Increase In Intracellular Calcium Levels Research Articles

Connexin 43 regulates pyroptosis by influencing intracellular calcium levels in X-ray induced vascular endothelial cell damage.

Our prior research has established that X-ray exposure induces pyroptosis in human umbilical vein endothelial cells (HUVECs), with Cx43 playing a regulatory role in this process. However, the precise mechanism by which Cx43 regulates pyroptosis remains unclear. The objective of this study is to assess the involvement of the calcium signaling pathway in Cx43-mediated regulation of X-ray-induced pyroptosis in HUVECs. HUVECs were exposed to 10 Gy X-ray radiation either alone or combined with Cx43 overexpression or knockdown. Calcium ions (Ca2+) were stained using Fluo-4/AM and analyzed via flow cytometry and confocal microscopy. Pyroptosis was assessed through flow cytometry by staining with FLICA (fluorescent-labeled inhibitor of caspase) and propidium iodide (PI). Calcium signaling was inhibited using BAPTA/AM, 2-APB, or nifedipine. Protein expression levels were assessed by western blotting. X-ray irradiation induced an increase in intracellular calcium levels in HUVECs in a dose- and time-dependent manner. The results demonstrated that regulating calcium release with BAPTA/AM, 2-APB, or nifedipine significantly reduced pyroptosis. Also, the overexpression of Cx43 significantly attenuated the increase in intracellular calcium. Conversely, Cx43 knockdown via siRNA significantly increased the intracellular calcium levels. Also, interfering with calcium signaling using BAPTA/AM, 2-APB, or nifedipine reduced the raised pyroptosis levels induced by Cx43 knockdown. Individual HUVECs exposed to high-dose X-ray irradiation exhibited an increase in intracellular calcium, leading to pyroptosis. Also, upregulating Cx43 expression reduced the pyroptosis levels by inhibiting intracellular calcium concentration. This study introduces new concepts for identifying targets for the prophylaxis and therapy of radiation-induced damage.

Ethanol Extract of Ampelopsis brevipedunculata Rhizomes Suppresses IgE-Mediated Mast Cell Activation and Anaphylaxis.

More than 20% of the world's population suffers from allergic diseases, including allergic asthma, rhinitis, and atopic dermatitis that severely reduce the patient's quality of life. The treatment of allergy has been developed, but there are still unmet needs. Ampelopsis brevipedunculata (Maxim.) Trautv. is a traditional medicinal herb with beneficial bioactivities, such as antioxidant, anti-hypertension, anti-viral, anti-mutagenic, and skin and liver (anti-hepatotoxic) protective actions. However, its anti-allergic effect has not been addressed. This study designed to investigate the pharmacological effect of an ethanol extract of A. brevipedunculata rhizomes (ABE) on mast cell and anaphylaxis models. For in vivo studies, we used ovalbumin-induced active systemic anaphylaxis (ASA) and immunoglobulin (Ig) E-mediated passive cutaneous anaphylaxis (PCA) models. In ASA model, oral administration of ABE (1, 10, and 100 mg/kg) attenuated the anaphylactic responses, such as hypothermia, serum histamine, and IgE productions. In PCA model, ABE also suppressed the plasma extravasation and swelling. The underlying mechanisms of action were identified in various mast cell types. In vitro, ABE (10, 30, and 60 µg/mL) inhibited the release of essential allergic mediators, such as histamine and β-hexosaminidase, in a concentration-dependent manner. ABE prevented the rapid increase in intracellular calcium levels induced by the DNP-HSA challenge. In addition, ABE downregulated the tumor necrosis factor-α and interleukin-4 by suppressing the activation of nuclear factor-κB. Collectively, this study is the first to identify the anti-allergic effect of ABE, suggesting that ABE is a promising candidate for treating allergic diseases.

The role of calcium in neuronal membrane tension and synaptic plasticity.

Calcium is a primary second messenger that plays a role in cellular functions including growth, movement and responses to drugs. The role that calcium plays in mediating communication between neurons by synaptic vesicle release is well established. This review focuses on the dependence of the physical properties of neuronal plasma membranes on calcium levels. After describing the key features of synaptic plasticity, we summarize the general role of calcium in cell function and the signaling pathways responsible for intracellular increase in calcium levels. We then present findings showing that increases in intracellular calcium levels cause neurites to contract and break synaptic connections by changes in membrane tension.

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.

A developmentally controlled cellular decompartmentalization process executes programmed cell death in the Arabidopsis root cap.

Programmed cell death (PCD) is a fundamental cellular process crucial to development, homeostasis, and immunity in multicellular eukaryotes. In contrast to our knowledge on the regulation of diverse animal cell death subroutines, information on execution of PCD in plants remains fragmentary. Here, we make use of the accessibility of the Arabidopsis (Arabidopsis thaliana) root cap to visualize the execution process of developmentally controlled PCD. We identify a succession of selective decompartmentalization events and ion fluxes as part of the terminal differentiation program that is orchestrated by the NO APICAL MERISTEM, ARABIDOPSIS THALIANA ACTIVATING FACTOR, CUP-SHAPED COTYLEDON (NAC) transcription factor SOMBRERO. Surprisingly, the breakdown of the large central vacuole is a relatively late and variable event, preceded by an increase of intracellular calcium levels and acidification, release of mitochondrial matrix proteins, leakage of nuclear and endoplasmic reticulum lumina, and release of fluorescent membrane reporters into the cytosol. In analogy to animal apoptosis, the plasma membrane remains impermeable for proteins during and after PCD execution. Elevated intracellular calcium levels and acidification are sufficient to trigger cell death execution specifically in terminally differentiated root cap cells, suggesting that these ion fluxes act as PCD-triggering signals. This detailed information on the cellular processes occurring during developmental PCD in plants is a pivotal prerequisite for future research into the molecular mechanisms of cell death execution.

Method for cryopreservation of trigeminal ganglion for establishing primary cultures of neurons and glia

BackgroundPrimary neuronal cultures are used to elucidate cellular and molecular mechanisms involved in disease pathology and modulation by pharmaceuticals and nutraceuticals, and to identify novel therapeutic targets. However, preparation of primary neuronal cultures from rodent embryos is labor-intensive, and it can be difficult to produce high-quality consistent cultures. To overcome these issues, cryopreservation can be used to obtain standardized, high-quality stocks of neuronal cultures. New methodIn this study, we present a simplified cryopreservation method for rodent primary trigeminal ganglion neurons and glia from Sprague-Dawley neonates, using a 90:10 (v/v) fetal bovine serum/dimethyl sulfoxide cell freezing medium. ResultsCryopreserved trigeminal ganglion cells stored for up to one year in liquid nitrogen exhibited similar neuronal and glial cell morphology to fresh cultures and retained high cell viability. Proteins implicated in inflammation and pain signaling were expressed in agreement with the reported subcellular localization. Additionally, both neurons and glial cells exhibited an increase in intracellular calcium levels in response to a depolarizing stimulus. Cryopreserved cells were also transiently transfected with reporter genes. Comparison with existing methodsOur method is simple, does not require special reagents or equipment, will save time and money, increase flexibility in study design, and produce consistent cultures. ConclusionsThis method for the preparation and cryopreservation of trigeminal ganglia results in primary cultures of neurons and glia similar in viability and morphology to fresh preparations that could be utilized for biochemical, cellular, and molecular studies, increase reproducibility, and save laboratory resources.

Isolation and electrophysiological recording of Ixodes ricinus synganglion neurons

The central nervous system of hard ticks (Ixodidae) consists of a concentrated merged nerve mass known as the synganglion. Although knowledge of tick neurobiology has dramatically improved over the last two decades, this is the first time that isolation and electrophysiological recordings have been carried out on tick neurons from the synganglion. Method: We developed a simple protocol for synganglion neuron isolation and used a whole-cell patch clamp to measure ionic currents induced by acetylcholine, nicotine and muscarine. Relatively large neurons (∼ 25 μm and ∼ 35 μm) were isolated and 1 mM acetylcholine was used to induce strong inward currents of −0.38 ± 0.1 nA and − 1.04 ± 0.1 nA, respectively, with the corresponding cell capacitances being at around 142 pF and 188 pF. In addition, successive application of 1 mM acetylcholine through ∼25 μm and ∼ 35 μm cells for increasing amounts of time resulted in a rapid reduction in current amplitudes. We also found that acetylcholine-evoked currents were associated with a reversible increase in intracellular calcium levels for each neuronal type. In contrast, 1 mM muscarine and nicotine induced a strong and non-reversible increase in intracellular calcium levels. This study serves as a proof of concept for the mechanical isolation of tick synganglion neurons followed by their electrophysiological recording. This approach will aid investigations into the pharmacological properties of tick neurons and provides the tools needed for the identification of drug-targeted sites and effective tick control measures.

Butylparaben promotes phosphatidylserine exposure and procoagulant activity of human red blood cells via increase of intracellular calcium levels

Parabens are widely used as preservatives, added to products commonly used by humans, and to which individuals are exposed orally or dermally. Once absorbed into the body, parabens move into the bloodstream and travel through the systemic circulation. We investigated the potential impact of parabens on the enhanced generation of thrombin by red blood cells (RBCs), which are the principal cellular components of blood. We tested the effects of methylparaben (MeP), ethylparaben (EtP), propylparaben (PrP), butylparaben (BuP), and p-hydroxybenzoic acid on freshly isolated human RBCs. BuP and simultaneous exposure to BuP and PrP significantly increased phosphatidylserine (PS) externalization to the outer membranes of RBCs. PS externalization by BuP was found to be mediated by increasing intracellular Ca2+ levels in RBCs. The morphological changes in BuP-treated RBCs were observed under an electron microscope. The BuP-exposed RBCs showed increased thrombin generation and adhesion to endothelial cells. Additionally, the externalization of PS exposure and thrombin generation in BuP-treated RBCs were more susceptible to high shear stress, which mimics blood turbulence under pathological conditions. Collectively, we observed that BuP induced morphological and functional changes in RBCs, especially under high shear stress, suggesting that BuP may contribute to the thrombotic risk via procoagulant activity in RBCs.

Riluzole treatment modulates KCC2 and EAAT-2 receptor expression and Ca2+ accumulation following ventral root avulsion injury

Avulsion injury results in motoneuron death due to the increased excitotoxicity developing in the affected spinal segments. This study focused on possible short and long term molecular and receptor expression alterations which are thought to be linked to the excitotoxic events in the ventral horn with or without the anti-excitotoxic riluzole treatment. In our experimental model the left lumbar 4 and 5 (L4, 5) ventral roots of the spinal cord were avulsed. Treated animals received riluzole for 2 weeks. Riluzole is a compound that acts to block voltage-activated Na+ and Ca2+ channels. In control animals the L4, 5 ventral roots were avulsed without riluzole treatment. Expression of astrocytic EAAT-2 and that of KCC2 in motoneurons on the affected side of the L4 spinal segment were detected after the injury by confocal and dSTORM imaging, intracellular Ca2+ levels in motoneurons were quantified by electron microscopy. The KCC2 labeling in the lateral and ventrolateral parts of the L4 ventral horn was weaker compared with the medial part of L4 ventral horn in both groups. Riluzole treatment dramatically enhanced motoneuron survival but was not able to prevent the down-regulation of KCC2 expression in injured motoneurons. In contrast, riluzole successfully obviated the increase of intracellular calcium level and the decrease of EAAT-2 expression in astrocytes compared with untreated injured animals. We conclude that KCC2 may not be an essential component for survival of injured motoneurons and riluzole is able to modulate the intracellular level of calcium and expression of EAAT-2.

Hexane fraction of Prunus japonica thunb. Seed extract enhances boar sperm motility via CatSper ion channel

IntroductionMammalian sperm motility is facilitated by flagellar beating, which depends on active ion movement through ion channels and their regulation. Prunus japonica Thunb., also known as oriental bush cherry, is a widely used traditional medicinal plant. However, its significance in improving fertility and sperm quality has not been fully elucidated yet. One of our previous reports revealed that P. japonica seed extract (PJE) can improve human sperm motility through intracellular pH modulation. Aim of the studyThe present study was designed to investigate the effects of PJE on boar spermatozoa and potential underlying mechanisms. Materials and methodsSperm motility changes were examined using a computer-assisted sperm analysis (CASA) system under both capacitated and non-capacitated conditions. Intracellular calcium concentration was measured using either confocal microscopy or a fluorescent microplate reader with Fluo-4AM calcium fluorescent dye. Sperm capacitation-related proteins were analyzed using western blotting. ResultsA significant increase in rapid motility, velocity, and linear displacement of sperm was observed in PJE-treated capacitated boar sperm, whereas the effect was insignificant in the non-capacitated counterparts. Intracellular calcium levels were significantly elevated upon PJE treatment (20–100 μg/L) in a concentration-dependent manner. The increase in intracellular calcium levels was inhibited when the sperm were treated with a CatSper (cation channel of sperm) channel inhibitor, 10 μM Mibefradil, indicating the involvement of the ion channel in the PJE modulatory mechanism. In addition, western blotting revealed an increased level of protein phosphorylation (p-tyrosine and p-PKA), which is a hallmark of sperm capacitation. ConclusionsPJE treatment resulted in a combination of increased motility, intracellular calcium concentration, and capacitation, thereby indicating its potential to ameliorate sperm motility parameters and induce capacitation of boar spermatozoa as a result of intracellular calcium elevation via the CatSper channel. Our observations further elaborate ion channel-related underlying mechanisms and show putative implications of the seed extract of traditionally used P. japonica Thunb. in ameliorating sperm quality.

Myo1b Promotes Premature Endothelial Senescence and Dysfunction via Suppressing Autophagy: Implications for Vascular Aging.

Endothelial cell (EC) senescence characterized by an irreversible growth arrest leading to endothelial dysfunction has been implicated in vascular aging and aging-associated cardiovascular diseases. Autophagy plays a crucial role in the modulation of cellular senescence. Our previous showed that myosin 1b (Myo1b), one family of nonfilamentous class-1 myosin, was reported to be involved in the modulation of human smooth muscle cell senescence. However, the role of Myo1b in the modulation of EC senescence with links to autophagy has yet to be elucidated. In this study, we sought to explore the role of Myo1b in endothelial senescence and further elucidate the underlying mechanisms. Here, we show prominent upregulation of Myo1b in senescent ECs in comparison with nonsenescence ECs in both mRNA and protein expression levels. Silencing Myo1b in senescent cells ameliorates endothelial dysfunctions and reverses endothelial senescence phenotypic changes such as senescence-associated-β-galactosidase activity, cyclin-dependent kinase inhibitor p21WAF1, expression of vascular adhesion molecule-1 (VCAM1) and intercellular adhesion molecule-1 (ICAM1), and the senescence-associated cytokines. In contrast, in nonsenescent cells, overexpressing Myo1b promotes endothelial senescence and suppresses autophagy through the impairment of autophagosome and lysosome fusion. The interaction between Myo1b and LRRK2 through Myo1b tail domain promotes intracellular calcium elevation, which results in the inhibition of autophagic flux. In vitro and in vivo aging models, Myo1b knockdown in senescent ECs and wild type-aged mice is able to enhance autophagy and ameliorate aging-associated endothelial dysfunction. Taken together, our studies reveal a new function for Myo1b, that is, to couple LRRK2 assembly to promote an increase in intracellular calcium level, which impairs the autophagosome-lysosome fusion, and ultimately the promotion of EC senescence and vascular aging.

The Weakened Interaction Between HECTD4 and GluN2B in Ischemic Stroke Promotes Calcium Overload and Brain Injury Through a Mechanism Involving the Decrease of GluN2B and MALT1 Ubiquitination.

Glutamate receptor ionotropic NMDA 2B (GluN2B) plays an essential role in calcium overload during excitotoxicity. Reverse-phase nano-liquid chromatography-tandem mass spectrometry has revealed an interaction between GluN2B and HECT domain E3 ubiquitin protein ligase 4 (HECTD4), an E3 ubiquitin ligase highly expressed in the brain. As a potential substrate for HECTD4, mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) acts as a scaffold with hydrolysis activity. This study explores the relationship between HECTD4, GluN2B, and MALT1, focusing on their role in brain injury in ischemic stroke. Rats were subjected to 2h-ischemia followed by 24-h reperfusion to establish an ischemic stroke model. We observed the downregulation of HECTD4 and the upregulation of MALT1. Additionally, an increased GluN2B phosphorylation was concomitant with weakened interactions between HECTD4 and GluN2B, followed by decreased striatal-enriched protein phosphatase (STEP61). Knockdown of HECTD4 exacerbated hypoxia- or NMDA-induced injury in nerve cells coincident with a decrease in GluN2B and MALT1 ubiquitination, and an increase in GluN2B phosphorylation as well as an increase in intracellular calcium level, which were counteracted by MALT1 siRNA. Blockage of MALT1 with its inhibitor or siRNA reduced STEP61 degradation, accompanied by a decrease in GluN2B phosphorylation, intracellular calcium concentration, and brain cell injury, which were reversed by overexpression of MALT1. Based on these observations, we conclude that the downregulation of HECTD4 in ischemic stroke rat brain accounts for calcium overload and brain injury due to activating GluN2B directly and indirectly through a mechanism involving the reduced ubiquitination of GluN2B and MALT1, respectively.

Design, synthesis and biological evaluation of a new series of arylidene indanones as small molecules for targeted therapy of non-small cell lung carcinoma and prostate cancer

In an attempt to identify small molecules for targeted therapy of non-small cell lung carcinoma (NSCLC) and prostate cancer (PCa), new arylidene indanones (1–10) were synthesized via the Claisen-Schmidt condensation of 5,6-methylenedioxy-1-indanone with p-substituted benzaldehyde. Compounds 1–10 were assessed for their cytotoxic effects on human lung adenocarcinoma (A549) and human pancreatic ductal carcinoma (PANC-1) cells as well as human normal lung fibroblast (CCD-19Lu) and human normal pancreatic ductal epithelial (hTERT-HPNE) cells. Among them, compounds 2, 4 and 10 were more effective on A549 and PANC-1 cells than cisplatin. Compounds 1 and 9 also showed more potent cytotoxic activity towards PANC-1 cells than cisplatin. In vitro assays were performed to assess their effects on DNA synthesis, apoptosis, caspase-3, mitochondrial membrane potential, intracellular calcium levels, morphological changes in cancer cells. Furthermore, all compounds were investigated for their inhibitory effects on cathepsin L (CatL) and cathepsin D (CatD). Compounds 2 and 4 exerted potent anti-NSCLC action through caspase-independent apoptosis induced by an increase in intracellular calcium level and correspondingly the disruption of the ΔΨm. These compounds also caused apoptotic morphological alterations in A549 cells. Compound 4 also inhibited both cathepsins but its inhibitory potency on CatL was more significant. Based on in vitro mechanistic assays, compound 4 was identified as a promising anticancer agent for targeted therapy of NSCLC. On the other hand, the marked anti- PCa activity of compound 1 mediated by apoptotic cell death is also noteworthy, but further enzymatic assays are required to elucidate its main mechanism of action.

Transcriptional Genetically Encoded Calcium Indicators in Drosophila.

Knowing which neurons are active during behavior is a crucial step toward understanding how nervous systems work. Neuronal activation is generally accompanied by an increase in intracellular calcium levels. Therefore, intracellular calcium levels are widely used as a proxy for neuronal activity. Many types of synthetic components and bioluminescent or fluorescent proteins that report transient and long-term changes in intracellular calcium levels have been developed over the past 60 years. Calcium indicators that enable imaging of the dynamic activity of a large ensemble of neurons in behaving animals have revolutionized the field of neuroscience. Among these, transcription-based genetically encoded calcium indicators (transcriptional GECIs) have proven easy to use and do not depend on sophisticated imaging systems, offering unique advantages over other types of calcium indicators. Here, we describe the two currently available fly transcriptional GECIs-calcium-dependent nuclear import of LexA (CaLexA) and transcriptional reporter of intracellular calcium (TRIC)-and review studies that have used them. In the accompanying protocol, we present step-by-step details for generating CaLexA- and TRIC-ready flies and for imaging CaLexA and TRIC signals in dissected brains after experimental manipulations of intact free-moving flies.

GB83, an Agonist of PAR2 with a Unique Mechanism of Action Distinct from Trypsin and PAR2-AP.

Protease-activated receptor 2 (PAR2) is a G-protein-coupled receptor (GPCR) activated by proteolytic cleavage of its N-terminal domain. Once activated, PAR2 is rapidly desensitized and internalized by phosphorylation and β-arrestin recruitment. Due to its irreversible activation mechanism, some agonists that rapidly desensitized PAR2 have been misconceived as antagonists, and this has impeded a better understanding of the pathophysiological role of PAR2. In the present study, we found that GB83, initially identified as a PAR2 antagonist, is a bona fide agonist of PAR2 that induces unique cellular signaling, distinct from trypsin and PAR2-activating peptide (AP). Activation of PAR2 by GB83 markedly elicited an increase in intracellular calcium levels and phosphorylation of MAPKs, but in a delayed and sustained manner compared to the rapid and transient signals induced by trypsin and PAR2-AP. Interestingly, unlike PAR2-AP, GB83 and trypsin induced sustained receptor endocytosis and PAR2 colocalization with β-arrestin. Moreover, the recovery of the localization and function of PAR2 was significantly delayed after stimulation by GB83, which may be the reason why GB83 is recognized as an antagonist of PAR2. Our results revealed that GB83 is a bona fide agonist of PAR2 that uniquely modulates PAR2-mediated cellular signaling and is a useful pharmacological tool for studying the pathophysiological role of PAR2.

Hyperoxia evokes pericyte-mediated capillary constriction

Oxygen supplementation is regularly prescribed to patients to treat or prevent hypoxia. However, excess oxygenation can lead to reduced cerebral blood flow (CBF) in healthy subjects and worsen the neurological outcome of critically ill patients. Most studies on the vascular effects of hyperoxia focus on arteries but there is no research on the effects on cerebral capillary pericytes, which are major regulators of CBF. Here, we used bright-field imaging of cerebral capillaries and modeling of CBF to show that hyperoxia (95% superfused O2) led to an increase in intracellular calcium level in pericytes and a significant capillary constriction, sufficient to cause an estimated 25% decrease in CBF. Although hyperoxia is reported to cause vascular smooth muscle cell contraction via generation of reactive oxygen species (ROS), endothelin-1 and 20-HETE, we found that increased cytosolic and mitochondrial ROS levels and endothelin release were not involved in the pericyte-mediated capillary constriction. However, a 20-HETE synthesis blocker greatly reduced the hyperoxia-evoked capillary constriction. Our findings establish pericytes as regulators of CBF in hyperoxia and 20-HETE synthesis as an oxygen sensor in CBF regulation. The results also provide a mechanism by which clinically administered oxygen can lead to a worse neurological outcome.

3-oxo-C12:2-HSL, quorum sensing molecule from human intestinal microbiota, inhibits pro-inflammatory pathways in immune cells via bitter taste receptors.

In the gut ecosystem, microorganisms regulate group behaviour and interplay with the host via a molecular system called quorum sensing (QS). The QS molecule 3-oxo-C12:2-HSL, first identified in human gut microbiota, exerts anti-inflammatory effects and could play a role in inflammatory bowel diseases where dysbiosis has been described. Our aim was to identify which signalling pathways are involved in this effect. We observed that 3-oxo-C12:2-HSL decreases expression of pro-inflammatory cytokines such as Interleukine-1β (− 35%) and Tumor Necrosis Factor-α (TNFα) (− 40%) by stimulated immune RAW264.7 cells and decreased TNF secretion by stimulated PBMC in a dose-dependent manner, between 25 to 100 µM. Transcriptomic analysis of RAW264.7 cells exposed to 3-oxo-C12:2-HSL, in a pro-inflammatory context, highlighted JAK-STAT, NF-κB and TFN signalling pathways and we confirmed that 3-oxo-C12:2-HSL inhibited JAK1 and STAT1 phosphorylation. We also showed through a screening assay that 3-oxo-C12:2-HSL interacted with several human bitter taste receptors. Its anti-inflammatory effect involved TAS2R38 as shown by pharmacologic inhibition and led to an increase in intracellular calcium levels. We thus unravelled the involvement of several cellular pathways in the anti-inflammatory effects exerted by the QS molecule 3-oxo-C12:2-HSL.

Short-term mechanisms activated by NGF to induce pulmonary arterial hyperreactivity

We have previously shown a pathophysiological role for NGF in pulmonary hypertension, particularly in pulmonary arterial (PA) hyperreactivity. Our experiments have suggested different mechanisms activated by NGF depending on a short- or a long-term PA treatment. The aim of the present study was to determine the mechanisms activated by a short-term NGF treatment (1 h) to induce PA hyperreactivity. Contractions of rat PA were induced ex vivo by endothelin-1 (ET-1, 10-12-10-6 M) in the absence or presence of NGF (100 ng/mL, 1 h), with or without K252a (TrkA kinase inhibitor, 300 nM) or Y–27632 (ROCK inhibitor, 10 μM). NGF–induced phosphorylation of myosin phosphatase target subunit 1 (MYPT1) was evaluated in vitro by Western blotting in primary PA smooth muscle cells (PASMC) from both rat and human origin. Basal and ET–1–induced (10-7 M) calcium responses were evaluated in vitro in PASMC using calcium imaging (Fura2, Fluo-4), in the absence or presence of NGF (100 ng/mL, 1 h). Calcium sources involved in NGF-dependent increase in calcium levels and response to ET-1 were determined using Thapsigargin and Cyclopiazonic acid (SERCA inhibitors, 1 or 10 μM, respectively) or verapamil and Nicardipine (calcium L–Type channels blocker, 2 μM). Ex vivo, NGF (1 h) induced rat PA hyperreactivity to ET-1 in a TrkA- and ROCK-dependent manner. In vitro, in both rat and human PASMC, NGF (1 h) enhanced basal calcium levels through activation of calcium L-type channels. NGF also enhanced ET–1–induced increase in intracellular calcium levels, with endoplasmic reticulum (ER) being the main calcium source contributing to these effects. Finally, in both rat and human cells, NGF also induced phosphorylation of MYPT1 in a TrkA- and ROCK-dependent manner. In conclusion, our results show that NGF induces PA hyperreactivity to ET-1 through activation of its TrkA receptor, leading to both increased calcium intracellular levels and increased ER calcium loading. NGF also activates ROCK-dependent calcium sensitization mechanisms. Such mechanisms may therefore contribute to NGF-induced PA hyperreactivity in pulmonary hypertension.

ID:15967 Differential Target Multiplexed SCS Programming Modulates Calcium-Mediated Signaling in an Animal Model of Neuropathic Pain

Calcium ion is an important messenger in chronic neuropathic pain. Increase of intracellular calcium levels in neurons and glial cells promotes inflammatory states leading to hypersensitization.1 Our group demonstrated that multiplexing electrical signals to differentially target glial and neuron cells is more effective than conventional SCS treatments at modulating gene expression in neuroinflammatory processes.2 This work provides an insight into the modulatory effect of this differential target multiplexed programming (DT) approach on proteins and phosphoproteins associated with calcium signaling in inflammation.

VI-116, A Novel Potent Inhibitor of VRAC with Minimal Effect on ANO1.

Volume-regulated anion channel (VRAC) is ubiquitously expressed and plays a pivotal role in vertebrate cell volume regulation. A heterologous complex of leucine-rich repeat containing 8A (LRRC8A) and LRRC8B-E constitutes the VRAC, which is involved in various processes such as cell proliferation, migration, differentiation, intercellular communication, and apoptosis. However, the lack of a potent and selective inhibitor of VRAC limits VRAC-related physiological and pathophysiological studies, and most previous VRAC inhibitors strongly blocked the calcium-activated chloride channel, anoctamin 1 (ANO1). In the present study, we performed a cell-based screening for the identification of potent and selective VRAC inhibitors. Screening of 55,000 drug-like small-molecules and subsequent chemical modification revealed 3,3′-((2-hydroxy-3-methoxyphenyl)methylene)bis(4-hydroxy-2H-chromen-2-one) (VI-116), a novel potent inhibitor of VRAC. VI-116 fully inhibited VRAC-mediated I− quenching with an IC50 of 1.27 ± 0.18 μM in LN215 cells and potently blocked endogenous VRAC activity in PC3, HT29 and HeLa cells in a dose-dependent manner. Notably, VI-116 had no effect on intracellular calcium signaling up to 10 μM, which completely inhibited VRAC, and showed high selectivity for VRAC compared to ANO1 and ANO2. However, DCPIB, a VRAC inhibitor, significantly affected ATP-induced increases in intracellular calcium levels and Eact-induced ANO1 activation. In addition, VI-116 showed minimal effect on hERG K+ channel activity up to 10 μM. These results indicate that VI-116 is a potent and selective VRAC inhibitor and a useful research tool for pharmacological dissection of VRAC.