Calcium Concentration In Cells Research Articles

High-Affinity Plasma Membrane Ca2+ Channel Cch1 Modulates Adaptation to Sodium Dodecyl Sulfate-Triggered Rise in Cytosolic Ca2+ Concentration in Ogataea parapolymorpha

The cytosolic calcium concentration ([Ca2+]cyt) in yeast cells is maintained at a low level via the action of different transporters sequestrating these cations in the vacuole. Among them, the vacuolar Ca2+ ATPase Pmc1 crucially contributes to this process. Its inactivation in Ogataea yeasts was shown to cause sodium dodecyl sulfate (SDS) hypersensitivity that can be alleviated by the inactivation of the plasma membrane high-affinity Ca2+ channel Cch1. Here, we show that SDS at low concentrations induces a rapid influx of external Ca2+ into cells, while the plasma membrane remains impermeable for propidium iodide. The inactivation of Pmc1 disturbs efficient adaptation to this activity of SDS. The inactivation of Cch1 partially restores the ability of pmc1 mutant cells to cope with an increased [Ca2+]cyt that correlates with the suppression of SDS hypersensitivity. At the same time, Cch1 is unlikely to be directly involved in SDS-induced Ca2+ influx, since its inactivation does not decrease the amplitude of the rapid [Ca2+]cyt elevation in the pmc1-Δ mutant. The obtained data suggest that the effects of CCH1 inactivation on SDS sensitivity and coping with increased [Ca2+]cyt are related to an additional Cch1 function beyond its direct involvement in Ca2+ transport.

Measuring GPCR-Induced Intracellular Calcium Signaling Using a Quantitative High-Throughput Assay.

Alterations in intracellular calcium are integral to signal transduction pathways for many G-protein-coupled receptors, but this signaling is not well studied. This is mostly due to a lack of reliable, robust, high-throughput, quantitative methods to monitor intracellular calcium concentrations in live cells. Recently, we developed a reliable, robust, quantitative method to measure intracellular calcium levels in which HEK293 cell suspensions loaded with Fura-2/AM are placed in 96-well plates. Minimum and maximum intracellular calcium levels, which are required for converting fluorescence into calcium concentrations, are calibrated using EGTA to chelate calcium and ionomycin to load calcium into cells, respectively. Fluorescence is monitored with a PHERAstar FS plate reader. We provide a detailed method for this high-throughput assay that can be used to quantitate intracellular calcium in endogenous and exogenously (stable or transient) expressed GPCRs in HEK293 cells.

Effect of Magnetic Field and Magnetic Nanoparticles on Choice of Endothelial Cell Phenotype

Background. Endothelial cells as participants in angiogenesis choose their phenotype as tip cells (leading, migratory) or stalk cells (following). It has been experimentally found and theoretically modeled that rapid oscillations in intracellular calcium concentration play a key role in controlling phenotype selection and possible vessel architecture. In addition, the intracellular calcium concentration in endothelial cells is known to be regulated by mechanical wall shear stress induced by blood flow, which controls mechanosensitive calcium ion channel gating. Experimental methods of controlling mechanosensitive ion channel gating in external magnetic fields with application of magnetic nanoparticles are developed that affect magnetic nanoparticles artificially attached to cell membranes. Objective. A key question is raised about the possibility of controlled selection of endothelial cell phenotype in external magnetic fields due to the presence of artificial or biogenic magnetic nanoparticles embedded in the cell membrane. Methods. The magnetic wall shear stress is calculated due to the influence of the external magnetic field on the magnetic nanoparticles embedded in the cell membrane, which controls the mechanosensitive calcium ion pathways. Numerical modeling of oscillations in intracellular calcium concentration in endothelial cells and determination of their final phenotype was carried out taking into account intercellular communication. The python programming language and scipy, py-pde, matplotlib packages of the python programming language were used for numerical modeling. Results. The magnetic field flux density and frequency ranges of a uniform rotating magnetic field, as well as the magnitude of the gradient and the frequency of a non-uniform oscillating magnetic field were calculated for controlling the amplitude and frequency of intracellular calcium concentration oscillations in endothelial cells, as well as the selection of their phenotype. It opens the perspective of controlling angiogenesis and vessel architecture. Conclusions. Phenotype selection by endothelial cells can be controlled in a uniform rotating external magnetic field, as well as in a non-homogeneous oscillating magnetic field.

Chromogranin B Protects Human Umbilical Endothelial Cells against Oxidative Stress

Chromogranin B (CgB) is involved in the control of the cardiovascular system through the regulation of catecholamine release. Whether CgB can exert direct actions on the endothelium has not yet been clarified. Here, we aimed to investigate the effects of CgB on cell viability, mitochondrial membrane potential, reactive oxygen species (ROS), glutathione (GSH), nitric oxide (NO) release, and the cytosolic calcium concentration ([Ca2+]c) in human vascular endothelial cells (HUVECs) cultured under both physiological and peroxidative conditions. In HUVECs, experiments were conducted to establish the proper concentration and timing of CgB stimulation. Thereafter, specific assays were used to evaluate the response of HUVECs cultured in physiologic or oxidative stress conditions to CgB in the presence or absence of β-adrenergic receptor agonists and antagonists and intracellular pathways blockers. Analysis of cell viability, mitochondrial membrane potential, and NO release revealed that CgB was able to cause increased effects in HUVECs cultured in physiological conditions. Additionally, the same analyses performed in HUVECs cultured with H2O2, showed protective effects exerted by CgB, which was also able to counteract ROS release and maintain GSH levels. Furthermore, CgB played a dual role on the [Ca2+]c depending on the physiological or peroxidative cell culturing conditions. In conclusion, our data provide new information about the direct role of CgB in the physiological regulation of endothelial function and highlight its potential as a protective agent against peroxidative conditions, such as those found in cardiovascular diseases.

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.

Potential Mechanism and Involvement of P120-Catenin in the Malignant Biology of Glioma.

This study analyzed the influence of p120-catenin (CTNND1) on the malignant characteristics of glioma and elucidated the potential underlying mechanism. The p120 expression level was assessed in the brain tissues of 42 glioma patients and 10 patients with epilepsy by using the immunohistochemical method. Meanwhile, quantitative PCR technology was employed to assess the expression of P120 in the brain tissues of 71 glioma patients and 13 epilepsy patients. LN229, U251, and U87 glioma cells were used for in vitro analysis and categorized into four treatment groups: siRNA-BC group (no RNA sequence was transfected), siRNA-NC group (transfected control RNA sequences with no effect), and siRNA-1 and siRNA-2 groups (two p120-specific interfering RNA transfection). p120 expression in these treatment groups was quantified by western blotting assay. The migratory and invasive capabilities of glioma cells were studied by wound healing assay and Transwell invasion assay, respectively, under different treatment conditions. MTT assay and cell cycle and apoptosis assay were used to determine glioma cell proliferation and apoptosis, respectively. Enzyme-labeled assay was performed to measure intracellular calcium ion concentration. Immunofluorescence assay was performed for determining microtubule formation and glioma cell distribution. Brain tissues of the glioma group exhibited a remarkable increase in the p120 expression level as compared to brain tissues of the nontumor group (P < 0.05). Furthermore, a strong positive correlation was noted between the malignancy degree in glioma brain tissues and p120 expression in Western blotting (r = 0.906, P = 0.00) and QT-PCR (F=830.6, P＜0.01). Compared to the BC and NC groups, the siRNA transfection groups showed a significant suppression in p120 expression in glioma cells (P < 0.05), with a marked attenuation in the invasive, migratory, and proliferative capabilities of glioma cells as well as an increase in apoptotic potential (P < 0.05). Enzyme-labeled assay showed a remarkable increase in calcium concentration in glioma cells after siRNA treatment. Immunofluorescence assay revealed that the microtubule formation ability of glioma cells reduced after siRNA treatment. p120 has a pivotal involvement in facilitating glioma cell invasion and proliferation by potentially modulating these processes through its involvement in microtubule formation and regulation of intracellular calcium ion levels.

High glucose impairs cognitive function through inducing mitochondrial calcium overload in Treg cells

High glucose has been proved to impair cognitive function in type 2 diabetes, but the underlying mechanisms remain elusive. Here, we found that high glucose increased transcription factors' SP1 O-GlcNAcylation in regulatory T (Treg) cells. Glycosylated SP1 further enhanced HDAC2 recruitment and histone deacetylation on Na+/Ca2+/Li+ exchanger (NCLX) promoter, which downregulated NCLX expression and led to mitochondrial calcium overload and oxidative damage, thereby promoting Treg cell dysfunction, M1 microglia polarization, and diabetes-associated cognitive impairment. Importantly, GLP-1 receptor agonist alleviated these deleterious effects via GLP-1-receptor-mediated upregulation of OGA and inhibition of SP1 O-GlcNAcylation in Treg cells. Our study highlighted a link between high-glucose-mediated SP1 O-GlcNAcylation and HDAC2/NCLX signaling in control of mitochondrial calcium concentrations in Treg cells. It also revealed a mechanism for linking Treg cell dysfunction and cognitive impairment in type 2 diabetes and provides an insight into the mechanism underlying the neuroprotective effects of GLP-1 receptor agonist.

Ethyl vinyl ketone activates oxidative and calcium burst and CML8-ACA8 participates in calcium recovery in Arabidopsis leaves

Plants produce ethyl vinyl ketone (evk) in response to biotic stress, but the evk's identification and downstream defense response remain unclear. In this paper, it is predicted by docking for the first time that evk can be recognized by RBOH protein and assist the electron transfer of RBOHD/RBOHF by binding to its FAD or NADPH binding site. Surface plasmon resonance (SPR) binding assay shows that evk indeed bind to RBOHD. Here, we show that evk treatment increased H2O2 and intracellular calcium concentrations in Arabidopsis thaliana mesophyll cells, as observed by confocal laser scanning microscopy and non-invasive micro-test technology, and that H2O2 signaling functioned upstream of Ca2+ signaling. Yeast two-hybrid, firefly luciferase complementation imaging, and in vitro pull-down assays demonstrated that the ACA8 (AUTOINHIBITED Ca2+-ATPASE, ISOFORM 8)–CML8 (CALMODULIN-LIKE 8) interaction promoted Ca2+ efflux to return Ca2+ levels to the resting state.

Application of In Silico Trials for the Investigation of Drug Effects on Cardiomyopathy-Diseased Heart Cycle Properties

In this paper, we present the abilities of an in silico platform used to simulate the effects of different drugs on heartbeat cycle performance. The platform is based on a finite element modelling approach with the fluid–solid interaction implemented using a loose coupling procedure. Active mechanical stresses are calculated using the Hunter excitation model while the passive mechanical stresses are calculated using a recently introduced experiment-based material model for the heart tissue. The applicability of the platform is illustrated using a simple parametric model of the left ventricle. The simulations are performed using parameters that are specific to drugs such as digoxin, mavacamten, 2-deoxy adenosine triphosphate, and disopyramide, with the concentration of calcium in the cardiac cells affected by these drugs given as an input function. The results are obtained for two geometries mimicking patients with hypertrophic and dilated cardiomyopathy, and also for different inlet/outlet boundary conditions simulating different drug effects at the macroscopic level. Using in silico simulations with virtual patients, it is possible to evaluate the influence of different drugs on cardiac output and ejection fraction. This approach can significantly reduce computational costs with an acceptable solution accuracy compared to approaches coupling finite element and biophysical muscle model methods that are used to calculate drug effects at the micro level.

TRPV4 enhances the synthesis of fatty acids to drive the progression of ovarian cancer through the calcium-mTORC1/SREBP1 signaling pathway

Transient receptor potential vanilloid 4 (TRPV4) is a nonselective cation channel activated by various stimuli, such as heat. A recent study reported that high expression of TRPV4 predicted a poor prognosis in ovarian cancer patients. This study demonstrated that TRPV4 was highly expressed in ovarian cancer and had the ability to promote proliferation and migration. Through RNA-seq and related experiments, we confirmed that the oncogenic pathway of TRPV4 in ovarian cancer may be related to the fatty acid synthesis. By correlation analysis and RNA-seq, we demonstrated that SREBP1 and mTORC1 were inseparably related to that. Therefore, we used inhibitors to perform experiments. Calcium fluorescent probe experiments suggest that the change of calcium content in ovarian cancer cells was related to the downstream mTORC1 signaling pathway and fatty acid synthesis. These results confirmed that TRPV4 affected the fatty acid synthesis through the calcium-mTOR/SREBP1 signaling pathway, thereby promoting ovarian cancer progression.

Investigation of calcium variations in single cells and the impact of Yoda1 on osteocytes by ICP-OES

BackgroundDetection of elements in individual cells by inductively coupled plasma (ICP) spectrometry has recently attracted significant interest in biological research, due to the unique ability of ICP spectrometry for trace element analysis. However, performing single-cell analysis using ICP optical emission spectrometry (ICP-OES) remains a challenge due to the small size and discrete nature of cells. This is while ICP-OES can serve as a cost-effective and label-free method for this purpose. Therefore, it is necessary to improve the current ICP-OES technique to facilitate the detection of elements in single cells, thereby unlocking novel applications. ResultsA new conical ICP torch, which has been illustrated to offer better analytical performance than the conventional ones, was applied to achieve the detection of calcium in single micro-sized cells. A new heated chamber was designed and coupled with a high-efficiency nebulizer as the sample introduction system. For the detection of single SiO2 particles, the number of particle events obtained by the new sample introduction system was found to be up to 9 times higher than that of the conventional system without sacrificing the signal intensity. Subsequently, calcium in human breast cancer cells (MDA-MB-231), mice breast cancer cells (Py8119), and mice osteocytes (MLO-Y4) was successfully detected using the new ICP-OES system. The cell detection efficiency turned out to be around 2%–3% which is much higher than that the reported values in previous single-cell ICP-OES research. Finally, as a new application, the effect of Yoda1, a recently identified activator of Piezo1 calcium channel, on osteocytes was investigated. The calcium content in Yoda1-treated MLO-Y4 cells was seen increase by 36% compared to the control sample. SignificanceThis research reveals the capability of ICP-OES in single-cell analysis for micro-sized cells which was made possible by the new conical ICP torch and the new sample introduction system. The ability to detect calcium in single mammalian cells enables the first ever application of this technique to assess the impact of the Yoda1 activator on the calcium level in osteocytes.

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.

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.

Reduction of TRPC1/TRPC3 mediated Ca2+-signaling protects oxidative stress-induced COPD

Oxidative stress is a predisposing factor in Chronic Obstructive Pulmonary Disease (COPD). Specifically, pulmonary epithelial (PE) cells reduce antioxidant capacity during COPD because of the continuous production of reactive oxygen species (ROS). However, the molecular pathogenesis that governs such ROS activity is unclear. Here we show that the dysregulation of intracellular calcium concentration ([Ca2+]i) in PE cells from COPD patients, compared to the healthy PE cells, is associated with the robust functional expressions of Transient Receptor Potential Canonical (TRPC)1 and TRPC3 channels, and Ca2+ entry (SOCE) components, Stromal Interaction Molecule 1 (STIM1) and ORAI1 channels. Additionally, the elevated expression levels of fibrotic, inflammatory, oxidative, and apoptotic markers in cells from COPD patients suggest detrimental pathway activation, thereby reducing the ability of lung remodeling. To further delineate the mechanism, we used human lung epithelial cell line, A549, since the behavior of SOCE and the expression patterns of TRPC1/C3, STIM1, and ORAI1 were much like PE cells. Notably, the knockdown of TRPC1/C3 in A549 cells substantially reduced the SOCE-induced [Ca2+]i rise, and reversed the ROS-mediated oxidative, fibrotic, inflammatory, and apoptotic responses, thus confirming the role of TRPC1/C3 in SOCE driven COPD-like condition. Higher TRPC1/C3, STIM1, and ORAI1 expressions, along with a greater Ca2+ entry, via SOCE in ROS-induced A549 cells, led to the rise in oxidative, fibrotic, inflammatory, and apoptotic gene expression, specifically through the extracellular signal-regulated kinase (ERK) pathway. Abatement of TRPC1 and/or TRPC3 reduced the mobilization of [Ca2+]i and reversed apoptotic gene expression and ERK activation, signifying the involvement of TRPC1/C3. Together these data suggest that TRPC1/C3 and SOCE facilitate the COPD condition through ROS-mediated cell death, thus implicating their likely roles as potential therapeutic targets for COPD. SummaryAlterations in Ca2+ signaling modalities in normal pulmonary epithelial cells exhibit COPD through oxidative stress and cellular injury, compromising repair, which was alleviated through inhibition of store-operated calcium entry. Subject areaCalcium, ROS, Cellular signaling, lung disease.

Costunolide covalently targets and inhibits CaMKII phosphorylation to reduce ischemia-associated brain damage

BackgroundChronic cerebral hypoperfusion (CCH) is a leading cause of disability and mortality worldwide. Restoring cerebral blood flow (CBF) through vasodilatation is particularly important in the treatment of CCH. Costunolide (Cos) is a natural sesquiterpenoid compound with vasodilatory effect, but its mechanism is unclear. PurposeThis study aimed to investigate the vasodilatory mechanism of Cos and provide a new therapeutic regimen for treating CCH. MethodsThe therapeutic effect of Cos on CCH was assessed in a rat model of permanent common carotid artery occlusion. The direct target protein for improving CBF was identified by drug affinity responsive target stability combined with quantitative differential proteomics analysis. The molecular mechanism of Cos acting on its target protein was analyzed by multidisciplinary approaches. The signalling involved was assessed using site-directed pharmacological intervention. ResultsCos has a significant therapeutic effect on ischemic brain injury by restoring CBF. Multifunctional calcium/calmodulin-dependent protein kinase II (CaMKII) was identified as a direct target of the natural small molecule Cos with a therapeutic effect on CCH. Mechanistic studies revealed that the α,β-unsaturated-γ-lactone ring of Cos covalently binds to the Cys116 residue of CaMKII. It then inhibits the phosphorylation of CaMKII and reduces the calcium concentration in vascular smooth muscle cells, thus playing a role in vasodilation and increasing CBF. Notably, this covalent binding between Cos and CaMKII can exert a long-term vasodilator activity. ConclusionWe reported for the first time that Cos reduced ischemia-associated brain damage by covalently binding to the Cys116 residue of CaMKII, inhibiting CaMKII phosphorylation, and exerting long-term vasodilatory activity. This study not only found a new covalent inhibitor against the phosphorylation of CaMKII but also suggested that pharmacologically targeting CaMKII is a promising therapeutic strategy for CCH.

Osteogenic Differentiation Effect of Human Periodontal Ligament Stem-Cell Initial Cell Density on Autologous Cells and Human Bone Marrow Stromal Cells.

Stem cells have differentiation and regulation functions. Here, we discussed the impact of cell culture density on stem cell proliferation, osteoblastogenesis, and regulation. To discuss the effect of the initial culture density of human periodontal ligament stem cells (hPDLSCs) on the osteogenic differentiation of autologous cells, we found that the hPDLSC proliferation rate decreased with an increase in the initial plating density (0.5-8 × 104 cells/cm2) for the 48 h culture cycle. After hPDLSCs induced osteogenic differentiation for 14 days with different initial cell culture densities, the expression of osteoprotegerin (OPG) and runt-related transcription factor 2(RUNX2) and the OPG/ Receptor Activator of Nuclear Factor-κ B Ligand (RANKL) ratio were the highest in the hPDLSCs initially plated at a density of 2 × 104 cells/cm2, and the average cell calcium concentration was also the highest. To study hPDLSCs regulating the osteoblastic differentiation of other cells, we used 50 μg/mL of secreted exosomes derived from hPDLSCs cultured using different initial cell densities to induce human bone marrow stromal cell (hBMSC) osteogenesis. After 14 days, the results indicated that the gene expression of OPG, Osteocalcin(OCN,)RUNX2, and osterix and the OPG/RANKL ratio were the highest in the 2 × 104 cells/cm2 initial cell density group, and the average calcium concentration was also the highest. This provides a new idea for the clinical application of stem cell osteogenesis.

Abstract 3825: Andrographis reverses gemcitabine-associated chemoresistance by regulating calcium signaling in pancreatic ductal adenocarcinoma

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the lethal malignancies due to the acquisition of intrinsic or acquired resistance to chemotherapeutic drugs, such as Gemcitabine (Gem). Recent evidence revealed that calcium signaling significantly contributes to Gem-associated chemoresistance (Gem resistance) in PDAC cells. Andrographis, a labdane diterpenoid isolated from the traditional herb Andrographis paniculate, is known to have a potent calcium channel-blocking potential. Accordingly, we hypothesized that Andrographis might facilitate the reversal of Gem resistance in PDAC. Methods: The critical regulatory pathways associated with the Gem resistance in PDAC were identified by analyzing publicly available cell line-based transcriptomic profiling. Gem resistant (Gem-R) PDAC cells (Gem-R MIA PaCa-2 and BxPC-3) were established by continuously culturing cells with Gem. A series of cell culture experiments were performed using Gem-R PDAC cells to evaluate the Andrographis-mediated reversal of Gem resistance in PDAC cells. At last, the cell culture findings were validated in patient-derived PDAC organoids. Results: Transcriptomic profiling identified the calcium signaling pathway as one of the key regulators of Gem resistance (Fold enrichment: 2.8, P =0.002). The combination of Andrographis and Gem revealed a superior anti-cancer potential in Gem-R PDAC cells on cell viability, clonogenicity, and migration compared to individual treatments with Andrographis or Gem. The combined treatment potentiated cellular apoptosis in Gem-R MIA PaCa-2 (Andro, Gem vs. Combination, 20.3%, 10.9% vs. 31.4%) and Gem-R BxPC-3 (Andro, Gem vs. Combination, 11.3%, 6.8% vs. 16.1%). The fluo-4 assay demonstrated that Andrographis decreased intracellular calcium concentration in Gem-R PDAC cells, while the combined treatment with Andrographis and Gem decreased its concentration more in Gem-R MIA PaCa-2 (fold change [FC] =0.79 vs. Andrographis; FC =0.54 vs. Gem) and Gem-R BxPC-3 (FC =0.80 vs. Andrographis; FC =0.73 vs. Gem). Finally, the combined treatment with Andrographis and Gem showed anti-cancer activity in patient-derived PDAC organoids. Conclusions: We have firstly investigated the Andrographis-mediated reversal of chemoresistance against Gem in PDAC cells, possibly through the regulation of cellular apoptosis and calcium signaling, using a systematic series of Gem resistant cell cultures and patient-derived tumor organoid models. Our findings could provide evidence of the combined treatment with Andrographis and Gem as a complementary therapeutic modality against PDAC. Citation Format: Keisuke Okuno, Caiming Xu, Masanori Tokunaga, Cristina Fillat, Haiyong Han, Yusuke Kinugasa, Ajay Goel. Andrographis reverses gemcitabine-associated chemoresistance by regulating calcium signaling in pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3825.

Finite volume simulation of calcium distribution in a cholangiocyte cell

Cholangiocytes are the cells of the liver having a major role in the conditioning of bile used in digestion. Other functions of cholangiocytes are in apoptosis and bicarbonate secretion. The Calcium in the intracellular environment of various cells including cholangiocytes regulates a large number of functions. This regulating mechanism in cholangiocytes has been poorly understood to date. In order to analyze the calcium regulation in cholangiocyte cells, a mathematical model for a one-dimensional steady-state case is constructed in this study. This involves a non-linear reaction-diffusion equation with appropriate boundary conditions. The influx from IP$_{3}$ receptor, ryanodine receptor (RYR), and plasma membrane as well as the efflux of calcium from SERCA pump and plasma membrane have been employed in the model. The finite volume method and Newton-Raphson method have been used to solve the problem. Numerical findings have been used to examine the effects of parameters like diffusion coefficient, rate of SERCA pump efflux, buffer, and influx from plasma membrane on calcium concentration in cholangiocyte cells. The information generated from the model can be useful for understanding the mechanism of cholestatic disorders which can be further useful in the diagnosis and treatment of these disorders.

Serum and Red Cell Magnesium and Calcium Concentrations in Normotensive and Pre-Eclamptic Pregnant Women in Lagos, Nigeria

Background: Pre-eclampsia is a significant cause of maternal and foetal morbidity and mortality. This study identified the relationship between serum and red cell concentrations of magnesium and calcium in pre-eclampsia. This is to justify the use of magnesium in the prevention of pre-eclampsia and eclampsia. Objective: To compare the serum and red cell levels of calcium and magnesium among pre-eclamptic and normotensive pregnant women. Method: A prospective, comparative study comparing serum and red cell concentrations of magnesium and calcium in sixty pre-eclamptic patients and sixty normotensive pregnant controls. Result: The mean serum calcium concentration among pre-eclamptic patients was 2.33±0.19 mmol/l, compared with 2.47±0.23 mmol/l in the normotensive group (p = 0.244). The mean serum magnesium concentration was significantly higher among normotensive pregnant women than pre-eclamptic women (1.18±0.31 mmol/l vs 0.91±0.23 mmol/l; p = 0.001). The mean red blood cell calcium concentration was also significantly higher among pre-eclamptic pregnant women compared to the normotensive group (4.34±0.62 mmol/l vs 3.95±0.79 mmol/l; p = 0.003), but the mean red cell magnesium concentration was lower in pre-eclamptic than the normotensive control group but without statistical significance (2.04±0.65 mmol/l vs 2.26±0.79 mmol/l; p = 0.098). Conclusion: Pre-eclampsia is associated with low concentrations of serum magnesium and calcium. Therefore, magnesium supplements may be beneficial in preventing pre-eclampsia.

Пригнічувальна дія загального анестетика кетаміну на транзієнти внутрішньоклітинної концентрації іонів кальцію та скорочення гладеньком’язових клітин тонкої кишки миші

The mechanisms of the negative consequences of general anaesthetics action on the nervous system have been studied in detail, but regarding smooth muscle function, such issues have not yet been sufficiently addressed. In this study, we investigated the effect of the general intravenous anaesthetic ketamine on the level of intracellular calcium in isolated ileum myocytes and the contractile activity of smooth muscle strips of the mouse small intestine. The concentration of intracellular calcium in cells was measured using the Ca2+-sensitive fluorescent dye Fura-2, and tensiometry was used to record the contractile activity of smooth muscles. It was shown that ketamine at a concentration of 100 µM significantly, by 40%, suppressed carbachol-induced contractile reactions of the ileum. The inhibitory effect correlated with the suppression of the intracellular calcium responses to carbachol in isolated smooth muscle cells after the addition of ketamine to the extracellular solution, which was by 65% on average. These results contribute to our better understanding of the possible membrane and intracellular mechanisms of the development of post-surgical intestinal motility disorders.