Elevation Of Intracellular Ca2 Research Articles

Furamidine-induced autophagy exerts an anti-mycobacterial effect in a SIRT1-pAMPK-FOXO3a-dependent manner by elevation of intracellular Ca2+ level expression

Mycobacterium tuberculosis (M. tb), the etiological agent of tuberculosis (TB), continues to be a major contributor to global mortality rates. To effectively combat this pandemic, TB control has to be enhanced in several areas, including point-of-care diagnostics, shorter and safer drug regimens, and preventative vaccination. The latest findings have highlighted autophagy as a host-defense mechanism that eradicates many invading bacteria, including M. tb. Thus, novel approaches like the stimulation of autophagy using various pharmaceutical drugs can be undertaken to deal with this noxious pathogen. The present study has been formulated to evaluate the anti-mycobacterial potential of Furamidine, a DNA minor groove binder (MGB). Initially, a non-cytotoxic concentration of Furamidine (10 µM) was used to assess its impact on the intracellular persistence of mycobacteria in differentiated THP-1 (dTHP-1) cells. Furamidine treatment compromised intracellular mycobacterial growth compared to control cells. Autophagy, a well-known host-defensive strategy, was investigated as a possible contributor to revealing the mechanism of action. Multiparametric approaches such as LC3-I to II conversion, protein level expression of different autophagic markers, and MDC staining were employed to study autophagic response that conclusively suggested the autophagy induction potential of Furamidine in dTHP-1 cells. Further, elevated LC3-II expression and increased autophagic vacuole accumulation under Baf-A1 treatment demonstrated the positive regulation of autophagic flux upon Furamidine treatment. Mechanistic investigations showed increased intracellular calcium (Ca2+) level expression, SIRT1, pAMPK, and FOXO3a activation upon its treatment. Inhibition of Ca2+ level expression suppressed Ca2+-mediated-FOXO3a level in Furamidine-treated cells. Furthermore, administering various inhibitors hampered the Furamidine-induced autophagy that impacted intracellular mycobacteria clearance. These results conclude that Furamidine triggered the Ca2+/pAMPK/SIRT1/FOXO3a pathway, causing less mycobacterial load in dTHP-1 cells.

The Structure Analysis and mRNA Expression of CaV2 Gene Responding to Hypoxia Stress in Anadara granosa

The blood clam (Anadara granosa) is an economic bivalve that is relatively tolerant to hypoxia, but its molecular mechanism of hypoxia tolerance is unclear. We found that a significant decrease in extracellular Ca2+ concentration and a marked increase in intracellular Ca2+ concentration was observed in the blood clam through the fluorescence probe method, under hypoxic conditions at 0.5 mg/L. Concomitantly, there was a downward trend in the expression level of CaV2 mRNA, whereas NFAT (nuclear factor of activated T cells) expression increased by qRT-PCR. These findings suggest that the elevated intracellular Ca2+ concentration may activate negative transcription factors of NFAT, which subsequently suppresses the transcription of CaV2, leading to its decreased expression. Then, the NFAT RNA interference experiments supported this hypothesis. Sequence analysis and 3D structure prediction revealed conserved and mutated residue sites in blood clam compared to other bivalves. Hypoxia-induced changes in intracellular and extracellular Ca2+ concentrations, activating transcription factor NFAT and suppressing CaV2 expression. This study highlights the key roles of CaV2 and NFAT in hypoxia adaptation, paving the way for further exploration of hypoxia tolerance mechanisms in mollusca.

TGF-β1 Induces Endoplasmic Reticulum Stress-Dependent Apoptosis in Human Placental Mesenchymal Stem Cells of Fetal Origin through PERK Signaling Pathway

Background: Mesenchymal Stem Cells (MSCs) are pivotal in immunomodulation, hematopoiesis, and tissue repair. The interplay between MSCs and the pathological microenvironment influences their proliferation and differentiation. Transforming Growth Factor-Beta 1 (TGF- β1) serves as a key cytokine in the MSC microenvironment. This study aimed to scrutinize the impact of TGF-β1 on human placenta-derived MSCs of fetal origin (fPMSCs) and elucidate its underlying mechanism. Methods: fPMSCs were isolated, and surface markers were identified by flow cytometry. Cell proliferation in fPMSCs was assessed using Cell Counting Kit-8 (CCK-8) and 5-Ethynyl-2’-Deoxy Uridine (EdU). Apoptosis was detected via Annexin V/PI staining, and apoptosis-related proteins were detected by western blot. Endoplasmic reticulum (ER) stress-related proteins were detected by western blot, and Flou-4 AM staining was utilized to assess intracellular Ca2+ levels under TGF-β1 exposure. The impact of 4-PBA treatment on ER stress and apoptosis was assessed by western blot and Annexin V/PI staining. Additionally, the PERK and p-PERK expressions were evaluated via Western blot. Results: CCK-8 and EdU assays revealed inhibited proliferation of fPMSCs under TGF-β1 exposure. Annexin V/PI staining demonstrated a significant induction of apoptosis in fPMSCs following TGF-β1 treatment. Furthermore, TGF-β1 treatment significantly elevated intracellular Ca2+ levels and the expressions of GRP78, p-eIF2α, and CHOP. Interruption of ER stress with 4-PBA mitigated TGF-β1-induced apoptosis in fPMSCs. Moreover, TGF-β1 increased p-PERK expression. Inhibition of PERK autophosphorylation with GSK2606414 suppressed TGF-β1-induced apoptosis and ER stress in fPMSCs. Conclusion: Our findings indicated that TGF-β1 induced ER stress-dependent apoptosis in fPMSCs through the PERK signaling pathway. These results offer insights into enhancing the therapeutic efficacy of fPMSCs by modulating TGF-β1-induced apoptosis.

Endothelial TRPV4/Cx43 Signaling Complex Regulates Vasomotor Tone in Resistance Arteries.

S-nitrosylation of Cx43 gap junction channels critically regulates communication between smooth muscle cells and endothelial cells. This posttranslational modification also induces the opening of undocked Cx43 hemichannels. However, its specific impact on vasomotor regulation remains unclear. Considering the role of endothelial TRPV4 channel activation in promoting vasodilation through nitric oxide (NO) production, we investigated the direct modulation of endothelial Cx43 hemichannels by TRPV4 channel activation. Using the proximity ligation assay, we identify that Cx43 and TRPV4 are found in close proximity in the endothelium of resistance arteries. In primary endothelial cell cultures from resistance arteries (ECs), GSK-induced TRPV4 activation enhances eNOS activity, increases NO production, and opens Cx43 hemichannels via direct S-nitrosylation. Notably, the elevated intracellular Ca2+ levels caused by TRPV4 activation were reduced by blocking Cx43 hemichannels. In ex vivo mesenteric arteries, inhibiting Cx43 hemichannels reduced endothelial hyperpolarization without affecting NO production in ECs, underscoring a critical role of TRPV4/Cx43 signaling in endothelial electrical behavior. We perturbed the proximity of Cx43/TRPV4 by disrupting lipid rafts in ECs using β-cyclodextrin. Under these conditions, hemichannel activity, Ca2+ influx, and endothelial hyperpolarization were blunted upon GSK stimulation. Intravital microscopy of mesenteric arterioles in vivo further demonstrated that inhibiting Cx43 hemichannels activity, NO production and disrupting endothelial integrity reduce TRPV4-induced relaxation. These findings underscore a new pivotal role of Cx43 hemichannel associated with TRPV4 signaling pathway in modulating endothelial electrical behavior and vasomotor tone regulation.

A PACAP-activated network for secretion requires coordination of Ca2+ influx and Ca2+ mobilization.

Chromaffin cells of the adrenal medulla transduce sympathetic nerve activity into stress hormone secretion. The two neurotransmitters principally responsible for coupling cell stimulation to secretion are acetylcholine and pituitary adenylate activating polypeptide (PACAP). In contrast to acetylcholine, PACAP evokes a persistent secretory response from chromaffin cells. However, the mechanisms by which PACAP acts are poorly understood. Here, it is shown that PACAP induces sustained increases in cytosolic Ca2+ which are disrupted when Ca2+ influx through L-type channels is blocked or internal Ca2+ stores are depleted. PACAP liberates stored Ca2+ via inositol trisphosphate receptors (IP3Rs) on the endoplasmic reticulum (ER), thereby functionally coupling Ca2+ mobilization to Ca2+ influx and supporting Ca2+-induced Ca2+-release. These Ca2+ influx and mobilization pathways are unified by an absolute dependence on phospholipase C epsilon (PLCε) activity. Thus, the persistent secretory response that is a defining feature of PACAP activity, in situ, is regulated by a signaling network that promotes sustained elevations in intracellular Ca2+ through multiple pathways.

Piezo1 Activation Drives Enhanced Collagen Synthesis in Aged Animal Skin Induced by Poly L-Lactic Acid Fillers.

Poly L-lactic acid (PLLA) fillers stimulate collagen synthesis by activating various immune cells and fibroblasts. Piezo1, an ion channel, responds to mechanical stimuli, including changes in extracellular matrix stiffness, by mediating Ca2+ influx. Given that elevated intracellular Ca2+ levels trigger signaling pathways associated with fibroblast proliferation, Piezo1 is a pivotal regulator of collagen synthesis and tissue fibrosis. The aim of the present study was to investigate the impact of PLLA on dermal collagen synthesis by activating Piezo1 in both an H2O2-induced cellular senescence model in vitro and aged animal skin in vivo. PLLA elevated intracellular Ca2+ levels in senescent fibroblasts, which was attenuated by the Piezo1 inhibitor GsMTx4. Furthermore, PLLA treatment increased the expression of phosphorylated ERK1/2 to total ERK1/2 (pERK1/2/ERK1/2) and phosphorylated AKT to total AKT (pAKT/AKT), indicating enhanced pathway activation. This was accompanied by upregulation of cell cycle-regulating proteins (CDK4 and cyclin D1), promoting the proliferation of senescent fibroblasts. Additionally, PLLA promoted the expression of phosphorylated mTOR/S6K1/4EBP1, TGF-β, and Collagen I/III in senescent fibroblasts, with GsMTx4 treatment mitigating these effects. In aged skin, PLLA treatment similarly upregulated the expression of pERK1/2/ERK1/2, pAKT/AKT, CDK4, cyclin D1, mTOR/S6K1/4EBP1, TGF-β, and Collagen I/III. In summary, our findings suggest Piezo1's involvement in PLLA-induced collagen synthesis, mediated by heightened activation of cell proliferation signaling pathways such as pERK1/2/ERK1/2, pAKT/AKT, and phosphorylated mTOR/S6K1/4EBP1, underscoring the therapeutic potential of PLLA in tissue regeneration.

Disruption of P2Y2 signaling promotes breast tumor cell dissemination by reducing ATP-dependent calcium elevation and actin localization to cell junctions.

The tumor microenvironment and wound healing after injury both contain extremely high concentrations of the extracellular signaling molecule, adenosine triphosphate (ATP) compared to normal tissue. P2Y2 receptor, an ATP-activated purinergic receptor, is typically associated with pulmonary, endothelial, and neurological cell signaling. Here we report its role and importance in breast epithelial cell signaling and how it is altered in metastatic breast cancer. In response to ATP activation, P2Y2 receptor signaling causes an increase of intracellular Ca 2+ in non-tumorigenic breast epithelial cells, while their tumorigenic and metastatic counterparts have significantly reduced Ca 2+ responses. The non-tumorigenic cells respond to increased Ca 2+ with actin polymerization and localization to cell edges, while the metastatic cells remained unaffected. The increase in intracellular Ca 2+ after ATP stimulation was blunted using a P2Y2 antagonist, which also prevented actin mobilization and caused cell dissemination from spheroids in non-tumorigenic breast epithelial cells. Furthermore, the lack of Ca 2+ concentration changes and actin mobilization in the metastatic breast cancer cells could be due to reduced P2Y2 expression, which correlates with poorer overall survival in breast cancer patients. This study elucidates rapid changes that occur after elevated intracellular Ca 2+ in breast epithelial cells and how metastatic cancer cells have adapted to evade this cellular response.

#788 Mitigation of tubular apoptosis and cystogenesis in ADPKD: the impact of TRPM8 through restriction of mitochondrial function

Abstract Background and Aims Autosomal dominant polycystic kidney disease (ADPKD), characterized by numerous fluid-filled cysts and a progressive decline in renal function, is a prevalent hereditary kidney disorder. Approximately 70% of individuals affected by ADPKD may develop end-stage renal disease, typically manifesting around the median age of 58 years. Despite its prevalence, effective clinical treatments for ADPKD remain elusive. Transient receptor potential melastatin member 8 (TRPM8), serving as a sensor for cold temperatures and activated by the cooling agent menthol, orchestrates cellular Ca2+ signaling by facilitating Ca2+ entry into the cytosol. However, the role of TRPM8 in ADPKD remains undefined. Method The WT 9-12, an epithelial cell line isolated from the proximal tubule of a patient with ADPKD, was utilized for in vitro experiments. Eight-week-old wild-type (WT), Pkd1 knockdown (pkd1kd), and Trpm8, Pkd1 double mutant (T-/-P) mice were used for experiments. Renal Trpm8 was activated by administration of a 0.5% menthol diet for 7 months in mice. The cyst index was measured by H&E stain and calculated using Image J. Apoptosis was evaluated by TUNEL assay (In Situ Cell Death Detection Kit, TMR red). Protein and mRNA expression of mice kidneys was detected by Western blotting and qRT-PCR, respectively. Blood urea nitrogen (BUN) was measured by HITACHI 7600 analyzer. Results The activation of TRPM8 by menthol treatment resulted in an elevation of intracellular Ca2+ levels, subsequently impeding proliferation in WT9-12 cells. Consistently, Trpm8 activation suppressed kidney weight, kidney weight-to-body weight ratio, BUN, fibrosis, and cystogenesis in Pkd1kd mice, while no such effects were observed in WT and T-/-P mice. This suggests a protective role of Trpm8 in ADPKD. Mechanistically, Trpm8 activation mitigated tubular apoptosis in the kidneys of Pkd1kd mice, as evidenced by reduced TUNEL-positive cells and downregulated Bax and cleaved caspase-3, with no such effects observed in WT and T-/-P mice. Intriguingly, the proliferation-associated signaling pathway, ERK, was also suppressed by Trpm8. Proteomic analysis of mice kidneys revealed that Trpm8 regulates mitochondrial dysfunction, EIF2 and Sirtuin signaling pathways. The result was verified in WT9-12 cells showing reduced mitochondrial respiration, particularly impacting oxidative phosphorylation by TRPM8. Conclusion Trpm8 inhibits tubular apoptosis and cyst formation through the restriction of mitochondrial function, subsequently retarding ADPKD development in mice. These findings illuminate a novel and promising strategy for intervening in ADPKD.

Non-neuronal cell-derived acetylcholine, a key modulator of the vascular endothelial function in health and disease.

Vascular endothelial cells play an important role in regulating peripheral circulation by modulating arterial tone in the microvasculature. Elevated intracellular Ca2+ levels are required in endothelial cells to induce smooth muscle relaxation via endothelium-dependent mechanisms such as nitric oxide production, prostacyclin, and endothelial cell hyperpolarization. It is well established that exogenous administration of acetylcholine can increase intracellular Ca2+ concentrations, followed by endothelium-dependent vasodilation. Although endogenous acetylcholine's regulation of vascular tone remains debatable, recent studies have reported that endogenously derived acetylcholine, but not neuronal cell-derived acetylcholine, is a key modulator of endothelial cell function. In this minireview, we summarize the current knowledge of the non-neuronal cholinergic system (NNCS) in vascular function, particularly vascular endothelial cell function, which contributes to blood pressure regulation. We also discuss the possible pathophysiological impact of endothelial NNCS, which may induce the development of vascular diseases due to endothelial dysfunction, and the potential of endothelial NNCS as a novel therapeutic target for endothelial dysfunction in the early stages of metabolic syndrome, diabetes, and hypertension.

Algal Oil Mitigates Sodium Taurocholate-Induced Pancreatitis by Alleviating Calcium Overload, Oxidative Stress, and NF-κB Activation in Pancreatic Acinar Cells.

Acute pancreatitis (AP) is characterized by elevated intracellular Ca2+ concentrations, mitochondrial dysfunction, and oxidative stress in pancreatic acinar cells. Algal oil (AO) has demonstrated antioxidant and anti-inflammatory properties. This study aims to explore the effects of algal oil on the microenvironment of AP. Rat pancreatic acinar AR42J cells were pretreated with AO containing 0, 50, 100, or 150 μM of docosahexaenoic acid (DHA) 2 h prior to AP induction using sodium taurocholate (STC). After 1 h of STC treatment, AR42J cells exhibited a significant increase in intracellular Ca2+ concentration and the production of amylase, lipase, reactive oxygen species, and pro-inflammatory mediators, including tumor necrosis factor-α and interleukin-6. These STC-induced increases were markedly reduced in cells pretreated with AO. In comparison to cells without AO, those treated with a high dose of AO before STC exposure demonstrated a significant increase in mitochondrial membrane potential and a decrease in lipid peroxidation. Furthermore, STC-activated nuclear factor kappa-B (NF-κB) was attenuated in AO-pretreated cells, as evidenced by a significant decrease in activated NF-κB. In conclusion, AO may prevent damage to pancreatic acinar cells by alleviating intracellular Ca2+ overload, mitigating mitochondrial dysfunction, reducing oxidative stress, and attenuating NF-κB-targeted inflammation.

An Investigation of Size Distribution and Calcium Signaling in Human Platelets.

Background Platelets are thin disc-shaped blood cells that play a major role in hemostasis, maintenance of vascular integrity, and blood coagulation. Large platelets are more reactive and seen in patients with cardiovascular disease. This study aims to analyze the changes in platelet size of ex vivo activated platelets which phenotypically simulates that of a patient at risk of cardiovascular disease and elucidate the calcium signaling pathway responsible for this change. Methodology Platelets were isolated from adult human blood by differential centrifugation. Calcium was mobilized into platelets by treatment with calcium ionophore A23187 in the presence of Ca2+. Platelet size distribution was analyzed using Coulter Counter Multisizer 4. The following signaling parameters were studied: intracellular Ca2+ measurement (using Fura-2/AM by fluorescence spectrophotometry), Ca2+-dependent thiol protease calpain assay (using fluorogenic substrate t-butoxycarbonyl-Leu-metchloromethylcoumarin in fluorescence microplate reader), platelet-derived microparticles (using FACS Calibur flow cytometry), and cytoskeletal protein talin expression (by western immunoblotting). Results When adult platelets were treated with A23187 and Ca2+, two subcellular populations (<2 µm and between 2-4 µm) were noted. The mean size of the second cell population was significantly higher than that of resting platelets (2.94 ± 0.13 µm vs. 2.82 ± 0.15 µm, t = 4.605, p = 0.00). A23187 treatment led to elevated intracellular Ca2+, release of platelet-derived microparticles, increase in calpain activity, and cytoskeletal talin degradation. These events were inhibited by calpeptin (a specific calpain inhibitor). Conclusions Elevated calcium caused talin degradation by calpain activity. Breakdown of this cytoskeletal protein leads to relative swelling of cells reflected by the increase in platelet size.

The MAP kinase FvHog1 regulates FB1 synthesis and Ca2+ homeostasis in Fusarium verticillioides

The high osmolarity glycerol 1 mitogen-activated protein kinase (Hog1-MAPK) cascade genes are important for diverse biological processes. The activated Hog1 upon multiple environmental stress stimuli enters into the nucleus where it directly phosphorylates transcription factors to regulate various physiological processes in phytopathogenic fungi. However, their roles have not been well-characterized in Fusarium verticillioides. In this study, FvHog1 is identified and functionally analyzed. The findings reveal that the phosphorylation level and nuclear localization of FvHog1 are increased in Fumonisin B1 (FB1)-inducing condition to regulate the expression of FB1 biosynthesis FUM genes. More importantly, the deletion mutants of Hog1-MAPK pathway show increased sensitivity to Ca2+ stress and elevated intracellular Ca2+ content. The phosphorylation level and nuclear localization of FvHog1 are increased with Ca2+ treatment. Furthermore, our results show that FvHog1 can directly phosphorylate Ca2+-responsive zinc finger transcription factor 1 (FvCrz1) to regulate Ca2+ homeostasis. In conclusion, our findings indicate that FvHog1 is required for FB1 biosynthesis, pathogenicity and Ca2+ homeostasis in F. verticillioides. It provides a theoretical basis for effective prevention and control maize ear and stalk rot disease.

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.

TRPM5 activation depends on a synergistic effect of calcium and PKC phosphorylation

Transient receptor potential melastatin 5 (TRPM5) is a calcium-activated monovalent-specific ion channel involved in insulin secretion and taste transduction, making it an attractive target for drug development in various pathologies. While TRPM5 activation involves ligand binding to Gq/G-protein coupled receptors (GPCR) and subsequent elevation of intracellular calcium levels, recent reports suggest the need for additional molecular determinants. Hence, the mechanism of TRPM5 activation remains to be elucidated. Here, we show that PKC phosphorylation and the elevation of intracellular Ca2+ levels are required for TRPM5 activation, with PKC phosphorylation being crucial for channel-evoked currents, primarily at physiological membrane potentials. In contrast, physiological relevant calcium levels alone only induce TRPM5 activation at positive voltages. Our findings highlight the necessity of coordinated intracellular calcium release and PKC phosphorylation for TRPM5 activation. Thus, our results suggest that regulation of PKC activity could be a promising therapeutic target for diseases associated with TRPM5 modulation.

Abstract A044: P2Y2 signaling disruption reduces ATP-dependent calcium elevation and actin localization to promote breast tumor cell dissemination

Abstract The tumor microenvironment and wound healing after injury both contain extremely high concentrations of the extracellular signaling molecule, adenosine triphosphate (ATP) compared to normal tissue. P2Y2 receptor, an ATP-activated purinergic receptor, is typically associated with pulmonary, endothelial, and neural cell signaling. Here we report the importance of ATP-dependent P2Y2 signaling in breast epithelial cells and how it is altered in metastatic breast cancer. In response to ATP activation, P2Y2 receptor signaling causes an increase of intracellular calcium (Ca2+) in non-tumorigenic breast epithelial cells, while their tumorigenic and metastatic counterparts have significantly reduced Ca2+ responses. The non-tumorigenic cells respond to increased Ca2+ with actin polymerization and localization to cell edges, while the metastatic cells remained unaffected. The increase in intracellular Ca2+ after ATP stimulation was blunted using a P2Y2 antagonist, which also prevented actin mobilization and caused cell dissemination from spheroids in non-tumorigenic breast epithelial cells. Furthermore, the lack of Ca2+ concentration changes and actin mobilization in the metastatic breast cancer cells could be due to reduced P2Y2 expression, which correlates with poorer overall survival in breast cancer patients. This study elucidates rapid changes that occur after elevated intracellular Ca2+ in breast epithelial cells and how metastatic cancer cells have adapted to evade this cellular response. Citation Format: Makenzy Mull, Stephen Pratt, Keyata Thompson, David Annis, Julia Ju, Megan Stemberger, Liron Boyman, Michele Vitolo, W. Jonathan Lederer, Stuart Martin. P2Y2 signaling disruption reduces ATP-dependent calcium elevation and actin localization to promote breast tumor cell dissemination [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A044.

Titanium dioxide nanoparticles induce apoptosis through ROS-Ca2+-p38/AKT/mTOR pathway in TM4 cells.

Titanium dioxide nanoparticles (TiO2 NPs) can cause apoptosis in TM4 cells; however, the underlying mechanism has not been entirely elucidated. The purpose of this study was to investigate the effects of TiO2 NPs on ROS, Ca2+ level, p38/AKT/mTOR pathway, and apoptosis in TM4 cells and to evaluate the role of Ca2+ in p38/AKT/mTOR pathway and apoptosis. After exposure to different concentrations (0, 50, 100, 150, and 200 μg/mL) of TiO2 NPs for 24 h, cell viability, ROS, Ca2+ level, Ca2+-ATPase activity, p38/AKT/mTOR pathway-related proteins, apoptosis rate, and apoptosis-related proteins (Bax, Bcl-2, Caspase 3, Caspase 9, and p53) were detected. The ROS scavenger NAC was used to determine the effect of ROS on Ca2+ level. The Ca2+ chelator BAPTA-AM was used to evaluate the role of Ca2+ in p38/AKT/mTOR pathway and apoptosis. TiO2 NPs significantly inhibited cell viability, increased ROS level, and elevated Ca2+ level while suppressing Ca2+-ATPase activity. TiO2 NPs regulated the p38/AKT/mTOR pathway via increasing p-p38 level and decreasing p-AKT and p-mTOR levels. TiO2 NPs significantly enhanced the apoptosis. NAC attenuated Ca2+ overload and reduction in Ca2+-ATPase activity caused by TiO2 NPs. BAPTA-AM alleviated TiO2 NPs-induced abnormal expression of p38/AKT/mTOR pathway-related proteins. BAPTA-AM assuaged the apoptosis caused by TiO2 NPs. Altogether, this study revealed that TiO2 NPs elevated intracellular Ca2+ level through ROS accumulation. Subsequently, the heightened intracellular Ca2+ level was observed to exert regulation over the p38/AKT/mTOR pathway, ultimately culminating in apoptosis. These results provides a complementary understanding to the mechanism of TiO2 NPs-induced apoptosis in TM4 cells.

Microglia regulate sleep through calcium-dependent modulation of norepinephrine transmission.

Sleep interacts reciprocally with immune system activity, but its specific relationship with microglia-the resident immune cells in the brain-remains poorly understood. Here, we show in mice that microglia can regulate sleep through a mechanism involving Gi-coupled GPCRs, intracellular Ca2+ signaling and suppression of norepinephrine transmission. Chemogenetic activation of microglia Gi signaling strongly promoted sleep, whereas pharmacological blockade of Gi-coupled P2Y12 receptors decreased sleep. Two-photon imaging in the cortex showed that P2Y12-Gi activation elevated microglia intracellular Ca2+, and blockade of this Ca2+ elevation largely abolished the Gi-induced sleep increase. Microglia Ca2+ level also increased at natural wake-to-sleep transitions, caused partly by reduced norepinephrine levels. Furthermore, imaging of norepinephrine with its biosensor in the cortex showed that microglia P2Y12-Gi activation significantly reduced norepinephrine levels, partly by increasing the adenosine concentration. These findings indicate that microglia can regulate sleep through reciprocal interactions with norepinephrine transmission.

Novel Pathogenic Mechanism of Late-onset Alzheimer's Disease by Deficiency of NMDA Receptor Subunit GluN3A

Late-onset Alzheimer’s disease (AD) and related dementia (ADRD) are serious neurodegenerative disorders among aging populations. The progress of AD/ADRD cultivates over years to decades in humans and several months in animal models. Ca&lt;sup&gt;2+&lt;/sup&gt; homeostasis is a core function of neurons where the N-methyl-D-aspartate (NMDA) receptor plays a major role in excitatory neuronal activities. The Ca&lt;sup&gt;2+&lt;/sup&gt; hypothesis of AD proposes that even slight but sustained Ca&lt;sup&gt;2+&lt;/sup&gt; dyshomeostasis in the brain is a critical pathophysiology or pathogenesis of AD. However, instigating factors like the trigger and time/duration of the Ca&lt;sup&gt;2+&lt;/sup&gt; dysregulation in AD progression have been largely obscure, while β-amyloid (Aβ) peptides are often indicated as the trigger. Hyperactivities of excitatory neurons and NMDARs have been implicated in AD as a main mediating mechanism caused by AD pathologies such as Aβ deposition. NMDAR overactivation is restrained by the unique regulatory GluN3 subunits (GluN3A and GluN3B; previously known as NR3A and NR3B). Expression of GluN3A in the receptor complex reduces NMDAR currents and Ca&lt;sup&gt;2+&lt;/sup&gt; influx, while deletion of GluN3A causes larger NMDA currents and elevated intracellular Ca&lt;sup&gt;2+&lt;/sup&gt;. Significant GluN3A levels are detected in both rodent and human adult/aging brains. We hypothesized that the “gatekeeper” role of GluN3A is constantly required for Ca&lt;sup&gt;2+&lt;/sup&gt; homeostasis and normal aging; its deficiency can lead to slowly evolved “degenerative excitotoxicity”. Our in vitro, ex vivo, and in vivo studies using GluN3A knockout (KO) mice of young and older ages revealed neuronal hyperactivity, moderate but sustained elevation of cytosolic Ca&lt;sup&gt;2+&lt;/sup&gt;, chronic inflammation, neuronal loss/apoptosis, synaptic impairments and progressive cognitive deficits. The AD hallmarks of Aβ and tau pathology were identified after, but not before, cognition decline. In the “gain of function” experiment, expression of GluN3A in the GluN3A KO brain prevented AD progression. Specific regional knockdown of GluN3A in the cortex and hippocampus of wild-type mice at the adult age (3 months old) resulted in similar AD/ADRD phenotypic alterations in the following 3-6 months. The NMDAR antagonist memantine (MEM) is approved by FDA as a symptomatic treatment for moderate-severe AD patients. According to the chronic neurohyperactivity in AD progression and the modified Ca&lt;sup&gt;2+&lt;/sup&gt; hypothesis that Ca&lt;sup&gt;2+&lt;/sup&gt; dysregulation is an early and “life-long” pathogenesis, the maintenance of NMDAR normal activity by MEM or other safe NMDAR antagonists could be a disease-modifying early treatment in preclinical/prodromal stages. This prediction is endorsed by clinical trials using MEM and other NMDAR antagonists in mild cognitive impairment (MCI) and early AD patients, showing beneficial effects of maintaining cognitive functions. Consistently, we showed that, in GluN3A KO mice and 5xFAD mice, early and chronic MEM treatment (started at 3-month of age and lasted for 3-6 months) prevented or attenuated AD phenotypes. Meanwhile, the chronic MEM therapy in mice showed preconditioning effect of neuroprotection against ischemic stroke that strikes over 50% AD patients. Large clinical trials of long-term and more systematic examinations on AD/ADRD progression and the comorbidity of stroke are warranted for this innovative therapy. Our results support the modification of Ca&lt;sup&gt;2+&lt;/sup&gt; hypothesis of AD with the novel amyloid-independent mechanism. Specifically, the deficiency of GluN3A alone causes lifelong Ca&lt;sup&gt;2+&lt;/sup&gt;-related progression of AD pathophysiology and amyloid pathology. The long-term GluN3A modulation of NMDARs signifies new therapeutic targets and possible preventive interventions for late-onset AD/ ADRD.

Pulsed ultrasound promotes secretion of anti-inflammatory extracellular vesicles from skeletal myotubes via elevation of intracellular calcium level.

The regulation of inflammatory responses is an important intervention in biological function and macrophages play an essential role during inflammation. Skeletal muscle is the largest organ in the human body and releases various factors which mediate anti-inflammatory/immune modulatory effects. Recently, the roles of extracellular vesicles (EVs) from a large variety of cells are reported. In particular, EVs released from skeletal muscle are attracting attention due to their therapeutic effects on dysfunctional organs and tissues. Also, ultrasound (US) promotes release of EVs from skeletal muscle. In this study, we investigated the output parameters and mechanisms of US-induced EV release enhancement and the potential of US-treated skeletal muscle-derived EVs in the regulation of inflammatory responses in macrophages. High-intensity US (3.0 W/cm2) irradiation increased EV secretion from C2C12 murine muscle cells via elevating intracellular Ca2+ level without negative effects. Moreover, US-induced EVs suppressed expression levels of pro-inflammatory factors in macrophages. miRNA sequencing analysis revealed that miR-206-3p and miR-378a-3p were especially abundant in skeletal myotube-derived EVs. In this study we demonstrated that high-intensity US promotes the release of anti-inflammatory EVs from skeletal myotubes and exert anti-inflammatory effects on macrophages.

Effects of green light-emitting diode irradiation on hepatic differentiation of hepatocyte-like cells generated from human adipose-derived mesenchymal cells

Light-emitting diode (LED) irradiation has been used in the differentiation of mesenchymal stem cells into a variety of cell types. This study investigated the effect of green LED (GLED) irradiation on the differentiation of adipocyte-derived mesenchymal cells into hepatocyte-like cells (HLCs) and the mechanism of its action. HLCs in the hepatocyte maturation phase were irradiated with GLED (520 nm, 21 W/m2, 5 min/day for 10 days). The cells were then assessed for expression of hepatocyte maturity genes and opsin 3 (OPN3), hepatocyte function, viability, apoptosis, and levels of reactive oxygen species (ROS), intracellular adenosine triphosphate (ATP) and calcium ions (Ca2+). GLED irradiation increased Alpha-1 antitrypsin and Ornithine transcarbamylase gene expression, promoted Cytochrome P450 3A4 activity and urea synthesis, and elevated intracellular ROS, ATP and Ca2+ levels. OPN3 expression was significantly more upregulated in GLED-irradiated HLCs than in the non-irradiated HLCs. No significant difference in cell viability or apoptosis was observed between GLED-irradiated and non-irradiated HLCs. GLED irradiation can promote hepatocyte maturation and functions through OPN3. GLED irradiation also stimulated mitochondrial function via Ca2+/ATP/ROS activation. GLED irradiation has potential to support cell-based transplantation in patients.