scholarly journals Activation of Cx43 Hemichannels Induces the Generation of Ca2+ Oscillations in White Adipocytes and Stimulates Lipolysis

2021 ◽  
Vol 22 (15) ◽  
pp. 8095
Author(s):  
Egor A. Turovsky ◽  
Elena G. Varlamova ◽  
Maria V. Turovskaya
Keyword(s):  
Intracellular Signaling ◽  
Connexin 43 ◽  
Lipid Droplets ◽  
External Environment ◽  
Atp Release ◽  
Complete Suppression ◽  
Bafilomycin A1 ◽  
Phosphoinositide Signaling ◽  
White Adipocytes ◽  
Connexin Hemichannels

The aim of the study was to investigate the mechanisms of Ca2+ oscillation generation upon activation of connexin-43 and regulation of the lipolysis/lipogenesis balance in white adipocytes through vesicular ATP release. With fluorescence microscopy it was revealed that a decrease in the concentration of extracellular calcium ([Ca2+]ex) results in two types of Ca2+ responses in white adipocytes: Ca2+ oscillations and transient Ca2+ signals. It was found that activation of the connexin half-channels is involved in the generation of Ca2+ oscillations, since the blockers of the connexin hemichannels—carbenoxolone, octanol, proadifen and Gap26—as well as Cx43 gene knockdown led to complete suppression of these signals. The activation of Cx43 in response to the reduction of [Ca2+]ex was confirmed by TIRF microscopy. It was shown that in response to the activation of Cx43, ATP-containing vesicles were released from the adipocytes. This process was suppressed by knockdown of the Cx43 gene and by bafilomycin A1, an inhibitor of vacuolar ATPase. At the level of intracellular signaling, the generation of Ca2+ oscillations in white adipocytes in response to a decrease in [Ca2+]ex occurred due to the mobilization of the Ca2+ ions from the thapsigargin-sensitive Ca2+ pool of IP3R as a result of activation of the purinergic P2Y1 receptors and phosphoinositide signaling pathway. After activation of Cx43 and generation of the Ca2+ oscillations, changes in the expression levels of key genes and their encoding proteins involved in the regulation of lipolysis were observed in white adipocytes. This effect was accompanied by a decrease in the number of adipocytes containing lipid droplets, while inhibition or knockdown of Cx43 led to inhibition of lipolysis and accumulation of lipid droplets. In this study, we investigated the mechanism of Ca2+ oscillation generation in white adipocytes in response to a decrease in the concentration of Ca2+ ions in the external environment and established an interplay between periodic Ca2+ modes and the regulation of the lipolysis/lipogenesis balance.

scholarly journals Activation of Cx-43 Hemichannels Induces the Generation of Ca2+-Oscillations in White Adipocytes and Stimulates Lipolysis.

2021 ◽  
Author(s):  
Maria Turovskaya ◽  
Egor Turovsky
Keyword(s):  
Intracellular Signaling ◽  
Lipid Droplets ◽  
Regulation Of Gene Expression ◽  
Signal Propagation ◽  
Complete Suppression ◽  
Atpase Inhibitor ◽  
Knock Down ◽  
White Adipocytes ◽  
Cx 43 ◽  
Connexin Hemichannels

Abstract With fluorescence microscopy, it was revealed that a decrease in the concentration of extracellular calcium ([Ca2+]ex) results in two types of Ca2+-responses in white adipocytes: Ca2+-oscillations and transient Ca2+-signals. Activation of connexin hemichannels is involved in the mechanism of generation of Ca2+-oscillations, since the blockers of connexin hemichannels - carbenoxolone, octanol and proadifen, as well as Cx43 gene knock-down lead to complete suppression of these signals. TIRF microscopy confirmed activation of Cx-43 in response to the reduction of [Ca2+]ex. In response to the activation of Cx-43, the secretion of ATP-containing vesicles from adipocytes occurs. And this ATP release is suppressed in adipocytes along with the Cx43 gene knock-down and is inhibited by Bafilomycin A1, a vacuolar ATPase inhibitor. At the level of intracellular signaling, the generation of Ca2+-oscillations in white adipocytes in response to a decrease in [Ca2+]ex takes place due to the mobilization of Ca2 + ions from the thapsigargin-sensitive Ca2+-pool in the endoplasmic reticulum via IP3R as a result of the activation of P2Y1 purinergic receptors and the phosphoinositide signaling pathway. Such paracrine activation of white adipose tissue in response to the opening of Cx43 hemichannels leads to local signal propagation and regulation of gene expression. At 24 hours after activation of Cx-43 and generation of Ca2+-oscillations in white adipocytes, there is a change in the expression of key genes involved in the regulation of lipolysis, which is accompanied by a decrease in the number of adipocytes that contained lipid droplets. Meanwhile, inhibition or knock-down of Cx-43 leads to inhibition of lipolysis and accumulation of lipid droplets. In this study, we elucidate and research the mechanism of generation of Ca2+-oscillations in white adipocytes in response to decreased concentration of Ca2 + ions in the external environment and show the correlation between periodic Ca2+-modes and lipolysis/lipogenesis balance regulation.

A2A Receptor-induced Overexpression of Pannexin-1 Hemichannels Indirectly Mediates Adenosine Fibrogenic Actions in Subcutaneous Human Fibroblasts by Favoring ATP Release

2021 ◽  
Author(s):  
Carina Herman-de-Sousa ◽  
Maria Adelina Costa ◽  
Rafaela Pedro Silva ◽  
Fátima Ferreirinha ◽  
Severino Ribeiro ◽  
...  
Keyword(s):  
Inflammatory Mediators ◽  
Connexin 43 ◽  
Myofascial Pain ◽  
Subcutaneous Tissue ◽  
Human Fibroblasts ◽  
Atp Release ◽  
Collagen Production ◽  
Pannexin 1 ◽  
A2a Receptors ◽  
Inflammatory Conditions

Abstract Disorganization of the subcutaneous tissue due to inflammation and fibrosis is a common feature in patients with myofascial pain. Dermal accumulation of adenosine favours collagen production by human subcutaneous fibroblasts (HSCF) via A2A receptors (A2AR) activation. Adenosine mimics the fibrogenic effect of inflammatory mediators (e.g. histamine, bradykinin), which act by promoting ATP release from HSCF via pannexin-1 (Panx1) and/or connexin-43 (Cx43) hemichannels. However, this mechanism was never implicated in the A2AR-mediated actions. NECA and CGS21680C, two enzymatically-stable A2AR agonists, increased Panx-1, but reduced Cx43, immunoreactivity in cultured HSCF. This effect was accompanied by increases in ATP release and collagen production by HSCF. Involvement of A2AR was verified upon blockage of NECA and CGS21680 effects with the selective A2AR antagonist, SCH442416. Inhibition of Panx1 hemichannels with probenecid also decreased ATP release and collagen production by HSCF under similar conditions. Superfluous ATP release by HSCF exposed to A2AR agonists overexpressing Panx1 hemichannels contributes to keep high [Ca2+]i levels in the presence of inflammatory mediators, like histamine. Adenosine A2AR-induced Panx1 overexpression was shown here for the first time; this feature indirectly implicates ATP release in the fibrogenic vicious cycle putatively operated by the nucleoside in subcutaneous tissue fibrosis and myofascial inflammatory conditions.

scholarly journals Purinergic responses of chondrogenic stem cells to dynamic loading

2013 ◽  
Vol 78 (12) ◽  
pp. 1865-1874 ◽  
Author(s):  
Ivana Gadjanski ◽  
Gordana Vunjak-Novakovic
Keyword(s):  
Stem Cells ◽  
Dynamic Loading ◽  
Lucifer Yellow ◽  
Atp Release ◽  
Human Mesenchymal Stem Cells ◽  
Cell Types ◽  
Flufenamic Acid ◽  
Release Assay ◽  
Extracellular Environment ◽  
Connexin Hemichannels

In habitually loaded tissues, dynamic loading can trigger ATP (adenosine 5?- triphosphate) release to extracellular environment, and result in calcium signaling via ATP binding to purine P2 receptors1. In the current study we have compared purinergic responses (ATP release) of two types of cells: bovine chondrocytes (bCHs) and human mesenchymal stem cells (hMSC) that were encapsulated in agarose and subjected to dynamic loading. Both cell types were cultured under chondrogenic conditions, and their responses to loading were evaluated by ATP release assay in combination with connexin (Cx)-sensitive fluorescent dye (Lucifer Yellow - LY) and a Cx-hemichannel blocker (Flufenamic acid - FFA). In response to dynamic loading, chondrogenic hMSCs released significantly higher amounts of ATP (5-fold) in comparison to the bCHs early in culture (day 2). Triggering of LY uptake in the bCHs and hMSCs by dynamic loading implies opening of the Cx-hemichannels. However, the number of LY-positive cells in hMSC-constructs was 2.5-fold lower compared to the loaded bCH-constructs, suggesting utilization of additional mechanisms of ATP release. Cx-reactive sites were detected in both bCHs and hMSCs-constructs. FFA application led to reduced ATP release both in bCHs and hMSCs, which confirms the involvement of connexin hemichannels, with more prominent effects in bCHs than in hMSCs, further implying the existence of additional mechanism of ATP release in chondrogenic hMSCs. Taken together, these results indicate stronger purinergic response to dynamic loading of chondrogenic hMSCs than primary chondrocytes, by activation of connexin hemichannels and additional mechanisms of ATP release.

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.

scholarly journals Endotoxin-induced autocrine ATP signaling inhibits neutrophil chemotaxis through enhancing myosin light chain phosphorylation

2017 ◽  
Vol 114 (17) ◽  
pp. 4483-4488 ◽  
Author(s):  
Xu Wang ◽  
Weiting Qin ◽  
Xiaohan Xu ◽  
Yuyun Xiong ◽  
Yisen Zhang ◽  
...  
Keyword(s):  
Light Chain ◽  
Connexin 43 ◽  
Myosin Light Chain ◽  
Calcium Influx ◽  
Neutrophil Migration ◽  
Atp Release ◽  
Human Neutrophils ◽  
Neutrophil Chemotaxis ◽  
P2x1 Receptor ◽  
Atp Signaling

Although the neutrophil recruitment cascade during inflammation has been well described, the molecular players that halt neutrophil chemotaxis remain unclear. In this study, we found that lipopolysaccharide (LPS) was a potent stop signal for chemotactic neutrophil migration. Treatment with an antagonist of the ATP receptor (P2X1) in primary human neutrophils or knockout of the P2X1 receptor in neutrophil-like differentiated HL-60 (dHL-60) cells recovered neutrophil chemotaxis. Further observations showed that LPS-induced ATP release through connexin 43 (Cx43) hemichannels was responsible for the activation of the P2X1 receptor and the subsequent calcium influx. Increased intracellular calcium stopped neutrophil chemotaxis by activating myosin light chain (MLC) through the myosin light chain kinase (MLCK)-dependent pathway. Taken together, these data identify a previously unknown function of LPS-induced autocrine ATP signaling in inhibiting neutrophil chemotaxis by enhancing MLC phosphorylation, which provides important evidence that stoppage of neutrophil chemotaxis at infectious foci plays a key role in the defense against invading pathogens.

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.

scholarly journals Osmotic surveillance mediates rapid wound closure through nucleotide release

2014 ◽  
Vol 207 (6) ◽  
pp. 767-782 ◽  
Author(s):  
William J. Gault ◽  
Balázs Enyedi ◽  
Philipp Niethammer
Keyword(s):  
Wound Repair ◽  
Interstitial Fluid ◽  
Wound Closure ◽  
External Environment ◽  
Atp Release ◽  
Nucleoside Triphosphate ◽  
Extracellular Nucleotide ◽  
Nucleoside Triphosphate Diphosphohydrolase ◽  
Larval Zebrafish ◽  
Zebrafish Larvae

Osmotic cues from the environment mediate rapid detection of epithelial breaches by leukocytes in larval zebrafish tail fins. Using intravital luminescence and fluorescence microscopy, we now show that osmolarity differences between the interstitial fluid and the external environment trigger ATP release at tail fin wounds to initiate rapid wound closure through long-range activation of basal epithelial cell motility. Extracellular nucleotide breakdown, at least in part mediated by ecto-nucleoside triphosphate diphosphohydrolase 3 (Entpd3), restricts the range and duration of osmotically induced cell migration after injury. Thus, in zebrafish larvae, wound repair is driven by an autoregulatory circuit that generates pro-migratory tissue signals as a function of environmental exposure of the inside of the tissue.

Brown adipocytes postnatally arise through both differentiation from progenitors and conversion from white adipocytes in Syrian hamster

2018 ◽  
Vol 124 (1) ◽  
pp. 99-108 ◽  
Author(s):  
Yuko Okamatsu-Ogura ◽  
Junko Nio-Kobayashi ◽  
Kazuki Nagaya ◽  
Ayumi Tsubota ◽  
Kazuhiro Kimura
Keyword(s):  
Adipose Tissue ◽  
Lipid Droplets ◽  
Brown Adipocyte ◽  
Fat Tissue ◽  
Brown Adipocytes ◽  
Syrian Hamsters ◽  
Monocarboxylate Transporter 1 ◽  
White Adipocytes ◽  
Proliferation And Differentiation ◽  
Adipocyte Progenitors

To investigate the postnatal development of brown adipose tissue (BAT) in Syrian hamsters, we histologically examined interscapular fat tissue from 5–16-day-old pups, focusing on how brown adipocytes arise. Interscapular fat of 5-day-old hamsters mainly consisted of white adipocytes containing large unilocular lipid droplets, as observed in typical white adipose tissue (WAT). On day 7, clusters of small, proliferative nonadipocytes with a strong immunoreactivity for Ki67 appeared near the edge of the interscapular fat tissue. The area of the Ki67-positive regions expanded to ~50% of the total tissue area by day 10. The interscapular fat showed the typical BAT feature by day 16. A brown adipocyte-specific marker, uncoupling protein-1, was clearly detected on day 10 and thereafter, while not detected on day 7. During conversion of interscapular fat from WAT to BAT, unilocular adipocytes completely and rapidly disappeared without obvious apoptosis. Dual immunofluorescence staining for Ki67 and monocarboxylate transporter 1 (MCT1), another selective marker for brown adipocytes, revealed that most of the proliferating cells were of the brown adipocyte lineage. Electron microscopic examination showed that some of the white adipocytes contained small lipid droplets in addition to the large droplet and expressed MCT1 as do progenitor and mature brown adipocytes, implying a direct conversion from white to brown adipocytes. These results suggest that BAT of Syrian hamsters develops postnatally through two different pathways: the proliferation and differentiation of brown adipocyte progenitors and the conversion of unilocular adipocytes to multilocular brown adipocytes. NEW & NOTEWORTHY Brown and white adipose tissues (BAT and WAT, respectively) are quite different in morphological features and function; however, the boundary between these tissues is obscure. In this study, we histologically evaluated the process of BAT development in Syrian hamsters, which shows postnatal conversion of WAT to BAT. Our results suggest that brown adipocytes arise through two different pathways: the proliferation and differentiation of brown adipocyte progenitors and the conversion from white adipocytes.

scholarly journals Connexin-43 dependent ATP release mediates macrophage activation during peritonitis

2018 ◽  
Author(s):  
Michel Dosch ◽  
Joël Zindel ◽  
Fadi Jebbawi ◽  
Nicolas Melin ◽  
Daniel Sanchez-Taltavull ◽  
...  
Keyword(s):  
Connexin 43 ◽  
Macrophage Activation ◽  
Purinergic Receptor ◽  
Atp Release ◽  
Peritoneal Macrophages ◽  
Dependent Manner ◽  
Autocrine Signaling ◽  
Cx43 Expression ◽  
Pulmonary Macrophages ◽  
And Control

ABSTRACTPeritonitis is the consequence of bacterial spillage into a sterile environment by gastrointestinal hollow-organ perforation that may lead to fulminant sepsis. Outcome of peritonitis-induced sepsis critically depends on macrophage activation by extracellular ATP release and associated para- and autocrine signaling via purinergic receptors. Mechanisms that mediate and control ATP release, however, are poorly understood. Here we show that TLR-2 and -4 agonists trigger ATP release via Connexin-43 (CX43) hemichannels in peritoneal macrophages leading to poor survival during sepsis. In humans, CX43 expression was upregulated on macrophages isolated from the peritoneal cavity in patients with intraperitoneal infection but not in healthy controls. Using a murine caecal ligation and puncture (CLP) model, we identified increased CX43 expression in activated infiltrating peritoneal, hepatic and pulmonary macrophages. Conditional MAC-CX43 KO Lyz2cre/creCx43flox/flox mice were developed to specifically assess the CX43 impact in macrophages. Both macrophage-specific CX43 deletion (using Lyz2cre/creCx43flox/flox mice) or pharmacological CX43 blockade were associated with reduced cytokine secretion by macrophages in response to LPS and CLP. This was ultimately resulting in increased survival in Lyz2cre/creCx43flox/flox mice and after pharmacological blockade. Specific inhibition of the purinergic receptor P2RY1 abrogated CX43 elicited cytokine responses. In conclusion, inhibition of autocrine ATP signaling via CX43 on macrophages and P2RY1 improves sepsis outcome in experimental peritonitis.Brief SummaryConnexin-43-mediated ATP release from macrophages in response to TLR-4 and -2 agonists modulates autocrine activation of macrophages in a P2Y1-dependent manner, ultimately determining sepsis survival.

scholarly journals Connexin-43-dependent ATP release mediates macrophage activation during sepsis

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Michel Dosch ◽  
Joël Zindel ◽  
Fadi Jebbawi ◽  
Nicolas Melin ◽  
Daniel Sanchez-Taltavull ◽  
...  
Keyword(s):  
Connexin 43 ◽  
Macrophage Activation ◽  
Purinergic Receptors ◽  
Atp Release ◽  
Healthy Controls ◽  
Sepsis Model ◽  
Hepatic Macrophages ◽  
Hollow Organ ◽  
Sepsis Survival ◽  
Autocrine Signalling

Bacterial spillage into a sterile environment following intestinal hollow-organ perforation leads to peritonitis and fulminant sepsis. Outcome of sepsis critically depends on macrophage activation by extracellular ATP-release and associated autocrine signalling via purinergic receptors. ATP-release mechanisms, however, are poorly understood. Here, we show that TLR-2 and −4 agonists trigger ATP-release via Connexin-43 hemichannels in macrophages leading to poor sepsis survival. In humans, Connexin-43 was upregulated on macrophages isolated from the peritoneal cavity in patients with peritonitis but not in healthy controls. Using a murine peritonitis/sepsis model, we identified increased Connexin-43 expression in peritoneal and hepatic macrophages. Conditional Lyz2cre/creGja1flox/flox mice were developed to specifically assess Connexin-43 impact in macrophages. Both macrophage-specific Connexin-43 deletion and pharmacological Connexin-43 blockade were associated with reduced cytokine secretion by macrophages in response to LPS and CLP, ultimately resulting in increased survival. In conclusion, inhibition of autocrine Connexin-43-dependent ATP signalling on macrophages improves sepsis outcome.

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