Intracellular Calcium Chelator Research Articles

Gintonin Stimulates Glucose Uptake in Myocytes: Involvement of Calcium and Extracellular Signal-Regulated Kinase Signaling.

Ginseng has anti-hyperglycemic effects. Gintonin, a glycolipoprotein derived from ginseng, also stimulates insulin release from pancreatic beta cells. However, the role of gintonin in glucose metabolism within skeletal muscle is unknown. Here, we showed the effect of gintonin on glucose uptake, glycogen content, glucose transporter (GLUT) 4 expression, and adenosine triphosphate (ATP) content in C2C12 myotubes. Gintonin (3-30 μg/mL) dose-dependently stimulated glucose uptake in myotubes. The expression of GLUT4 on the cell membrane was increased by gintonin treatment. Treatment with 1-3 μg/mL of gintonin increased glycogen content in myotubes, but the content was decreased at 30 μg/mL of gintonin. The ATP content in myotubes increased following treatment with 10-100 μg/mL gintonin. Gintonin transiently elevated intracellular calcium concentrations and increased the phosphorylation of extracellular signal-regulated kinase (ERK). Gintonin-induced transient calcium increases were inhibited by treatment with the lysophosphatidic acid receptor inhibitor Ki16425, the phospholipase C inhibitor U73122, and the inositol 1,4,5-trisphosphate receptor antagonist 2-aminoethoxydiphenyl borate. Gintonin-stimulated glucose uptake was decreased by treatment with U73122, the intracellular calcium chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester, and the ERK inhibitor PD98059. These results show that gintonin plays a role in glucose metabolism by increasing glucose uptake through transient calcium increases and ERK signaling pathways. Thus, gintonin may be beneficial for glucose metabolism control.

The bidirectional role of GABAA and GABAB receptors during the differentiation process of neural precursor cells of the subventricular zone.

The intricate process of neuronal differentiation integrates multiple signals to induce transcriptional, morphological, and electrophysiological changes that reshape the properties of neural precursor cells during their maturation and migration process. An increasing number of neurotransmitters and biomolecules have been identified as molecular signals that trigger and guide this process. In this sense, taurine, a sulfur-containing, non-essential amino acid widely expressed in the mammal brain, modulates the neuronal differentiation process. In this study, we describe the effect of taurine acting via the ionotropic GABAA receptor and the metabotropic GABAB receptor on the neuronal differentiation and electrophysiological properties of precursor cells derived from the subventricular zone of the mouse brain. Taurine stimulates the number of neurites and favors the dendritic complexity of the neural precursor cells, accompanied by changes in the somatic input resistance and the strength of inward and outward membranal currents. At the pharmacological level, the blockade of GABAA receptors inhibits these effects, whereas the stimulation of GABAB receptors has no positive effects on the taurine-mediated differentiation process. Strikingly, the blockade of the GABAB receptor with CGP533737 stimulates neurite outgrowth, dendritic complexity, and membranal current kinetics of neural precursor cells. The effects of taurine on the differentiation process involve Ca2+ mobilization and the activation of intracellular signaling cascades since chelation of intracellular calcium with BAPTA-AM, and inhibition of the CaMKII, ERK1/2, and Src kinase inhibits the neurite outgrowth of neural precursor cells of the subventricular zone.

IP3R-Mediated Calcium Release Promotes Ferroptotic Death in SH-SY5Y Neuroblastoma Cells.

Ferroptosis is an iron-dependent cell death pathway that involves the depletion of intracellular glutathione (GSH) levels and iron-mediated lipid peroxidation. Ferroptosis is experimentally caused by the inhibition of the cystine/glutamate antiporter xCT, which depletes cells of GSH, or by inhibition of glutathione peroxidase 4 (GPx4), a key regulator of lipid peroxidation. The events that occur between GPx4 inhibition and the execution of ferroptotic cell death are currently a matter of active research. Previous work has shown that calcium release from the endoplasmic reticulum (ER) mediated by ryanodine receptor (RyR) channels contributes to ferroptosis-induced cell death in primary hippocampal neurons. Here, we used SH-SY5Y neuroblastoma cells, which do not express RyR channels, to test if calcium release mediated by the inositol 1,4,5-trisphosphate receptor (IP3R) channel plays a role in this process. We show that treatment with RAS Selective Lethal Compound 3 (RSL3), a GPx4 inhibitor, enhanced reactive oxygen species (ROS) generation, increased cytoplasmic and mitochondrial calcium levels, increased lipid peroxidation, and caused cell death. The RSL3-induced calcium signals were inhibited by Xestospongin B, a specific inhibitor of the ER-resident IP3R calcium channel, by decreasing IP3R levels with carbachol and by IP3R1 knockdown, which also prevented the changes in cell morphology toward roundness induced by RSL3. Intracellular calcium chelation by incubation with BAPTA-AM inhibited RSL3-induced calcium signals, which were not affected by extracellular calcium depletion. We propose that GPx4 inhibition activates IP3R-mediated calcium release in SH-SY5Y cells, leading to increased cytoplasmic and mitochondrial calcium levels, which, in turn, stimulate ROS production and induce lipid peroxidation and cell death in a noxious positive feedback cycle.

The Secretion of Inflammatory Cytokines Triggered by TLR2 Through Calcium‐Dependent and Calcium‐Independent Pathways in Keratinocytes

Keratinocytes can be activated by Cutibacterium acnes, leading to the production of proinflammatory cytokines via toll‐like receptors (TLRs) 2 and 4. Although several studies have investigated keratinocytes, the mechanism of calcium‐mediated activation remains unclear. Herein, we investigated whether calcium influx via TLR2 and TLR4 stimulation was involved in cytokine secretion by keratinocytes in HaCaT cells. Although TLR2 stimulation by peptidoglycan (PGN) increased intracellular calcium influx, TLR4 stimulation by lipopolysaccharide (LPS) did not increase it, as analyzed using flow cytometry with the calcium indicator Fluo‐3. However, activation by either TLR2 or TLR4 ligands upregulated the intracellular calcium influx in THP‐1 monocytes. Additionally, the expression of major proinflammatory cytokines and chemokines, such as interleukin (IL)‐6, IL‐8, IL‐1α, granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), and monocyte chemoattractant protein‐1 (MCP‐1), was significantly increased by TLR2 in HaCaT cells. Moreover, treatment with the intracellular calcium chelator, BAPTA‐AM, disrupted PGN‐mediated induction of IL‐6, IL‐8, and MCP‐1 production. Real‐time quantitative polymerase chain reaction (PCR) and western blotting revealed that TLR2 stimulation induced expression of the epidermal differentiation marker keratin 1. In conclusion, TLR2‐induced intracellular calcium influx plays a pivotal role in the secretion of proinflammatory cytokines, such as IL‐6 and MCP‐1, in keratinocytes. Moreover, the continuous influx of calcium via TLR2 activation leads to keratinization. In vitro studies using HaCaT cells provide basic research on the effect of TLR2‐induced calcium on C. acnes‐mediated inflammation in keratinocytes. These studies are limited in their ability to clinically predict what happens in human keratinocytes. Clinical studies on patients with acne, including three‐dimensional (3D) cultures of primary keratinocytes, are required to develop new diagnostic markers for determining the severity of acne vulgaris.

Arresting calcium-regulated sperm metabolic dynamics enables prolonged fertility in poultry liquid semen storage

The preservation of liquid semen is pivotal for both industrial livestock production and genetic management/conservation of species with sperm that are not highly cryo-tolerant. Nevertheless, with regard to poultry semen, even brief in vitro storage periods can lead to a notable decline in fertility, despite the in vivo capacity to maintain fertility for several weeks when within the hen’s sperm storage tubules. For fertility in sperm, intracellular calcium ions ([Ca2+]i) play a key role in signaling towards modifying energy metabolism. While reducing [Ca2+]i has been found to enhance the preservation of sperm fertility in some mammals, the connection between semen fertility and calcium availability in avian sperm has received limited attention. In this study, we demonstrate that the use of extracellular and intracellular calcium chelators in liquid semen extenders, specifically EGTA and EGTA-AM, has distinct effects on prolonging the fertility of chicken sperm. These results were validated through in vivo fertility tests. Mechanistically, the effects observed were linked to coordination of mitochondrial metabolism and ATP catabolism. Despite both calcium chelators inducing hypoxia, they differentially regulated mitochondrial respiration and ATP accumulation. This regulation was closely linked to a bimodal control of dynein ATPase activity; a direct initial activation with reduction in [Ca2+]i, and subsequent suppression by cytoplasmic acidification caused by lactic acid. These findings not only contribute to advancing poultry liquid semen preservation techniques, but also elucidates biologically relevant mechanisms that may underlie storage within the female reproductive tract in birds.

SARS-CoV-2 spike protein receptor-binding domain perturbates intracellular calcium homeostasis and impairs pulmonary vascular endothelial cells

Exposure to the spike protein or receptor-binding domain (S-RBD) of SARS-CoV-2 significantly influences endothelial cells and induces pulmonary vascular endotheliopathy. In this study, angiotensin-converting enzyme 2 humanized inbred (hACE2 Tg) mice and cultured pulmonary vascular endothelial cells were used to investigate how spike protein/S-RBD impacts pulmonary vascular endothelium. Results show that S-RBD leads to acute-to-prolonged induction of the intracellular free calcium concentration ([Ca2+]i) via acute activation of TRPV4, and prolonged upregulation of mechanosensitive channel Piezo1 and store-operated calcium channel (SOCC) key component Orai1 in cultured human pulmonary arterial endothelial cells (PAECs). In mechanism, S-RBD interacts with ACE2 to induce formation of clusters involving Orai1, Piezo1 and TRPC1, facilitate the channel activation of Piezo1 and SOCC, and lead to elevated apoptosis. These effects are blocked by Kobophenol A, which inhibits the binding between S-RBD and ACE2, or intracellular calcium chelator, BAPTA-AM. Blockade of Piezo1 and SOCC by GsMTx4 effectively protects the S-RBD-induced pulmonary microvascular endothelial damage in hACE2 Tg mice via normalizing the elevated [Ca2+]i. Comparing to prototypic strain, Omicron variants (BA.5.2 and XBB) of S-RBD induces significantly less severe cell apoptosis. Transcriptomic analysis indicates that prototypic S-RBD confers more severe acute impacts than Delta or Lambda S-RBD. In summary, this study provides compelling evidence that S-RBD could induce persistent pulmonary vascular endothelial damage by binding to ACE2 and triggering [Ca2+]i through upregulation of Piezo1 and Orai1. Targeted inhibition of ACE2-Piezo1/SOCC-[Ca2+]i axis proves a powerful strategy to treat S-RBD-induced pulmonary vascular diseases.

Secretion of Interleukin 6 in Human Skeletal Muscle Cultures Depends on Ca2+ Signalling.

The systemic effects of physical activity are mediated by the release of IL-6 and other myokines from contracting muscle. Although the release of IL-6 from muscle has been extensively studied, the information on the cellular mechanisms is fragmentary and scarce, especially regarding the role of Ca2+ signals. The aim of this study was to characterize the role of the main components of Ca2+ signals in human skeletal muscle cells during IL-6 secretion stimulated by the Ca2+ mobilizing agonist ATP. Primary cultures were prepared from surgical samples, fluorescence microscopy was used to evaluate the Ca2+ signals and the stimulated release of IL-6 into the medium was determined using ELISA. Intracellular calcium chelator Bapta, low extracellular calcium and the Ca2+ channels blocker La3+ reduced the ATP-stimulated, but not the basal secretion. Secretion was inhibited by blockers of L-type (nifedipine, verapamil), T-type (NNC55-0396) and Orai1 (Synta66) Ca2+ channels and by silencing Orai1 expression. The same effect was achieved with inhibitors of ryanodine receptors (ryanodine, dantrolene) and IP3 receptors (xestospongin C, 2-APB, caffeine). Inhibitors of calmodulin (calmidazolium) and calcineurin (FK506) also decreased secretion. IL-6 transcription in response to ATP was not affected by Bapta or by the T channel blocker. Our results prove that ATP-stimulated IL-6 secretion is mediated at the post-transcriptional level by Ca2+ signals, including the mobilization of calcium stores, the activation of store-operated Ca2+ entry, and the subsequent activation of voltage-operated Ca2+ channels and calmodulin/calcineurin pathways.

ERK MAPK signaling pathway inhibition as a potential target to prevent autophagy alterations in Spinal Muscular Atrophy motoneurons

Spinal Muscular Atrophy (SMA) is a severe genetic neuromuscular disorder that occurs in childhood and is caused by misexpression of the survival motor neuron (SMN) protein. SMN reduction induces spinal cord motoneuron (MN) degeneration, which leads to progressive muscular atrophy and weakness. The link between SMN deficiency and the molecular mechanisms altered in SMA cells remains unclear. Autophagy, deregulation of intracellular survival pathways and ERK hyperphosphorylation may contribute to SMN-reduced MNs collapse, offering a useful strategy to develop new therapies to prevent neurodegeneration in SMA. Using SMA MN in vitro models, the effect of pharmacological inhibition of PI3K/Akt and ERK MAPK pathways on SMN and autophagy markers modulation was studied by western blot analysis and RT-qPCR. Experiments involved primary cultures of mouse SMA spinal cord MNs and differentiated SMA human MNs derived from induced pluripotent stem cells (iPSCs). Inhibition of the PI3K/Akt and the ERK MAPK pathways reduced SMN protein and mRNA levels. Importantly, mTOR phosphorylation, p62, and LC3-II autophagy markers protein level were decreased after ERK MAPK pharmacological inhibition. Furthermore, the intracellular calcium chelator BAPTA prevented ERK hyperphosphorylation in SMA cells. Our results propose a link between intracellular calcium, signaling pathways, and autophagy in SMA MNs, suggesting that ERK hyperphosphorylation may contribute to autophagy deregulation in SMN-reduced MNs.

Abstract B021: Piezo1 and ROCK2 promote fast amoeboid migration in confined environments

Abstract Cell migration through confined environments may induce a phenotypic transition to fast amoeboid (leader bleb-based) migration. However, the molecular mechanism(s) controlling this phenotypic transition remain poorly understood. Here, we show that regulation of intracellular calcium levels by the plasma membrane tension sensor, Piezo1, promotes the Leader Bleb-Based Migration (LBBM) of melanoma cells. Using a ratiometric assay, intracellular calcium is shown to rise with increasing levels of confinement. Chelation of extracellular and intracellular calcium by BAPTA and BAPTA-AM, respectively, inhibits LBBM. Moreover, in highly motile cells, we found intracellular calcium levels to be dramatically increased at the cell rear. Using the Piezo1 inhibitor, GsMTx4, and RNAi, we can inhibit the phenotypic transition to fast amoeboid (leader bleb-based) migration. Therefore, we wondered if Piezo1 through calcium/calmodulin activates Myosin Light Chain Kinase (MLCK) to promote actomyosin contractility and amoeboid migration. Using a microchannel based assay, we find that ROCK2 and not MLCK, promotes amoeboid migration. Altogether, our work reveals an unanticipated collaboration between Piezo1 and ROCK2 in amoeboid migrating melanoma cells. Citation Format: Alexa Caruso, Neelakshi Kar, Jeremy Logue. Piezo1 and ROCK2 promote fast amoeboid migration in confined environments [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr B021.

Mechanism of Oxytocin-Induced Contraction in Rat Gastric Circular Smooth Muscle

Oxytocin produces an excitatory effect on gastric muscle through the activation of receptors present on stomach smooth muscle cells. However, the intracellular mechanisms that mediate oxytocin excitatory effects are still largely unknown. Therefore, we aimed to investigate the signaling pathways involved in oxytocin-induced contractions in gastric smooth muscle, shedding light on phospholipase C (PLC)-β1 signaling and its downstream molecules, including inositol 1,4,5- trisphosphate (IP3) and myosin light chain kinase (MLCK). The contractions of gastric smooth muscle from male rats were measured in an organ bath set up in response to exogenous oxytocin 10-7 M, in the presence and absence of inhibitors of the indicated signaling molecules. Oxytocin (10-9-10-5 M) induced dose-dependent stomach smooth muscle contraction. Pre-incubation with atosiban, an oxytocin receptor inhibitor, abolished the oxytocin-induced contraction. Moreover, PLC β1 inhibitor (U73122) and IP3 inhibitor Xestospongin C inhibited oxytocin-induced muscle contraction to various degrees. Verapamil, a calcium channel blocker, inhibited oxytocin-induced contraction, and pre-incubation of the strips, with both verapamil and Xestospongin C, further inhibited the excitatory effect of oxytocin. Chelation of intracellular calcium with BAPT-AM (1,2-bis-(o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid) significantly inhibited the effect of oxytocin on muscle contraction. Finally, pre-incubation of the strips with the Ca2+/calmodulin-dependent protein kinase selective inhibitor STO-609 significantly inhibited the contraction induced by oxytocin. These results suggest that oxytocin directly stimulates its cell surface receptor to activate PLC β1, which in turn liberates IP3, which eventually elevates intracellular calcium, the prerequisite for smooth muscle contraction.

1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic Acid Acetoxymethyl Ester Loaded Reactive Oxygen Species Responsive Hyaluronic Acid-Bilirubin Nanoparticles for Acute Kidney Injury Therapy via Alleviating Calcium Overload Mediated Endoplasmic Reticulum Stress.

Calcium overload is one of the early determinants of the core cellular events that contribute to the pathogenesis of acute kidney injury (AKI), which include oxidative stress, ATP depletion, calcium overload, and inflammatory response with self-amplifying and interactive feedback loops that ultimately lead to cellular injury and renal failure. Excluding adjuvant therapy, there are currently no approved pharmacotherapies for the treatment of AKI. Using an adipic dihydride linker, we modified the hyaluronic acid polymer chain with a potent antioxidant, bilirubin, to produce an amphiphilic conjugate. Subsequently, we developed a kidney-targeted and reactive oxygen species (ROS)-responsive drug delivery system based on the flash nanocomplexation method to deliver a well-known intracellular calcium chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM, BA), with the goal of rescuing renal cell damage via rapidly scavenging of intracellularly overloaded Ca2+. In the ischemia-reperfusion (I/R) induced AKI rat model, a single dose of as-prepared formulation (BA 100 μg·kg-1) 6 h post-reperfusion significantly reduced renal function indicators by more than 60% within 12 h, significantly alleviated tissular pathological changes, ameliorated tissular oxidative damage, significantly inhibited apoptosis of renal tubular cells and the expression of renal tubular marker kidney injury molecule 1, etc., thus greatly reducing the risk of kidney failure. Mechanistically, the treatment with BA-loaded NPs significantly inhibited the activation of the ER stress cascade response (IRE1-TRAF2-JNK, ATF4-CHOP, and ATF6 axis) and regulated the downstream apoptosis-related pathway while also reducing the inflammatory response. The BA-loaded NPs hold great promise as a potential therapy for I/R injury-related diseases.

Calcium is a noncompetitive inhibitor of DMT1 on the intestinal iron absorption process: empirical evidence and mathematical modeling analysis.

Iron absorption is a complex and highly controlled process where DMT1 transports nonheme iron through the brush-border membrane of enterocytes to the cytoplasm but does not transport alkaline-earth metals such as calcium. However, it has been proposed that high concentrations of calcium in the diet could reduce iron bioavailability. In this work, we investigate the effect of intracellular and extracellular calcium on iron uptake by Caco-2 cells, as determined by calcein fluorescence quenching. We found that extracellular calcium inhibits iron uptake by Caco-2 cells in a concentration-dependent manner. Chelation of intracellular calcium with BAPTA did not affect iron uptake, which indicates that the inhibitory effect of calcium is not exerted through intracellular calcium signaling. Kinetic studies performed, provided evidence that calcium acts as a reversible noncompetitive inhibitor of the iron transport activity of DMT1. Based on these experimental results, a mathematical model was developed that considers the dynamics of noncompetitive inhibition using a four-state mechanism to describe the inhibitory effect of calcium on the DMT1 iron transport process in intestinal cells. The model accurately predicts the calcein fluorescence quenching dynamics observed experimentally after an iron challenge. Therefore, the proposed model structure is capable of representing the inhibitory effect of extracellular calcium on DMT1-mediated iron entry into the cLIP of Caco-2 cells. Considering the range of calcium concentrations that can inhibit iron uptake, the possible inhibition of dietary calcium on intestinal iron uptake is discussed.

Enhanced recruitment of glutamate receptors underlies excitotoxicity of mitral cells in acute hyperammonemia.

Hepatic encephalopathy (HE)-a major complication of liver disease-has been found to increase the risk of olfactory dysfunction, which may be attributed to elevated levels of ammonia/ammonium in the blood and cerebrospinal fluid. However, the cellular mechanisms underlying hyperammonemia-induced olfactory dysfunction remain unclear. By performing patch-clamp recordings of mitral cells (MCs) in the mouse olfactory bulb (OB), we found that 3 mM ammonium (NH4 +) increased the spontaneous firing frequency and attenuated the amplitude, but synaptic blockers could prevent the changes, suggesting the important role of glutamate receptors in NH4 +-induced hyperexcitability of MCs. We also found NH4 + reduced the currents of voltage-gated K+ channel (Kv), which may lead to the attenuation of spontaneous firing amplitude by NH4 +. Further studies demonstrated NH4 + enhanced the amplitude and integral area of long-lasting spontaneous excitatory post-synaptic currents (sEPSCs) in acute OB slices. This enhancement of excitatory neurotransmission in MCs occurred independently of pre-synaptic glutamate release and re-uptake, and was prevented by the exocytosis inhibitor TAT-NSF700. In addition, an NH4 +-induced increasement in expression of NR1 and GluR1 was detected on cytoplasmic membrane, indicating that increased trafficking of glutamate receptors on membrane surface in MCs is the core mechanism. Moreover, NH4 +-induced enhanced activity of glutamate receptors in acute OB slices caused cell death, which was prevented by antagonizing glutamate receptors or chelating intracellular calcium levels. Our study demonstrates that the enhancement of the activity and recruitment of glutamate receptor directly induces neuronal excitotoxicity, and contributes to the vulnerability of OB to acute hyperammonemia, thus providing a potential pathological mechanism of olfactory defects in patients with hyperammonemia and HE.

TRPC6 mediates high glucose-induced mitochondrial fission through activation of CDK5 in cultured human podocytes.

Mitochondrial abnormalities contribute to the development of diabetic nephropathy (DN). However, the precise mechanisms of mitochondrial dysfunction in DN remain unclear. Transient receptor potential canonical channel-6 (TRPC6), a non-selective cation channel permeable to Ca2+, has been shown to regulate mitochondrial dynamics. This study was therefore aimed to explore the regulatory role and mechanisms of TRPC6 in high glucose (HG)-induced mitochondrial dysfunction in podocytes. Here we found that TRPC6 expression and TRPC6-induced Ca2+ influx were increased in HG-treated podocytes. Furthermore, the TRPC6 inhibitor and TRPC6 siRNA ameliorated mitochondrial dysfunction and apoptosis in HG-treated podocytes. BAPTA-AM, an intracellular calcium chelating agent, attenuated mitochondrial fission under HG conditions as well. Then, we found the activity of calpain and cyclin-dependent kinase 5 (CDK5) was markedly enhanced in HG-treated podocytes, which can be blocked by pretreatment with the TRPC6 inhibitor. Calpain-1 inhibition by calpeptin or by calpain-1 siRNA transfection not only attenuated HG-induced mitochondrial fission but also reduced the activity of CDK5. Additionally, the CDK5 inhibitor and its siRNA decreased mitochondrial fragmentation in HG-treated podocytes. Collectively, we revealed the essential role of TRPC6 in regulating HG-induced mitochondrial fission and apoptosis through the calpain-1/CDK5 pathway in human podocytes, which may provide new insights into the pathogenesis of DN.

Thrombin Induced Apoptosis through Calcium-Mediated Activation of Cytosolic Phospholipase A2 in Intestinal Myofibroblasts.

Thrombin is a serine protease that participates in a variety of biological signaling through protease-activated receptors. Intestinal myofibroblasts play central roles in maintaining intestinal homeostasis. In this study, we found that thrombin-induced apoptosis is mediated by the calcium-mediated activation of cytosolic phospholipase A2 in the CCD-18Co cell. Thrombin reduced cell viability by inducing apoptosis and proteinase-activated receptor-1 antagonist attenuated thrombin-induced cell death. Endogenous ceramide did not affect the cell viability itself, but a ceramide-mediated pathway was involved in thrombin-induced cell death. Thrombin increased intracellular calcium levels and cytosolic phospholipase A2 activity. The ceramide synthase inhibitor Fumonisin B1, intracellular calcium chelator BAPTA-AM, and cytosolic phospholipase A2 inhibitor AACOCF3 inhibited thrombin-induced cell death. Thrombin stimulated arachidonic acid release and reactive oxygen species generation, which was blocked by AACOCF3, BAPTA-AM, and the antioxidant reagent Trolox. Taken together, thrombin triggered apoptosis through calcium-mediated activation of cytosolic phospholipase A2 in intestinal myofibroblasts.

Capsaicin ameliorates intermittent high glucose-mediated endothelial senescence via the TRPV1/SIRT1 pathway

BackgroundPatients with diabetes have accelerated vascular aging when compared with healthy individuals. Hyperglycemia, especially intermittent high glucose (IHG), is the main cause of vascular endothelial senescence. Capsaicin, a major component of chili pepper is thought to contribute to cardiovascular protection by spicy food. ObjectiveTo investigate the pathway related with the effects of capsaicin on endothelial cell senescence induced by IHG. MethodsHUVECs were exposed to IHG (5 mM or 33 mM glucose, alternating every 12 hours for 3 days) and treated with capsaicin at 0.3, 1 and 3 μM. To determine endothelial cell senescence, we examined the senescence-related β-galactosidase staining, cell cycle arrest, cell viability, as well as production of reactive oxygen species (ROS). To evaluate the involvement of TRPV1/[Ca2+]i/CaMKII/AMPK/SIRT1 pathway in anti- senescence effects of capsaicin, HUVECs were treated with CAPZ (a TRPV1 antagonist), BAPTA-AM (an intracellular calcium chelator), KN62 (a CaMKII antagonist), compound C (an AMPK inhibitor), or EX527 (a SIRT1 inhibitor). To knockdown TRPV1, HUVECs were transfected with shRNA lentivirus targeting TRPV1. The levels of SIRT1, p21, TRPV1, AMPK and phospho-AMPK were evaluated by western blotting. ResultsIHG suppressed the levels of SIRT1 and enhanced endothelial senescence. Capsaicin upregulated SIRT1 expression and downregulated the senescence marker, p21, thereby protecting endothelial cells from IHG-induced senescence as indicated by relieved G0/G1 phase arrest, improved cell viabilities, and reduced counts of senescent cells and ROS production. Pre-treatment with CAPZ, BAPTA-AM, KN62 or compound C abrogated the anti-senescence effects of capsaicin. Capsaicin restored AMPK phosphorylation and IHG-inhibited TRPV1 expression. Moreover, TRPV1 silencing suppressed SIRT1 expression and abolished the anti-senescence effects of capsaicin. ConclusionCapsaicin elevates SIRT1 levels through TRPV1/[Ca2+]i/CaMKII/AMPK pathway and suppresses IHG-mediated endothelial cell senescence. This study provides initial evidence that capsaicin is a potential candidate for the prevention of vascular aging in diabetes.

A mitochondria cluster at the proximal axon initial segment controls axodendritic TAU trafficking in rodent primary and human iPSC-derived neurons

Loss of neuronal polarity and missorting of the axonal microtubule-associated-protein TAU are hallmarks of Alzheimer’s disease (AD) and related tauopathies. Impairment of mitochondrial function is causative for various mitochondriopathies, but the role of mitochondria in tauopathies and in axonal TAU-sorting is unclear. The axon-initial-segment (AIS) is vital for maintaining neuronal polarity, action potential generation, and—here important—TAU-sorting. Here, we investigate the role of mitochondria in the AIS for maintenance of TAU cellular polarity. Using not only global and local mitochondria impairment via inhibitors of the respiratory chain and a locally activatable protonophore/uncoupler, but also live-cell-imaging and photoconversion methods, we specifically tracked and selectively impaired mitochondria in the AIS in primary mouse and human iPSC-derived forebrain/cortical neurons, and assessed somatic presence of TAU. Global application of mitochondrial toxins efficiently induced tauopathy-like TAU-missorting, indicating involvement of mitochondria in TAU-polarity. Mitochondria show a biased distribution within the AIS, with a proximal cluster and relative absence in the central AIS. The mitochondria of this cluster are largely immobile and only sparsely participate in axonal mitochondria-trafficking. Locally constricted impairment of the AIS-mitochondria-cluster leads to detectable increases of somatic TAU, reminiscent of AD-like TAU-missorting. Mechanistically, mitochondrial impairment sufficient to induce TAU-missorting results in decreases of calcium oscillation but increases in baseline calcium, yet chelating intracellular calcium did not prevent mitochondrial impairment-induced TAU-missorting. Stabilizing microtubules via taxol prevented TAU-missorting, hinting towards a role for impaired microtubule dynamics in mitochondrial-dysfunction-induced TAU-missorting. We provide evidence that the mitochondrial distribution within the proximal axon is biased towards the proximal AIS and that proper function of this newly described mitochondrial cluster may be essential for the maintenance of TAU polarity. Mitochondrial impairment may be an upstream event in and therapeutic target for AD/tauopathy.

Prevention of glutamate excitotoxicity in lateral habenula alleviates ethanol withdrawal-induced somatic and behavioral effects in ethanol dependent mice

Ethanol withdrawal commonly leads to anxiety-related disorder, a central factor toward negative reinforcement leading to relapse. The lateral habenula (LHb), an epithalamic nucleus, has emerged to be critical for both reward and aversion processing. Recent studies have also implicated the hyperactivity of LHb, adding to the emergence of negative emotional states during withdrawal from addictive drugs. Herein, we have studied the effects of glutamate transporter inhibitor (PDC), GluN2B-containing NMDAR antagonist (Ro25-6981), and intracellular calcium chelator (BAPTA-AM) injection in LHb on ethanol withdrawal symptoms. We found that ethanol 4 g/kg 20 % w/v intragastric (i.g.) for 10 days followed by 24 h of withdrawal showed a significant increase in somatic signs characterized by vocalization, shaking, and scratching. It also increased locomotor activity and anxiety-like behavior, collectively showing expression of ethanol withdrawal symptoms. The intra-LHb administration of PDC (0.5 ng) worsened the effect of ethanol withdrawal, whereas Ro25-6981 (2 and 4 ng) and BAPTA-AM (6.5 and 13 ng) significantly reversed ethanol withdrawal-induced behavior evident by a decrease in somatic signs, locomotor activity, and anxiety-like behavior. Further, pretreatment of Ro25-6981 and BAPTA-AM reduced the neuronal loss, whereas PDC increased it compared to the vehicle-treated group, as evidenced by NeuN staining. Altogether, our results suggest that increased glutamate, GluN2B activation, and likely calcium increase indicative of glutamate excitotoxicity-induced neuronal loss in LHb possibly endorse the emergence of ethanol withdrawal symptoms, while their inhibition might help in alleviating the ethanol withdrawal symptoms.

Cortisol modulates calcium release-activated calcium channel gating in fish hepatocytes

Glucocorticoids (GCs) are rapidly released in response to stress and play an important role in the physiological adjustments to re-establish homeostasis. The mode of action of GCs for stress coping is mediated largely by the steroid binding to the glucocorticoid receptor (GR), a ligand-bound transcription factor, and modulating the expression of target genes. However, GCs also exert rapid actions that are independent of transcriptional regulation by modulating second messenger signaling. However, a membrane-specific protein that transduces rapid GCs signal is yet to be characterized. Here, using freshly isolated hepatocytes from rainbow trout (Oncorhynchus mykiss) and fura2 fluorescence microscopy, we report that stressed levels of cortisol rapidly stimulate the rise in cytosolic free calcium ([Ca2+]i). Pharmacological manipulations using specific extra- and intra-cellular calcium chelators, plasma membrane and endoplasmic reticulum channel blockers and receptors, indicated extracellular Ca2+ entry is required for the cortisol-mediated rise in ([Ca2+]i). Particularly, the calcium release-activated calcium (CRAC) channel gating appears to be a key target for the rapid action of cortisol in the ([Ca2+]i) rise in trout hepatocytes. To test this further, we carried out in silico molecular docking studies using the Drosophila CRAC channel modulator 1 (ORAI1) protein, the pore forming subunit of CRAC channel that is highly conserved. The result predicts a putative binding site on CRAC for cortisol to modulate channel gating, suggesting a direct, as well as an indirect regulation (by other membrane receptors) of CRAC channel gating by cortisol. Altogether, CRAC channel may be a novel cortisol-gated Ca2+ channel transducing rapid nongenomic signalling in hepatocytes during acute stress.

Effects of fetuin-A-containing calciprotein particles on posttranslational modifications of fetuin-A in HepG2 cells

Fetuin-A is an inhibitor of ectopic calcification that is expressed mainly in hepatocytes and is secreted into the circulation after posttranslational processing, including glycosylation and phosphorylation. The molecular weight (MW) of fully modified fetuin-A (FM-fetuin-A) is approximately 60 kDa in an immunoblot, which is much higher than the estimated MW by amino acid sequence. Under conditions of calcification stress such as advanced stage chronic kidney disease, fetuin-A prevents calcification by forming colloidal complexes, which are referred to as calciprotein particles (CPP). Since the significance of CPP in this process is unclear, we investigated the effect of synthetic secondary CPP on the level of FM-fetuin-A in HepG2 cells. Secondary CPP increased the level of FM-fetuin-A in dose- and time-dependent manners, but did not affect expression of mRNA for fetuin-A. Treatment with O- and/or N-glycosidase caused a shift of the 60 kDa band of FM-fetuin-A to a lower MW. Preincubation with brefeldin A, an inhibitor of transport of newly synthesized proteins from the endoplasmic reticulum to the Golgi apparatus, completely blocked the secondary CPP-induced increase in FM-fetuin-A. Treatment with BAPTA-AM, an intracellular calcium chelating agent, also inhibited the CPP-induced increase in the FM-fetuin-A level. Secondary CPP accelerate posttranslational processing of fetuin-A in HepG2 cells.