Calcium Levels In Cells Research Articles

Preventing Donepezil-Induced Adverse Effects Through N-acetylcysteine Co-Administration.

Drug-induced adverse symptoms affect patients' quality of life (QoL) during treatment. Understanding the underlying mechanisms of drug-induced adverse effects could help prevent them. As current drugs have limited effects in halting the progress of Alzheimer's disease (AD), patients are required to take these drugs over a long period. The main obstacles to long-term compliance are drug-elicited side effects that deteriorate patient QoL. Donepezil, the most popular acetylcholinesterase inhibitor (AChEI) drug for AD, induces various side effects, especially at high doses. This study aimed to identify a drug that can attenuate the side effects of donepezil and investigate the underlying mechanisms. Five-week-old Sprague-Dawley rats received daily oral donepezil and N-acetylcysteine (NAC) for four weeks. General symptoms following administration were monitored daily to address drug-related adverse effects. Cytosolic calcium influx and generation of reactive oxygen species (ROS) after drug treatment were measured in vitro using C2C12 myotubes. High-dose donepezil induced numerous adverse symptoms in male and female rats, which were markedly attenuated by co-treatment with NAC. NAC significantly reduced both acute and chronic muscle-related symptoms caused by donepezil. Additionally, in vitro studies showed that high-dose donepezil increased ROS and intracellular calcium ([Ca2+]i) levels in muscle cells, contributing to these adverse effects. NAC co-treatment dramatically reduced ROS and [Ca2+]i levels in muscle cells. Combined treatment with NAC effectively diminishes the adverse effects elicited by donepezil by regulating ROS and [Ca2+]i levels in the skeletal muscle, which could contribute to improving donepezil treatment in patients.

Heightened mitochondrial respiration in CF cells is normalised by triple CFTR modulator therapy through mechanisms involving calcium

BackgroundCystic fibrosis (CF) is associated with increased resting energy expenditure. However, the introduction of elexacaftor/tezacaftor/ivacaftor (ETI) has resulted in a paradigm shift in nutritional status for many people with CF, with increase body mass index and reduction in the need for nutritional support. While these changes are likely to reflect improved clinical status and an associated downregulation of energy expenditure, they may also reflect drug-induced alterations in metabolic perturbations within CF cells. We hypothesise that some of these changes relate to normalisation of mitochondrial respiration in CF. MethodsUsing wild-type (WT) and F508del/F508del CFTR human bronchial epithelial cell lines (HBE cell lines) and baby hamster kidney (BHK) cells we examined the impact of ETI on cellular metabolism. We monitored mitochondrial respiration, using Seahorse extracellular flux assays and monitored mitochondrial reactive oxygen species (mROS) and intracellular calcium levels by flow cytometry. ResultsIncreased mitochondrial respiration was found in HBE cell lines and BHK cells expressing CFTR F508del/F508del when assessing basal, maximal, spare respiratory capacities and ATP production, as well as increased mitochondrial ROS generated via forward electron transport. ETI significantly decreased basal, maximal, spare respiratory capacity and ATP production to WT levels or below. Calcium blocker, BAPTA-AM normalised mitochondrial respiration, suggesting a calcium-mediated mechanism. ETI decreased intracellular calcium levels in CF cells to the same extent as BAPTA-AM, highlighting the importance of calcium and chloride in mitochondrial respiration in CF. ConclusionsCF cell lines exhibit increased mitochondrial respiration, which can be downregulated by ETI therapy through mechanisms involving calcium.

The functional subclasses of AT1 receptor autoantibody in patients with coronary heart disease

Recently, the identification of autoantibodies (AT1-AA) targeting the second extracellular loop of angiotensin II type 1 receptor (AT1R-ECII) in patients with coronary heart disease (CHD) offers a novel perspective on the interplay between immunity and cardiovascular disease. However, much remains unknown regarding the functional diversity of AT1-AA. In this study, we measured the levels of AT1-AA in the sera of 306 CHD patients and purified AT1-AA from patient’s sera (n = 127). The subclasses of AT1-AA were categorized based on their impact on intracellular calcium ([Ca2+]i) levels in mouse arterial smooth muscle cells (MASMCs). Our findings revealed 4 distinct [Ca2+]i response patterns indicating the existence of 4 functional subclasses named H1-, H2-, H3-, and H4-AT1-AA. The correlation analysis demonstrated a positive association between H1-AT1-AA and endogenous coagulation, as well as between H2-AT1-AA and exogenous coagulation; no significant correlation was observed between H3-AT1-AA and the indicators we analyzed. Conversely, H4-AT1-AA exhibited a negative correlation with both leukocyte number and bile acid levels. Logistic regression analysis showed that H2-AT1-AA possessed predictive value for severe CHD. Furthermore, in vitro experiments indicated that both H1- and H2-AT1-AA exerted cytotoxic effects on MASMCs, while H4-AT1-AA increased cell viability. Additionally, an AT1-AA-positive rat model was established by subcutaneously injecting with AT1R-ECII peptide, which produced four similar functional subclasses of rat AT1-AA upon active immunization. This study suggested that classifying different functional subclasses of AT1-AAs can facilitate more accurate evaluation of the condition and prognosis in patients with CHD, thereby providing a novel basis for clinical diagnosis and treatment.

Chrysophanol Induces Cell Death and Inhibits Invasiveness through Alteration of Calcium Levels in HepG2 Human Liver Cancer Cells.

To investigate the effect of chrysophanol, a phytochemical derived from Radix et Rhizoma Rhei on HepG2 liver cancer cells. HepG2 cell line was treated with different concentrations chrysophanol (0-100 μmol/L) for 24 h. The cell counting kit 8 assay was employed to assess cell viability. Intracellular calcium levels were examined using Fluo-4 AM and Mag-fluo-4 AM staining, followed by flow cytometry analysis. Mitochondrial membrane potential was measured with JC-1 assay kit. Additionally, the expressions of key proteins such as p-JNK, Bax, cytochrome c (Cyt C), cleaved caspase-3 (cCaspase-3), and caspase-8 were analyzed by Western blot. The inhibitory effects of chrysophanol on the invasion of cells were determined using a Transwell assay. Analysis of invasiveness was conducted by wound healing assay. Chrysophanol significantly reduced the proliferation of HepG2 liver cancer cells by affecting intracellular calcium distribution, diminishing mitochondrial membrane potential, and enhancing the expressions of p-JNK, Bax, Cyt C, cCaspase-3, and caspase-8 in the groups treated with 75 or 100 μmol/L chrysophanol compared to the control group (P<0.05). Additionally, 75 and 100 μmol/L chrysophanol exhibited inhibitory effects on cell migration and wound healing. Chrysophanol demonstrates potential against HepG2 liver cancer cells, suggesting its potential use as a therapeutic agent for liver cancer treatment.

Abstract A032: The role of ASIC2 and calcium influx in epithelial ovarian cancer pathogenesis

Abstract Objective: Inflammation associated with incessant ovulation plays a key role in epithelial ovarian cancer (EOC) pathogenesis. Ion channels, such as acid-sensing ion channel-2 or ASIC2 is known to be upregulated in inflammatory conditions and may play a role in calcium ion dysregulation. Here, we describe a potential relationship between ASIC2 and intracellular calcium levels in EOC development and progression. Methods: EOC cell lines OVCAR-8, ES-2 and SKOV-3 were grown to sub-confluence prior to experimentation. Global extracellular pH was changed with the use of hydrochloric acid (HCl) over various time points at hours (h): 2 h, 4 h, 8 h, 12 h and 24 h. ASIC2 expression was subsequently measured via western blot. Intracellular pH was measured with the use of pH-sensitive pHrodo red dye. Cytosolic pH was determined across all cell lines at baseline and after treatment with a known ASIC2 inhibitor: Diminazene (10 μM) over 30 minutes. Intracellular calcium levels were measured with a Fluo Calcium Indicator at baseline and with similar ASIC2 inhibition. Live cell imaging was utilized on a Nikon A1r confocal microscope. Results: Changing global, extracellular pH to 6.0 and 6.5 across various time points from 2-24 h did not affect ASIC2 protein expression via western blot analysis. We then utilized a pH-sensitive assay to determine whether intracellular pH changes were associated with ASIC2 expression. Interestingly, intracellular pH was noted to increase across most cell lines with ASIC2 inhibition via Diminazene treatment. There was a statistically significant increase in pH in ES2 and OVCAR8 cell lines. Because ASIC2 is known to result in influx of various ions, assessment of intracellular calcium levels was performed first after 30 minutes of Diminazene treatment. There was a corresponding significant increase in intracellular calcium levels in ES2 cells after ASIC2 inhibition. And there was a small trend, albeit not significant, toward a similar increase across SKOV-3 and OVCAR-8 cells. Conclusion: There seems to be a role for ASIC2 in EOC, particularly in the context of inflammation as noted by changes in intracellular pH levels. It is likely that ASIC2 may mediate changes in intracellular calcium signaling and promote tumorigenesis in ovarian cancer. Citation Format: Tanvi Joshi, Annelise Wilhite, Sagar Chokshi, Mary Howard Singleton, Jennifer Scalici, Kevin Lee. The role of ASIC2 and calcium influx in epithelial ovarian cancer pathogenesis [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr A032.

Analysis of cholesterol-recognition motifs of the plasma membrane Ca2+-ATPase

The plasma membrane Ca2+-ATPase (PMCA) is crucial for the fine tuning of intracellular calcium levels in eukaryotic cells. In this study, we show the presence of CARC sequences in all human and rat PMCA isoforms and we performed further analysis by molecular dynamics simulations. This analysis focuses on PMCA1, containing three CARC motifs, and PMCA4, with four CARC domains. In PMCA1, two CARC motifs reside within transmembrane domains, while the third is situated at the intracellular interface. The simulations depict more stable RMSD values and lower RMSF fluctuations in the presence of cholesterol, emphasizing its potential stabilizing effect. In PMCA4, a distinct dynamic was found. Notably, the total energy differences between simulations with cholesterol and phospholipids are pronounced in PMCA4 compared to PMCA1. RMSD values for PMCA4 indicate a more energetically favorable conformation in the presence of cholesterol, suggesting a robust interaction between CARCs and this lipid in the membranes. Furthermore, RMSF analysis for CARCs in both PMCA isoforms exhibit lower values in the presence of cholesterol compared to POPC alone. The analysis of H-bond occupancy and total energy values strongly suggests the potential interaction of CARCs with cholesterol. Given the crucial role of PMCAs in physiological calcium regulation and their involvement in diverse pathological processes, this study underscores the significance of CARC motifs and their interaction with cholesterol in elucidating PMCA function. These insights into the energetic preferences associated with CARC-cholesterol interactions offer valuable implications for understanding PMCA function in maintaining calcium homeostasis and addressing potential associated pathologies.

CRISPLD1 promotes gastric cancer progression by regulating the Ca2+/PI3K-AKT signaling pathway

Gastric cancer (GC) is a malignant tumor with poor prognosis. Studies have shown that cysteine-rich secretory protein LCCL domain containing 1 (CRISPLD1) is associated with tumor progression. However, its role in GC is unclear. The present study aimed to determine the pathogenic mechanism of CRISPLD1 in GC. Analysis of public databases revealed high mRNA expression of CRISPLD1 in GC, which was associated with poor prognosis. Additionally, CRISPLD1 expression levels showed significant correlations with T stage, overall survival events, and stage. Knockdown of CRISPLD1 reduced cell proliferation, invasion, and migration. Furthermore, CRISPLD1 knockdown decreased intracellular calcium levels in GC cells and inhibited the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)-protein kinase B (AKT) signaling pathway. Treatment with an AKT activator reversed the inhibitory effect of CRISPLD1 knockdown on GC cell migration and invasion. Our findings suggest that CRISPLD1 promotes tumor cell progression in GC by mediating intracellular calcium levels and activating the PI3K-AKT pathway, highlighting CRISPLD1 as a potential therapeutic target for GC.

Roles of sphingosine‐1‐phosphate in follicle development and oocyte maturation

AbstractSphingosine‐1‐phosphate (S1P), a lipid messenger, propagates its signals by interacting with its intracellular targets or is transported to autocrine/paracrine to activate its cell surface receptors. In the female reproductive system, the homeostasis of S1P plays an important role in ovarian follicular development. Our recent studies show that S1P emerges as a functional mediator of LH‐EGFR signaling from cumulus cells to oocytes: elevating calcium levels in cumulus cells to induce oocyte meiotic maturation, and activating Akt/mTOR cascade reaction to promote oocyte developmental competence. Thus, S1P might be applied to promote oocyte maturation in animals and humans.

Decreased pyruvate dehydrogenase activity in Tafazzin-deficient cells is caused by dysregulation of pyruvate dehydrogenase phosphatase 1 (PDP1)

Cardiolipin (CL), the signature lipid of the mitochondrial inner membrane, is critical for maintaining optimal mitochondrial function and bioenergetics. Disruption of CL metabolism, caused by mutations in the CL remodeling enzyme TAFAZZIN, results in the rare and life-threatening disorder Barth syndrome (BTHS). While the clinical manifestations of BTHS, such as dilated cardiomyopathy and skeletal myopathy, point to defects in mitochondrial bioenergetics, the disorder is also characterized by broad metabolic dysregulation, including abnormal levels of metabolites associated with the tricarboxylic acid (TCA) cycle. In line with this, recent studies have identified inhibition of pyruvate dehydrogenase (PDH), the gatekeeper enzyme for TCA cycle carbon influx, as a key deficiency in various BTHS model systems. However, the molecular mechanisms linking aberrant CL remodeling, particularly the primary, direct consequence of reduced tetralinoleoyl-CL (TLCL) levels, to PDH activity deficiency are not yet understood. This knowledge gap has limited our understanding of lipid-mediated metabolic regulation in BTHS and hindered the development of effective treatment strategies. In the current study, we provide evidence that remodeled TLCL promotes PDH function by directly binding to and enhancing the activity of PDH phosphatase 1 (PDP1). This is supported by our findings that TLCL uniquely activates PDH in a dose-dependent manner, TLCL binds to PDP1 in vitro, TLCL-mediated PDH activation is attenuated in the presence of phosphatase inhibitor, and PDP1 activity is decreased in Tafazzin-knockout (TAZ-KO) C2C12 myoblasts. Additionally, we observed decreased mitochondrial calcium levels in TAZ-KO cells, which may affect the calcium-sensitive activity of PDP1. Treating TAZ-KO cells with calcium lactate (CaLac) increases mitochondrial calcium and restores PDH activity and mitochondrial oxygen consumption rate. Based on our findings, we conclude that reduced mitochondrial calcium levels and decreased binding of PDP1 to TLCL contribute to decreased PDP1 activity in TAZ-KO cells.

Tunneling nanotube-transmitted mechanical signal and its cellular response

Tunneling nanotubes (TNTs) are elastic tubular structures that physically link cells, facilitating the intercellular transfer of organelles, chemical signals, and electrical signals. Despite TNTs serving as a multifunctional pathway for cell-cell communication, the transmission of mechanical signals through TNTs and the response of TNT-connected cells to these forces remain unexplored. In this study, external mechanical forces were applied to induce TNT bending between rat kidney (NRK) cells using micromanipulation. These forces, transmitted via TNTs, induced reduced curvature of the actin cortex and increased membrane tension at the TNT-connected sites. Additionally, TNT bending results in an elevation of intracellular calcium levels in TNT-connected cells, a response attenuated by gadolinium ions, a non-selective mechanosensitive calcium channel blocker. The degree of TNT deflection positively correlated with decreased actin cortex curvature and increased calcium levels. Furthermore, stretching TNT due to the separation of TNT-connected cells resulted in decreased actin cortex curvature and increased intracellular calcium in TNT-connected cells. The levels of these cellular responses depended on the length changes of TNTs. Moreover, TNT connections influence cell migration by regulating cell rotation, which involves the activation of mechanosensitive calcium channels. In conclusion, our study revealed the transmission of mechanical signals through TNTs and the subsequent responses of TNT-connected cells, highlighting a previously unrecognized communication function of TNTs. This research provides valuable insights into the role of TNTs in long-distance intercellular mechanical signaling.

Abused drug-induced intracranial self-stimulation is correlated with the alteration of dopamine transporter availability in the medial prefrontal cortex and nucleus accumbens of mice

Intracranial self-stimulation (ICSS) of the medial forebrain bundle in mice is an experimental model use to assess the relative potential of reward-seeking behaviors. Here, we used the ICSS model to evaluate the abuse potential of 18 abused drugs: 3-Fluoroethamphetamine (3-FEA); methylphenidate; cocaine; dextroamphetamine; alpha-Pyrrolidinobutyrophenone (α-PBT); 4'-Fluoro-4-methylaminorex (4-FPO); methamphetamine; larocaine; phentermine; paramethoxymethamphetamine (PMMA); phendimetrazine; N-(1-adamantyl)-1-pentyl-1H-indazole-3-carboxamide (AKB-48); Naphthalen-1-yl-(4-pentyloxynaphthalen-1-yl)methanone (CB-13); 4-Ethylnaphthalen-1-yl-(1-pentylindol-3-yl)methanone (JWH-210); Naphthalen-1-yl-(1-pentylindol-3-yl)methanone (JWH-018); N-(ortho-methoxybenzyl)-4-ethylamphetamine (4-EA-NBOMe); N-[(2-Methoxyphenyl)methyl]-N-methyl-1-(4-methylphenyl)propan-2-amine (4-MMA-NBOMe); and 1-[1-(4-methoxyphenyl)cyclohexyl]piperidine (4-MeO-PCP). We determined dopamine transporter (DAT) availability in the medial prefrontal cortex (mPFC), striatum, and nucleus accumbens (NAc) after drug treatment. DAT availability in the mPFC and NAc significantly correlated with the ICSS threshold after drug treatment. Extracellular dopamine and calcium levels in PC-12 cells were measured following drug treatment. After drug treatment, Spearman rank and Pearson correlation analyses showed a significant difference between the extracellular dopamine level and the ICSS threshold. After drug treatment, Spearman rank correlation analysis showed a significant correlation between Ca2+ signaling and the ICSS threshold. A positive correlation exists between the ICSS threshold and DAT availability in the mPFC and NAc provoked by abused drugs. The relative potential of drug-induced reward-seeking behavior may be related to DAT availability-mediated extracellular dopamine levels in the mPFC and NAc.

Bifenox compromises porcine trophectoderm and luminal epithelial cells in early pregnancy by arresting cell cycle progression and impairing mitochondrial and calcium homeostasis

Bifenox is a widely used herbicide that contains a diphenyl ether group. However its global usage, the cell physiological effects that induce toxicity have not been elucidated. In this study, the effect of bifenox was examined in porcine trophectoderm and uterine epithelial cells to investigate the potential toxicity of the implantation process. To uncover the toxic effects of bifenox, cell viability and apoptosis following treatment with bifenox were evaluated. To investigate the underlying cellular mechanisms, mitochondrial and calcium homeostasis were investigated in both cell lines. In addition, the dysregulation of cell signal transduction and transcriptional alterations were also demonstrated. Bifenox reduced cell viability and significantly increased the number of cells arrested at the sub-G1 stage. Moreover, bifenox depolarized the mitochondrial membrane and upregulated the calcium flux into the mitochondria in both cell lines. Cytosolic calcium flux increased in porcine trophectoderm (pTr) cells and decreased in porcine luminal epithelium (pLE) cells. In addition, bifenox activated the mitogen-activated protein kinase and phosphoinositide 3-kinase signaling pathways. Furthermore, bifenox inhibited the expression of retinoid receptor genes, such as RXRA, RXRB, and RXRG. Chemokine CCL8 was also downregulated at the mRNA level, whereas CCL5 expression remained unchanged. Overall, the results of this study suggest that bifenox deteriorates cell viability by arresting cell cycle progression, damaging mitochondria, and controlling calcium levels in pTr and pLE cells. The present study indicates the toxic potential of bifenox in the trophectoderm and luminal epithelial cells, which can lead to implantation disorders in early pregnancy.

Tunicamycin induced inhibition of calpain 1 and 2 enzyme activity in ovarian cancer cells

Background: Tunicamycin (TN) is an antitumor agent and induced intracellular calcium levels in many cells, however its molecular mechanism is still needed to be explored. Calpeptin (Calp) is an inhibitor of both calpain 1 and 2 (CAPN-1/2) enzymes, and plays a fundamental role in tumor mechanism. In this study, the effects of TN and Calp were investigated on CAPN-1/2 enzyme activity in normal and ovarian cancer cells adhered to fibronectin. Methods: 24uM TN, 50uM Calp, and combined TN and Calp (TN+Calp) were applied for 1 and 12 hours to FN-bound (FN+) and non-FN-bound (FN-) normal human ovarian epithelial (IHOSE) and ovarian cancer (SKOV-3) cells. The activation of CAPN-1/2 was measured by the luminescent method and the significance of the results was analyzed with the t-test. Results: CAPN-1/2 enzyme activity (at 12 hour) was present in both cell lines, but the level of enzyme is higher in IHOSE cells compared to SKOV-3 cells. The results showed that 1 hour TN and TN+Calp applications stimulated CAPN-1/2 enzyme activity in IHOSE cells but did not show any stimulating effect in SKOV-3 cells. After 12-hour of treatment, the cells with TN, Calp or TN+Calp showed an inhibitory effect on CAPN-1/2 enzyme in both FN+IHOSE and SKOV-3 cells. At 12-hour TN+Calp administration was determined to be the most effective inhibitor in FN+ SKOV-3 since it inhibited CAPN1/2 activity statistically significantly more than both Calp and TN administrations. Conclusions: The effects of TN, Calp and TN+Calp applications on the CAPN-1/2 enzyme varied according to the cell type, normal or cancer cells, and whether the cell was bound to FN and the incubation period. 12 h administrations of TN, Calp or TN+Calp inhibited the CAPN-1/2 enzyme in both FN+ IHOSE and SKOV-3 cells.

Identification and Biological Evaluation of a Water-Soluble Fullerene Nanomaterial as BTK Kinase Inhibitor.

Thanks to recent advances in synthetic methodology, water-soluble fullerene nanomaterials that interfere with biomolecules, especially DNA/RNA and selected proteins, have been found with tremendous potential for applications in nanomedicine. Herein, we describe the synthesis and evaluation of a water-soluble glycine-derived [60]fullerene hexakisadduct (HDGF) with T h symmetry, which is a first-in-class BTK protein inhibitor. We synthesized and characterized glycine derived [60]fullerene using NMR, ESI-MS, and ATR-FT-IR. DLS and zeta potential were measured and high-resolution transmission electron microscopy (HRTEM) observations were performed. The chemical composition of the water-soluble fullerene nanomaterial was examined by X-ray photoelectron spectrometry. To observe aggregate formation, the cryo-TEM analysis was carried out. The docking studies and molecular dynamic simulations were performed to determine interactions between HDGF and BTK. The in vitro cytotoxicity was evaluated on RAJI and K562 blood cancer cell lines. Subsequently, we examined the induction of cell death by autophagy and apoptosis by determining the expression levels of crucial genes and caspases. We investigated the direct association of HDGF on inhibition of the BTK signalling pathway by examining changes in the calcium levels in RAJI cells after treatment. The inhibitory potential of HDGF against non-receptor tyrosine kinases was evaluated. Finally, we assessed the effects of HDGF and ibrutinib on the expression of the BTK protein and downstream signal transduction in RAJI cells following anti-IgM stimulation. Computational studies revealed that the inhibitory activity of the obtained [60]fullerene derivative is multifaceted: it hampers the BTK active site, interacting directly with the catalytic residues, rendering it inaccessible to phosphorylation, and binds to residues that form the ATP binding pocket. The anticancer activity of produced carbon nanomaterial revealed that it inhibited the BTK protein and its downstream pathways, including PLC and Akt proteins, at the cellular level. The mechanistic studies suggested the formation of autophagosomes (increased gene expression of LC3 and p62) and two caspases (caspase-3 and -9) were responsible for the activation and progression of apoptosis. These data illustrate the potential of fullerene-based BTK protein inhibitors as nanotherapeutics for blood cancer and provide helpful information to support the future development of fullerene nanomaterials as a novel class of enzyme inhibitors.

Voltage to Calcium Transformation Enhances Direction Selectivity in Drosophila T4 Neurons.

An important step in neural information processing is the transformation of membrane voltage into calcium signals leading to transmitter release. However, the effect of voltage to calcium transformation on neural responses to different sensory stimuli is not well understood. Here, we use in vivo two-photon imaging of genetically encoded voltage and calcium indicators, ArcLight and GCaMP6f, respectively, to measure responses in direction-selective T4 neurons of female Drosophila Comparison between ArcLight and GCaMP6f signals reveals calcium signals to have a significantly higher direction selectivity compared with voltage signals. Using these recordings, we build a model which transforms T4 voltage responses into calcium responses. Using a cascade of thresholding, temporal filtering and a stationary nonlinearity, the model reproduces experimentally measured calcium responses across different visual stimuli. These findings provide a mechanistic underpinning of the voltage to calcium transformation and show how this processing step, in addition to synaptic mechanisms on the dendrites of T4 cells, enhances direction selectivity in the output signal of T4 neurons. Measuring the directional tuning of postsynaptic vertical system (VS)-cells with inputs from other cells blocked, we found that, indeed, it matches the one of the calcium signal in presynaptic T4 cells.SIGNIFICANCE STATEMENT The transformation of voltage to calcium influx is an important step in the signaling cascade within a nerve cell. While this process has been intensely studied in the context of transmitter release mechanism, its consequences for information transmission and neural computation are unclear. Here, we measured both membrane voltage and cytosolic calcium levels in direction-selective cells of Drosophila in response to a large set of visual stimuli. We found direction selectivity in the calcium signal to be significantly enhanced compared with membrane voltage through a nonlinear transformation of voltage to calcium. Our findings highlight the importance of an additional step in the signaling cascade for information processing within single nerve cells.

Activation of the human TRPA1 channel by different alkylating sulfur and nitrogen mustards and structurally related chemotherapeutic drugs

An important target in toxicology is the ion channel known as human transient receptor potential ankyrin 1 (hTRPA1). It is triggered by a variety of chemicals, including the alkylating chemical warfare agent sulfur mustard (SM). The activation potentials of structural analogs including O- and sesquimustard, nitrogen mustards (HN1, HN2, and HN3), and related chemotherapeutic drugs (bendamustine, cycylophosphamide, and ifosfamide) were examined in the current study. The aequorin assay was used to measure changes in intracellular calcium levels in human hTRPA1 overexpressing HEK293 cells. The XTT assay was used to determine cytotoxicity. The data presented here highlight that all investigated alkylating substances, with the exception of cyclophosphamide and ifosfamide, cause the activation of hTRPA1. Cytotoxicity and activation of hTRPA1 were found to be related. Compounds with high reactivity had higher cytotoxicity and vice versa. However, inhibiting hTRPA1 with the specific inhibitor AP18 could not reduce the cytotoxicity induced by alkylating agents. As a result, hTRPA1 does not play a significant role in the cytotoxicity of alkylating agents.

Antiviral activity of soybean GL 2626/96 (Glycine max) ethanolic extract against influenza A virus in vitro and in vivo

Influenza viruses cause respiratory infections in humans with high morbidity and mortality rates. Neuraminidase inhibitors such as oseltamivir and peramivir are the most commonly used drugs for influenza virus infections. However, the emergence of resistant viruses necessitates the urgent need to develop next-generation anti-influenza drugs. Soybean (Glycine max L. Merr.) is widely cultivated and used as food worldwide. In addition, soybean has long been used as a nutritional supplement and herbal medicine. However, the potential anti-influenza properties of the soybean cultivar “GL 2626/96″ (SG2626) are yet to be investigated. Herein, we determined whether the ethanolic extract of SG2626 (SG2626E) has anti-viral activity through performing SG2626E pre-, co-, and post-treatment assays, using the influenza green fluorescent protein (GFP)-tagged influenza A/PR/8/34 (A/PR/8/34-GFP) virus. SG2626E showed anti-influenza virus activity in pre- and co-treated cells in a dose-dependent manner, but not in post-treated cells. SG2626E imparted a considerable inhibitory effect on influenza A virus (IAV) infection through blocking viral attachment. SG2626E inhibited the activity of viral hemagglutinin, but not viral neuraminidase of the IAV. SG2626E inhibited IAV infection by reducing intracellular calcium levels in infected human lung epithelial A549 cells. Additionally, SG2626E reduced body weight loss, decreased mortality, and increased the survival rate through reducing viral replication in the lungs of IAV-infected mice. Overall, these results suggest that SG2626E inhibits IAV infection and is a potential novel anti-influenza agent.

Effects of mitochondrial reactive oxygen species-induced NLRP3 inflammasome activation on trichloroethylene-mediated kidney immune injury.

This study aimed to investigate the activating mechanism of the NLRP3 inflammasome in trichloroethylene-sensitized mice. In total, 88 BALB/c female mice were used to establish the trichloroethylene (TCE)-sensitized mouse model. Some of the mice received MitoTEMPO, MCC 950 or soluble recombinant CD59-Cys to inhibit mitochondrial reactive oxygen species (mtROS) production, NLRP3 assembly, or C5b-9 formation. Mouse tubular epithelial cell expression levels of NLRP3, ASC, Caspase 1, IL-1β, IL-18 and mitochondrial antiviral signaling protein (MAVS) were detected by western blot. Mitochondrial numbers, membrane potential (ΔΨm) and mtROS were detected by using MitoScene Green II, JC-1 dye and MitoSOX Red indicator, respectively. Tubular epithelial cell calcium levels were detected by a Fluo-8 no wash calcium assay kit. Human kidney-2 (HK-2) cells were cultured and stimulated by C5b6 and normal human serum (NHS) to verify the role of C5b-9-induced mitochondrial ROS in activating NLRP3 inflammasome. Urine α1-MG, β2-MG, and mtROS production and calcium levels were increased, while mitochondrial numbers were decreased in TCE-sensitized positive mice. After treatment with MitoTEMPO, renal tubular injury was alleviated, JC-1 fluorescence and mitochondrial numbers were significantly increased, and mitochondrial ROS were inhibited. The NLRP3 inflammasome was activated in TCE-sensitized positive mice, while Mito TEMPO inhibited MAVS expression and NLRP3 inflammasome activation. The in vitro studies proved that C5b-9 can induce mtROS release and activate the assembly of NLRP3 inflammasome in HK-2 cells. In conclusion, in TCE-sensitized positive mouse renal tubular epithelial cells, C5b-9 caused calcium influx and thus induced mitochondrial injury and mtROS overexpression, finally inducing MAVS expression and NLRP3 inflammasome activation and kidney injury.

Abstract 045: Soluble Protein Oligomers Induce Endoplasmic Reticulum Stress In Endothelial Cells And Lead To Decreased Calcium Levels In The Endoplasmic Reticulum.

Soluble protein oligomers (SPOs) have powerful vascular effects suggesting a link between Alzheimer’s disease (AD) and cardiovascular diseases (CVD). SPOs accumulation induces endoplasmic reticulum (ER) stress hence cell death in several CVDs. Our previous data showed that in mesenteric resistance arteries (MRA), SPOs induce ER stress leading to pathologically exacerbated endothelium-dependent vasodilation. We therefore hypothesized that the SPOs will cause ER calcium dysregulation in endothelial cells isolated from MRA. To confirm the presence of ER stress and SPO subcellular localization, endothelial cells were treated with SPOs (0.1 υM) in the presence and absence of the ER stress inhibitor 4-PBA (2 mM) and vehicle (F-12 media). Immunostaining with KDEL antibody specific for ER resident chaperones Grp78/94 was performed for 19 hr prior to visualization. Endothelial cells treated with SPOs showed increased anti-KDEL fluorescence (AU: Vehicle: 4.74 ± 0.3 vs. SPO: 15.74 ± 1.7; n= 3; p= 0.018, one-way ANOVA). This is consistent with the increased Grp78/94 protein concentration during ER stress. Interestingly, 4-PBA treated cells reduced anti-KDEL fluorescence to the same level as the control (AU: Vehicle: 4.74 ± 0.3 vs. 4-PBA: 8.74 ± 2.9; n= 3; p= 0.39; one-way ANOVA) suggesting that SPOs bind to the ER-anchored proteins during ER stress. Additionally, changes in Ca 2+ concentrations have been implicated as a mediator between ER stress and SPOs. Calcium levels in endothelial cells was investigated by transfecting a plasmid expressing a Ca 2+ indicator pCMV G-CEPIA1 er for 72 hr . Cells were treated with vehicle or SPOs in the presence or absence of 4-PBA (45 min) prior to fluorescence imaging. Cells treated with SPOs+ACh had a decreased fluorescence in the ER from the basal Ca 2+ levels (%Δ Ff-F0/F0; SPO: 53% reduction vs. vehicle), but treatment with 4-PBA+ACh did not improve this response (%Δ F f -F 0 /F 0 ; SPO+4-PBA: 48.18% reduction vs. vehicle n=2). This reveals that SPOs induce ER stress and depletion of ER calcium. However, ER stress inhibitor did not improve Ca 2+ levels. These data suggest that ER stress, is the mechanism by which SPO’s exacerbate vasodilation in the mesenteric resistance artery endothelial cells regardless of changes in Ca 2+ levels.

Cell-To-Cell Communication in the Resistance Vasculature.

The arterial vasculature can be divided into large conduit arteries, intermediate contractile arteries, resistance arteries, arterioles, and capillaries. Resistance arteries and arterioles primarily function to control systemic blood pressure. The resistance arteries are composed of a layer of endothelial cells oriented parallel to the direction of blood flow, which are separated by a matrix layer termed the internal elastic lamina from several layers of smooth muscle cells oriented perpendicular to the direction of blood flow. Cells within the vessel walls communicate in a homocellular and heterocellular fashion to govern luminal diameter, arterial resistance, and blood pressure. At rest, potassium currents govern the basal state of endothelial and smooth muscle cells. Multiple stimuli can elicit rises in intracellular calcium levels in either endothelial cells or smooth muscle cells, sourced from intracellular stores such as the endoplasmic reticulum or the extracellular space. In general, activation of endothelial cells results in the production of a vasodilatory signal, usually in the form of nitric oxide or endothelial-derived hyperpolarization. Conversely, activation of smooth muscle cells results in a vasoconstriction response through smooth muscle cell contraction. © 2022 American Physiological Society. Compr Physiol 12: 1-35, 2022.