Mediating ATP Release Research Articles

Pkd1RC/RC mice demonstrate remodeling of purinergic receptors in ADPKD cysts

Polycystic kidney diseases (PKD) are characterized by development of multiple cysts, dilations of nephron segments, which replace normal tissues and lead to kidney insuffciency. To identify new gene pathways affected by cyst development in collecting ducts, we used a bulk RNAseq approach comparing gene expression of normal microdissected cortical collecting ducts (n=3 mice) vs cysts microdissected from Pkd1 RC/RC mice (n=4). Bulk-RNA analysis identified 18,000 genes and allowed statistical comparison of over 15,000 genes. Our data reveals that although cysts originate from normal collecting ducts, cystic epithelium show 2692 down-regulated and 2278 up-regulated genes (p<0.05 pAdj. FDR). Ingenuity Pathways Analysis identifies the following intracellular mechanisms mostly affected by transition: Rac and Rho signaling, fibrosis signaling, epithelial-to-mesenchymal transition, cytoskeleton rearrangement and ERK/MAPK signaling. Several of the differentially expressed genes were identified in GWAS of PKD, chronic kidney diseases and hypertension. Our previous publication reported that development of cysts in an autosomal recessive model of PKD is associated with a shift of P2Y to P2X receptor abundance. In the current study we found that in the autosomal dominant Pkd1 RC/RC mice purinergic signaling undergoes similar remodeling. The most abundant ionotropic receptors with reduced expression were P2ry2 -2.25; P2ry4 -1.37, log2 fold change, whereas ionotropic receptors P2rx5 and P2rx7 increased expression (2.88 and 1.53, log2 fold change, respectively). Additionally, analysis detected elevated abundance of P2ry6 and P2ry12 RNA level. We used a model of cystogenesis to test how P2X signaling affects growth of cysts formed by mpkCCDcl4 cells in Matrigel. Stimulation of P2X receptors with α,βMe-ATP significantly increases cyst growth. We hypothesize that the physiological significance of the predominant P2X signaling in the cysts include their role in regulation of ATP release via pannexin-1 channels. Abnormal ATP accumulation in the cyst space was shown earlier to contribute in cystogenesis and we previously showed that pannexin-1 mediates ATP release to the cyst lumen. Co-immunoprecipitatation experiments in mpkCCDcl4 cells revealed that P2X5 and P2X7 receptors bind pannexin-1 whereas P2Y6 and P2Y12 do not interact with pannexin-1. Interestingly, stimulation of cystogenesis with α,βMe-ATP was accompanied by increased abundance of pannexin-1. Lack of functional P2X5 in humans prioritizes P2X7 receptors as a therapeutic target in ADPKD. We conclude that development of ADPKD cysts involves massive transcriptome remodeling of collecting ducts which include a shift in purinergic signaling that facilitates pathogenic pannexin-1 hyperactivity. DK123266 and DK131114. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Connexin-43 hemichannels orchestrate NOD-like receptor protein-3 (NLRP3) inflammasome activation and sterile inflammation in tubular injury

BackgroundWithout a viable cure, chronic kidney disease is a global health concern. Inflammatory damage in and around the renal tubules dictates disease severity and is contributed to by multiple cell types. Activated in response to danger associated molecular patterns (DAMPs) including ATP, the NOD-like receptor protein-3 (NLRP3) inflammasome is integral to this inflammation. In vivo, we have previously observed that increased expression of Connexin 43 (Cx43) is linked to inflammation in chronic kidney disease (CKD) whilst in vitro studies in human proximal tubule cells highlight a role for aberrant Cx43 hemichannel mediated ATP release in tubule injury. A role for Cx43 hemichannels in priming and activation of the NLRP3 inflammasome in tubule epithelial cells remains to be determined.MethodsUsing the Nephroseq database, analysis of unpublished transcriptomic data, examined gene expression and correlation in human CKD. The unilateral ureteral obstruction (UUO) mouse model was combined with genetic (tubule-specific Cx43 knockout) and specific pharmacological blockade of Cx43 (Peptide5), to explore a role for Cx43-hemichannels in tubule damage. Human primary tubule epithelial cells were used as an in vitro model of CKD.ResultsIncreased Cx43 and NLRP3 expression correlates with declining glomerular filtration rate and increased proteinuria in biopsies isolated from patients with CKD. Connexin 43-tubule deletion prior to UUO protected against tubular injury, increased expression of proinflammatory molecules, and significantly reduced NLRP3 expression and downstream signalling mediators. Accompanied by a reduction in F4/80 macrophages and fibroblast specific protein (FSP1+) fibroblasts, Cx43 specific hemichannel blocker Peptide5 conferred similar protection in UUO mice. In vitro, Peptide5 determined that increased Cx43-hemichannel activity primes and activates the NLRP3 inflammasome via ATP-P2X7 receptor signalling culminating in increased secretion of chemokines and cytokines, each of which are elevated in individuals with CKD. Inhibition of NLRP3 and caspase 1 similarly decreased markers of tubular injury, whilst preventing the perpetual increase in Cx43-hemichannel activity.ConclusionAberrant Cx43-hemichannel activity in kidney tubule cells contributes to tubule inflammation via activation of the NLRP3 inflammasome and downstream paracrine mediated cell signalling. Use of hemichannel blockers in targeting Cx43-hemichannels is an attractive future therapeutic target to slow or prevent disease progression in CKD.-uWW98vNU7-T3Uscg54sCsVideo

Conformational rearrangements associated with Panx1 channel gating.

Pannexins (Panx1-3) are ATP permeable membrane channels that are widely expressed in the human body. It has been demonstrated that pannexins play central roles in ATP release from a wide range of cell types including airway epithelia, blood cells, glial cells, and neurons. Pannexin mediated ATP release contributes to blood pressure regulation, neurotransmission, and apoptotic cell clearance. Our group and others determined structures of Panx1 using cryo-EM, which uncovered that Panx1 assembles into a unique heptameric channel with a potential pathway for the ATP molecule. However, how Panx1 controls ATP migration through the pore remains unclear. In fact, it is still uncertain whether the currently available Panx1 structures represent open or closed conformations. We recently discovered a cell metabolite as a bona fide Panx1 agonist. We performed cryo-EM single particle reconstructions in the presence or absence of this agonist to shed light on the Panx1 channel gating mechanism. We found a major conformational rearrangement in a region surrounding the ATP permeation pathway. Cysteine accessibility studies using whole cell patch clamp electrophysiology support that the conformational rearrangement in this region is important for Panx1 channel gating. This is in contrast to the proposed-gating mechanism in apoptotic cells, in which caspase-dependent C-terminal cleavage unplugs the pore for permeant molecules. Based on the current study, we provide a novel mechanism underlying Panx1 channel gating in non-apoptotic cells.

Activation of cardiac connexin 43 hemichannels by shear stress and subsequent contribution of pannexins in left-side cardiac myocytes

Cardiac myocytes are exposed to shear stress under physiological conditions and hemodynamic disturbances. Shear stress has been thought to trigger global Ca2+ waves in atrial myocytes through autocrine activation of P2 receptors by connexin43 (Cx43)-mediated ATP release. We examined whether shear stress activates Cx43 hemichannels and pannexins in atrial and ventricular myocytes from right and left side of heart chambers. The activities of these hemichannels were measured as an efflux of large fluorescence dye (calcein-red/orange) to permeate these channels using two-dimensional confocal imaging method in single isolated myocytes of murine hearts.

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.

Association Between Adenosine A2A Receptors and Connexin 43 Regulates Hemichannels Activity and ATP Release in Astrocytes Exposed to Amyloid-β Peptides.

Increasing evidence implicates astrocytes and the associated purinergic modulation in Alzheimer's disease (AD), characterized by cognitive deficits involving the extracellular amyloid-β peptides (Aβ) accumulation. Aβ can affect astrocytic gliotransmitters release, namely ATP, which is rapidly metabolized into adenosine by ecto-5'-nucleotidase, CD73, resulting in adenosine A2A receptors (A2AR) activation that bolsters neurodegeneration. AD's brains exhibit an upregulation of A2AR and of connexin 43 (Cx43), which in astrocytes forms hemichannels that can mediate ATP release. However, a coupling between astrocytic A2AR and Cx43 remains to be established. This was now investigated using astrocytic primary cultures exposed to Aβ1-42 peptides. Aβ triggered ATP release through Cx43 hemichannels, a process blocked by A2AR antagonists and mimicked by selective A2AR activation. A2AR directly regulated hemichannels activity and prevented Cx43 upregulation and phosphorylation observed in Aβ1-42-exposed astrocytes. Moreover, a proximity ligand assay revealed a physical association between astrocytic A2AR and Cx43. Finally, the blockade of CD73-mediated extracellular formation of ATP-derived adenosine prevented the Aβ-induced increase of Cx43 hemichannel activity and of ATP release. Overall, the data identify a feed-forward loop involving astrocytic A2AR and Cx43 hemichannels, whereby A2AR increase Cx43 hemichannel activity leading to increased ATP release, which is converted into adenosine by CD73, sustaining the increased astrocytic A2AR activity in AD-like conditions.

Pannexin 1 as a driver of inflammation and ischemia-reperfusion injury.

Pannexin 1 (Panx1) is a ubiquitously expressed protein forming large conductance channels that are central to many distinct inflammation and injury responses. There is accumulating evidence showing ATP released from Panx1 channels, as well as metabolites, provide effective paracrine and autocrine signaling molecules that regulate different elements of the injury response. As channels with a broad range of permselectivity, Panx1 channels mediate the secretion and uptake of multiple solutes, ranging from calcium to bacterial derived molecules. In this review, we describe how Panx1 functions in response to different pro-inflammatory stimuli, focusing mainly on signaling coordinated by the vasculature. How Panx1 mediates ATP release by injured cells is also discussed. The ability of Panx1 to serve as a central component of many diverse physiologic responses has proven to be critically dependent on the context of expression, post-translational modification, interacting partners, and the mode of stimulation.

PANX2 and brain lower grade glioma genesis: A bioinformatic analysis.

PANX2 forms large-pore channels mediating ATP release in response to physiological and pathological stimuli. Although PANX2 shows involvements in glioma genesis, the underlying mechanism remains unclear. PANX2 mRNA expression was analyzed via Oncomine and was confirmed via Gene Expression Profiling Interactive Analysis (GEPIA). The influence of PANX2 on overall survival (OS) of glioma was evaluated using LinkedOmics and further assessed through Cox regression analysis. The correlated genes with PANX2 acquired from LinkedOmics were validated through GEPIA and cBioPortal. Protein-protein interaction (PPI) of these genes was then obtained using Search Tool for the Retrieval of Interacting Genes (STRING) and Cytoscape with MCODE plug-in. All the PANX2-related genes underwent Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. The correlation between PANX2 and cancer immune infiltrates was evaluated via Tumor Immune Estimation Resource (TIMER). A higher expression of PANX2 only revealed a better OS in brain low grade glioma (LGG). PANX2-related genes in LGG functionally enriched in neuroactive ligand-receptor interaction, synaptic vesicle cycle, and calcium signaling. The hub genes from highest module of PPI were mainly linked to chemical synaptic transmission, plasma membrane, neuropeptide, and the pathway of neuroactive ligand-receptor interaction. Besides, PANX2 expression was negatively associated with infiltrating levels of macrophage, dendritic cells, and CD4+ T cells. This study demonstrated that PANX2 likely participated in LGG pathogenesis by affecting multiple molecular pathways and immune-related processes. PANX2 was associated with LGG prognosis and might become a promising therapeutic target of LGG.

Basic and clinical science posters: Basic science of diabetes complications

Aims: Glucose evoked changes in profibrotic transforming growth factor-beta1 (TGFs1), increase connexin hemichannel mediated ATP release in kidney proximal tubule cells. With ATP linked to inflammation, we evaluated the efficacy of hemichannel blocker Peptide 5 in negating ATP induced inflammatory cytokine secretion in proximal tubule cells. Methods: Primary human proximal tubular epithelial cells (hPTECs), and immortalised human kidney (HK2) cells were treated with TGFs1 (10ng/ml) ± Peptide5 (25µM) for 48hrs. Carboxyfluorescein (200µM) dye uptake and ATP biosensing determined hemichannel activity and ATP release respectively. Proteome arrays determined cytokine secretion. Results: In TGFs1 treated hPTECs, carboxyfluorescein dye uptake decreased from 347.4±11.9%, (P<0.001, N=3) to 158.6±7.9% when co-incubated with Peptide 5. Parallel to this, ATP release increased from 0.16±0.1µM to 2.5±0.14µM (P<0.001, N=3) in TGF1 treated cells as compared to control. Peptide 5 significantly blunted this response, to 0.62±0.17µM (P<0.001, N=3). A proteome array determined the impact of Peptide 5 on secretion of 105 candidate chemokines and cytokines in TGFs1 treated primary cells. As an example, Peptide 5 significantly restored secretion of adiponectin from 163±8.0% (P<0.001, N=4) to 136±6.1% (P<0.05, N=4), granulocyte-macrophage colony-stimulating factor from 183±8.0% (P<0.001, N=4) to 111.3±5.9% (P<0.001, N=4) and interleukin 1-beta from 189±14.9 (P<0.001, N=4) to 109±9.6% (P<0.001, N=4) in TGF-s1 treated hPTECs, as compared to control. Conclusion: Glucose evoked changes in TGFs1 increase hemichannel mediated ATP release. Hemichannel blocker; Peptide 5 blocks ATP release and downstream cytokine and chemokine secretion, suggesting that Peptide 5 may be of future therapeutic potential in targeting inflammation of the diabetic kidney.

Non-Selective Cation Currents Mediated by Cx43 Hemichannel-P2X4 Receptor Signaling Pathway in Rat Atrial Myocytes under Shear Stress

Cardiac myocytes are exposed to shear stress under physiological conditions and hemodynamic disturbances. Shear stress has been thought to trigger two distinct global Ca2+ waves in atrial myocytes through autocrine activation of P2X4 receptor (P2X4R) and P2Y1-receptor (P2Y1R) via connexin 43 (Cx43)-mediated ATP release. In this study, we examined whether such shear stress-triggered Cx43-ATP-P2 receptor signaling generates depolarizing currents in atrial myocytes. Whole-cell patch clamp and dye-flux assays were used with genetic and pharmacological interventions in rat atrial myocytes and HL-1 cells. We found that shear stress activated a non-selective inward cation (Cs+) current (IS,Cs) at resting potential which was eliminated by Cx43 blockade using siRNA, antibodies, carbenoxolone or La3+. IS,Cs was enhanced by removal of external Ca2+, whereas internal Ca2+ buffering suppressed IS,Cs. Hemichannel-mediated dye (calcein red-orange) flux was induced by shear stress. This response was augmented by removing external Ca2+ but was not affected by pannexin blocker (probenecid). The shear-activated current was significantly reduced when Cs+ was replaced with NMDG+, and this was enhanced by quinine and suppressed by La3+. P2X4R antagonists (5-BDBD and PSB-12054), P2XR-blocker (iso-PPADS) and anti-P2X4R antibodies each blocked IS,Cs by approximately 50%. About 25% of IS,Cs was suppressed by P2Y1R inhibitor (MRS2179) and/or transient receptor potential melastatin 4 blocker (9-phenanthrol). P2X4R, but not P2X5R, was co-localized with Cx43 at the cell ends. P2X4R-dependent IS,Cs was augmented in atrial myocytes from hypertrophied rat hearts by short-term aortic constriction. Our data suggest that shear stress triggers Cx43 hemichannels to gate P2X4Rs in their vicinity. This shear stress signaling may play an important role in membrane depolarization at negative potentials and in atrial remodeling under increased afterload.

Mechanically induced integrin ligation mediates intracellular calcium signaling with single pulsating cavitation bubbles.

Therapeutic ultrasound or shockwave has shown its great potential to stimulate neural and muscle tissue, where cavitation microbubble induced Ca2+ signaling is believed to play an important role. However, the pertinent mechanisms are unknown, especially at the single-cell level. Particularly, it is still a major challenge to get a comprehensive understanding of the effect of potential mechanosensitive molecular players on the cellular responses, including mechanosensitive ion channels, purinergic signaling and integrin ligation by extracellular matrix.Methods: Here, laser-induced cavitation microbubble was used to stimulate individual HEK293T cells either genetically knocked out or expressing Piezo1 ion channels with different normalized bubble-cell distance. Ca2+ signaling and potential membrane poration were evaluated with a real-time fluorescence imaging system. Integrin-binding microbeads were attached to the apical surface of the cells at mild cavitation conditions, where the effect of Piezo1, P2X receptors and integrin ligation on single cell intracellular Ca2+ signaling was assessed.Results: Ca2+ responses were rare at normalized cell-bubble distances that avoided membrane poration, even with overexpression of Piezo1, but could be increased in frequency to 42% of cells by attaching integrin-binding beads. We identified key molecular players in the bead-enhanced Ca2+ response: increased integrin ligation by substrate ECM triggered ATP release and activation of P2X—but not Piezo1—ion channels. The resultant Ca2+ influx caused dynamic changes in cell spread area.Conclusion: This approach to safely eliciting a Ca2+ response with cavitation microbubbles and the uncovered mechanism by which increased integrin-ligation mediates ATP release and Ca2+ signaling will inform new strategies to stimulate tissues with ultrasound and shockwaves.

Deletion of TRPV4 enhances in vitro wound healing of murine esophageal keratinocytes

Transient receptor potential vanilloid 4 (TRPV4) is a non-selective cation channel that is widely expressed in different body tissues and plays several physiological roles. This channel is highly expressed in esophageal keratinocytes where its activation mediates ATP release. However, whether TRPV4 has a role in wound healing of esophageal keratinocytes is unclear. In this study, we demonstrated that both cell migration and proliferation were slower in wild-type esophageal keratinocytes compared to cells having TRPV4 knockout. Our results suggest that TRPV4-mediated release of ATP from esophageal keratinocytes contributes to a decrease in the rate of in vitro wound healing via the ATP degradation product adenosine, which acts on A2B adenosine receptors.

Pannexin-1 mediates fluid shear stress-sensitive purinergic signaling and cyst growth in polycystic kidney disease.

Tubular ATP release is regulated by mechanosensation of fluid shear stress (FSS). Polycystin-1/polycystin-2 (PC1/PC2) functions as a mechanosensory complex in the kidney. Extracellular ATP is implicated in polycystic kidney disease (PKD), where PC1/PC2 is dysfunctional. This study aims to provide new insights into the ATP signaling under physiological conditions and PKD. Microfluidics, pharmacologic inhibition, and loss-of-function approaches were combined to assess the ATP release in mouse distal convoluted tubule 15 (mDCT15) cells. Kidney-specific Pkd1 knockout mice (iKsp-Pkd1-/- ) and zebrafish pkd2 morphants (pkd2-MO) were as models for PKD. FSS-exposed mDCT15 cells displayed increased ATP release. Pannexin-1 inhibition and knockout decreased FSS-modulated ATP release. In iKsp-Pkd1-/- mice, elevated renal pannexin-1 mRNA expression and urinary ATP were observed. In Pkd1-/- mDCT15 cells, elevated ATP release was observed upon the FSS mechanosensation. In these cells, increased pannexin-1 mRNA expression was observed. Importantly, pannexin-1 inhibition in pkd2-MO decreased the renal cyst growth. Our results demonstrate that pannexin-1 channels mediate ATP release into the tubular lumen due to pro-urinary flow. We present pannexin-1 as novel therapeutic target to prevent the renal cyst growth in PKD.

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

Extracellular ATP signalling is involved in many physiological and pathophysiological processes in several tissues, including adipose tissue. Adipocytes have crucial functions in lipid and glucose metabolism and they express purinergic receptors. However, the sources of extracellular ATP in adipose tissue are not well characterized. In the present study, we investigated the mechanism and regulation of ATP release in white adipocytes, and evaluated the role of extracellular ATP as potential autocrine and paracrine signal. Online ATP release was monitored in C3H10T1/2 cells and freshly isolated murine adipocytes. The ATP release mechanism and its regulation were tested in cells exposed to adrenergic agonists, insulin, glucose load and pharmacological inhibitors. Cell metabolism was monitored using Seahorse respirometry and expression analysis of pannexin-1 was performed on pre- and mature adipocytes. The ATP signalling was evaluated in live cell imaging (Ca2+ , pore formation), glycerol release and its effect on macrophages was tested in co-culture and migration assays. Here, we show that upon adrenergic stimulation white murine adipocytes release ATP through the pannexin-1 pore that is regulated by a cAMP-PKA-dependent pathway. The ATP release correlates with increased cell metabolism and is sensitive to glucose. Extracellular ATP induces Ca2+ signalling and lipolysis in adipocytes and promotes macrophage migration. Importantly, ATP release is markedly inhibited by insulin, which operates via the activation of phosphodiesterase 3. Our findings reveal an insulin-pannexin-1-purinergic signalling crosstalk in adipose tissue and we propose that deregulation of this signalling may contribute to adipose tissue inflammation and type 2 diabetes.

A Complex Neuroprotective Effect of Alpha-2-Adrenergic Receptor Agonists in a Model of Cerebral Ischemia–Reoxygenation In Vitro

The mechanism of the neuroprotective action of α2-adrenergic receptor agonists in a model of oxygen-glucose deprivation (OGD) in vitro was investigated. Using fluorescent microscopy, immunostaining, inhibitor analysis, and real-time PCR analysis, we demonstrated that OGD evokes a biphasic [Ca2+]i increase in all cells. Initial Ca2+ impulse in astrocytes and Ca2+ oscillations in neurons were followed by a slower global increase of [Ca2+]i in both cell populations. Accumulation of pro-inflammatory factors, such as IL1b and TNFα, was observed during 40-min OGD. It was established that reoxygenation is followed by hyperexcitation of neurons, caspase-3 activation, and subsequent cell death. We showed that a 24-h pretreatment of cell cultures with selective α2-adrenergic receptor agonists guanfacine and UK-14,304 abolished the global [Ca2+]i increase in astrocytes and neurons but did not suppress the first phase of the OGD-induced Ca2+ impulse in astrocytes. In addition, the number of dead cells after OGD was decreased in cell cultures pretreated with the α2-agonists. Guanfacine inhibited caspase-3 activation and suppressed apoptosis in our experiments. In particular, the expression of antiapoptotic genes Stat3 and Bcl-2 was enhanced after the pretreatment with guanfacine. On the contrary, the expression of proapoptotic genes (Socs-3, p53, fas, and Ikk) was decreased. Moreover, application of guanfacine evoked Ca2+ response in astrocytes and led to Ca2+-mediated ATP release and this way suppressed hyperexcitation of the neurons. Thus, activation of astrocytes and Ca2+-mediated ATP release possibly contribute to the complex neuroprotective effects of the α2-adrenergic receptor agonists.

Constitutive SRC-mediated phosphorylation of pannexin 1 at tyrosine 198 occurs at the plasma membrane

Pannexin 1 (PANX1)-mediated ATP release in vascular smooth muscle coordinates α1-adrenergic receptor (α1-AR) vasoconstriction and blood pressure homeostasis. We recently identified amino acids 198-200 (YLK) on the PANX1 intracellular loop that are critical for α1-AR-mediated vasoconstriction and PANX1 channel function. We report herein that the YLK motif is contained within an SRC homology 2 domain and is directly phosphorylated by SRC proto-oncogene, nonreceptor tyrosine kinase (SRC) at Tyr198 We demonstrate that PANX1-mediated ATP release occurs independently of intracellular calcium but is sensitive to SRC family kinase (SFK) inhibition, suggestive of channel regulation by tyrosine phosphorylation. Using a PANX1 Tyr198-specific antibody, SFK inhibitors, SRC knockdown, temperature-dependent SRC cells, and kinase assays, we found that PANX1-mediated ATP release and vasoconstriction involves constitutive phosphorylation of PANX1 Tyr198 by SRC. We specifically detected SRC-mediated Tyr198 phosphorylation at the plasma membrane and observed that it is not enhanced or induced by α1-AR activation. Last, we show that PANX1 immunostaining is enriched in the smooth muscle layer of arteries from hypertensive humans and that Tyr198 phosphorylation is detectable in these samples, indicative of a role for membrane-associated PANX1 in small arteries of hypertensive humans. Our discovery adds insight into the regulation of PANX1 by post-translational modifications and connects a significant purinergic vasoconstriction pathway with a previously identified, yet unexplored, tyrosine kinase-based α1-AR constriction mechanism. This work implicates SRC-mediated PANX1 function in normal vascular hemodynamics and suggests that Tyr198-phosphorylated PANX1 is involved in hypertensive vascular pathology.

Roles of volume-regulatory anion channels, VSOR and Maxi-Cl, in apoptosis, cisplatin resistance, necrosis, ischemic cell death, stroke and myocardial infarction.

Two types of anion channels are directly activated by osmotic swelling and are involved in the regulatory volume decrease (RVD) in most types of mammalian cells, and they include the volume-sensitive outwardly rectifying anion channel (VSOR), also called the volume-regulated anion channel (VRAC), and the large-conductance maxi-anion channel (Maxi-Cl). In cardiomyocytes, a splice variant of cystic fibrosis transmembrane conductance regulator anion channel (cardiac CFTR) participates in the RVD mechanism under β-adrenergic stimulation. VSOR and Maxi-Cl are also involved in facilitation of the RVD process by releasing extracellular autocrine/paracrine signals, glutamate and ATP. Apoptotic cell death starts with cell shrinkage, called the apoptotic volume decrease (AVD), which is also caused by activation of VSOR. Since VSOR is implicated not only in the AVD induction but also in the uptake of an anti-cancer drug, cisplatin, downregulation of VSOR activity is causatively involved in acquisition of cisplatin resistance in cancer cells. Necrotic cell death exhibits persistent cell swelling, called the necrotic volume increase (NVI), which is coupled to RVD dysfunction due to impaired VSOR function. Acidotoxic and lactacidosis-induced necrotic cell death is induced both by glutamate release mediated by astroglial VSOR and Maxi-Cl and by exaggerated Cl- influx mediated by neuronal VSOR under prolonged depolarization caused by activation of ionotropic glutamate receptor (iGluR) cation channels. Both VSOR and Maxi-Cl are elaborately involved, in a manner as double-edged swords, in ischemia- and ischemia-reperfusion-induced apoptotic or necrotic cell death in cerebral and myocardial cells by mediating not only Cl- transport but also release of glutamate and/or ATP. Cardiac CFTR exerts a protective action against ischemia(-reperfusion)-induced cardiac injury, called myocardial infarction (MI), which is largely necrotic. Cardiac Maxi-Cl activity may participate in protection against ischemia(-reperfusion) injury by mediating ATP release.

VNUT and VMAT2 segregate within sympathetic varicosities and localize near preferred Cav2 isoforms in the rat tail artery.

ATP and norepinephrine (NE) are coreleased from peripheral sympathetic nerve terminals. Whether they are stored in the same vesicles has been debated for decades. Preferential dependence of NE or ATP release on Ca2+ influx through specific voltage-gated Ca2+ channel (Cav2) isoforms suggests that NE and ATP are stored in separate vesicle pools, but simultaneous imaging of NE and ATP containing vesicles within single varicosities has not been reported. We conducted an immunohistochemical study of vesicular monoamine transporter 2 (VMAT2/SLC18A2) and vesicular nucleotide translocase (VNUT/SLC17A9) as markers of vesicles containing NE and ATP in sympathetic nerves of the rat tail artery. A large fraction of varicosities exhibited neighboring, rather than overlapping, VNUT and VMAT2 fluorescent puncta. VMAT2, but not VNUT, colocalized with synaptotagmin 1. Cav2.1, Cav2.2, and Cav2.3 are expressed in nerves in the tunica adventitia. VMAT2 preferentially localized adjacent to Cav2.2 and Cav2.3 rather than Cav2.1. VNUT preferentially localized adjacent to Cav2.3 > Cav2.2 >> Cav2.1. With the use of wire myography, inhibition of field-stimulated vasoconstriction with the Cav2.3 blocker SNX-482 (0.25 µM) mimicked the effects of the P2X inhibitor suramin (100 µM) rather than the α-adrenergic inhibitor phentolamine (10 µM). Variable sensitivity to SNX-482 and suramin between animals closely correlated with Cav2.3 staining. We concluded that a majority of ATP and NE stores localize to separate vesicle pools that use different synaptotagmin isoforms and that localize near different Cav2 isoforms to mediate vesicle release. Cav2.3 appears to play a previously unrecognized role in mediating ATP release in the rat tail artery. NEW & NOTEWORTHY Immunofluorescence imaging of vesicular nucleotide translocase and vesicular monoamine transporter 2 in rat tail arteries revealed that ATP and norepinephrine, classical cotransmitters, localize to well-segregated vesicle pools. Furthermore, vesicular nucleotide translocase and vesicular monoamine transporter 2 exhibit preferential localization with specific Cav2 isoforms. These novel observations address long-standing debates regarding the mechanism(s) of sympathetic neurotransmitter corelease.

Inhibition of pannexin-1 channel activity by adiponectin in podocytes: Role of acid ceramidase activation

The pannexin-1 (Panx1) channel has been reported to mediate the release of ATP that is involved in local tissue inflammation, obesity, and many chronic degenerative diseases. It remains unknown whether Panx1 is present in podocytes and whether this channel in podocytes mediates ATP release leading to glomerular inflammation or fibrosis. To answer these questions, we first characterized the expression of Panx channels in podocytes. Among the three known pannexins, Panx1 was the most enriched in podocytes, either cultured or native in mouse glomeruli. Using a Port-a-Patch planar patch-clamp system, we recorded a large voltage-gated outward current through podocyte membrane under the Cs+in/Na+out gradient. Substitution of gluconate or aspartate for chloride in the bath solution blocked voltage-gated outward currents and shifted the reversal potential of Panx1 currents to the right, indicating the anion permeability of this channel. Pharmacologically, the recorded voltage-gated outward currents were substantially attenuated by specific Panx1 channel inhibitors. Given the anti-inflammatory and intracellular ATP restorative effects of adiponectin, we tested whether this adipokine inhibits Panx1 channel activity to block ATP release. Adiponectin blocked Panx1 channel activity in podocytes. Mechanistically, inhibition of acid ceramidase (AC) remarkably enhanced Panx1 channel activity under control conditions and prevented the inhibition of Panx1 channel by adiponectin. Correspondingly, intracellular addition of AC products, sphingosine or sphingosine-1-phosphate (S1P), blocked Panx1 channel activity, while elevation of intracellular ceramide had no effect on Panx1 channel activity. These results suggest that adiponectin inhibits Panx1 channel activity in podocytes through activation of AC and associated elevation of intracellular S1P.

Human erythrocytes release ATP by a novel pathway involving VDAC oligomerization independent of pannexin-1

We previously demonstrated that the translocase protein TSPO2 together with the voltage-dependent anion channel (VDAC) and adenine nucleotide transporter (ANT) were involved in a membrane transport complex in human red blood cells (RBCs). Because VDAC was proposed as a channel mediating ATP release in RBCs, we used TSPO ligands together with VDAC and ANT inhibitors to test this hypothesis. ATP release was activated by TSPO ligands, and blocked by inhibitors of VDAC and ANT, while it was insensitive to pannexin-1 blockers. TSPO ligand increased extracellular ATP (ATPe) concentration by 24–59% over the basal values, displaying an acute increase in [ATPe] to a maximal value, which remained constant thereafter. ATPe kinetics were compatible with VDAC mediating a fast but transient ATP efflux. ATP release was strongly inhibited by PKC and PKA inhibitors as well as by depleting intracellular cAMP or extracellular Ca2+, suggesting a mechanism involving protein kinases. TSPO ligands favoured VDAC polymerization yielding significantly higher densities of oligomeric bands than in unstimulated cells. Polymerization was partially inhibited by decreasing Ca2+ and cAMP contents. The present results show that TSPO ligands induce polymerization of VDAC, coupled to activation of ATP release by a supramolecular complex involving VDAC, TSPO2 and ANT.