Cells upon Prolonged Activation of Acetylcholine Receptors.

Pharmacological adaptation is a common phenomenon observed during prolonged drug exposure and often leads to drug resistance. Understanding the cellular events involved in adaptation could provide new strategies to circumvent this resistance issue. We used the nematode Caenorhabditis elegans to analyze the adaptation to levamisole, an ionotropic acetylcholine receptor agonist, used for decades to treat nematode parasitic infections. Genetic screens in C. elegans identified "adapting mutants" that initially paralyze upon exposure to levamisole as the wild type (WT), but recover locomotion after a few hours whereas WT remain paralyzed. Here, we show that levamisole induces a sustained increase in cytosolic calcium concentration in the muscle cells of adapting mutants, lasting several hours and preceding a decrease in levamisole-sensitive acetylcholine receptors (L-AChR) at the muscle plasma membrane. This decrease correlated with a drop in calcium concentration, a relaxation of the animal's body and a resumption of locomotion. The decrease in calcium and L-AChR content depends on calcineurin activation in muscle cells. We also showed that levamisole adaptation triggers homeostatic mechanisms in muscle cells including mitochondria remodeling, lysosomal tubulation and an increase in autophagic activity. Levamisole adaptation thus provides a new experimental paradigm for studying how cells cope with calcium stress.

CDPK2A and CDPK1 form a signaling module upstream of Toxoplasma motility.

This work uncovers interactions between various signaling pathways that govern Toxoplasma gondii egress. Specifically, we compare the function of three canonical calcium-dependent protein kinases (CDPKs) using chemical-genetic and conditional-depletion approaches. We describe the function of a previously uncharacterized CDPK, CDPK2A, in the Toxoplasma lytic cycle, demonstrating that it contributes to parasite fitness through regulation of microneme discharge, gliding motility, and egress from infected host cells. Comparison of analog-sensitive kinase alleles and conditionally depleted alleles uncovered epistasis between CDPK2A and CDPK1, implying a partial functional redundancy. Understanding the topology of signaling pathways underlying key events in the parasite life cycle can aid in efforts targeting kinases for anti-parasitic therapies.

Interaction of calcium responsive proteins and transcriptional factors with the PHO regulon in yeasts and fungi.

Phosphate and calcium ions are nutrients that play key roles in growth, differentiation and the production of bioactive secondary metabolites in filamentous fungi. Phosphate concentration regulates the biosynthesis of hundreds of fungal metabolites. The central mechanisms of phosphate transport and regulation, mediated by the master Pho4 transcriptional factor are known, but many aspects of the control of gene expression need further research. High ATP concentration in the cells leads to inositol pyrophosphate molecules formation, such as IP3 and IP7, that act as phosphorylation status reporters. Calcium ions are intracellular messengers in eukaryotic organisms and calcium homeostasis follows elaborated patterns in response to different nutritional and environmental factors, including cross-talking with phosphate concentrations. A large part of the intracellular calcium is stored in vacuoles and other organelles forming complexes with polyphosphate. The free cytosolic calcium concentration is maintained by transport from the external medium or by release from the store organelles through calcium permeable transient receptor potential (TRP) ion channels. Calcium ions, particularly the free cytosolic calcium levels, control the biosynthesis of fungal metabolites by two mechanisms, 1) direct interaction of calcium-bound calmodulin with antibiotic synthesizing enzymes, and 2) by the calmodulin-calcineurin signaling cascade. Control of very different secondary metabolites, including pathogenicity determinants, are mediated by calcium through the Crz1 factor. Several interactions between calcium homeostasis and phosphate have been demonstrated in the last decade: 1) The inositol pyrophosphate IP3 triggers the release of calcium ions from internal stores into the cytosol, 2) Expression of the high affinity phosphate transporter Pho89, a Na+/phosphate symporter, is controlled by Crz1. Also, mutants defective in the calcium permeable TRPCa7-like of Saccharomyces cerevisiae shown impaired expression of Pho89. This information suggests that CrzA and Pho89 play key roles in the interaction of phosphate and calcium regulatory pathways, 3) Finally, acidocalcisomes organelles have been found in mycorrhiza and in some melanin producing fungi that show similar characteristics as protozoa calcisomes. In these organelles there is a close interaction between orthophosphate, pyrophosphate and polyphosphate and calcium ions that are absorbed in the polyanionic polyphosphate matrix. These advances open new perspectives for the control of fungal metabolism.