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Pannexin1 (Panx1) ATP channels are important in adipocyte biology, potentially influencing energy storage and expenditure. We compared the metabolic phenotype of young (14 weeks old) and mature (20 weeks old) wild-type (WT) and Panx1−/− mice exposed or not to cold (6 °C) during 28 days, a condition promoting adipocyte browning. Young Panx1−/− mice weighed less and exhibited increased fat mass, improved glucose tolerance, and lower insulin sensitivity than WT mice. Their energy expenditure and respiratory exchange ratio (RER) were increased, and their fatty acid oxidation decreased. These metabolic effects were no longer observed in mature Panx1−/− mice. The exposure of mature mice to cold exacerbated their younger metabolic phenotype. The white adipose tissue (WAT) of cold-exposed Panx1−/− mice contained more small-sized adipocytes, but, in contrast to WT mice, white adipocytes did not increase their expression of Ucp1 nor of other markers of browning adipocytes. Interestingly, Glut4 expression was already enhanced in the WAT of young Panx1−/− mice kept at 22 °C as compared to WT mice. Thus, Panx1 deletion exerts overall beneficial metabolic effects in mice that are pre-adapted to chronic cold exposure. Panx1−/− mice show morphological characteristics of WAT browning, which are exacerbated upon cold exposure, an effect that appears to be associated with Ucp1-independent thermogenesis. [ABSTRACT FROM AUTHOR]

The lymphatic network of capillaries and collecting vessels ensures tissue fluid homeostasis, absorption of dietary fats and trafficking of immune cells. Pannexin1 (Panx1) channels allow for the passage of ions and small metabolites between the cytosol and extracellular environment. Panx1 channels regulate the pathophysiological function of several tissues in a sex‐dependent manner. Here, we studied the role of Panx1 in lymphatic function, and potential sex‐dependent differences therein, in Prox1‐CreERT2Panx1fl/fl and Panx1fl/fl control mice. Panx1 expression was higher in lymphatic endothelial cells (LECs) of male mice. Lymphatic vessel morphology was not affected in Prox1‐CreERT2Panx1fl/fl male and female mice. Lymphatic drainage was decreased by 25% in male Prox1‐CreERT2Panx1fl/fl mice, but was similar in females of both genotypes. Accordingly, only male Prox1‐CreERT2Panx1fl/fl mice exhibited tail swelling, pointing to interstitial fluid accumulation in males upon Panx1 deletion in LECs. Moreover, serum triglyceride and free fatty acid levels raised less in Prox1‐CreERT2Panx1fl/fl mice of both sexes in an oral lipid tolerance test. Finally, the percentage of migratory dendritic cells arriving in draining lymph nodes was increased in Prox1‐CreERT2Panx1fl/fl female mice, but was comparable between male mice of both genotypes. Our results point to a LEC‐specific role for Panx1 in the functions of the lymphatic system. [ABSTRACT FROM AUTHOR]

Many immunological diseases can be treated by regulating neurobehavior, in which extracellular ATP is a vital member of endogenous danger-associated molecular pattern signaling molecule that plays a crucial part in innate neuro-related immunity. It is actively released through pannexin (Panx) and connexin (Cx) hemichannels from activated or stressed cells during inflammation, injury, or apoptosis. In addition to participating in ATP release, Panxs and Cxs also have crucial immune functions. In this study, pannexin1, three connexin32 isoforms and connexin43 were identified and characterized in spotted sea bass (Lateolabrax maculatus), which were named LmPanx1, LmCx32.2, LmCx32.3, LmCx32.7, and LmCx43. Their similar topological structures were discovered by sequence analysis: a relatively unconserved C-terminal region and four highly conserved transmembrane (TM) domains, and so on. Each extracellular (ECL) region of Panx1 has two conserved cysteine residues. Unlike Panx1, each ECL region of Cx32 and Cx43 contains three conserved cysteine residues, forming two conserved motifs: CX6CX3C motif in ECL1 and CX4CX5C motif in ECL2. Furthermore, Panx1 and Cx43 share similar genomic organization and synteny with their counterparts in selected vertebrates. Cx32 and CX43 were located in the same locus in fish, but diverged into two loci from amphibian. Moreover, despite varying expression levels, the identified genes were constitutively expressed in all examined tissues. All genes were upregulated by PAMP [lipopolysaccharide and poly(I:C)] stimulation or bacterial infection in vivo and in vitro, but they were downregulated in the brain at 6 or 12 h after stimulation. Especially, the three LmCx32 isoforms and LmCx43 were upregulated by ATP stimulation in primary head kidney leukocytes; however, downregulation of LmCx32.3 and LmCx43 expression were noted at 12 h. Conversely, ATP treatment inhibited the expression of LmPanx1. Importantly, we showed that the spotted sea bass Panx1, Cx43, and Cx32 were localized on the cellular membrane and involved in inflammation-induced ATP release. Taken together, our results demonstrated that Panx1, Cx32, and Cx43 are important neurorelated immune response genes involved in inflammation-induced ATP release. [ABSTRACT FROM AUTHOR]

Summary: Neonatal platelets present a reduced response to the platelet agonist, thrombin (Thr), thus resulting in a deficient Thr‐induced aggregation. These alterations are more pronounced in premature newborns. Here, our aim was to uncover the causes underneath the impaired Ca2+ homeostasis described in neonatal platelets. Both Ca2+ mobilization and Ca2+ influx in response to Thr are decreased in neonatal platelets compared to maternal and control woman platelets. In neonatal platelets, we observed impaired Ca2+ mobilization in response to the PAR‐1 agonist (SFLLRN) or by blocking SERCA3 function with tert‐butylhydroquinone. Regarding SOCE, the STIM1 regulatory protein, SARAF, was found overexpressed in neonatal platelets, promoting an increase in STIM1/SARAF interaction even under resting conditions. Additionally, higher interaction between SARAF and PDCD61/ALG2 was also observed, reducing SARAF ubiquitination and prolonging its half‐life. These results were reproduced by overexpressing SARAF in MEG01 and DAMI cells. Finally, we also observed that pannexin 1 permeability is enhanced in response to Thr in control woman and maternal platelets, but not in neonatal platelets, hence, leading to the deregulation of the Ca2+ entry found in neonatal platelets. Summarizing, we show that in neonatal platelets both Ca2+ accumulation in the intracellular stores and Thr‐evoked Ca2+ entry through either capacitative channels or non‐selective channels are altered in neonatal platelets, contributing to deregulated Ca2+ homeostasis in neonatal platelets and leading to the altered aggregation observed in these subjects. [ABSTRACT FROM AUTHOR]

Abstract The lymphatic network of capillaries and collecting vessels ensures tissue fluid homeostasis, absorption of dietary fats and trafficking of immune cells. Pannexin1 (Panx1) channels allow for the passage of ions and small metabolites between the cytosol and extracellular environment. Panx1 channels regulate the pathophysiological function of several tissues in a sex‐dependent manner. Here, we studied the role of Panx1 in lymphatic function, and potential sex‐dependent differences therein, in Prox1‐CreERT2Panx1fl/fl and Panx1fl/fl control mice. Panx1 expression was higher in lymphatic endothelial cells (LECs) of male mice. Lymphatic vessel morphology was not affected in Prox1‐CreERT2Panx1fl/fl male and female mice. Lymphatic drainage was decreased by 25% in male Prox1‐CreERT2Panx1fl/fl mice, but was similar in females of both genotypes. Accordingly, only male Prox1‐CreERT2Panx1fl/fl mice exhibited tail swelling, pointing to interstitial fluid accumulation in males upon Panx1 deletion in LECs. Moreover, serum triglyceride and free fatty acid levels raised less in Prox1‐CreERT2Panx1fl/fl mice of both sexes in an oral lipid tolerance test. Finally, the percentage of migratory dendritic cells arriving in draining lymph nodes was increased in Prox1‐CreERT2Panx1fl/fl female mice, but was comparable between male mice of both genotypes. Our results point to a LEC‐specific role for Panx1 in the functions of the lymphatic system.

To explore the processes of epileptogenesis/ictogenesis, this study determined the age-dependent development of the functional abnormalities in astroglial transmission associated with pannexin1-hemichannel using a genetic rat model of autosomal dominant sleep-related hypermotor epilepsy (ADSHE) named 'S286L-TG'. Pannexin1 expression in the plasma membrane of primary cultured cortical astrocytes and the orbitofrontal cortex (OFC), which is an ADSHE focus region, were determined using capillary immunoblotting. Astroglial D-serine releases induced by artificial high-frequency oscillation (HFO)-evoked stimulation, the removal of extracellular Ca2+, and the P2X7 receptor agonist (BzATP) were determined using ultra-high performance liquid chromatography (UHPLC). The expressions of pannexin1 in the plasma membrane fraction of the OFC in S286L-TG at four weeks old were almost equivalent when compared to the wild type. The pannexin1 expression in the OFC of the wild type non-statistically decreased age-dependently, whereas that in S286L-TG significantly increased age-dependently, resulting in relatively increasing pannexin1 expression from the 7- (at the onset of interictal discharge) and 10-week-old (after the ADSHE seizure onset) S286L-TG compared to the wild type. However, no functional abnormalities of astroglial pannexin1 expression or D-serine release through the pannexin1-hemichannels from the cultured astrocytes of S286L-TG could be detected. Acutely HFO-evoked stimulation, such as physiological ripple burst (200 Hz) and epileptogenic fast ripple burst (500 Hz), frequency-dependently increased both pannexin1 expression in the astroglial plasma membrane and astroglial D-serine release. Neither the selective inhibitors of pannexin1-hemichannel (10PANX) nor connexin43-hemichannel (Gap19) affected astroglial D-serine release during the resting stage, whereas HFO-evoked D-serine release was suppressed by both inhibitors. The inhibitory effect of 10PANX on the ripple burst-evoked D-serine release was more predominant than that of Gap19, whereas fast ripple burst-evoked D-serine release was predominantly suppressed by Gap19 rather than 10PANX. Astroglial D-serine release induced by acute exposure to BzATP was suppressed by 10PANX but not by Gap19. These results suggest that physiological ripple burst during the sleep spindle plays important roles in the organization of some components of cognition in healthy individuals, but conversely, it contributes to the initial development of epileptogenesis/ictogenesis in individuals who have ADSHE vulnerability via activation of the astroglial excitatory transmission associated with pannexin1-hemichannels. [ABSTRACT FROM AUTHOR]

Abstract Background The opening of pannexin1 channels is considered as a key event in inflammation. Pannexin1 channel-mediated release of adenosine triphosphate triggers inflammasome signaling and activation of immune cells. By doing so, pannexin1 channels play an important role in several inflammatory diseases. Although pannexin1 channel inhibition could represent a novel clinical strategy for treatment of inflammatory disorders, therapeutic pannexin1 channel targeting is impeded by the lack of specific, potent and/or in vivo-applicable inhibitors. The goal of this study is to generate nanobody-based inhibitors of pannexin1 channels. Results Pannexin1-targeting nanobodies were developed as potential new pannexin1 channel inhibitors. We identified 3 cross-reactive nanobodies that showed affinity for both murine and human pannexin1 proteins. Flow cytometry experiments revealed binding capacities in the nanomolar range. Moreover, the pannexin1-targeting nanobodies were found to block pannexin1 channel-mediated release of adenosine triphosphate. The pannexin1-targeting nanobodies were also demonstrated to display anti-inflammatory effects in vitro through reduction of interleukin 1 beta amounts. This anti-inflammatory outcome was reproduced in vivo using a human-relevant mouse model of acute liver disease relying on acetaminophen overdosing. More specifically, the pannexin1-targeting nanobodies lowered serum levels of inflammatory cytokines and diminished liver damage. These effects were linked with alteration of the expression of several NLRP3 inflammasome components. Conclusions This study introduced for the first time specific, potent and in vivo-applicable nanobody-based inhibitors of pannexin1 channels. As demonstrated for the case of liver disease, the pannexin1-targeting nanobodies hold great promise as anti-inflammatory agents, yet this should be further tested for extrahepatic inflammatory disorders. Moreover, the pannexin1-targeting nanobodies represent novel tools for fundamental research regarding the role of pannexin1 channels in pathological and physiological processes. Graphical Abstract

Pannexin1 (Panx1) ATP channels are important in adipocyte biology, potentially influencing energy storage and expenditure. We compared the metabolic phenotype of young (14 weeks old) and mature (20 weeks old) wild-type (WT) and Panx1−/− mice exposed or not to cold (6 °C) during 28 days, a condition promoting adipocyte browning. Young Panx1−/− mice weighed less and exhibited increased fat mass, improved glucose tolerance, and lower insulin sensitivity than WT mice. Their energy expenditure and respiratory exchange ratio (RER) were increased, and their fatty acid oxidation decreased. These metabolic effects were no longer observed in mature Panx1−/− mice. The exposure of mature mice to cold exacerbated their younger metabolic phenotype. The white adipose tissue (WAT) of cold-exposed Panx1−/− mice contained more small-sized adipocytes, but, in contrast to WT mice, white adipocytes did not increase their expression of Ucp1 nor of other markers of browning adipocytes. Interestingly, Glut4 expression was already enhanced in the WAT of young Panx1−/− mice kept at 22 °C as compared to WT mice. Thus, Panx1 deletion exerts overall beneficial metabolic effects in mice that are pre-adapted to chronic cold exposure. Panx1−/− mice show morphological characteristics of WAT browning, which are exacerbated upon cold exposure, an effect that appears to be associated with Ucp1-independent thermogenesis.

Background: The opening of pannexin1 channels is considered as a key event in inflammation. Pannexin1 channel-mediated release of adenosine triphosphate triggers inflammasome signaling and activation of immune cells. By doing so, pannexin1 channels play an important role in several inflammatory diseases. Although pannexin1 channel inhibition could represent a novel clinical strategy for treatment of inflammatory disorders, therapeutic pannexin1 channel targeting is impeded by the lack of specific, potent and/or in vivo-applicable inhibitors. The goal of this study is to generate nanobody-based inhibitors of pannexin1 channels. Results: Pannexin1-targeting nanobodies were developed as potential new pannexin1 channel inhibitors. We identified 3 cross-reactive nanobodies that showed affinity for both murine and human pannexin1 proteins. Flow cytometry experiments revealed binding capacities in the nanomolar range. Moreover, the pannexin1-targeting nanobodies were found to block pannexin1 channel-mediated release of adenosine triphosphate. The pannexin1-targeting nanobodies were also demonstrated to display anti-inflammatory effects in vitro through reduction of interleukin 1 beta amounts. This anti-inflammatory outcome was reproduced in vivo using a human-relevant mouse model of acute liver disease relying on acetaminophen overdosing. More specifically, the pannexin1-targeting nanobodies lowered serum levels of inflammatory cytokines and diminished liver damage. These effects were linked with alteration of the expression of several NLRP3 inflammasome components. Conclusions: This study introduced for the first time specific, potent and in vivo-applicable nanobody-based inhibitors of pannexin1 channels. As demonstrated for the case of liver disease, the pannexin1-targeting nanobodies hold great promise as anti-inflammatory agents, yet this should be further tested for extrahepatic inflammatory disorders. Moreover, the pannexin1-targeting nanobodies represent novel tools for fundamental research regarding the role of pannexin1 channels in pathological and physiological processes. [ABSTRACT FROM AUTHOR]

Aims No effective therapy is available in clinics to protect the heart from ischaemia/reperfusion (I/R) injury. Endothelial cells are activated after I/R, which may drive the inflammatory response by releasing ATP through pannexin1 (Panx1) channels. Here, we investigated the role of Panx1 in cardiac I/R. Methods and results Panx1 was found in cardiac endothelial cells, neutrophils, and cardiomyocytes. After in vivo I/R, serum Troponin-I, and infarct size were less pronounced in Panx1−/− mice, but leukocyte infiltration in the infarct area was similar between Panx1−/− and wild-type mice. Serum Troponin-I and infarct size were not different between mice with neutrophil-specific deletion of Panx1 and Panx1fl/fl mice, suggesting that cardioprotection by Panx1 deletion rather involved cardiomyocytes than the inflammatory response. Physiological cardiac function in wild-type and Panx1−/− hearts was similar. The time to onset of contracture and time to maximal contracture were delayed in Panx1−/− hearts, suggesting reduced sensitivity of these hearts to ischaemic injury. Moreover, Panx1−/− hearts showed better recovery of left ventricle developed pressure, cardiac contractility, and relaxation after I/R. Ischaemic preconditioning failed to confer further protection in Panx1−/− hearts. Panx1 was found in subsarcolemmal mitochondria (SSM). SSM in WT or Panx1−/− hearts showed no differences in morphology. The function of the mitochondrial permeability transition pore and production of reactive oxygen species in SSM was not affected, but mitochondrial respiration was reduced in Panx1−/− SSM. Finally, Panx1−/− cardiomyocytes had a decreased mitochondrial membrane potential and an increased mitochondrial ATP content. Conclusion Panx1−/− mice display decreased sensitivity to cardiac I/R injury, resulting in smaller infarcts and improved recovery of left ventricular function. This cardioprotective effect of Panx1 deletion seems to involve cardiac mitochondria rather than a reduced inflammatory response. Thus, Panx1 may represent a new target for controlling cardiac reperfusion damage. [ABSTRACT FROM AUTHOR]

Pannexin1 proteins form communication channels at the cell plasma membrane surface, which allow the transfer of small molecules and ions between the intracellular compartment and extracellular environment. In this way, pannexin1 channels play an important role in various cellular processes and diseases. Indeed, a plethora of human pathologies is associated with the activation of pannexin1 channels. The present paper reviews and summarizes the structure, life cycle, regulation and (patho)physiological roles of pannexin1 channels, with a particular focus on the relevance of pannexin1 channels in liver diseases.

Apoptotic cell death is a critical step in organism development and tissue homeostasis. Apoptotic cells affect immune cell activities in normal tissues. It is not clear whether similar cell death machinery causes tumor environments to evade anti-tumor immune responses. Here, using a mouse transplant model, we found a large number of tumor cells undergoing intrinsic apoptosis in tumors derived from the 4T1 breast cancer cell line, where neutrophils significantly accumulated. Interestingly, these apoptotic 4T1 tumor cells directly induced neutrophil extracellular traps (NETs) in a pannexin 1 (Panx1) channel-dependent manner, and knockdown of Panx1 in 4T1 cells led to a reduction in tumor size. Spermidine released through Panx1 from apoptotic 4T1 cells induced NETs in bone marrow-derived neutrophils in vitro. In addition, inhibition of spermidine synthesis suppressed tumor growth in the mouse transplant model. Collectively, our data suggested a new immune-escape mechanism for tumors by Panx1-mediated secretome from intrinsic apoptotic cells, which may provide a new therapeutic target for cancer. • 4T1 breast tumor cells undergo a high frequency of intrinsic apoptosis. • Secretome from apoptotic 4T1 cells induce neutrophils extracellular traps. • Pannexin1 is indispensable to architect the unique tumor immune microenvironments. [ABSTRACT FROM AUTHOR]

This document is a corrigendum published in the journal Frontiers in Immunology. It corrects an error in Figure 8 of a previously published article titled "Characterization of pannexin1, connexin32, and connexin43 in spotted sea bass (Lateolabrax maculatus): they are important neuro-related immune response genes involved in inflammation-induced ATP release." The authors apologize for the error and state that it does not change the scientific conclusions of the article. The corrected figure and its caption are provided in the corrigendum. [Extracted from the article]

Although many efforts have been made to elucidate the pathogenesis of COVID-19, the underlying mechanisms are yet to be fully uncovered. However, it is known that a dysfunctional immune response and the accompanying uncontrollable inflammation lead to troublesome outcomes in COVID-19 patients. Pannexin1 channels are put forward as interesting drug targets for the treatment of COVID-19 due to their key role in inflammation and their link to other viral infections. In the present study, we selected a panel of drugs previously tested in clinical trials as potential candidates for the treatment of COVID-19 early on in the pandemic, including hydroxychloroquine, chloroquine, azithromycin, dexamethasone, ribavirin, remdesivir, favipiravir, lopinavir, and ritonavir. The effect of the drugs on pannexin1 channels was assessed at a functional level by means of measurement of extracellular ATP release. Immunoblot analysis and real-time quantitative reversetranscription polymerase chain reaction analysis were used to study the potential of the drugs to alter pannexin1 protein and mRNA expression levels, respectively. Favipiravir, hydroxychloroquine, lopinavir, and the combination of lopinavir with ritonavir were found to inhibit pannexin1 channel activity without affecting pannexin1 protein or mRNA levels. Thusthree new inhibitors of pannexin1 channels were identified that, though currently not being used anymore for the treatment of COVID-19 patients, could be potential drug candidates for other pannexin1-related diseases. [ABSTRACT FROM AUTHOR]

Many immunological diseases can be treated by regulating neurobehavior, in which extracellular ATP is a vital member of endogenous danger-associated molecular pattern signaling molecule that plays a crucial part in innate neuro-related immunity. It is actively released through pannexin (Panx) and connexin (Cx) hemichannels from activated or stressed cells during inflammation, injury, or apoptosis. In addition to participating in ATP release, Panxs and Cxs also have crucial immune functions. In this study, pannexin1, three connexin32 isoforms and connexin43 were identified and characterized in spotted sea bass (Lateolabrax maculatus), which were named Lm Panx1, Lm Cx32.2, Lm Cx32.3, Lm Cx32.7, and Lm Cx43. Their similar topological structures were discovered by sequence analysis: a relatively unconserved C-terminal region and four highly conserved transmembrane (TM) domains, and so on. Each extracellular (ECL) region of Panx1 has two conserved cysteine residues. Unlike Panx1, each ECL region of Cx32 and Cx43 contains three conserved cysteine residues, forming two conserved motifs: CX6CX3C motif in ECL1 and CX4CX5C motif in ECL2. Furthermore, Panx1 and Cx43 share similar genomic organization and synteny with their counterparts in selected vertebrates. Cx32 and CX43 were located in the same locus in fish, but diverged into two loci from amphibian. Moreover, despite varying expression levels, the identified genes were constitutively expressed in all examined tissues. All genes were upregulated by PAMP [lipopolysaccharide and poly(I:C)] stimulation or bacterial infection in vivo and in vitro , but they were downregulated in the brain at 6 or 12 h after stimulation. Especially, the three Lm Cx32 isoforms and Lm Cx43 were upregulated by ATP stimulation in primary head kidney leukocytes; however, downregulation of Lm Cx32.3 and Lm Cx43 expression were noted at 12 h. Conversely, ATP treatment inhibited the expression of Lm Panx1. Importantly, we showed that the spotted sea bass Panx1, Cx43, and Cx32 were localized on the cellular membrane and involved in inflammation-induced ATP release. Taken together, our results demonstrated that Panx1, Cx32, and Cx43 are important neuro-related immune response genes involved in inflammation-induced ATP release. [ABSTRACT FROM AUTHOR]

Many immunological diseases can be treated by regulating neurobehavior, in which extracellular ATP is a vital member of endogenous danger-associated molecular pattern signaling molecule that plays a crucial part in innate neuro-related immunity. It is actively released through pannexin (Panx) and connexin (Cx) hemichannels from activated or stressed cells during inflammation, injury, or apoptosis. In addition to participating in ATP release, Panxs and Cxs also have crucial immune functions. In this study, pannexin1, three connexin32 isoforms and connexin43 were identified and characterized in spotted sea bass (Lateolabrax maculatus), which were named LmPanx1, LmCx32.2, LmCx32.3, LmCx32.7, and LmCx43. Their similar topological structures were discovered by sequence analysis: a relatively unconserved C-terminal region and four highly conserved transmembrane (TM) domains, and so on. Each extracellular (ECL) region of Panx1 has two conserved cysteine residues. Unlike Panx1, each ECL region of Cx32 and Cx43 contains three conserved cysteine residues, forming two conserved motifs: CX6CX3C motif in ECL1 and CX4CX5C motif in ECL2. Furthermore, Panx1 and Cx43 share similar genomic organization and synteny with their counterparts in selected vertebrates. Cx32 and CX43 were located in the same locus in fish, but diverged into two loci from amphibian. Moreover, despite varying expression levels, the identified genes were constitutively expressed in all examined tissues. All genes were upregulated by PAMP [lipopolysaccharide and poly(I:C)] stimulation or bacterial infection in vivo and in vitro, but they were downregulated in the brain at 6 or 12 h after stimulation. Especially, the three LmCx32 isoforms and LmCx43 were upregulated by ATP stimulation in primary head kidney leukocytes; however, downregulation of LmCx32.3 and LmCx43 expression were noted at 12 h. Conversely, ATP treatment inhibited the expression of LmPanx1. Importantly, we showed that the spotted sea bass Panx1, Cx43, and Cx32 were localized on the cellular membrane and involved in inflammation-induced ATP release. Taken together, our results demonstrated that Panx1, Cx32, and Cx43 are important neuro-related immune response genes involved in inflammation-induced ATP release.

Pannexin1 hemichannels (Panx1 HCs) are found in the membrane of most mammalian cells and communicate the intracellular and extracellular spaces, enabling the passive transfer of ions and small molecules. They are involved in physiological and pathophysiological conditions. During apoptosis, the C-terminal tail of Panx1 is proteolytically cleaved, but the permeability features of hemichannels and their role in cell death remain elusive. To address these topics, HeLa cells transfected with full-length human Panx1 (fl-hPanx1) or C-terminal truncated hPanx1 (Δ371hPanx1) were exposed to alkaline extracellular saline solution, increasing the activity of Panx1 HCs. The Δ371hPanx1 HC was permeable to DAPI and Etd + , but not to propidium iodide, whereas fl-hPanx1 HC was only permeable to DAPI. Furthermore, the cytoplasmic Ca 2+ signal increased only in Δ371hPanx1 cells, which was supported by bioinformatics approaches. The influx of Ca 2+ through Δ371hPanx1 HCs was necessary to promote cell death up to about 95% of cells, whereas the exposure to alkaline saline solution without Ca 2+ failed to induce cell death, and the Ca 2+ ionophore A23187 promoted more than 80% cell death even in fl-hPanx1 transfectants. Moreover, cell death was prevented with carbenoxolone or 10 Panx1 in Δ371hPanx1 cells, whereas it was undetectable in HeLa Panx1 -/- cells. Pretreatment with Ferrostatin-1 and necrostatin-1 did not prevent cell death, suggesting that ferroptosis or necroptosis was not involved. In comparison, zVAD-FMK, a pancaspase inhibitor, reduced death by ~60%, suggesting the involvement of apoptosis. Therefore, alkaline pH increases the activity of Δ371hPanx1HCs, leading to a critical intracellular free-Ca 2+ overload that promotes cell death., Competing Interests: Competing interests statement:V.M.B., a reviewer, and J.C.S. were on a retrospective for Michael Bennett together this year (https://pubmed.ncbi.nlm.nih.gov/38278514/).

Neonatal platelets present a reduced response to the platelet agonist, thrombin (Thr), thus resulting in a deficient Thr-induced aggregation. These alterations are more pronounced in premature newborns. Here, our aim was to uncover the causes underneath the impaired Ca 2+ homeostasis described in neonatal platelets. Both Ca 2+ mobilization and Ca 2+ influx in response to Thr are decreased in neonatal platelets compared to maternal and control woman platelets. In neonatal platelets, we observed impaired Ca 2+ mobilization in response to the PAR-1 agonist (SFLLRN) or by blocking SERCA3 function with tert-butylhydroquinone. Regarding SOCE, the STIM1 regulatory protein, SARAF, was found overexpressed in neonatal platelets, promoting an increase in STIM1/SARAF interaction even under resting conditions. Additionally, higher interaction between SARAF and PDCD61/ALG2 was also observed, reducing SARAF ubiquitination and prolonging its half-life. These results were reproduced by overexpressing SARAF in MEG01 and DAMI cells. Finally, we also observed that pannexin 1 permeability is enhanced in response to Thr in control woman and maternal platelets, but not in neonatal platelets, hence, leading to the deregulation of the Ca 2+ entry found in neonatal platelets. Summarizing, we show that in neonatal platelets both Ca 2+ accumulation in the intracellular stores and Thr-evoked Ca 2+ entry through either capacitative channels or non-selective channels are altered in neonatal platelets, contributing to deregulated Ca 2+ homeostasis in neonatal platelets and leading to the altered aggregation observed in these subjects., (© 2023 The Authors. British Journal of Haematology published by British Society for Haematology and John Wiley & Sons Ltd.)

Abstract It is generally considered that enteric neuropathy is one of the causative factors in diabetic gastroparesis. Our previous study demonstrated that there is a loss of NOS neurons in diabetic mice. However, the underlying mechanism remains unclear. The present study was designed to clarify the relationship between neuronal P2X7R and NOS neuron damage. The effect of P2X7R on diabetes-induced gastric NOS neurons damage and its mechanism were investigated by using quantitative RT-PCR，immunofluorescence, western blot, isometric force recording, intracellular calcium ([Ca2+]i) measurement and whole-cell patch clamp techniques. The immunohistochemistry and western blot results showed that nNOS expression was significantly down-regulated in diabetic mice, meanwhile, electric field stimulation-induced NOS sensitive relaxation was significantly suppressed. Myenteric neurons expressed P2X7R and pannexin1, and the mRNA and protein level of P2X7R and pannexin1 were up-regulated in diabetic mice. BzATP, a P2X7R activator, evoked [Ca2+]i increase in Hek293 cells with heterologous expression of P2X7R (Hek293-P2X7R cells) and the same dose of ATP-induced [Ca2+]i was more obvious in Hek293-P2X7R cells than in Hek293 cells. Application of BzATP activated an inward current of Hek293-P2X7R in a dose dependent manner. Hek293-P2X7R but not untransfected Hek293 cells could take up of YO-PRO-1. In addition, the uptake of YO-PRO-1 by Hek293-P2X7R was blocked by oxATP, a P2X7 antagonist and CBX, a pannexin1 inhibitor. The results suggest that the P2X7R of enteric neurons may be involved in diabetes-induced NOS neuron damage via combining with pannexin-1 to form transmembrane pores which induce macromolecular substances and calcium into the cells. [ABSTRACT FROM AUTHOR]

ATP released from immune cells plays an important role in activation of host innate immunity. However, the molecular mechanisms for pathogen infection-induced ATP release in fish remains unclear. Pannexin1 (Panx1) is a recently identified ATP release channel important for controlling immune responses. The immune relevance of Panx1 in fish, however, is still poorly understood. In this study, we characterized a Panx1 gene homologue (termed tPanx1 ) from Nile tilapia ( Oreochromis niloticus ) and analyzed its expression in response to different immune challenges. We also investigated the role of tPanx1 channel in bacterial infection-induced ATP release. Real-time quantitative PCR analysis revealed that tPanx1 gene is expressed in all tested tissues with predominant expression in intestine. Immune challenges with lipopolysaccharide, polyinosinic-polycytidylic acid and zymosan led to increased gene expression of tPanx1 in tilapia head kidney cells and peripheral blood leucocytes. In addition, tPanx1 gene was up-regulated in hepatopancreas, muscle, spleen, gill, head kidney and blood after Aeromonas hydrophila infection. Furthermore, pharmacological inhibition of tPanx1 channel activity with Panx1 channel inhibitor, carbenoxolone, significantly attenuated A. hydrophila infection-induced ATP release in tilapia head kidney cells. Taken together, our findings suggested that tPanx1 is an important immune response gene involved in bacterial infection-induced ATP release in tilapia O. niloticus . [ABSTRACT FROM AUTHOR]

Liver fibrosis is the final common pathway for almost all causes of chronic liver injury. This chronic disease is characterized by excessive deposition of extracellular matrix components mainly due to transdifferentiation of quiescent hepatic stellate cell into myofibroblasts-like cells, which in turn is driven by cell death and inflammation. In the last few years, paracrine signaling through pannexin1 channels has emerged as a key player in the latter processes. The current study was set up to investigate the role of pannexin1 signaling in liver fibrosis. Wild-type and whole body pannexin1 knock-out mice were treated with carbon tetrachloride or subjected to bile duct ligation. Evaluation of the effects of pannexin1 deletion was based on a number of clinically relevant read-outs, including markers of liver damage, histopathological analysis, oxidative stress, inflammation and regenerative capacity. In parallel, to elucidate the molecular pathways affected by pannexin1 deletion as well as to mechanistically anchor the clinical observations, whole transcriptome analysis of liver tissue was performed. While pannexin1 knock-out mice treated with carbon tetrachloride displayed reduced collagen content, hepatic stellate cell activation, inflammation and hepatic regeneration, bile duct ligated counterparts showed increased hepatocellular injury and antioxidant enzyme activity with a predominant immune response. Gene expression profiling revealed a downregulation of fibrotic and immune responses in pannexin1 knock-out mice treated with carbon tetrachloride, whereas bile duct ligated pannexin1-deficient animals showed a pronounced inflammatory profile. This study shows for the first time an etiology-dependent role for pannexin1 signaling in experimental liver fibrosis. [ABSTRACT FROM AUTHOR]

Pannexins are a three-member family of vertebrate plasma membrane spanning molecules that have homology to the invertebrate gap junction forming proteins, the innexins. However, pannexins do not form gap junctions but operate as plasma membrane channels. The best-characterized member of these proteins, Pannexin1 (Panx1) was suggested to be functionally associated with purinergic P2X and N-methyl-D-aspartate (NMDA) receptor channels. Activation of these receptor channels by their endogenous ligands leads to cross-activation of Panx1 channels. This in turn potentiates P2X and NMDA receptor channel signaling. Two potentiation concepts have been suggested: enhancement of the current responses and/or sustained receptor channel activation by ATP released through Panx1 pore and adenosine generated by ectonucleotidase-dependent dephosphorylation of ATP. Here we summarize the current knowledge and hypotheses about interactions of Panx1 channels with P2X and NMDA receptor channels. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve. [ABSTRACT FROM AUTHOR]

Cisplatin (DDP) is the most commonly used drug in testicular cancer. However, drug resistance severely limits its clinical use and the underlying mechanisms need to be further clarified. The aim of present study was to investigate the role of ATP/IP 3 pathway mediated by pannexin1 (Panx-1) channels on DDP-induced apoptosis and to reveal the potential mechanisms of DDP-resistance in testicular cancer. We found that the expression of Panx-1 in I-10/DDP cells (DDP-resistance) was decreased compared with parental I-10 cells determined by western blotting and immunofluorescence assay. To further clarify the role of Panx-1 in DDP resistance, Panx-1 function was modulated by overexpression and knockdown of Panx-1 expression. Panx-1 overexpression increased DDP-induced apoptosis, ATP release and IP 3 levels. On the contrary, Panx-1 silencing decreased DDP-induced apoptosis, ATP release and IP 3 levels. Apyrase (hydrolyzing extracellular ATP) or xestospongin C (antagonizing IP 3 receptor) also decreased DDP-induced apoptosis. Our findings demonstrate that Panx-1 is involved in DDP-resistance and ATP/IP 3 pathway mediated by Panx-1 channels participates in DDP-induced apoptosis in testicular cancer. Panx-1 modulation may be interesting to amplify the clinical effect of DDP and reverse the resistance of testicular cancer cells to DDP. [ABSTRACT FROM AUTHOR]

Pannexin1 (Panx1), a membrane channel-forming protein permitting the passage of small-sized molecules, such as ATP, is expressed in human platelets. Recently, we showed that inhibiting Panx1 affects collagen-induced platelet aggregation but not aggregation triggered by other agonists. We also found that a single nucleotide polymorphism (SNP; rs1138800) in thePanx1gene encoded for a gain-of-function channel (Panx1-400C)and was associated with enhanced collagen-induced platelet reactivity. Here, we assessed the association of this SNP with platelet reactivity in a cohort of 758 stable cardiovascular patients from the ADRIE study treated with aspirin and/or clopidogrel. We found that presence of thePanx1-400Callele was not associated with platelet reactivity in stable cardiovascular patients, irrespective of the platelet aggregation agonist used (collagen, ADP or arachidonic acid) or the anti-platelet drug regimen. Moreover, thePanx1-400A > CSNP did also not affect the re-occurrence of cardiac ischemic events in the same stable cardiovascular patient cohort. [ABSTRACT FROM PUBLISHER]

Pannexin1 proteins form communication channels at the cell plasma membrane surface, which allow the transfer of small molecules and ions between the intracellular compartment and extracellular environment. In this way, pannexin1 channels play an important role in various cellular processes and diseases. Indeed, a plethora of human pathologies is associated with the activation of pannexin1 channels. The present paper reviews and summarizes the structure, life cycle, regulation and (patho)physiological roles of pannexin1 channels, with a particular focus on the relevance of pannexin1 channels in liver diseases., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Van Campenhout, Caufriez, Tabernilla, Maerten, De Boever, Sanz-Serrano, Kadam and Vinken.)

The elabela-apelin/angiotensin domain type 1 receptor-associated protein (APJ) system is an important regulator in certain thrombosis-related diseases such as atherosclerosis, myocardial infarction, and cerebral infarction. Our previous reports have revealed that apelin exacerbates atherosclerotic lesions. However, the relationship between the elabela-apelin/APJ system and platelet aggregation and atherothrombosis is unclear. The results of the present study demonstrate that elabela and other endogenous ligands such as apelin-12, -17, and -36 induce platelet aggregation and thrombosis by activating the pannexin1(PANX1)-P2X7 signaling pathway. Interestingly, the diuretic, spironolactone, a novel PANX1 inhibitor, alleviated elabela- and apelin isoforms-induced platelet aggregation and thrombosis. Significantly, two potential antithrombotic drugs were screened out by targeting APJ receptors, including the anti-HIV ancillary drug cobicistat and the traditional Chinese medicine monomer Schisandrin A. Both cobicistat and Schisandrin A abolished the effects of elabela and apelin isoforms on platelet aggregation, thrombosis, and cerebral infarction. In addition, cobicistat significantly attenuated thrombosis in a ponatinib-induced zebrafish trunk model. Overall, the elabela-apelin/APJ axis mediated platelet aggregation and thrombosis via the PANX1-P2X7 signaling pathway in vitro and in vivo. Blocking the APJ receptor with cobicistat/Schisandrin A or inhibiting PANX1 with spironolactone may provide novel therapeutic strategies against thrombosis., (© 2023 Wiley Periodicals LLC.)

Skeletal muscles of patients with Duchenne muscular dystrophy (DMD) show numerous alterations including inflammation, apoptosis, and necrosis of myofibers. However, the molecular mechanism that explains these changes remains largely unknown. Here, the involvement of hemichannels formed by connexins (Cx HCs) was evaluated in skeletal muscle of mdx mouse model of DMD. Fast myofibers of mdx mice were found to express three connexins (39, 43 and 45) and high sarcolemma permeability, which was absent in myofibers of mdx Cx43Cx45:Myo-Cre mice (deficient in skeletal muscle Cx43/Cx45 expression). These myofibers did not show elevated basal intracellular free Ca levels, immunoreactivity to phosphorylated p65 (active NF-κB), eNOS and annexin V/active Caspase 3 (marker of apoptosis) but presented dystrophin immunoreactivity. Moreover, muscles of mdx Cx43Cx45:Myo-Cre mice exhibited partial decrease of necrotic features (big cells and high creatine kinase levels). Accordingly, these muscles showed similar macrophage infiltration as control mdx muscles. Nonetheless, the hanging test performance of mdx Cx43Cx45:Myo-Cre mice was significantly better than that of control mdx Cx43Cx45 mice. All three Cxs found in skeletal muscles of mdx mice were also detected in fast myofibers of biopsy specimens from patients with muscular dystrophy. Thus, reduction of Cx expression and/or function of Cx HCs may be potential therapeutic approaches to abrogate myofiber apoptosis in DMD. [ABSTRACT FROM AUTHOR]

The World Health Organization has predicted that by 2040 neurodegenerative diseases will overtake cancer to become the world’s second leading cause of death after cardiovascular disease. This has sparked the development of several European and American brain research initiatives focusing on elucidating the underlying cellular and molecular mechanisms of neurodegenerative diseases. Connexin (Cx) and pannexin (Panx) membrane channel proteins are conduits through which neuronal, glial, and vascular tissues interact. In the brain, this interaction is highly critical for homeostasis and brain repair after injury. Understanding the molecular mechanisms by which these membrane channels function, in health and disease, might be particularly influential in establishing conceptual frameworks to develop new therapeutics against Cx and Panx channels. This review focuses on current insights and emerging concepts, particularly the impact of connexin43 and pannexin1, under neuroprotective and neurodegenerative conditions within the context of astrocytes. [ABSTRACT FROM AUTHOR]

The ubiquitous pannexin 1 (Panx1) ion- and metabolitepermeable channelmediates the release ofATP, a potent signalling molecule. In the present study, we provide striking evidence that ATP, in turn, stimulates internalization of Panx1 to intracellular membranes. These findings hold important implications for understanding the regulation of Panx1 when extracellular ATP is elevated. In the nervous system, this includes phenomena such as synaptic plasticity, pain, precursor cell development and stroke; outside of the nervous system, this includes things like skeletal and smooth muscle activity and inflammation. Within 15 min, ATP led to significant Panx1-EGFP internalization. In a series of experiments, we determined that hydrolysable ATP is the most potent stimulator of Panx1 internalization. We identified two possible mechanisms for Panx1 internalization, including activation of ionotropic purinergic (P2X) receptors and involvement of a putative ATP-sensitive residue in the first extracellular loop of Panx1 (Trp74). Internalization was cholesterol-dependent, but clathrin, caveolin and dynamin independent. Detailed analysis of Panx1 at specific endosome sub-compartments confirmed that Panx1 is expressed in endosome membranes of the classical degradation pathway under basal conditions and that elevation of ATP levels diverts a sub-population to recycling endosomes. This is the first report detailing endosome localization of Panx1 under basal conditions and the potential for ATP regulation of its surface expression. Given the ubiquitous expression profile of Panx1 and the importance of ATP signalling, these findings are of critical importance for understanding the role of Panx1 in health and disease. [ABSTRACT FROM AUTHOR]

Recruitment of monocytes in the liver is a key pathogenic feature of hepatic inflammation in nonalcoholic steatohepatitis (NASH), but the mechanisms involved are poorly understood. Here, we studied migration of human monocytes in response to supernatants obtained from liver cells after inducing lipoapoptosis with saturated free fatty acids (FFA). Lipoapoptotic supernatants stimulated monocyte migration with the magnitude similar to a monocyte chemoattractant protein, CCL2 (MCP-1). Inhibition of c-Jun NH-terminal kinase (JNK) in liver cells with SP600125 blocked migration of monocytes in a dose-dependent manner, indicating that JNK stimulates release of chemoattractants in lipoapoptosis. Notably, treatment of supernatants with Apyrase to remove ATP potently inhibited migration of THP-1 monocytes and partially blocked migration of primary human monocytes. Inhibition of the CCL2 receptor (CCR2) on THP-1 monocytes with RS102895, a specific CCR2 inhibitor, did not block migration induced by lipoapoptotic supernatants. Consistent with these findings, lipoapoptosis stimulated pathophysiological extracellular ATP (eATP) release that increased supernatant eATP concentration from 5 to ~60 nM. Importantly, inhibition of Panx1 expression in liver cells with short hairpin RNA (shRNA) decreased supernatant eATP concentration and inhibited monocyte migration, indicating that monocyte migration is mediated in part by Panx1-dependent eATP release. Moreover, JNK inhibition decreased supernatant eATP concentration and inhibited Pannexin1 activation, as determined by YoPro-1 uptake in liver cells in a dose-dependent manner. These results suggest that JNK regulates activation of Panx1 channels, and provide evidence that Pannexin1-dependent pathophysiological eATP release in lipoapoptosis is capable of stimulating migration of human monocytes, and may participate in the recruitment of monocytes in chronic liver injury induced by saturated FFA. [ABSTRACT FROM AUTHOR]

Hepatic encephalopathy (HE) represents a nervous system disorder caused due to liver dysfunction. HE is broadly classified as acute/overt and moderate-minimal HE. Since HE syndrome severely affects quality of life of the patients and it may be life threatening, it is important to develop effective therapeutic strategy against HE. Mainly ammonia neurotoxicity is considered accountable for HE. Increased level of ammonia in the brain activates glutamate-NMDA (N-methyl-D-aspartate) receptor (NMDAR) pathway leading to Ca influx, energy deficit and oxidative stress in the post synaptic neurons. Moreover, NMDAR blockage has been found to be a poor therapeutic option, as this neurotransmitter receptor plays important role in maintaining normal neurophysiology of the brain. Thus, searching new molecular players in HE pathogenesis is of current concern. There is an evolving concept about roles of the trans-membrane channels in the pathogenesis of a number of neurological complications. Pannexin1 (Panx1) is one of them and has been described to be implicated in stroke, epilepsy and ischemia. Importantly, the pathogenesis of these complications relates to some extent with NMDAR over activation. Thus, it is speculated that HE pathogenesis might also involve Panx1. Indeed, some recent observations in the animal models of HE provide support to this argument. Since opening of Panx1 channel is mostly associated with the neuronal dysfunctions, down regulation of this channel could serve as a relevant therapeutic strategy without producing any serious side effects. In the review article an attempt has been made to summarize the current information on implication of Panx1 in the brain disorders and its prospects for being examined as pharmacological target in HE pathogenesis. [ABSTRACT FROM AUTHOR]

Pannexin1 channels facilitate the extracellular release of a number of messengers, including adenosine triphosphate. Although fulfilling some physiological functions, pannexin1 channel communication has to date been primarily studied in the context of inflammation and cell death. In the past decade, a variety of chemical substances have been reported to induce pannexin1 channel opening, including metals, chelating agents, particulate matter, nanoparticles and drugs. While the pathophysiological aspects of pannexin1 channel communication have been reviewed on many previous occasions, the present paper intends to provide a short perspective in order to motivate research that will advance mechanistic understanding of the roles of pannexin1 signaling in chemical toxicity. [ABSTRACT FROM AUTHOR]

Background and Objective Pannexin 1 (Panx1) has been found to form nonjunctional hemichannels. It is also proposed to combine with the P2X7 receptor, forming a complex involved in adenosine triphosphate ( ATP)-induced interleukin-1beta ( IL-1β) release in macrophages. Previously, we reported that mechanical stress induced IL-1β expression via the ATP/P2X7 receptor-dependent pathway in human periodontal ligament ( HPDL) cells and that ATP was released through the connexin 43 (Cx43) hemichannel. In the present work, we examined the role of Panx1 in stress-induced IL-1β induction in HPDL cells. Material and Methods Cultured HPDL cells were treated with compressive loading or ATP to stimulate IL-1β expression. Inhibitors, antagonists and the small interfering RNA technique were used to investigate the involvement of Panx1 in IL-1β induction. Co-immunoprecipitation (Co- IP) and immunostaining were used to determine the association of Panx1 with the P2X7 receptor. The IL-1β release mechanism was analyzed using inhibitors. Results Blocking Panx1 significantly decreased ATP release, as well as IL-1β up-regulation, upon stimulation with stress or ATP. Co- IP revealed the association of Panx1 and the P2X7 receptor in HPDL cells, which was increased in response to mechanical loading. Pretreatment with vesicular trafficking inhibitors significantly reduced the amount of IL-1β released from stimulated cells, suggesting that IL-1β might be released through vesicles. Conclusion We clearly illustrated the contribution of Panx1 in ATP release, as well as in IL-1β induction in HPDL cells. The association of Panx1 and the P2X7 receptor might be required for IL-1β induction, and their possible novel role in IL-1β vesicular release was indicated. [ABSTRACT FROM AUTHOR]

Pannexin1 is ubiquitously expressed in vertebrate tissues, but the role it plays in vascular tone regulation remains unclear. We found that Pannexin1 expression level is much higher in the endothelium relative to smooth muscle of saphenous artery. The ability of endothelium-intact arteries for dilation was significantly impaired whereas contractile responses were considerably increased in mice with genetic ablation of Pannexin1. No such increased contractile responses were detected in the endothelium-denuded arteries. Combined, our findings suggest a new function of Pannexin1 as an important player in normal endotheliumdependent regulation of arterial tone, where it facilitates vessel dilation and attenuates constriction. [ABSTRACT FROM AUTHOR]

Pannexins are channel proteins displaying functional similarities to gap junctions in vertebrates and are regarded as transmembrane ATP-releasing channels. A member of this family, denominate pannexin1, has been detected in the epidermis and cutaneous adnexal structures. Here we used immunohistochemistry to investigate whether human digital Merkel cells express this protein since ATP is postulated as a neurotransmitter in the Merkel cell-axon complexes low-threshold mecahoreceptors. Pannexin1 immunoreactivity was found in cytokeratine 20-, chromogranin A- and synaptophysin-positive cells placed at the basal layer of the epidermis. Cell displaying pannexin1 immunoreactivities were thus identified as Merkel cells and showed close contact with nerve profiles. Light pannexin1 immunoreactivity in dermal blood vessels was also verified. Present results demonstrate for the first time the expression of pannexin1 in human digital Merkel cells supporting the idea that ATP can be involved directly or indirectly in the mechanotransductional process at Merkel-axon complexes. [ABSTRACT FROM AUTHOR]

Until recently, members of the connexin gene family were believed to comprise the sole molecular component forming gap junction channels in vertebrates. The recent discovery of the pannexin gene family has challenged this view, as these genes may encode for a putative second class of gap junction proteins in vertebrates. The expression of pannexin genes overlaps with those cellular networks known to exhibit a high degree of gap junctional coupling. We investigated the spatio-temporal mRNA distribution of one member of this gene family, pannexin1 (Panx1), in the brain and retina of mice using quantitative real-time polymerase chain reaction and a combination of in situ hybridization and immunohistochemistry for cellular resolution. Our results demonstrate a widespread expression of Panx1 in the brain, retina and other non-neuronal tissues. In the cortex, cerebellum and eye, Panx1 is expressed at early embryonic time points and peaks around embryonic day 18 followed by a decline towards adulthood. Most notably, Panx1 is detectable in neurons of many brain nuclei, which are known to be coupled by gap junctions as well as in previously unrecognized areas. Abundant expression was found in the adult hippocampal and neocortical pyramidal cells and interneurons, neurons of the reticular thalamus, the inferior olive, magnocellular hypothalamic neurons, midbrain and brain stem motoneurons, Purkinje cells and the retina. [ABSTRACT FROM AUTHOR]

Sepsis-induced acute kidney injury (SI-AKI) is the fifth most common cause of hospital-acquired acute kidney injury. Pannexin1 (Panx1) triggers inflammation and apoptosis which act as crucial factors in the pathogenesis of SI-AKI. We aimed to investigate the expression of Panx1 and its role on the inflammation and apoptosis in SI-AKI. SI-AKI model was established by lipopolysaccharide (LPS) injection in mice and LPS-treated HK-2 cells in vitro. Panx1 was inhibited by pretreating with carbenoxolone (CBX) or small interfering RNA in vivo and vitro, respectively. The expression of Panx1 was determined by qPCR, western blot and immunohistochemistry (IHC). Kidney damage was evaluated by kidney function, histopathological examination and AKI biomarkers. Inflammatory cytokines were detected by qPCR and ELISA. Apoptosis was detected by TUNEL staining and the expression of apoptosis-related proteins. The activation of nucleotide-binding domain-like receptor protein 3 (NLRP3) inflammasome was measured by western blot. Panx1 increased in LPS-induced SI-AKI mice and HK-2 cells, as well as in SI-AKI patients. CBX alleviated the renal function and pathological damage, as well as decreased the mRNA of kidney injury molecule (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL). Inhibiting Panx1 decreased the production of IL-1β, IL-6 and TNF-α, as well as tubular cell apoptosis in SI-AKI. Inhibiting Panx1 suppressed inflammatory cytokines and apoptosis via inhibiting NLRP3 inflammasome activation and regulating apoptotic protein Bax and Bcl2 expression, respectively. These observations suggest that pharmacological inhibition of Panx1 might be a potential approach in the clinical therapy of SI-AKI. [ABSTRACT FROM AUTHOR]

• Pristine MWCNTs altered tight junction integrity, and elevated paracellular permeability in murine brain microvascular endothelial cells. • Pristine MWCNT-induced elevation in endothelial permeability was regulated by connexin 43- and pannexin 1-based hemichannels. • Connexin 43- and Pannexin 1- mediated ATP release may contribute to the endothelial permeability elevation in pristine MWCNT-treated cells. • Carboxyl functionalization may weaken MWCNT-induced effects on endothelial permeability and hemichannel activity. Multi-walled carbon nanotubes (MWCNTs) have promising applications in neurology depending on their unique physicochemical properties. However, there is limited understanding of their impacts on brain microvascular endothelial cells, the cells lining the vessels and maintaining the low and selective permeability of the blood-brain barrier. In this study, we examined the influence of pristine MWCNT (p-MWCNT) and carboxylated MWCNT (c-MWCNT) on permeability and tight junction tightness of murine brain microvascular endothelial cells, and investigated the potential mechanisms in the sight of hemichannel activity. Treatment with p-MWCNT for 24 h at subtoxic concentration (20 μg/mL) decreased the protein expression of occludin, disrupted zonula occludens-1 continuity, and elevated monolayer permeability as quantified by transendothelial electrical resistance and paracellular flux of 4000 Da fluorescein isothiocyanate-dextran conjugates. Moreover, p-MWCNT exposure also increased hemichannel activity with upregulated protein expression and altered subcellular localization of connexin (Cx)43 and pannexin (Panx)1. p-MWCNT-induced elevation in endothelial permeability could be prevented by hemichannel inhibitor carbenoxolone and peptide blocker of Cx43 and Panx1, indicating the crucial role of activated Cx43 and Panx1 hemichannels. Furthermore, Cx43 and Panx1 hemichannel-mediated ATP release might be involved in p-MWCNT-induced rise in endothelial permeability. In contrast, the above effects caused by p-MWCNT were not observed in cells treated with c-MWCNT, the functionalized form with more stable dispersion and a lower tendency to aggregate. Our study contributes further understanding of the impact of MWCNTs on brain endothelial tightness and permeability, which may have important implications for the safety application of MWCNTs in nanomedicine. [ABSTRACT FROM AUTHOR]

We profiled the transcriptomes of primary mouse cortical astrocytes cultured alone or co-cultured with immortalized precursor oligodendrocytes (Oli-neu cells). Filters between the cell types prevented formation of hetero-cellular gap junction channels but allowed for free exchange of the two culture media. We previously reported that major functional pathways in the Oli-neu cells are remodeled by the proximity of non-touching astrocytes and that astrocytes and oligodendrocytes form a panglial transcriptomic syncytium in the brain. Here, we present evidence that the astrocyte transcriptome likewise changes significantly in the proximity of non-touching Oli-neu cells. Our results indicate that the cellular environment strongly modulates the transcriptome of each cell type and that integration in a heterocellular tissue changes not only the expression profile but also the expression control and networking of the genes in each cell phenotype. The significant decrease of the overall transcription control suggests that in the co-culture astrocytes are closer to their normal conditions from the brain. The Oli-neu secretome regulates astrocyte genes known to modulate neuronal synaptic transmission and remodels calcium, chemokine, NOD-like receptor, PI3K-Akt, and thyroid hormone signaling, as well as actin-cytoskeleton, autophagy, cell cycle, and circadian rhythm pathways. Moreover, the co-culture significantly changes the gene hierarchy in the astrocytes. [ABSTRACT FROM AUTHOR]

Stressed or injured cells release ATP into the extracellular milieu via the pannexin1 (Panx1) channels, which is the basis of inflammation in a variety of conditions, including allergic lung inflammation. Although the role of Panx1 in mediating inflammation has been well established, the role of its mimetic peptide, 10 Panx1, which inhibits ATP release from Panx1 channels, in allergic asthma remains understudied. The aim of this study was to evaluate the effects of using 10 Panx1 to inhibit Panx1 channel in a murine model of ovalbumin (OVA)-induced asthma. We demonstrate that blockade of Panx1 significantly attenuated goblet cell hyperplasia and inflammatory cell infiltration into the lungs of OVA-sensitized mice. Inhibition of Panx1 also reduced the total and eosinophil cell numbers in the bronchoalveolar lavage fluid (BALF) and reduced expression of CCL11 and CCL2 in lung tissues from mice. Moreover, we detected lower levels of IL-5 and IL-13 in the culture supernatant of OVA-restimulated splenocytes from 10 Panx1-treated mice. Collectively, our findings suggest that Panx1 inhibition of allergen-mediated lung inflammation has the potential to suppress allergic responses in asthma., Competing Interests: None., (AJTR Copyright © 2020.)

Loss of connexin-mediated cell-cell communication is a hallmark of breast cancer progression. Pannexin1 (PANX1), a glycoprotein that shares structural and functional features with connexins and engages in cell communication with its environment, is highly expressed in breast cancer metastatic foci; however, PANX1 contribution to metastatic progression is still obscure. Here we report elevated expression of PANX1 in different breast cancer (BRCA) subtypes using RNA-seq data from The Cancer Genome Atlas (TCGA). The elevated PANX1 expression correlated with poorer outcomes in TCGA BRCA patients. In addition, gene set enrichment analysis (GSEA) revealed that epithelial-to-mesenchymal transition (EMT) pathway genes correlated positively with PANX1 expression. Pharmacological inhibition of PANX1, in MDA-MB-231 and MCF-7 breast cancer cells, or genetic ablation of PANX1, in MDA-MB-231 cells, reverted the EMT phenotype, as evidenced by decreased expression of EMT markers. In addition, PANX1 inhibition or genetic ablation decreased the invasiveness of MDA-MB-231 cells. Our results suggest PANX1 overexpression in breast cancer is associated with a shift towards an EMT phenotype, in silico and in vitro, attributing to it a tumor-promoting effect, with poorer clinical outcomes in breast cancer patients. This association offers a novel target for breast cancer therapy.

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 (Ca 2+ 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., (© 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.)