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.

scholarly journals Structure versus function: Are new conformations of pannexin 1 yet to be resolved?

2021 ◽  
Vol 153 (5) ◽  
Author(s):  
Carsten Mim ◽  
Guy Perkins ◽  
Gerhard Dahl
Keyword(s):  
Structural Information ◽  
Purinergic Signaling ◽  
Atp Release ◽  
Strong Support ◽  
Decisive Role ◽  
Chloride Ions ◽  
Pannexin 1 ◽  
Selective Membrane ◽  
Selective Channel ◽  
Structure Versus

Pannexin 1 (Panx1) plays a decisive role in multiple physiological and pathological settings, including oxygen delivery to tissues, mucociliary clearance in airways, sepsis, neuropathic pain, and epilepsy. It is widely accepted that Panx1 exerts its role in the context of purinergic signaling by providing a transmembrane pathway for ATP. However, under certain conditions, Panx1 can also act as a highly selective membrane channel for chloride ions without ATP permeability. A recent flurry of publications has provided structural information about the Panx1 channel. However, while these structures are consistent with a chloride selective channel, none show a conformation with strong support for the ATP release function of Panx1. In this Viewpoint, we critically assess the existing evidence for the function and structure of the Panx1 channel and conclude that the structure corresponding to the ATP permeation pathway is yet to be determined. We also list a set of additional topics needing attention and propose ways to attain the large-pore, ATP-permeable conformation of the Panx1 channel.

scholarly journals Pannexin-1 mediated ATP release in adipocytes is sensitive to glucose and insulin and modulates lipolysis and macrophage migration

2018 ◽  
Author(s):  
Marco Tozzi ◽  
Jacob B. Hansen ◽  
Ivana Novak
Keyword(s):  
Adipose Tissue ◽  
Purinergic Receptors ◽  
Purinergic Signaling ◽  
Cell Metabolism ◽  
Atp Release ◽  
Extracellular Atp ◽  
Adrenergic Stimulation ◽  
Pannexin 1 ◽  
White Adipocytes ◽  
Obesity And Diabetes

One-sentence summaryInsulin inhibits ATP release in adipocytesAbstractExtracellular ATP signaling is involved in many physiological and pathophysiological processes, and purinergic receptors are targets for drug therapy in several diseases, including obesity and diabetes. Adipose tissue has crucial functions in lipid and glucose metabolism and adipocytes express purinergic receptors. However, the sources of extracellular ATP in adipose tissue are not yet characterized.Here, we show that upon adrenergic stimulation white adipocytes release ATP through the pannexin-1 pore that is regulated by a cAMP-PKA dependent pathway. The ATP release correlates with increased cell metabolism, and extracellular ATP induces Ca2+ signaling and lipolysis in adipocytes and promotes macrophages migration. Most importantly, ATP release is markedly inhibited by insulin, and thereby auto/paracrine purinergic signaling in adipose tissue would be attenuated. Furthermore, we define the signaling pathway for insulin regulated ATP release.Our findings reveal the insulin-pannexin-1-purinergic signaling cross-talk in adipose tissue and we propose that deregulation of this signaling may underlie adipose tissue inflammation and type-2 diabetes.

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.

scholarly journals HIV gp120 Protein Increases the Function of Connexin 43 Hemichannels and Pannexin-1 Channels in Astrocytes: Repercussions on Astroglial Function

2020 ◽  
Vol 21 (7) ◽  
pp. 2503 ◽  
Author(s):  
Rosario Gajardo-Gómez ◽  
Cristian A. Santibañez ◽  
Valeria C. Labra ◽  
Gonzalo I. Gómez ◽  
Eliseo A. Eugenin ◽  
...  
Keyword(s):  
Connexin 43 ◽  
Molecular Mechanisms ◽  
Ex Vivo ◽  
Purinergic Signaling ◽  
Atp Release ◽  
P38 Map Kinase ◽  
Motor Deficits ◽  
Pannexin 1 ◽  
Channel Opening ◽  
Wide Range

At least half of human immunodeficiency virus (HIV)-infected individuals suffer from a wide range of cognitive, behavioral and motor deficits, collectively known as HIV-associated neurocognitive disorders (HAND). The molecular mechanisms that amplify damage within the brain of HIV-infected individuals are unknown. Recently, we described that HIV augments the opening of connexin-43 (Cx43) hemichannels in cultured human astrocytes, which result in the collapse of neuronal processes. Whether HIV soluble viral proteins such as gp120, can regulate hemichannel opening in astrocytes is still ignored. These channels communicate the cytosol with the extracellular space during pathological conditions. We found that gp120 enhances the function of both Cx43 hemichannels and pannexin-1 channels in mouse cortical astrocytes. These effects depended on the activation of IL-1β/TNF-α, p38 MAP kinase, iNOS, cytoplasmic Ca2+ and purinergic signaling. The gp120-induced channel opening resulted in alterations in Ca2+ dynamics, nitric oxide production and ATP release. Although the channel opening evoked by gp120 in astrocytes was reproduced in ex vivo brain preparations, these responses were heterogeneous depending on the CA1 region analyzed. We speculate that soluble gp120-induced activation of astroglial Cx43 hemichannels and pannexin-1 channels could be crucial for the pathogenesis of HAND.

Extracellular ATP signaling and P2X nucleotide receptors in monolayers of primary human vascular endothelial cells

2002 ◽  
Vol 282 (2) ◽  
pp. C289-C301 ◽  
Author(s):  
Lisa M. Schwiebert ◽  
William C. Rice ◽  
Brian A. Kudlow ◽  
Amanda L. Taylor ◽  
Erik M. Schwiebert
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Blood Vessels ◽  
Vascular Tone ◽  
Vascular Endothelial Cells ◽  
Purinergic Signaling ◽  
Primary Cultures ◽  
Atp Release ◽  
Vascular Endothelial ◽  
Multiple Blood

ATP and its metabolites regulate vascular tone; however, the sources of the ATP released in vascular beds are ill defined. As such, we tested the hypothesis that all limbs of an extracellular purinergic signaling system are present in vascular endothelial cells: ATP release, ATP receptors, and ATP receptor-triggered signal transduction. Primary cultures of human endothelial cells derived from multiple blood vessels were grown as monolayers and studied using a bioluminescence detection assay for ATP released into the medium. ATP is released constitutively and exclusively across the apical membrane under basal conditions. Hypotonic challenge or the calcium agonists ionomycin and thapsigargin stimulate ATP release in a reversible and regulated manner. To assess expression of P2X purinergic receptor channel subtypes (P2XRs), we performed degenerate RT-PCR, sequencing of the degenerate P2XR product, and immunoblotting with P2XR subtype-specific antibodies. Results revealed that P2X4and P2X5are expressed abundantly by endothelial cell primary cultures derived from multiple blood vessels. Together, these results suggest that components of an autocrine purinergic signaling loop exist in the endothelial cell microvasculature that may allow for “self-regulation” of endothelial cell function and modulation of vascular tone.

scholarly journals Methyl mercury triggers endothelial leukocyte adhesion and increases expression of cell adhesion molecules and chemokines

2021 ◽  
pp. 153537022110338
Author(s):  
Joshua Fowler ◽  
Martin Tsz-Ki Tsui ◽  
Jessica Chavez ◽  
Safeera Khan ◽  
Hassan Ahmed ◽  
...  
Keyword(s):  
Cardiovascular Disease ◽  
Endothelial Cells ◽  
Methyl Mercury ◽  
Leukocyte Adhesion ◽  
Flow Cytometric Analysis ◽  
Toxic Metal ◽  
Initial Step ◽  
Intercellular Adhesion Molecule ◽  
Intercellular Adhesion Molecule 1 ◽  
Monocyte Chemoattractant Protein 1

Cardiovascular disease is the leading cause of morbidity, mortality, and health care costs in the USA, and around the world. Among the various risk factors of cardiovascular disease, environmental and dietary exposures to methyl mercury, a highly toxic metal traditionally labeled as a neurotoxin, have been epidemiologically linked to human cardiovascular disease development. However, its role in development and promotion of atherosclerosis, an initial step in more immediately life-threatening cardiovascular diseases, remains unclear. This study was conducted to examine the role that methyl mercury plays in the adhesion of monocytes to human microvascular endothelial cells (HMEC-1), and the underlying mechanisms. Methyl mercury treatment significantly induced the adhesion of monocyte to HMEC-1 endothelial cells, a critical step in atherosclerosis, while also upregulating the expression of proinflammatory cytokines interleukin-6, interleukin-8. Further, methyl mercury treatment also upregulated the chemotactic cytokine monocyte chemoattractant protein-1 and intercellular adhesion molecule-1. These molecules are imperative for the firm adhesion of leukocytes to endothelial cells. Additionally, our results further demonstrated that methyl mercury stimulated a significant increase in NF-κB activation. These findings suggest that NF-κB signaling pathway activation by methyl mercury is an important factor in the binding of monocytes to endothelial cells. Finally, by using flow cytometric analysis, methyl mercury treatment caused a significant increase in necrotic cell death only at higher concentrations without initiating apoptosis. This study provides new insights into the molecular actions of methyl mercury that can lead to endothelial dysfunction, inflammation, and subsequent atherosclerotic development.

A dual voltage clamp technique to study gap junction hemichannels in astrocytes cultured from neonatal rodent spinal cords

2021 ◽  
Author(s):  
Juan Mauricio Garre ◽  
Feliksas F Bukauskas ◽  
Michael V Bennett
Keyword(s):  
Voltage Clamp ◽  
Connexin 43 ◽  
Single Channel ◽  
Purinergic Signaling ◽  
Atp Release ◽  
Single Channels ◽  
P2x7 Receptors ◽  
Voltage Clamp Technique ◽  
Pannexin 1 ◽  
Clamp Technique

Astrocytes express surface channels involved in purinergic signaling, and among these channels, pannexin-1 (Px1) and connexin-43 (Cx43) hemichannels (HCs) mediate ATP release that acts directly, or through its derivatives, on neurons and glia via purinergic receptors. Although HCs are functional, i.e., open and close, under physiological and pathological conditions, single channel conductance of Px1 HCs is not well defined. Here, we developed a dual voltage clamp technique in HeLa cells overexpressing human Px1-YFP, and then applied this system to rodent spinal astrocytes. Single channels were recorded in cell attached patches and evoked with ramp cycles of 2 s duration and -/+ 80-100 mV amplitude or rectangular pulses through another pipette in whole cell clamp. Conductance of Px1 HC openings recorded during ramp stimuli ranged 25-110 pS. Based on their single channel conductances, Px1 HCs could be distinguished from Cx43 HCs and P2X7 receptors (P2X7Rs) in spinal astrocytes during dual voltage clamp experiments. Furthermore, we found that single channel activity of Cx43 HCs and P2X7Rs was increased, and that of Px1 HCs was decreased, in spinal astrocytes treated for 7h with FGF-1, a growth factor implicated in neurodevelopment, repair and inflammation.

scholarly journals Pannexin-1 channels on endothelial cells mediate vascular inflammation during lung ischemia-reperfusion injury

2018 ◽  
Vol 315 (2) ◽  
pp. L301-L312 ◽  
Author(s):  
Ashish K. Sharma ◽  
Eric J. Charles ◽  
Yunge Zhao ◽  
Adishesh K. Narahari ◽  
Pranav K. Baderdinni ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Lung Transplantation ◽  
Vascular Permeability ◽  
Ischemia Reperfusion ◽  
Vascular Inflammation ◽  
Atp Release ◽  
Neutrophil Infiltration ◽  
Wild Type ◽  
Pannexin 1 ◽  
Lung Dysfunction

Ischemia-reperfusion (I/R) injury (IRI), which involves inflammation, vascular permeability, and edema, remains a major challenge after lung transplantation. Pannexin-1 (Panx1) channels modulate cellular ATP release during inflammation. This study tests the hypothesis that endothelial Panx1 is a key mediator of vascular inflammation and edema after I/R and that IRI can be blocked by Panx1 antagonism. A murine hilar ligation model of IRI was used whereby left lungs underwent 1 h of ischemia and 2 h of reperfusion. Treatment of wild-type mice with Panx1 inhibitors (carbenoxolone or probenecid) significantly attenuated I/R-induced pulmonary dysfunction, edema, cytokine production, and neutrophil infiltration versus vehicle-treated mice. In addition, VE-Cad-CreERT2+/Panx1fl/fl mice (tamoxifen-inducible deletion of Panx1 in vascular endothelium) treated with tamoxifen were significantly protected from IRI (reduced dysfunction, endothelial permeability, edema, proinflammatory cytokines, and neutrophil infiltration) versus vehicle-treated mice. Furthermore, extracellular ATP levels in bronchoalveolar lavage fluid is Panx1-mediated after I/R as it was markedly attenuated by Panx1 antagonism in wild-type mice and by endothelial-specific Panx1 deficiency. Panx1 gene expression in lungs after I/R was also significantly elevated compared with sham. In vitro experiments demonstrated that TNF-α and/or hypoxia-reoxygenation induced ATP release from lung microvascular endothelial cells, which was attenuated by Panx1 inhibitors. This study is the first, to our knowledge, to demonstrate that endothelial Panx1 plays a key role in mediating vascular permeability, inflammation, edema, leukocyte infiltration, and lung dysfunction after I/R. Pharmacological antagonism of Panx1 activity may be a novel therapeutic strategy to prevent IRI and primary graft dysfunction after lung transplantation.

Endogenously released ATP mediates shear stress-induced Ca2+influx into pulmonary artery endothelial cells

2003 ◽  
Vol 285 (2) ◽  
pp. H793-H803 ◽  
Author(s):  
Kimiko Yamamoto ◽  
Takaaki Sokabe ◽  
Norihiko Ohura ◽  
Hideki Nakatsuka ◽  
Akira Kamiya ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
Pulmonary Artery ◽  
Atp Release ◽  
Atpase Inhibitor ◽  
Intracellular Ca ◽  
Human Pulmonary Artery ◽  
Stress Dependent ◽  
Dose Dependent ◽  
Balanced Solution

The mechanisms by which flow-imposed shear stress elevates intracellular Ca2+in cultured endothelial cells (ECs) are not fully understood. Here we report finding that endogenously released ATP contributes to shear stress-induced Ca2+responses. Application of flow of Hanks' balanced solution to human pulmonary artery ECs (HPAECs) elicited shear stress-dependent increases in Ca2+concentrations. Chelation of extracellular Ca2+with EGTA completely abolished the Ca2+responses, whereas the phospholipase C inhibitor U-73122 or the Ca2+-ATPase inhibitor thapsigargin had no effect, which thereby indicates that the response was due to the influx of extracellular Ca2+. The Ca2+influx was significantly suppressed by apyrase, which degrades ATP, or antisense oligonucleotide targeted to P2X4purinoceptors. A luciferase luminometric assay showed that shear stress induced dose-dependent release of ATP. When the ATP release was inhibited by the ATP synthase inhibitors angiostatin or oligomycin, the Ca2+influx was markedly suppressed but was restored by removal of these inhibitors or addition of extracellular ATP. These results suggest that shear stress stimulates HPAECs to release ATP, which activates Ca2+influx via P2X4receptors.

scholarly journals Inhibition of Pannexin 1 Reduces the Tumorigenic Properties of Human Melanoma Cells

Cancers ◽  
2019 ◽  
Vol 11 (1) ◽  
pp. 102 ◽  
Author(s):  
Taylor Freeman ◽  
Samar Sayedyahossein ◽  
Danielle Johnston ◽  
Rafael Sanchez-Pupo ◽  
Brooke O’Donnell ◽  
...  
Keyword(s):  
Cell Lines ◽  
Purinergic Signaling ◽  
Atp Release ◽  
Melanoma Cells ◽  
Human Melanoma ◽  
Channel Blockers ◽  
Melanoma Tumor ◽  
Cell Growth And Migration ◽  
Pannexin 1 ◽  
Protein Levels

Pannexin 1 (PANX1) is a channel-forming glycoprotein expressed in many tissues including the skin. PANX1 channels allow the passage of ions and molecules up to 1 kDa, including ATP and other metabolites. In this study, we show that PANX1 is highly expressed in human melanoma tumors at all stages of disease progression, as well as in patient-derived cells and established melanoma cell lines. Reducing PANX1 protein levels using shRNA or inhibiting channel function with the channel blockers, carbenoxolone (CBX) and probenecid (PBN), significantly decreased cell growth and migration, and increased melanin production in A375-P and A375-MA2 cell lines. Further, treatment of A375-MA2 tumors in chicken embryo xenografts with CBX or PBN significantly reduced melanoma tumor weight and invasiveness. Blocking PANX1 channels with PBN reduced ATP release in A375-P cells, suggesting a potential role for PANX1 in purinergic signaling of melanoma cells. In addition, cell-surface biotinylation assays indicate that there is an intracellular pool of PANX1 in melanoma cells. PANX1 likely modulates signaling through the Wnt/β-catenin pathway, because β-catenin levels were significantly decreased upon PANX1 silencing. Collectively, our findings identify a role for PANX1 in controlling growth and tumorigenic properties of melanoma cells contributing to signaling pathways that modulate melanoma progression.

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