Panx1 Channels Research Articles

NMDA and P2X7 Receptors Require Pannexin 1 Activation to Initiate and Maintain Nociceptive Signaling in the Spinal Cord of Neuropathic Rats.

Pannexin 1 (Panx1) is involved in the spinal central sensitization process in rats with neuropathic pain, but its interaction with well-known, pain-related, ligand-dependent receptors, such as NMDA receptors (NMDAR) and P2X7 purinoceptors (P2X7R), remains largely unexplored. Here, we studied whether NMDAR- and P2X7R-dependent nociceptive signaling in neuropathic rats require the activation of Panx1 channels to generate spinal central sensitization, as assessed by behavioral (mechanical hyperalgesia) and electrophysiological (C-reflex wind-up potentiation) indexes. Administration of either a selective NMDAR agonist i.t. (NMDA, 2 mM) or a P2X7R agonist (BzATP, 150 μM) significantly increased both the mechanical hyperalgesia and the C-reflex wind-up potentiation, effects that were rapidly reversed (minutes) by i.t. administration of a selective pannexin 1 antagonist (10panx peptide, 300 μM), with the scores even reaching values of rats without neuropathy. Accordingly, 300 μM 10panx completely prevented the effects of NMDA and BzATP administered 1 h later, on mechanical hyperalgesia and C-reflex wind-up potentiation. Confocal immunofluorescence imaging revealed coexpression of Panx1 with NeuN protein in intrinsic dorsal horn neurons of neuropathic rats. The results indicate that both NMDAR- and P2X7R-mediated increases in mechanical hyperalgesia and C-reflex wind-up potentiation require neuronal Panx1 channel activation to initiate and maintain nociceptive signaling in neuropathic rats.

Oscillatory shear stress augments endothelial pannexin1 by inhibiting macro-autophagy

Abstract Funding Acknowledgements Type of funding sources: None. Introduction Atherosclerotic lesions preferentially develop in arterial regions exposed to disturbed blood flow, which are characterized by a dysfunctional proinflammatory endothelial cell (EC) phenotype. ECs release ATP in response to changes in wall shear stress (WSS), which subsequently regulates the inflammatory response. ATP can be released from cells in a controlled manner through Pannexin1 (Panx1) channels. Objective To study the expression of Panx1 in response to WSS and its role in the endothelium. Methods Human ECs (HUVECs or EA.hy926) were exposed to physiological high laminar shear stress (HLSS) and atheroprone oscillatory shear stress (OSS) for 48h using an orbital shaker. Panx1 transcript and protein levels were determined by qPCR and Western blot, respectively. mRNA was extracted for RNAseq. WSS-modifying casts were placed for 1 week around the carotid artery of mice and Panx1 expression was analyzed en face. Carotid WSS-modifying casts were also placed in Tie2CreTgPanx1fl/flApoE-/- and Panx1fl/flApoE-/- mice followed by 9 weeks high fat diet. CD68+ cells were quantified in OSS-induced atherosclerotic lesions. Results We found that Panx1 expression was increased in carotid regions exposed to OSS as compared to HLSS regions. These results were confirmed in vitro in HUVECs. In silico analysis of the promotor of Panx1 revealed binding sites for the WSS-sensitive transcription factors NF-kB and CREB. NF-kB and CREB mRNA levels were similar under OSS and HLSS, however NF-kB activation (phospho-NF-kB) was detected in ECs under OSS. Surprisingly, Panx1 mRNA level was not affected under these conditions, suggesting that the increased Panx1 protein observed under OSS may be due to decreased Panx1 degradation rather than to increased synthesis of the protein. Unbiased analysis of differential gene expression in ECs exposed to HLSS or OSS revealed 320 up-regulated and 353 down-regulated genes under OSS. Down-regulated genes included proteins involved in macro-autophagy. Inhibition of macro-autophagy in ECs by exposure to chloroquine for 6h increased the expression of glycosylated Panx1, suggestive for more Panx1 channels at the plasma membrane under these conditions. Finally, we observed that atherosclerotic lesions in OSS regions of Tie2CreTgPanx1fl/flApoE-/- mice contained more CD68+ cells than Panx1fl/flApoE-/- controls. Conclusion Endothelial Panx1 expression is upregulated in response to OSS. Absence of OSS effects on NF-kB, CREB and Panx1 mRNA revealed that the upregulation of Panx1 protein is not due to transcriptional effects of OSS. Expression of genes involved in the macro-autophagic process were down-regulated under OSS, and chemical inhibition of macro-autophagy augmented the plasma membranous form of Panx1. OSS-induced decrease in autophagic flux may enhance ATP release through Panx1 channels, thereby counterbalancing leukocyte recruitment in atherosclerosis.

Panx1 channels promote both anti- and pro-seizure-like activities in the zebrafish via p2rx7 receptors and ATP signaling

The molecular mechanisms of excitation/inhibition imbalances promoting seizure generation in epilepsy patients are not fully understood. Evidence suggests that Pannexin1 (Panx1), an ATP release channel, modulates the excitability of the brain. In this report, we performed electrophysiological, behavioral, and molecular phenotyping experiments on zebrafish larvae bearing genetic or pharmacological knockouts of Panx1a and Panx1b channels, each homologous to human PANX1. When Panx1a function is lost, or both channels are under pharmacological blockade, seizures with ictal-like events and seizure-like locomotion are reduced in the presence of pentylenetetrazol. Transcriptome profiling by RNA-seq demonstrates a spectrum of distinct metabolic and cell signaling states which correlate with the loss of Panx1a. Furthermore, the pro- and anticonvulsant activities of both Panx1 channels affect ATP release and involve the purinergic receptor P2rx7. Our findings suggest a subfunctionalization of Panx1 enabling dual roles in seizures, providing a unique and comprehensive perspective to understanding seizure mechanisms in the context of this channel.

Abstract 194: Pharmacologic Inhibition By Spironolactone Attenuates Experimental Abdominal Aortic Aneurysms

Introduction: Abdominal aortic aneurysms (AAA) are characterized by vascular inflammation and remodeling that can lead to aortic rupture resulting in significant risk of mortality. Spironolactone has been recently described as a pannexin-1 (Panx1) channel modulator. Our hypothesis focuses on the role of spironolactone on Panx1 channels to attenuate AAA formation. Methods: A topical elastase (0.4 U/mL type 1 porcine pancreatic elastase) or heat inactivated elastase (control) AAA model was used in C57BL/6 (wild-type; WT) male mice. Mice were administered either a vehicle control (saline) or spironolactone (1.25 mg/kg) i.p. daily for 14 days. On day 14, the abdominal aortic diameter was measured by video micrometry and expressed as percentage increase over baseline. In a second model, mice were subjected to β-aminopropionitrile (BAPN) two days prior to elastase treatment and everyday thereafter. A vehicle control (saline) or spironolactone was given i.p. daily starting from day 14. Immunohistochemistry was used to analyze immune cell infiltration and elastic fiber disruption (Verhoeff Van Gieson staining). Groups were compared using ANOVA and data is displayed as mean ± SEM with p<0.05 as statistically significant. Results: Aortic diameter was significantly increased in elastase-treated WT mice compared to controls (202.5 ± 4.2% vs 0.5 ± 1.9%; n=9/group, p<0.001). Elastase-treated WT mice administered spironolactone had a significant, dose-independent decrease in AAA formation compared to elastase-treated WT controls (139.8 ± 9.9% vs 202.5 ± 4.2%; n=10/group, p<0.001). Elastase-treated mice given BAPN and administered spironolactone had significant attenuation of AAA formation compared to controls on day 28 (420.2 ± 30.5% vs 643.8 ± 41.3%; n=8/group, p<0.001). A significant decrease in immune cell infiltration and elastic fiber disruption occurred in all mice treated with spironolactone compared to respective controls. Conclusion: These results suggest spironolactone mitigates aortic remodeling to attenuate AAA formation as well as decrease growth of preformed aneurysms. Further studies aim to characterize the molecular mechanisms of specific intercellular interactions involving Panx1-mediated AAA formation by spironolactone.

A Critical Role for Pannexin 1 in Heart Failure Induced by Acute and Chronic Isoproterenol Administration

Pannexin 1 (Panx1), a ubiquitously expressed ATP release membrane channel, has been shown to play a role in inflammation and blood pressure regulation. Panx1 channel function is inhibited by spironolactone, a frontline therapy for heart failure patients. Despite this, the function of Panx1 in cardiomyocytes and a possible role in heart failure has not yet been studied. To investigate if Panx1 is involved in the development of cardiac dysfunction and fibrosis, we use a model of chronic adrenergic stimulation to induce heart failure in two novel mouse models. We have generated mice with constitutive deletion of Panx1 in cardiomyocytes by crossing mice expressing loxP‐flanked alleles of Panx1 to mice expressing Cre recombinase under the control of the cardiomyocyte specific MyHC6 promoter (Panx1MyHC6‐Cre). Using the same approach with mice expressing the tamoxifen‐inducible MerCreMer system under the control of the Tnnt2 promoter we generated mice with tamoxifen‐inducible deletion of Panx1 in cardiomyocytes (Panx1Tnnt2‐MerCreMer). Constitutive deletion of Panx1 in cardiomyocytes had no effect on heart function at baseline as measured by echocardiography. However, after acute isoproterenol treatment (15 mg/kg/day, i.p.), Panx1MyHC6‐Cre mice were protected from systolic and diastolic dysfunction and cardiac hypertrophy. In vitro, treatment of H9c2 cardiomyocytes with isoproterenol led to Panx1‐dependent release of ATP. Furthermore, siRNA‐mediated knock down of Panx1 in H9c2 cells attenuated basal and maximal extracellular acidification rate (a measure of glycolysis) after 24 hours of isoproterenol treatment, indicating a role for Panx1 in regulation of cardiomyocyte metabolism. Taken together, these data demonstrates that Panx1 deficiency protects against isoproterenol‐induced cardiac dysfunction and pathological glycolytic shift in stressed cardiomyocytes. Further elucidating the role of Panx1 in cardiomyocytes in regulating cardiac function and fibrosis will identify a novel therapeutic target to prevent heart failure.

TLR2/caspase-5/Panx1 pathway mediates necrosis-induced NLRP3 inflammasome activation in macrophages during acute kidney injury

Acute kidney injury (AKI) is characterized by necroinflammation formed by necrotic tubular epithelial cells (TECs) and interstitial inflammation. In necroinflammation, macrophages are key inflammatory cells and can be activated and polarized into proinflammatory macrophages. Membranous Toll-like receptors (TLRs) can cooperate with intracellular NOD-like receptor protein 3 (NLRP3) to recognize danger signals from necrotic TECs and activate proinflammatory macrophages by assembling NLRP3 inflammasome. However, the cooperation between TLRs and NLRP3 is still unclear. Using conditioned medium from necrotic TECs, we confirmed that necrotic TECs could release danger signals to activate NLRP3 inflammasome in macrophages. We further identified that necrotic TECs-induced NLRP3 inflammasome activation was dependent on ATP secretion via Pannexin-1 (Panx1) channel in macrophages. Next, we verified that TLR2 was required for the activation of Panx1 and NLRP3 in macrophages. Mechanistically, we indicated that caspase-5 mediated TLR2-induced Panx1 activation. In addition, we showed that necrotic TECs-induced activation of TLR2/caspase-5/Panx1 axis could be decreased in macrophages when TECs was protected by N-acetylcysteine (NAC). Overall, we demonstrate that danger signals from necrotic TECs could activate NLRP3 inflammasome in macrophages via TLR2/caspase-5/Panx1 axis during AKI.

Pannexin1 channels regulate mechanically stimulated but not spontaneous adenosine release.

Fast-scan cyclic voltammetry (FSCV)is a rapid technique to measure neuromodulators, and using FSCV, two modes of rapid adenosine have been discovered. Spontaneous transients occur randomly in the brain, while mechanical stimulation also causes a rapid adenosine event. Pannexin1 channels are membrane channels that transport ions, including ATP, out of the cell where it is rapidly broken down into adenosine. Pannexin 1 channels (Panx1) have a flickering mode of rapid opening and are also mechanically stimulated. Here, we test the extent to which pannexin channels, specifically pannexin1 (Panx1) channels, are responsible for rapid adenosine events. Spontaneous adenosine release or mechanosensitive adenosine release were measured using fast-scan cyclic voltammetry in hippocampal (CA1) brain slices. In global Panx1KO mice, there is no significant difference in the frequency or concentration of spontaneous adenosine release, indicating Panx1 is not a release mechanism for spontaneous adenosine. Spontaneous adenosine frequency decreased slightly after administration of a large (100µM) dose of carbenoxolone, a nonspecific inhibitor of many pannexin and connexin channels, suggesting other hemichannels only play a small role at most. For mechanically stimulated adenosine release, the concentration of each adenosine event significantly decreased 30% in Panx1KO mice and the frequency of stimulations that evoked adenosine also decreased. The response was similar in WT mice with carbenoxolone. Thus, Panx1 is a release mechanism for mechanically stimulated adenosine release, but not the only mechanism. These results demonstrate that pannexin channels differentially regulate rapid adenosine release and could be targeted to differentially affect mechanically stimulated adenosine due to brain damage.

Stem cell conversion to the cardiac lineage requires nucleotide signalling from apoptosing cells.

Pluripotent stem cells can be driven by manipulation of Wnt signaling through a series of states similar to those that occur during early embryonic development, transitioning from an epithelial phenotype into the cardiogenic mesoderm lineage and ultimately into functional cardiomyocytes. Strikingly, we observed that initiation of differentiation in induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) triggers widespread apoptosis, followed by a synchronous epithelial-mesenchymal transition (EMT). Apoptosis is caused by absence of bFGF from the differentiation medium. EMT requires induction of transcription factors SNAI1/SNAI2 downstream of MESP1 expression, and double knock-out of SNAI1/2, or loss of MESP1 in iPSCs blocks EMT and prevents cardiac differentiation. Remarkably, blockade of early apoptosis chemically or by ablation of pro-apoptotic genes also completely prevents the EMT, suppressing even the earliest events in mesoderm conversion, including BRA/T, TBX6, and MESP1 induction. Conditioned medium from WNT-activated WT iPSCs overcomes the block to EMT by cells incapable of apoptosis (Apop-), suggesting involvement of soluble factors from apoptotic cells in mesoderm conversion. Knockout of the PANX1 channel blocked EMT, while treatment with a purinergic P2 receptor inhibitor or addition of apyrase demonstrated a requirement for nucleotide triphosphate signaling. ATP and/or UTP was sufficient to induce a partial EMT in Apop- cells treated with WNT activator. Notably, knockout of the ATP/UTP-specific P2Y2 receptor blocked EMT and mesoderm induction. We conclude that nucleotides, in addition to acting as chemo-attractants for clearance of apoptotic cells can function as essential paracrine signals that, with WNT signaling, create a logical AND gate for mesoderm specification.

Endothelial pannexin-1 channels modulate macrophage and smooth muscle cell activation in abdominal aortic aneurysm formation

Pannexin-1 (Panx1) channels have been shown to regulate leukocyte trafficking and tissue inflammation but the mechanism of Panx1 in chronic vascular diseases like abdominal aortic aneurysms (AAA) is unknown. Here we demonstrate that Panx1 on endothelial cells, but not smooth muscle cells, orchestrate a cascade of signaling events to mediate vascular inflammation and remodeling. Mechanistically, Panx1 on endothelial cells acts as a conduit for ATP release that stimulates macrophage activation via P2X7 receptors and mitochondrial DNA release to increase IL-1β and HMGB1 secretion. Secondly, Panx1 signaling regulates smooth muscle cell-dependent intracellular Ca2+ release and vascular remodeling via P2Y2 receptors. Panx1 blockade using probenecid markedly inhibits leukocyte transmigration, aortic inflammation and remodeling to mitigate AAA formation. Panx1 expression is upregulated in human AAAs and retrospective clinical data demonstrated reduced mortality in aortic aneurysm patients treated with Panx1 inhibitors. Collectively, these data identify Panx1 signaling as a contributory mechanism of AAA formation.

Divalent cations promote Panx1 channel activity through its C-terminal domain

Pannexin-1 (Panx1) is an ion channel that releases ATP and other important signaling molecules. However, the stimuli that directly act on Panx1 to promote channel opening are largely unknown. Using patch clamp electrophysiology, we show that the human Panx1 channel opens when certain divalent cations (Co2+, Mn2+ and Mg2+, but not Ca2+ or Zn2+) are applied at millimolar concentrations to the cytoplasmic side of the channel. The effect only occurs in excised patches and is much greater in the inside-out than the outside-out configuration.

Pannexin Channel Regulation of Cell Migration: Focus on Immune Cells.

The role of Pannexin (PANX) channels during collective and single cell migration is increasingly recognized. Amongst many functions that are relevant to cell migration, here we focus on the role of PANX-mediated adenine nucleotide release and associated autocrine and paracrine signaling. We also summarize the contribution of PANXs with the cytoskeleton, which is also key regulator of cell migration. PANXs, as mechanosensitive ATP releasing channels, provide a unique link between cell migration and purinergic communication. The functional association with several purinergic receptors, together with a plethora of signals that modulate their opening, allows PANX channels to integrate physical and chemical cues during inflammation. Ubiquitously expressed in almost all immune cells, PANX1 opening has been reported in different immunological contexts. Immune activation is the epitome coordination between cell communication and migration, as leukocytes (i.e., T cells, dendritic cells) exchange information while migrating towards the injury site. In the current review, we summarized the contribution of PANX channels during immune cell migration and recruitment; although we also compile the available evidence for non-immune cells (including fibroblasts, keratinocytes, astrocytes, and cancer cells). Finally, we discuss the current evidence of PANX1 and PANX3 channels as a both positive and/or negative regulator in different inflammatory conditions, proposing a general mechanism of these channels contribution during cell migration.

Pannexin-1 channel opening is critical for COVID-19 pathogenesis

SummarySevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) rapidly rampaged worldwide, causing a pandemic of coronavirus disease (COVID -19), but the biology of SARS-CoV-2 remains under investigation. We demonstrate that both SARS-CoV-2 spike protein and human coronavirus 229E (hCoV-229E) or its purified S protein, one of the main viruses responsible for the common cold, induce the transient opening of Pannexin-1 (Panx-1) channels in human lung epithelial cells. However, the Panx-1 channel opening induced by SARS-CoV-2 is greater and more prolonged than hCoV-229E/S protein, resulting in an enhanced ATP, PGE2, and IL-1β release. Analysis of lung lavages and tissues indicate that Panx-1 mRNA expression is associated with increased ATP, PGE2, and IL-1β levels. Panx-1 channel opening induced by SARS-CoV-2 spike protein is angiotensin-converting enzyme 2 (ACE-2), endocytosis, and furin dependent. Overall, we demonstrated that Panx-1 channel is a critical contributor to SARS-CoV-2 infection and should be considered as an alternative therapy.

Abstract 12129: Vascular Endothelial Barrier Protection May Prevent Arrhythmias in Atrial Fibrillation and Myocardial Infarction

Vascular leak is a major sequela of inflammation, which is associated with arrhythmic pathologies such as atrial fibrillation (AF) and myocardial infarction (MI). We recently demonstrated that the vascular leak-inducing cytokine vascular endothelial growth factor (VEGF; 90-580 pg/ml - levels found in AF patients) induces acute remodeling (30-60 minutes) of sodium channel (Na V 1.5) -rich intercalated disk (ID) nanodomains, disrupting their ultrastructure and prompting translocation of Na V 1.5 from these sites. This in turn disrupted impulse propagation and promoted arrhythmias in murine atria. Here, we tested the hypotheses that i) similar acute pro-arrhythmic remodeling occurs in the ventricles of MI patients, and ii) protecting the vascular barrier may prevent arrhythmias following an acute inflammatory insult. First, we examined myocardial samples from five human MI patients. VEGF was overexpressed in both cardiomyocytes and vascular endothelium in the border zone surrounding <6 month-old infarcts. Notably, co-localization analysis showed significantly reduced Na V 1.5 near both connexin43 and N-cadherin within the border zone in 1-, 3-, and 9-day-old infarcts, paralleling our observations in mouse atria. Next, we returned to our murine model of AF induced by acute inflammatory insult (100 pg/ml VEGF for 60 minutes) to test the antiarrhythmic efficacy of protecting the vascular endothelial barrier. Overall, median in vivo arrhythmia burden was higher in VEGF-treated mice relative to vehicle controls (7.5±11 vs. 0±6 s/hr). We tested two strategies shown to prevent vascular barrier breakdown: Blocking connexin43 hemichannels (αCT11 peptide) decreased in vivo arrhythmia burden to 0 ± 6.07 s/hr. Panx1-IL2 (a peptide inhibitor of Panx1 channels) treatment decreased also in vivo arrhythmia burden (0 ± 15.57 s/hr with 1.6 μM Panx1-IL2). Similar antiarrhythmic efficacy was also achieved with small molecule inhibitors of Cx43 and Panx1. These results highlight VEGF-induced vascular leak as a novel mechanism for acute arrhythmias both in the early stage AF and following MI. Indeed, this mechanism may contribute to post-MI AF. Importantly, vascular-barrier protection may be a viable strategy to prevent these arrhythmias.

Probenecid affects sarcoplasmic reticulum Ca2+ release and depresses contractile activation in mouse skeletal muscle

Pannexins are plasma membrane heptameric channels mediating ATP release from the cytosol to the extracellular space. Skeletal muscle activity is associated with Pannexin 1 (Panx1) channels activation, ATP release out to the extracellular space and subsequent activation of purinergic signaling pathways. In agreement, recent evidence has shown molecular and functional interactions between Panx1 and the excitation-contraction (EC) coupling machinery of skeletal muscle. In this framework, we tested whether pharmacological effectors of Panx1 affect EC coupling in differentiated muscle fibers. Using confocal detection of cytosolic Ca2+ in voltage-clamped mouse muscle fibers, we found that the Panx1 blocker probenecid (1 mM) affects intracellular Ca2+ handling and EC coupling: acute application of probenecid generates a rise in resting Ca2+ that also occurs in nominally Ca2+-free extracellular medium. This effect is associated with a reduction of Ca2+ release through the sarcoplasmic reticulum (SR) Ca2+ channel RYR1. The effect of probenecid persists with time, with muscle fibers incubated for 30 min in the presence of the drug exhibiting a 40% reduction in peak SR Ca2+ release. Under the same conditions, the other Panx1 blocker carbenoxolone (50 µM) produced a 70% reduction in peak SR Ca2+ release. Application of probenecid on electrically stimulated whole mouse muscle induced a slight rise in resting tension and a >50% reduction of tetanic force after 30 min of incubation. Our results provide further support for the strong links between Panx1 function and EC coupling. Because probenecid is used both in the clinic for several types of therapeutic benefits and as a hiding agent for doping in sport, our results question whether potential adverse muscular effects may have, so far, been overlooked.

Estrogen depletion on In vivo osteocyte calcium signaling responses to mechanical loading.

Microstructural adaptation of bone in response to mechanical stimuli is diminished with estrogen deprivation. Here we tested in vivo whether ovariectomy (OVX) alters the acute response of osteocytes, the principal mechanosensory cells of bone, to mechanical loading in mice. We also used super resolution microscopy (Structured Illumination microscopy or SIM) in conjunction with immunohistochemistry to assess changes in the number and organization of "osteocyte mechanosomes" - complexes of Panx1 channels, P2X7 receptors and CaV3 voltage-gated Ca2+ channels clustered around αvβ3 integrin foci on osteocyte processes. Third metatarsals bones of mice expressing an osteocyte-targeted genetically encoded Ca2+ indicator (DMP1-GCaMP3) were cyclically loaded in vivo to strains from 250 to 3000 με and osteocyte intracellular Ca2+ signaling responses were assessed in mid-diaphyses using multiphoton microscopy. The number of Ca2+ signaling osteocytes in control mice increase monotonically with applied strain magnitude for the physiological range of strains. The relationship between the number of Ca2+ signaling osteocytes and loading was unchanged at 2days post-OVX. However, it was altered markedly at 28days post-OVX. At loads up to 1000 με, there was a dramatic reduction in number of responding (i.e. Ca2+ signaling) osteocytes; however, at higher strains the numbers of Ca2+ signaling osteocytes were similar to control mice. OVX significantly altered the abundance, make-up and organization of osteocyte mechanosome complexes on dendritic processes. Numbers of αvβ3 foci also staining with either Panx 1, P2X7R or CaV3 declined by nearly half after OVX, pointing to a loss of osteocyte mechanosomes on the dendritic processes with estrogen depletion. At the same time, the areas of the remaining foci that stained for αvβ3 and channel proteins increased significantly, a redistribution of mechanosome components suggesting a potential compensatory response. These results demonstrate that the deleterious effects of estrogen depletion on skeletal mechanical adaptation appear at the level of mechanosensation; osteocytes lose the ability to sense small (physiological) mechanical stimuli. This decline may result at least partly from changes in the structure and organization of osteocyte mechanosomes, which contribute to the distinctive sensitivity of osteocytes (particularly their dendritic processes) to mechanical stimulation.

Design and synthesis of the first indole-based blockers of Panx-1 channel.

Panx-1 is a membrane channel protein involved in some pathologies such as ischemic stroke, cancer and neuropathic pain, thus representing a promising therapeutic target. We present here a study aimed at obtaining the first class of selective Panx-1 blockers, a new topic for pharmaceutical chemistry, since all compounds used so far for the study of this channel have different primary targets. Among various scaffolds analyzed, the indole nucleous emerged, whose elaboration yielded interesting Panx-1 blockers, such as the potent 5-sulfamoyl derivatives 14c and 15b (I%=100at 50μM). Invivo tests performed in the mouse model of oxaliplatin-induced neuropathy, demonstrated that the hypersensitivity was completely reverted by treatment with 15b (1nmol, administered intrathecally), suggesting a relationship between this effect and the channel blocking ability. Finally, we decided to perform a virtual screening study on compounds 5b, 6l and 14c using a recently resolved cryo-EM structure of hPanx-1 channel, to try to relate the potency of our new inhibitors.

Leukotoxin (LtxA/Leukothera) induces ATP expulsion via pannexin-1 channels and subsequent cell death in malignant lymphocytes

Leukotoxin (LtxA) (Trade name, Leukothera) is a protein that is secreted from the oral bacterium Aggregatibacter actinomycetemcomitans, which targets and kills activated white blood cells (WBCs) by binding to lymphocyte function associated antigen-1 (LFA-1). Interaction between LtxA and Jurkat T-cells results in cell death and is characterized by increased intracellular Ca2+, activation of caspases, clustering of LtxA and LFA-1 within lipid rafts, and involvement of the Fas death receptor. Here, we show that LtxA can kill malignant lymphocytes via apoptotic and necrotic forms of cell death. We show that LtxA causes activation of caspases and PARP, cleavage of pannexin-1 (Panx1) channels, and expulsion of ATP, ultimately leading to cell death via apoptosis and necrosis. CRISPR-Cas9 mediated knockout (K/O) of Panx1 in Jurkat cells prevented ATP expulsion and resulted in resistance to LtxA for both apoptotic and necrotic forms of death. Resistance to necrosis could only be overcome when supplementing LtxA with endogenous ATP (bzATP). The combination of LtxA and bzATP promoted only necrosis, as no Panx1 K/O cells stained positive for phosphatidylserine (PS) exposure following the combined treatment. Inhibition of LtxA/bzATP-induced necrosis was possible when pretreating Jurkat cells with oATP, a P2X7R antagonist. Similarly, blockage of P2X7Rs with oATP prevented the intracellular mobilization of Ca2+, an important early step in LtxA induced cell death. We show that LtxA is able to kill malignant lymphocytes through an apoptotic death pathway which is potentially linked to a Panx1/P2X7R mediated necrotic form of death. Thus, inhibition of ATP release appears to significantly delay the onset of LtxA induced apoptosis while completely disabling the necrotic death pathway in T-lymphocytes, demonstrating the crucial role of ATP release in LtxA-mediated cell death.

Endothelial pannexin 1-TRPV4 channel signaling lowers pulmonary arterial pressure in mice.

Pannexin 1 (Panx1), an ATP-efflux pathway, has been linked with inflammation in pulmonary capillaries. However, the physiological roles of endothelial Panx1 in the pulmonary vasculature are unknown. Endothelial transient receptor potential vanilloid 4 (TRPV4) channels lower pulmonary artery (PA) contractility and exogenous ATP activates endothelial TRPV4 channels. We hypothesized that endothelial Panx1-ATP-TRPV4 channel signaling promotes vasodilation and lowers pulmonary arterial pressure (PAP). Endothelial, but not smooth muscle, knockout of Panx1 increased PA contractility and raised PAP in mice. Flow/shear stress increased ATP efflux through endothelial Panx1 in PAs. Panx1-effluxed extracellular ATP signaled through purinergic P2Y2 receptor (P2Y2R) to activate protein kinase Cα (PKCα), which in turn activated endothelial TRPV4 channels. Finally, caveolin-1 provided a signaling scaffold for endothelial Panx1, P2Y2R, PKCα, and TRPV4 channels in PAs, promoting their spatial proximity and enabling signaling interactions. These results indicate that endothelial Panx1-P2Y2R-TRPV4 channel signaling, facilitated by caveolin-1, reduces PA contractility and lowers PAP in mice.

Abstract P333: The Vascular Barrier: A Common Anti-arrhythmic Target In Atrial Fibrillation And Myocardial Infarction

Vascular leak is a major sequela of inflammation, which is associated with arrhythmic pathologies such as atrial fibrillation (AF) and myocardial infarction (MI). We recently demonstrated that the vascular leak-inducing cytokine vascular endothelial growth factor (VEGF; 90-580 pg/ml - levels found in AF patients) induces acute remodeling (30-60 minutes) of sodium channel (Na V 1.5) -rich intercalated disk (ID) nanodomains, disrupting their ultrastructure and prompting translocation of Na V 1.5 from these sites. This in turn disrupted impulse propagation and promoted arrhythmias in murine atria. Here, we tested the hypotheses that i) similar acute pro-arrhythmic remodeling occurs in the ventricles of MI patients, and ii) protecting the vascular barrier may prevent arrhythmias following an acute inflammatory insult. First, we examined myocardial samples from five human MI patients. VEGF was overexpressed in both cardiomyocytes and vascular endothelium in the border zone surrounding <6 month-old infarcts. Notably, co-localization analysis showed significantly reduced Na V 1.5 near both connexin43 and N-cadherin within the border zone in 1-, 3-, and 9-day-old infarcts, paralleling our observations in mouse atria. Next, we returned to our murine model of AF induced by acute inflammatory insult (100 pg/ml VEGF for 60 minutes) to test the antiarrhythmic efficacy of protecting the vascular endothelial barrier. Overall, median in vivo arrhythmia burden was higher in VEGF-treated mice relative to vehicle controls (7.5±11 vs. 0±6 s/hr). We tested two strategies shown to prevent vascular barrier breakdown: Blocking connexin43 hemichannels (αCT11 peptide) decreased in vivo arrhythmia burden to 0 ± 6.07 s/hr. Panx1-IL2 (a peptide inhibitor of Panx1 channels) treatment decreased also in vivo arrhythmia burden (0 ± 15.57 s/hr with 1.6 μM Panx1-IL2). Similar antiarrhythmic efficacy was also achieved with small molecule inhibitors of Cx43 and Panx1. These results highlight VEGF-induced vascular leak as a novel mechanism for acute arrhythmias both in the early stage AF and following MI. Indeed, this mechanism may contribute to post-MI AF. Importantly, vascular-barrier protection may be a viable strategy to prevent these arrhythmias.

Pannexin 1 plays a pro-survival role by attenuating P2X7 receptor-mediated Ca2+ influx

Extracellular ATP works as an autocrine and/or paracrine signaling molecule by activating plasma membrane-localized purinergic receptors. Stimulation of purinergic P2X7 receptor (P2X7R) increases cytosolic Ca2+ ([Ca2+]c), which in turn activates Pannexin 1 (Panx1) channel. In earlier studies, Panx1 and P2X7R have been shown to interact physically. Also, both the channels have been implicated in similar pathophysiological processes. In this study, we investigated the effect of Panx1 on P2X7R-mediated Ca2+influx. Panx1 attenuated P2X7R-mediated [Ca2+]c rise in CHO-K1 and HEK-293 cells. [Ca2+]c rise was higher in Panx1 knockdown astrocytes. The inhibitory effect was unaffected in the presence of Panx1 blocker, carbenoxolone. The region between 350th and 386th amino acid residues in the carboxyl terminus (CT) of Panx1 was found to be crucial for inhibiting P2X7R. Like full-length Panx1, the CT (350th to 426th amino acids) alone was able to attenuate the [Ca2+]c rise. Further, CT prevented cell death caused by P2X7R overactivation. Based on our results, we propose a novel pro-survival role of Panx1 exerted by modulating P2X7R.