P2X7 receptor cross-talk regulates ATP-induced pannexin 1 internalization

2017 ◽  
Vol 474 (13) ◽  
pp. 2133-2144 ◽  
Author(s):  
Andrew K.J. Boyce ◽  
Leigh Anne Swayne

In the nervous system, extracellular ATP levels transiently increase in physiological and pathophysiological circumstances, effecting key signalling pathways in plasticity and inflammation through purinergic receptors. Pannexin 1 (Panx1) forms ion- and metabolite-permeable channels that mediate ATP release and are particularly enriched in the nervous system. Our recent study demonstrated that elevation of extracellular ATP triggers Panx1 internalization in a concentration- and time-dependent manner. Notably, this effect was sensitive to inhibition of ionotropic P2X7 purinergic receptors (P2X7Rs). Here, we report our novel findings from the detailed investigation of the mechanism underlying P2X7R–Panx1 cross-talk in ATP-stimulated internalization. We demonstrate that extracellular ATP triggers and is required for the clustering of P2X7Rs and Panx1 on Neuro2a cells through an extracellular physical interaction with the Panx1 first extracellular loop (EL1). Importantly, disruption of P2X7R–Panx1 clustering by mutation of tryptophan 74 within the Panx1 EL1 inhibits Panx1 internalization. Notably, P2X7R–Panx1 clustering and internalization are independent of P2X7R-associated intracellular signalling pathways (Ca2+ influx and Src activation). Further analysis revealed that cholesterol is required for ATP-stimulated P2X7R–Panx1 clustering at the cell periphery. Taken together, our data suggest that extracellular ATP induces and is required for Panx1 EL1-mediated, cholesterol-dependent P2X7R–Panx1 clustering and endocytosis. These findings have important implications for understanding the role of Panx1 in the nervous system and provide important new insights into Panx1–P2X7R cross-talk.

2018 ◽  
Author(s):  
Marco Tozzi ◽  
Jacob B. Hansen ◽  
Ivana Novak

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.


2014 ◽  
Vol 82 (12) ◽  
pp. 5076-5085 ◽  
Author(s):  
Hua Ren ◽  
Yunfei Teng ◽  
Binghe Tan ◽  
Xiaoyu Zhang ◽  
Wei Jiang ◽  
...  

ABSTRACTExtracellular ATP (eATP), released as a “danger signal” by injured or stressed cells, plays an important role in the regulation of immune responses, but the relationship between ATP release and innate immune responses is still uncertain. In this study, we demonstrated that ATP was released through Toll-like receptor (TLR)-associated signaling in bothEscherichia coli-infected mice and lipopolysaccharide (LPS)- or Pam3CSK4-treated macrophages. This ATP release could be blocked completely only byN-ethylmaleimide (NEM), not by carbenoxolone (CBX), flufenamic acid (FFA), or probenecid, suggesting the key role of exocytosis in this process. Furthermore, LPS-induced ATP release could also be reduced dramatically through suppressing calcium mobilization by use of U73122, caffeine, and thapsigargin (TG). In addition, the secretion of interleukin-1β (IL-1β) and CCL-2 was enhanced significantly by ATP, in a time- and dose-dependent manner. Meanwhile, macrophage-mediated phagocytosis of bacteria was also promoted significantly by ATP stimulation. Furthermore, extracellular ATP reduced the number of invading bacteria and protected mice from peritonitis by activating purinergic receptors. Mechanistically, phosphorylation of AKT and ERK was overtly increased by ATP in antibacterial immune responses. Accordingly, if we blocked the P2X- and P2Y-associated signaling pathway by using suramin and pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid), tetrasodium salt (PPADS), the ATP-enhanced immune response was restrained significantly. Taken together, our findings reveal an internal relationship between danger signals and TLR signaling in innate immune responses, which suggests a potential therapeutic significance of calcium mobilization-mediated ATP release in infectious diseases.


1989 ◽  
Vol 67 (9) ◽  
pp. 495-502 ◽  
Author(s):  
Chakib El-Moatassim ◽  
Nicole Bernad ◽  
Jean-Claude Mani ◽  
Jacques Dornand

We have previously demonstrated that extracellular ATP can give medullary thymocytes the calcium message required for the induction of their blastogenesis, without mobilization of intracellular calcium. We describe here the effects of extracellular nucleotides on membrane permeability to monovalent and divalent cations in mouse thymocytes. Among all nucleotides tested, under physiological conditions, only ATP and, to a lesser extent, 2-methylthio-ATP, adenosine 5′-O-(3-thio-triphosphate), and ADP were able to depolarize thymocyte plasma membranes and to induce Na+ and Ca2+ influxes into thymocytes; other nonhydrolysable ATP analogs were only effective in the absence of Mg2+. The ATP-induced effects were inhibited in a dose-dependent manner by Mg2+ and greatly potentiated in its absence, which suggests that the tetrabasic ATP4− is probably the active species and that a phosphotransferase activity is not involved in its effects. These ATP-mediated changes in ion fluxes result from an increase in nonspecific permeability of thymocyte membranes, probably by pore formation. These ion flux changes might be responsible for the mitogenic induction of phorbol 12-myristate 13-acetate treated medullary thymocytes. The potency order for the adenine derivatives to affect these fluxes (ATP>ADP> >AMP>adenosine) suggests the presence of ATP specific receptors (P2 purinergic receptors) on thymocyte plasma membranes.Key words: purinergic receptors, extracellular ATP, membrane potential, cation fluxes, thymocytes.


2011 ◽  
Vol 208 (9) ◽  
pp. 1823-1834 ◽  
Author(s):  
Claire Séror ◽  
Marie-Thérèse Melki ◽  
Frédéric Subra ◽  
Syed Qasim Raza ◽  
Marlène Bras ◽  
...  

Extracellular adenosine triphosphate (ATP) can activate purinergic receptors of the plasma membrane and modulate multiple cellular functions. We report that ATP is released from HIV-1 target cells through pannexin-1 channels upon interaction between the HIV-1 envelope protein and specific target cell receptors. Extracellular ATP then acts on purinergic receptors, including P2Y2, to activate proline-rich tyrosine kinase 2 (Pyk2) kinase and transient plasma membrane depolarization, which in turn stimulate fusion between Env-expressing membranes and membranes containing CD4 plus appropriate chemokine co-receptors. Inhibition of any of the constituents of this cascade (pannexin-1, ATP, P2Y2, and Pyk2) impairs the replication of HIV-1 mutant viruses that are resistant to conventional antiretroviral agents. Altogether, our results reveal a novel signaling pathway involved in the early steps of HIV-1 infection that may be targeted with new therapeutic approaches.


2008 ◽  
Vol 40 (1) ◽  
pp. 19 ◽  
Author(s):  
Dong Reoyl Seo ◽  
Soo Yoon Kim ◽  
Kyung You Kim ◽  
Hwan Goo Lee ◽  
Ju Hyun Moon ◽  
...  

1996 ◽  
Vol 320 (2) ◽  
pp. 499-504 ◽  
Author(s):  
Carlo FORESTA ◽  
Marco ROSSATO ◽  
Andrea NOGARA ◽  
Francesco GOTTARDELLO ◽  
Paola BORDON ◽  
...  

The present study investigated the effects of extracellular ATP on the intracellular calcium ion concentration ([Ca2+]i) and testosterone production in isolated adult rat Leydig cells. This nucleotide caused an increase in [Ca2+]i, with a maximal effect at a concentration of 100 µM ATP, comprising a rapid initial spike followed by a long-lasting plateau. The first rapid spike was dependent on the release of Ca2+ from internal stores, since it also occurred in Ca2+-free medium and was abolished after depletion of internal stores with thapsigargin. The second, long-lasting, phase was dependent on the influx of Ca2+ from the extracellular medium. After 3 h of incubation, extracellular ATP stimulated testosterone secretion in a dose-dependent manner, with a maximal effect at 100 µM. Activation of steroidogenesis by ATP was fully dependent on the presence of Ca2+ in the external medium. Among different nucleotides, only ATP, adenosine 5´-[γ-thio]triphosphate, UTP, benzoylbenzoic-ATP and 2-methylthio-ATP were effective in inducing both the rise in [Ca2+]i and testosterone secretion. These effects were blocked by preincubation of Leydig cells with oxidized ATP, an inhibitor of the P2Z-purinergic receptor subtype. These results show that rat Leydig cells possess P2-purinergic receptors whose activation triggers an increase in [Ca2+]i due to the release of Ca2+ from internal stores and Ca2+ influx from the external medium. The stimulatory effect of extracellular ATP on testosterone secretion seems to be coupled to the influx of Ca2+ from the external medium.


2009 ◽  
Vol 297 (5) ◽  
pp. L954-L964 ◽  
Author(s):  
Heather N. Woodward ◽  
Adil Anwar ◽  
Suzette Riddle ◽  
Laimute Taraseviciene-Stewart ◽  
Miguel Fragoso ◽  
...  

We recently reported that vasa vasorum expansion occurs in the pulmonary artery (PA) adventitia of chronically hypoxic animals and that extracellular ATP is a pro-angiogenic factor for isolated vasa vasorum endothelial cells (VVEC). However, the sources of extracellular ATP in the PA vascular wall, as well as the molecular mechanisms underlying its release, remain elusive. Studies were undertaken to explore whether VVEC release ATP in response to hypoxia and to determine signaling pathways involved in this process. We found that hypoxia (1–3% O2) resulted in time- and O2-dependent ATP release from VVEC. Preincubation with the inhibitors of vesicular transport (monensin, brefeldin A, and N-ethylmaleimide) significantly decreased ATP accumulation in the VVEC conditioned media, suggesting that hypoxia-induced ATP release occurs through vesicular exocytosis. Additionally, both hypoxia and exogenously added ATP resulted in the activation of PI3K and accumulation of GTP-bound RhoA in a time-dependent manner. Pharmacological inhibition of PI3K and ROCK or knockout of RhoA by small interfering RNA significantly abolished hypoxia-induced ATP release from VVEC. Moreover, RhoA and ROCK play a critical role in ATP-induced increases in VVEC DNA synthesis, migration, and tube formation, indicating a functional contribution of PI3K, Rho, and ROCK to both the autocrine mechanism of ATP release and ATP-mediated angiogenic activation of VVEC. Taken together, our findings provide novel evidence for the signaling mechanisms that link hypoxia-induced increases in extracellular ATP and vasa vasorum expansion.


2012 ◽  
Vol 303 (10) ◽  
pp. C1034-C1044 ◽  
Author(s):  
Feng Xiao ◽  
Shar L. Waldrop ◽  
Al-karim Khimji ◽  
Gordan Kilic

Hepatocyte lipoapoptosis induced by saturated free fatty acids (FFA) contributes to hepatic inflammation in lipotoxic liver injury, and the cellular mechanisms involved have not been defined. Recent studies have shown that apoptosis in nonhepatic cells stimulates ATP release via activation of pannexin1 (panx1), and extracellular ATP functions as a proinflammatory signal for recruitment and activation of the inflammatory cells. However, it is not known whether lipoapoptosis stimulates ATP release in liver cells. We found that lipoapoptosis induced by saturated FFA stimulated ATP release in liver cells that increased extracellular ATP concentration by more than fivefold above the values observed in healthy cells. This sustained pathophysiological ATP release was not dependent on caspase-3/7 activation. Inhibition of c-Jun NH2-terminal kinase (JNK), a key mediator of lipoapoptosis, with SP600125 blocked pathophysiological ATP release in a dose-dependent manner. RT-PCR analysis indicated that panx1 is expressed in hepatocytes and multiple liver cell lines. Notably, inhibition of panx1 expression with short hairpin (sh)RNA inhibited in part pathophysiological ATP release. Moreover, lipoapoptosis stimulated uptake of a membrane impermeable dye YoPro-1 (indicative of panx1 activation), which was inhibited by panx1 shRNA, probenecid, and mefloquine. These results suggest that panx1 contributes to pathophysiological ATP release in lipoapoptosis induced by saturated FFA. Thus panx1 may play an important role in hepatic inflammation by mediating an increase in extracellular ATP concentration in lipotoxic liver injury.


10.1085/ecc41 ◽  
2021 ◽  
Vol 154 (9) ◽  
Author(s):  
Mariana Casas ◽  
Gonzalo Jorquera ◽  
Camilo Morales ◽  
Enrique Jaimovich

An important question in neuromuscular biology is how skeletal muscle cells decipher the stimulation pattern coming from motoneurons to define their phenotype-activating transcriptional changes in a process named excitation–transcription coupling. We have shown in adult muscle fibers that 20 Hz electrical stimulation (ES) activates a signaling cascade that starts with Cav1.1 activation, ATP release trough pannexin-1 channel, activation of purinergic receptors, and IP3-dependent Ca2+ signals inducing transcriptional changes related to muscle plasticity from fast to slow phenotype. Extracellular addition of 30 µM ATP mimics transcriptional changes induced by ES at 20 Hz. ATP release occurs in two peaks, the first around 15 s after ES and a second around 300 s after ES. In the present work, we used apyrase to hydrolyze ATP 60 s after ES, maintaining the first peak and eliminating the second peak. In this condition, transcriptional changes were abolished, indicating that the second peak is the one crucial to activate transcription. Additionally, we observed a small depolarization of fibers after ES. The addition of 30 to 100 µM external ATP also induced depolarization of muscle fibers. This depolarization was unable to activate contraction but was able to induce transcriptional changes induced by 20 Hz ES. These changes were completely inhibited by the IP3R blocker xestospongin B, suggesting that IP3-dependent events are triggered at these membrane depolarization values. Moreover, transcriptional changes induced by addition of 30 µM extracellular ATP was blocked by incubation of fibers with 25 µM Nifedipine. These results suggest that the second ATP peak observed after 20 Hz ES is responsible for transcriptional activation by inducing small depolarizations of fiber membranes that are also sensed by Cav1.1. Finally, we show evidence that downstream of purinergic receptors, PKC is activated, likely causing phosphorylation of ClC-1 chloride channels, possibly responsible for depolarization after 20 Hz.


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