scholarly journals Superoxide Flux in Endothelial Cells via the Chloride Channel-3 Mediates Intracellular Signaling

2007 ◽  
Vol 18 (6) ◽  
pp. 2002-2012 ◽  
Author(s):  
Brian J. Hawkins ◽  
Muniswamy Madesh ◽  
C. J. Kirkpatrick ◽  
Aron B. Fisher
Keyword(s):  
Endothelial Cells ◽  
Plasma Membrane ◽  
Chloride Channel ◽  
Intracellular Signaling ◽  
Anion Channel ◽  
Transmembrane Flux ◽  
Cell Plasma ◽  
Intracellular Ca ◽  
Cellular Apoptosis ◽  
Extracellular Side

Reactive oxygen species (ROS) have been implicated in both cell signaling and pathology. A major source of ROS in endothelial cells is NADPH oxidase, which generates superoxide (O2.−) on the extracellular side of the plasma membrane but can result in intracellular signaling. To study possible transmembrane flux of O2.−, pulmonary microvascular endothelial cells were preloaded with the O2.−-sensitive fluorophore hydroethidine (HE). Application of an extracellular bolus of O2.−resulted in rapid and concentration-dependent transient HE oxidation that was followed by a progressive and nonreversible increase in nuclear HE fluorescence. These fluorescence changes were inhibited by superoxide dismutase (SOD), the anion channel blocker DIDS, and selective silencing of the chloride channel-3 (ClC-3) by treatment with siRNA. Extracellular O2.−triggered Ca2+release in turn triggered mitochondrial membrane potential alterations that were followed by mitochondrial O2.−production and cellular apoptosis. These “signaling” effects of O2.−were prevented by DIDS treatment, by depletion of intracellular Ca2+stores with thapsigargin and by chelation of intracellular Ca2+. This study demonstrates that O2.−flux across the endothelial cell plasma membrane occurs through ClC-3 channels and induces intracellular Ca2+release, which activates mitochondrial O2.−generation.

FEATURES OF CALCIUM HOMEOSTASIS AND CALCIUM SIGNALING IN NEURONS

Vestnik ◽  
2021 ◽  
pp. 208-214
Author(s):  
Б.К. Кайрат ◽  
С.Т. Тулеуханов ◽  
В.П. Зинченко
Keyword(s):  
Plasma Membrane ◽  
Calcium Signaling ◽  
Calcium Binding ◽  
Intracellular Signaling ◽  
Cell Damage ◽  
Compensatory Mechanisms ◽  
Physiological Processes ◽  
Intracellular Ca ◽  
Ca Ions ◽  
Ca Homeostasis

Ионы Са являются основным мессенджером в регуляции физиологических функций клеток. Внутриклеточном пространстве ионы Ca могут свободно состоянии диффундироваться в различных частях цитоплазмы, в то же время значительное количество Ca в связанном виде накапливается в различных внутриклеточных депо или в составе кальций-связывающих белков. Регуляция физиологических процессов с ионами внутриклеточного Са происходит в диапазоне концентраций 10 М, тогда как концентрация Са во внеклеточном пространстве выше и составляет 10 М, для поддержании градиента концентраций в клетках имеются важные Са транспортирующие системы плазматической мембраны, эндоплазматического ретикулума и митохондрий. В нейронах функционируют внутриклеточные ферменты и белки плазматической мембраны для поддержания Са-гомеостаза и реализации механизмов внутриклеточной сигнализации для обеспечения жизнедеятельности в выживании клеток. Нарушение или гиперактивация одного или нескольких механизмов кальциевой сигнализации может привести к повреждению и гибели нейронов в случае отсутствия компенсаторных механизмов. Ca ions are a key messenger for the regulation of most of the physiological functions of cells. Inside the cell, Ca ions can freely diffuse in various parts of the cytoplasm, but a significant amount of Ca is also bound in various intracellular depots or in the form of calcium-binding proteins. The regulation of physiological processes by intracellular Ca ions occurs in the concentration range of 10 M, and the concentration of Ca in the extracellular space is higher and is 10 M, and to maintain this concentration gradient, cells have Ca-transporting systems of the plasma membrane, endoplasmic reticulum and mitochondria. In neurons, a large number of intracellular enzymes and plasma membrane proteins function to maintain Ca-homeostasis and implement intracellular signaling mechanisms to ensure vital activity in the survival of cells. Violation or hyperactivation of one or more mechanisms of calcium signaling can lead to cell damage and death in the absence of compensatory mechanisms.

Oxidative burst and NO generation as initial response to ischemia in flow-adapted endothelial cells

2001 ◽  
Vol 280 (5) ◽  
pp. H2126-H2135 ◽  
Author(s):  
Yefim Manevich ◽  
Abu Al-Mehdi ◽  
Vladimir Muzykantov ◽  
Aron B. Fisher
Keyword(s):  
Endothelial Cells ◽  
Plasma Membrane ◽  
Oxidative Burst ◽  
Initial Response ◽  
Membrane Depolarization ◽  
No Synthase ◽  
Potential Production ◽  
Perfusion Chamber ◽  
Intracellular Ca ◽  
Static Conditions

Shear stress modulates endothelial physiology, yet the effect(s) of flow cessation is poorly understood. The initial metabolic responses of flow-adapted bovine pulmonary artery endothelial cells to the abrupt cessation of flow (simulated ischemia) was evaluated using a perfusion chamber designed for continuous spectroscopy. Plasma membrane potential, production of reactive O2 species (ROS), and intracellular Ca2+ and nitric oxide (NO) levels were measured with fluorescent probes. Within 15 s after flow cessation, flow-adapted cells, but not cells cultured under static conditions, showed plasma membrane depolarization and an oxidative burst with generation of ROS that was inhibited by diphenyleneiodonium. EGTA-inhibitable elevation of intracellular Ca2+ and NO were observed at ∼30 and 60 s after flow cessation, respectively. NO generation was decreased in the presence of inhibitors of NO synthase and calmodulin. Thus flow-adapted endothelial cells sense the altered hemodynamics associated with flow cessation and respond by plasma membrane depolarization, activation of NADPH oxidase, Ca2+ influx, and activation of Ca2+/calmodulin-dependent NO synthase. This signaling response is unrelated to cellular anoxia.

A mathematical model of plasma membrane electrophysiology and calcium dynamics in vascular endothelial cells

2007 ◽  
Vol 293 (1) ◽  
pp. C277-C293 ◽  
Author(s):  
Haroldo S. Silva ◽  
Adam Kapela ◽  
Nikolaos M. Tsoukias
Keyword(s):  
Mathematical Model ◽  
Endothelial Cells ◽  
Plasma Membrane ◽  
Vascular Tone ◽  
Vascular Endothelial Cells ◽  
Anion Channel ◽  
Ionic Species ◽  
Vascular Endothelial ◽  
Health And Disease ◽  
Vascular Autoregulation

Vascular endothelial cells (ECs) modulate smooth muscle cell (SMC) contractility, assisting in vascular tone regulation. Cytosolic Ca2+ concentration ([Ca2+]i) and membrane potential ( Vm) play important roles in this process by controlling EC-dependent vasoactive signals and intercellular communication. The present mathematical model integrates plasmalemma electrophysiology and Ca2+ dynamics to investigate EC responses to different stimuli and the controversial relationship between [Ca2+]i and Vm. The model contains descriptions for the intracellular balance of major ionic species and the release of Ca2+ from intracellular stores. It also expands previous formulations by including more detailed transmembrane current descriptions. The model reproduces Vm responses to volume-regulated anion channel (VRAC) blockers and extracellular K+ concentration ([K+]o) challenges, predicting 1) that Vm changes upon VRAC blockade are [K+]o dependent and 2) a biphasic response of Vm to increasing [K+]o. Simulations of agonist-induced Ca2+ mobilization replicate experiments under control and Vm hyperpolarization blockade conditions. They show that peak [Ca2+]i is governed by store Ca2+ release while Ca2+ influx (and consequently Vm) impacts more the resting and plateau [Ca2+]i. The Vm sensitivity of rest and plateau [Ca2+]i is dictated by a [Ca2+]i “buffering” system capable of masking the Vm-dependent transmembrane Ca2+ influx. The model predicts plasma membrane Ca2+-ATPase and Ca2+ permeability as main players in this process. The heterogeneous Vm impact on [Ca2+]i may elucidate conflicting reports on how Vm influences EC Ca2+. The present study forms the basis for the development of multicellular EC-SMC models that can assist in understanding vascular autoregulation in health and disease.

Intracellular Ca2+signaling in endothelial cells by the angiogenesis inhibitors endostatin and angiostatin

2001 ◽  
Vol 280 (5) ◽  
pp. C1140-C1150 ◽  
Author(s):  
Lianwei Jiang ◽  
Vivekanand Jha ◽  
Mohanraj Dhanabal ◽  
Vikas P. Sukhatme ◽  
Seth L. Alper
Keyword(s):  
Endothelial Cells ◽  
Growth Factor ◽  
Intracellular Signaling ◽  
Angiogenesis Inhibitors ◽  
Vascular Endothelial ◽  
Entry Inhibitors ◽  
Acute Elevation ◽  
Intracellular Ca ◽  
Nih 3T3 ◽  
Endothelial Growth Factor

Intracellular signaling mechanisms by the angiogenesis inhibitors endostatin and angiostatin remain poorly understood. We have found that endostatin (2 μg/ml) and angiostatin (5 μg/ml) elicited transient, approximately threefold increases in intracellular Ca2+concentration ([Ca2+]i). Acute exposure to angiostatin or endostatin nearly abolished subsequent endothelial [Ca2+]iresponses to carbachol or to thapsigargin; conversely, thapsigargin attenuated the Ca2+signal elicited by endostatin. The phospholipase C inhibitor U-73122 and the inositol trisphosphate (IP3) receptor inhibitor xestospongin C both inhibited endostatin-induced elevation in [Ca2+]i, and endostatin rapidly elevated endothelial cell IP3levels. Pertussis toxin and SB-220025 modestly inhibited the endostatin-induced Ca2+signal. Removal of extracellular Ca2+inhibited the endostatin-induced rise in [Ca2+]i, as did a subset of Ca2+-entry inhibitors. Peak Ca2+responses to endostatin and angiostatin in endothelial cells exceeded those in epithelial cells and were minimal in NIH/3T3 cells. Overnight pretreatment of endothelial cells with endostatin reduced the subsequent acute elevation in [Ca2+]iin response to vascular endothelial growth factor or to fibroblast growth factor by ∼70%. Intracellular Ca2+signaling may initiate or mediate some of the cellular actions of endostatin and angiostatin.

scholarly journals MLC1: A New Calcium-regulated Protein Conferring Calcium Dependence to Volume-regulated Anion Channels (VRAC) in Astrocytes.

2022 ◽  
Author(s):  
Maria Stefania Brignone ◽  
Angela Lanciotti ◽  
Antonio Michelucci ◽  
Cinzia Mallozzi ◽  
Serena Camerini ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Brain Edema ◽  
Intracellular Signaling ◽  
Regulatory Volume Decrease ◽  
Physiological Role ◽  
Anion Channel ◽  
Effector Proteins ◽  
Astrocyte Swelling ◽  
Cultured Astrocytes ◽  
Intracellular Ca

Abstract MLC1 is a membrane protein highly expressed by brain perivascular astrocytes. Mutations in the MLC1 gene account for megalencephalic leukoencephalopathy with subcortical cysts (MLC), an incurable leukodystrophy characterized by macrocephaly, brain edema and cysts, myelin vacuolation and astrocyte swelling, causing cognitive and motor dysfunctions. It has been demonstrated that MLC1 mutations affect the swelling-activated Cl - currents (I Cl,swell ) mediated by volume-regulated anion channel (VRAC) and the consequent regulatory volume decrease (RVD) and lead to abnormal activation of intracellular signaling pathways linked to inflammation/osmotic stress. Despite this knowledge, the MLC1 physiological role and MLC molecular pathogenesis are still elusive. Following the observations that Ca 2+ regulates all the MLC1-modulated processes and that intracellular Ca 2+ homeostasis is altered in MLC1-defective cells, we applied a multidisciplinary approach including biochemistry, molecular biology, video imaging, electrophysiology and proteomic techniques on cultured astrocytes to uncover new Ca 2+ -dependent signaling pathways controlling MLC1 function. Here, we revealed that MLC1 binds the Ca 2+ effector proteins calmodulin (CaM) and Ca 2+ /CaM-dependent protein kinase II (CaMKII) and, as result, changes its assembly, localization and functional properties in response to Ca 2+ changes. Noteworthy, CaM binding to the COOH terminal promotes MLC1 trafficking to the plasma membrane, while CaMKII phosphorylation of the NH 2 -terminal potentiates MLC1 activation of I Cl,swell . Overall, these results revealed that MLC1 is a Ca 2+ -regulated protein linking VRAC function and, possibly, volume regulation to Ca 2+ signaling in astrocytes. These findings open new avenues of investigations aimed at clarifying the abnormal molecular pathways underlying MLC and other diseases characterized by astrocyte swelling and brain edema.

scholarly journals Endothelial pannexin 1 channels control inflammation by regulating intracellular calcium

2019 ◽  
Author(s):  
Yang Yang ◽  
Leon Delalio ◽  
Angela K Best ◽  
Edgar Macal ◽  
Jenna Milstein ◽  
...  
Keyword(s):  
Cardiovascular Disease ◽  
Endothelial Cells ◽  
Plasma Membrane ◽  
Intracellular Calcium ◽  
Tumor Necrosis ◽  
Purinergic Signaling ◽  
Flow Cytometric Analysis ◽  
Atp Release ◽  
Pannexin 1 ◽  
Intracellular Ca

In BriefInterleukine-1 beta (IL-1β) has been identified as a critical factor that contributes to the inflammatory response in cardiovascular disease (e.g., atherosclerosis). Pannexin 1 (Panx1) channel activity in endothelial cells regulates localized inflammatory cell recruitment. In response to prolonged tumor necrosis factor alpha (TNF) treatment, Yang et al. found that the Panx1 channel is targeted to the plasma membrane, where it facilitates an increase in intracellular calcium to control the production and release of cytokines including IL-1β.GRAPHICAL ABSTRACTAbstractThe proinflammatory cytokine IL-1β is a significant risk factor in cardiovascular disease that can be targeted to reduce major cardiovascular events. IL-1β expression and release are tightly controlled by changes in intracellular Ca2+. In addition, purinergic signaling through ATP release has also been reported to promote IL-1β production. Despite this, the mechanisms that control IL-1β synthesis and expression have not been identified. The pannexin 1 (Panx1) channel has canonically been implicated in ATP release, especially during inflammation. However, resolution of purinergic signaling occurs quickly due to blood flow and the presence of ectonucleotidases. We examined Panx1 in human endothelial cells following treatment with the pro-inflammatory cytokine tumor necrosis alpha (TNF). In response to long-term TNF treatment, we identified a dramatic increase in Panx1 protein expression at the plasma membrane. Analysis by whole transcriptome sequencing (RNA-seq), qPCR, and treatment with specific kinase inhibitors, revealed that TNF signaling induced NFκβ-associated Panx1 transcription. Genetic inhibition of Panx1 reduced the expression and secretion of IL-1β. We initially hypothesized that increased Panx1-mediated ATP release acted in a paracrine fashion to control cytokine expression. However, our data demonstrate that IL1-β expression was not altered after direct ATP stimulation, following degradation of ATP by apyrase, or after pharmacological blockade of P2 receptors. These data suggest that non-purinergic pathways, involving Panx1, control IL-1β production. Because Panx1 forms a large pore channel, we hypothesized it may act to passively diffuse Ca2+ into the cell upon opening to regulate IL-1β. High-throughput flow cytometric analysis demonstrated that TNF treatments lead to elevated intracellular Ca2+. Genetic or pharmacological inhibition of Panx1 reduced TNF-associated increases in intracellular Ca2+, and IL-1β transcription. Furthermore, we found that the Ca2+-sensitive NFκβ-p65 protein failed to localize to the nucleus after genetic or pharmacological block of Panx1. Taken together, our study provides the first evidence that intracellular Ca2+ regulation via the Panx1 channel induces a feed-forward effect on NFκβ to regulate IL-1β synthesis and release in endothelium during inflammation.

ATP regulates anion channel-mediated organic osmolyte release from cultured rat astrocytes via multiple Ca2+-sensitive mechanisms

2005 ◽  
Vol 288 (1) ◽  
pp. C204-C213 ◽  
Author(s):  
Alexander A. Mongin ◽  
Harold K. Kimelberg
Keyword(s):  
Intracellular Signaling ◽  
Anion Channel ◽  
Cell Swelling ◽  
Organic Osmolytes ◽  
Signaling Mechanism ◽  
Pharmacological Agents ◽  
Organic Osmolyte ◽  
Primary Astrocyte ◽  
Intracellular Ca ◽  
Rat Astrocytes

Ubiquitously expressed volume-regulated anion channels (VRACs) are activated in response to cell swelling but may also show limited activity in nonswollen cells. VRACs are permeable to inorganic anions and small organic osmolytes, including the amino acids aspartate, glutamate, and taurine. Several recent reports have demonstrated that neurotransmitters or hormones, such as ATP and vasopressin, induce or strongly potentiate astrocytic whole cell Cl− currents and amino acid release, which are inhibited by VRAC blockers. In the present study, we explored the intracellular signaling mechanisms mediating the effects of ATP on d-[3H]aspartate release via the putative VRAC pathway in rat primary astrocyte cultures. Cells were exposed to moderate (5%) or substantial (30%) reductions in medium osmolarity. ATP strongly potentiated d-[3H]aspartate release in both moderately swollen and substantially swollen cells. These ATP effects were blocked (≥80% inhibition) by intracellular Ca2+ chelation with BAPTA-AM, calmodulin inhibitors, or a combination of the inhibitors of protein kinase C (PKC) and calmodulin-dependent kinase II (CaMK II). In contrast, control d-[3H]aspartate release activated by the substantial hyposmotic swelling showed little (≤25% inhibition) sensitivity to the same pharmacological agents. These data indicate that ATP regulates VRAC activity via two separate Ca2+-sensitive signaling cascades involving PKC and CaMK II and that cell swelling per se activates VRACs via a separate Ca2+/calmodulin-independent signaling mechanism. Ca2+-dependent organic osmolyte release via VRACs may contribute to the physiological functions of these channels in the brain, including astrocyte-to-neuron intercellular communication.

Multimodal Atomic Force Imaging of Open Hemichannelinduced Modulation of Cell Volume and Viscoelastic Properties

1999 ◽  
Vol 5 (S2) ◽  
pp. 998-999
Author(s):  
Seung K. Rhee ◽  
Arjan P. Quist ◽  
Hai Lin ◽  
Nils Almqvist ◽  
Ratneshx Lai
Keyword(s):  
Plasma Membrane ◽  
Cell Volume ◽  
Lucifer Yellow ◽  
Single Cells ◽  
Dye Uptake ◽  
Aortic Endothelial Cells ◽  
Atomic Force ◽  
Cell Plasma ◽  
Uptake Assay ◽  
Gap Junctional

Hemichannels from two single cells can join upon contact between these cells to form gap junctions - an intercellular pathway for the direct exchange of ions and small metabolites. Using techniques of fluorescent dye-uptake assay, laser confocal fluorescence imaging and atomic force microscopy (AFM), we have examined the role of hemichannels, present in the non-junctional regions of single cell plasma membrane, in the modulation of cell volume.Antibodies against a gap junctional protein connexin43, were immunolocalized to nonjunctional plasma membrane regions of single BICR-MlRk k (breast tumor epithelial) cells, KOM-1 (bovine aortic endothelial) cells, and GM04260 (AD-free human) fibroblast cells. In the absence of extracellular calcium, cytoplasmic uptake of Lucifer yellow (LY) but not of dextran-conjugated LY was observed in single cells. Dye uptake was prevented by gap junctional inhibitors, ẞ-glycyrrhetinic acid (ẞGCA) and oleamide.

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