Activation Of Purinergic Receptors Research Articles

Interplay between renal endothelin and purinergic signaling systems.

Alterations in extracellular fluid volume regulation and sodium balance may result in the development and maintenance of salt-dependent hypertension, a major risk factor for cardiovascular disease. Numerous pathways contribute to the regulation of sodium excretion and blood pressure, including endothelin and purinergic signaling. Increasing evidence suggests a link between purinergic receptor activation and endothelin production within the renal collecting duct as a means of promoting natriuresis. A better understanding of the relationship between these two systems, especially in regard to sodium homeostasis, will fill a significant knowledge gap and may provide novel antihypertensive treatment options. Therefore, this review focuses on the cross talk between endothelin and purinergic signaling as it relates to the renal regulation of sodium and blood pressure homeostasis.

High-throughput screen detects calcium signaling dysfunction in typical sporadic autism spectrum disorder

Autism spectrum disorder (ASD) is a heterogeneous group of neurodevelopmental disorders without any defined uniting pathophysiology. Ca2+ signaling is emerging as a potential node in the genetic architecture of the disorder. We previously reported decreased inositol trisphosphate (IP3)-mediated Ca2+ release from the endoplasmic reticulum in several rare monogenic syndromes highly comorbid with autism – fragile X and tuberous sclerosis types 1 and 2 syndromes. We now extend those findings to a cohort of subjects with sporadic ASD without any known mutations. We developed and applied a high throughput Fluorometric Imaging Plate Reader (FLIPR) assay to monitor agonist-evoked Ca2+ signals in human primary skin fibroblasts. Our results indicate that IP3 -mediated Ca2+ release from the endoplasmic reticulum in response to activation of purinergic receptors is significantly depressed in subjects with sporadic as well as rare syndromic forms of ASD. We propose that deficits in IP3-mediated Ca2+ signaling represent a convergent hub function shared across the spectrum of autistic disorders – whether caused by rare highly penetrant mutations or sporadic forms – and holds promise as a biomarker for diagnosis and novel drug discovery.

Müller Cell-Microglia Cross Talk Drives Neuroinflammation in Diabetic Retinopathy.

Diabetic retinopathy (DR) is the most common complication of diabetes and a leading cause of vision loss worldwide (1). Unfortunately, there are no treatments targeting early stages of the disease prior to the onset of sight-threatening vascular defects such as macular edema or neovascularization. A better understanding of the etiology of DR is needed to identify therapeutic targets to halt early disease progression. To this end, numerous studies demonstrated that a low-grade inflammation occurs in retinas of diabetic animal models and suggest that inflammation contributes a role in DR progression. Various mechanisms leading to retinal inflammation in DR have been described, with the majority of studies implicating retinal Muller glial cells and microglia as the initiators of retinal inflammation (for review, see ref. 2). However, seldom does a study make a strong connection between these two cell types. In this issue of Diabetes , Portillo et al. (3) describe a mechanism in which retinal inflammation in diabetic mice is dependent upon expression of the cluster of differentiation gene 40 (CD40) receptor by Muller cells (Fig. 1). The study suggests that CD40 activation induces Muller cells to release ATP, leading to activation of P2X7 purinergic receptors on retinal microglia and their subsequent expression of inflammatory cytokines. Importantly, the requirement of Muller cell–specific CD40 expression to recapitulate the appearance of acellular capillaries in diabetic retinas also suggests that inflammation is necessary for the loss of vascular cells associated with DR pathology. Figure 1 Proposed …

Evaluation of P2X7 receptor expression in peripheral lymphocytes and immune profile from patients with indeterminate form of Chagas disease.

Chagas disease (CD) is caused by Trypanosoma cruzi, an intracellular protozoan which is a potent stimulator of cell-mediated immunity. In the indeterminate form of CD (IFCD) a modulation between pro- and anti-inflammatory responses establishes a host-parasite adaptation. It was previously demonstrated that purinergic ecto-enzymes regulates extracellular ATP and adenosine levels, influencing immune and inflammatory processes during IFCD. In inflammatory sites ATP, as well as its degradation product, adenosine, function as signaling molecules and immunoregulators through the activation of purinergic receptors. In this work, it was analyzed the gene and protein expression of P2X7 purinergic receptor in peripheral lymphocytes and serum immunoregulatory cytokines from IFCD patients. Gene and protein expression of P2X7 receptor (P2X7R), and serum cytokines (IL-2, IL-10, IL-17 and IFN-γ) were unaltered. However, IFCD group showed significantly higher IL-4 and IL-6 levels while TNF-α was significantly decreased. These results indicate that imune profile of IFCD patients displays anti-inflammatory characteristics, consistent with the establishment of an immunomodulatory response. Further study about the molecular knowledge of P2X7R in IFCD is useful to clarify the participation of purinergic system in the regulatory mechanism which avoid the progression of CD.

Paradoxical induction of growth arrest and apoptosis by EGF via the up-regulation of PTEN by activating Redox factor-1/Egr-1 in human lung cancer cells.

Epidermal growth factor (EGF) signaling promotes cell proliferation and survival in several types of cancer. Here, however, we showed that EGF inhibits proliferation and promotes programmed cell death in non-small cell lung cancer (NSCLC) cells. In A549 cells, EGF increased redox factor-1 (Ref-1) expression and the association of Ref-1 with zinc finger-containing transcriptional regulator (EGR1) via activation of p22phox, RAC1, and an NADPH oxidase subunit. EGF increased p22phox and RAC1 expression through activation of purinergic receptors (P2Y). Elevated Ref-1/EGR1 levels increased phosphatase and tensin homolog (PTEN) levels, leading to inhibition of the Akt pathway. EGF-induced PTEN upregulation increased apoptosis and autophagy-induced damage in A549 cells, whereas Ref-1 knockdown blocked EGF-induced PTEN upregulation in an NADPH oxidase p22phox subunit-independent manner. In addition, p22phox knockdown restored EGF-induced effects, implying that changes in P2Y activity caused by EGF, which activates NADPH oxidase via RAC1, influenced Ref-1-mediated redox regulation. Finally, EGF similarly attenuated cell proliferation and promoted autophagy and apoptosis in vivo in a xenograft model using A549 cells. These findings reveal that EGF-induced redox signaling is linked to Ref-1-induced death in NSCLC cells.

Erratum: Activation of P2X7 and P2Y11 purinergic receptors inhibits migration and normalizes tumor-derived endothelial cells via cAMP signaling.

Scientific Reports 6: Article number: 32602; published online: 02 September 2016; updated: 11 November 2016

2',3'-O-Substituted ATP derivatives as potent antagonists of purinergic P2X3 receptors and potential analgesic agents.

Blocking membrane currents evoked by the activation of purinergic P2X3 receptors localized on nociceptive neurons represents a promising strategy for the development of agents useful for the treatment of chronic pain conditions. Among compounds endowed with such antagonistic action, 2',3'-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP) is an ATP analogue, whose inhibitory activity on P2X receptors has been previously reported. Based on the results of molecular modelling studies performed with homology models of the P2X3 receptor, novel adenosine nucleotide analogues bearing cycloalkyl or arylalkyl substituents replacing the trinitrophenyl moiety of TNP-ATP were designed and synthesized. These new compounds were functionally evaluated on native P2X3 receptors from mouse trigeminal ganglion (TG) sensory neurons using patch clamp recordings under voltage clamp configuration. Our data show that some of these molecules are potent (nanomolar range) and reversible inhibitors of P2X3 receptors, without any apparent effect on trigeminal GABAA and 5-HT3 receptors, whose membrane currents were unaffected by the tested compounds.

Neuronal NTPDase3 Mediates Extracellular ATP Degradation in Trigeminal Nociceptive Pathway.

ATP induces pain via activation of purinergic receptors in nociceptive sensory nerves. ATP signaling is terminated by ATP hydrolysis mediated by cell surface-localized ecto-nucleotidases. Using enzymatic histochemical staining, we show that ecto-ATPase activity is present in mouse trigeminal nerves. Using immunofluorescence staining, we found that ecto-NTPDase3 is expressed in trigeminal nociceptive neurons and their projections to the brainstem. In addition, ecto-ATPase activity and ecto-NTPDase3 are also detected in the nociceptive outermost layer of the trigeminal subnucleus caudalis. Furthermore, we demonstrate that incubation with anti-NTPDase3 serum reduces extracellular ATP degradation in the nociceptive lamina of both the trigeminal subnucleus caudalis and the spinal cord dorsal horn. These results are consistent with neuronal NTPDase3 activity modulating pain signal transduction and transmission by affecting extracellular ATP hydrolysis within the trigeminal nociceptive pathway. Thus, disruption of trigeminal neuronal NTPDase3 expression and localization to presynaptic terminals during chronic inflammation, local constriction and injury may contribute to the pathogenesis of orofacial neuropathic pain.

Two different mechanosensitive calcium responses in Müller glial cells of the guinea pig retina: Differential dependence on purinergic receptor signaling.

Tractional forces or mechanical stimulation are known to induce calcium responses in retinal glial cells. The aim of the study was to determine the characteristics of calcium responses in Müller glial cells of the avascular guinea pig retina induced by focal mechanical stimulation. Freshly isolated retinal wholemounts were loaded with Mitotracker Deep Red (to fill Müller cells) and the calcium-sensitive dye Fluo-4/AM. The inner retinal surface was mechanically stimulated with a micropipette tip for 10 ms. Stimulation induced two different cytosolic calcium responses in Müller cells with different kinetics in dependence on the distance from the stimulation site. Müller cells near the stimulation site displayed an immediate and long-lasting calcium response with high amplitude. This response was mediated by calcium influx from the extracellular space likely triggered by activation of ATP-insensitive P2 receptors. More distant Müller cells displayed, with a delay of 2.4 s, transient calcium responses which propagated laterally in a wave-like fashion. Propagating calcium waves were induced by a calcium-independent release of ATP from Müller cells near the stimulation site, and were mediated by a release of calcium from internal stores triggered by ATP, acting in part at P2Y1 receptors. The data suggest that mechanically stimulated Müller cells of the guinea pig retina release ATP which induces a propagating calcium wave in surrounding Müller cells. Propagating calcium waves may be implicated in the spatial regulation of the neuronal activity and homeostatic glial functions, and may transmit gliosis-inducing signals across the retina. Mechanical stimulation of guinea pig Müller cells induces two calcium responses: an immediate response around the stimulation site and propagating calcium waves. Both responses are differentially mediated by activation of purinergic receptors. GLIA 2016 GLIA 2017;65:62-74.

Subfailure Overstretch Injury Leads to Reversible Functional Impairment and Purinergic P2X7 Receptor Activation in Intact Vascular Tissue.

Vascular stretch injury is associated with blunt trauma, vascular surgical procedures, and harvest of human saphenous vein for use in vascular bypass grafting. A model of subfailure overstretch in rat abdominal aorta was developed to characterize surgical vascular stretch injury. Longitudinal stretch of rat aorta was characterized ex vivo. Stretch to the haptic endpoint, where the tissues would no longer lengthen, occurred at twice the resting length. The stress produced at this length was greater than physiologic mechanical forces but well below the level of mechanical disruption. Functional responses were determined in a muscle bath, and this subfailure overstretch injury led to impaired smooth muscle function that was partially reversed by treatment with purinergic receptor (P2X7R) antagonists. These data suggest that vasomotor dysfunction caused by subfailure overstretch injury may be due to the activation of P2X7R. These studies have implications for our understanding of mechanical stretch injury of blood vessels and offer novel therapeutic opportunities.

Spontaneous Ca2+ transients in mouse microglia

Microglia are the resident immune cells in the central nervous system and many of their physiological functions are known to be linked to intracellular calcium (Ca2+) signaling. Here we show that isolated and purified mouse microglia—either freshly or cultured—display spontaneous and transient Ca2+ elevations lasting for around ten to twenty seconds and occurring at frequencies of around five to ten events per hour and cell. The events were absent after depletion of internal Ca2+ stores, by phospholipase C (PLC) inhibition or blockade of inositol-1,4,5-trisphosphate receptors (IP3Rs), but not by removal of extracellular Ca2+, indicating that Ca2+ is released from endoplasmic reticulum intracellular stores. We furthermore provide evidence that autocrine ATP release and subsequent activation of purinergic P2Y receptors is not the trigger for these events. Spontaneous Ca2+ transients did also occur after stimulation with Lipopolysaccharide (LPS) and in glioma-associated microglia, but their kinetics differed from control conditions. We hypothesize that spontaneous Ca2+ transients reflect aspects of cellular homeostasis that are linked to regular and patho-physiological functions of microglia.

Activation of P2X7 and P2Y11 purinergic receptors inhibits migration and normalizes tumor-derived endothelial cells via cAMP signaling.

Purinergic signaling is involved in inflammation and cancer. Extracellular ATP accumulates in tumor interstitium, reaching hundreds micromolar concentrations, but its functional role on tumor vasculature and endothelium is unknown. Here we show that high ATP doses (>20 μM) strongly inhibit migration of endothelial cells from human breast carcinoma (BTEC), but not of normal human microvascular EC. Lower doses (1–10 mm result ineffective. The anti-migratory activity is associated with cytoskeleton remodeling and is significantly prevented by hypoxia. Pharmacological and molecular evidences suggest a major role for P2X7R and P2Y11R in ATP-mediated inhibition of TEC migration: selective activation of these purinergic receptors by BzATP mimics the anti-migratory effect of ATP, which is in turn impaired by their pharmacological or molecular silencing. Downstream pathway includes calcium-dependent Adenilyl Cyclase 10 (AC10) recruitment, cAMP release and EPAC-1 activation. Notably, high ATP enhances TEC-mediated attraction of human pericytes, leading to a decrease of endothelial permeability, a hallmark of vessel normalization. Finally, we provide the first evidence of in vivo P2X7R expression in blood vessels of murine and human breast carcinoma. In conclusion, we have identified a purinergic pathway selectively acting as an antiangiogenic and normalizing signal for human tumor-derived vascular endothelium.

Abstract 095: Ovariectomy Restores Purinergic Receptor Activation of Endothelin-dependent Natriuresis

We have recently reported interplay between renal medullary endothelin-1 (ET-1) and purinergic (P2) systems, which play central roles in controlling Na + homeostasis in male rats. Evidence suggests that sex hormones regulate ET-1 and P2 systems. To test whether activation of the renal medullary P2 receptors promotes ET-dependent natriuresis in females and whether ovariectomy (OVX) modulates this potential interaction, we studied the effect of medullary NaCl loading on Na + excretion in adult intact female and OVX SD rats in the presence and absence of P2 or ET receptor antagonism. Isosmotic saline (284 mOsmol/kg H 2 O) was infused into the renal medullary interstitium during a baseline urine collection period, followed by isosmotic or hyperosmotic saline (1800 mOsmol/kg H 2 O) infusion. Blood pressure, renal blood flow, urine Na + , K + and osmolality were measured. Medullary NaCl loading significantly enhanced Na + excretion in intact females and OVX (from 0.8±0.2 to 6.2±1.6 and from 0.7±0.1 to 5.6±0.8 μmol/min, respectively, n=6-8, p<0.05). The natriuretic effect of NaCl loading in intact females was not attenuated by P2 or ET receptor blockade. Whereas, intramedullary infusion of the P2 receptor antagonist, suramin, inhibited the natriuresis induced by medullary NaCl loading in OVX (from 0.4±0.2 to 0.9±0.4 μmol/min, n=6). Additionally, combined ET A/B receptor blockade (ABT-627 + A-192621) abolished the natriuretic response to medullary NaCl load in OVX rats (from 0.2±0.1 to 1.4±0.7 μmol/min, n=4). Activation of medullary purinergic (P2Y 2/4 ) receptors by UTP infusion had no significant effect in intact females, but enhanced Na + excretion in OVX rats (from 0.5±0.1 to 2.3±0.8 μmol/min, n=5, p<0.05). Combined ET A/B receptor blockade significantly inhibited the natriuretic response to UTP observed in OVX rats. These data suggest that increased medullary NaCl loading induces ET-independent and P2-independent natriuresis in intact females. In OVX, activation of medullary P2 receptors promotes ET-dependent natriuresis, similar to our previous findings in male rats and suggests that OVX restores the interplay between the renal ET-1 and purinergic (P2) signaling systems to facilitate Na + excretion. Funded by AHA 15POST25090329 to EYG and P01 HL95499 to DMP

Dorsal horn neurons release extracellular ATP in a VNUT-dependent manner that underlies neuropathic pain.

Activation of purinergic receptors in the spinal cord by extracellular ATP is essential for neuropathic hypersensitivity after peripheral nerve injury (PNI). However, the cell type responsible for releasing ATP within the spinal cord after PNI is unknown. Here we show that PNI increases expression of vesicular nucleotide transporter (VNUT) in the spinal cord. Extracellular ATP content ([ATP]e) within the spinal cord was increased after PNI, and this increase was suppressed by exocytotic inhibitors. Mice lacking VNUT did not show PNI-induced increase in [ATP]e and had attenuated hypersensitivity. These phenotypes were recapitulated in mice with specific deletion of VNUT in spinal dorsal horn (SDH) neurons, but not in mice lacking VNUT in primary sensory neurons, microglia or astrocytes. Conversely, ectopic VNUT expression in SDH neurons of VNUT-deficient mice restored PNI-induced increase in [ATP]e and pain. Thus, VNUT is necessary for exocytotic ATP release from SDH neurons which contributes to neuropathic pain.

Purinergic A2b Receptor Activation by Extracellular Cues Affects Positioning of the Centrosome and Nucleus and Causes Reduced Cell Migration

The tight, relative positioning of the nucleus and centrosome in mammalian cells is important for the regulation of cell migration. Under pathophysiological conditions, the purinergic A2b receptor can regulate cell motility, but the underlying mechanism remains unknown. Expression of A2b, normally low, is increased in tissues experiencing adverse physiological conditions, including hypoxia and inflammation. ATP is released from such cells. We investigated whether extracellular cues can regulate centrosome-nucleus positioning and cell migration. We discovered that hypoxia as well as extracellular ATP cause a reversible increase in the distance between the centrosome and nucleus and reduced cell motility. We uncovered the underlying pathway: both treatments act through the A2b receptor and specifically activate the Epac1/RapGef3 pathway. We show that cells lacking A2b do not respond in this manner to hypoxia or ATP but transfection of A2b restores this response, that Epac1 is critically involved, and that Rap1B is important for the relative positioning of the centrosome and nucleus. Our results represent, to our knowledge, the first report demonstrating that pathophysiological conditions can impact the distance between the centrosome and nucleus. Furthermore, we identify the A2b receptor as a central player in this process.

Heterologous, PKC-Mediated Desensitization of Human Histamine H3 Receptors Expressed in CHO-K1 Cells.

Desensitization is a major mechanism to regulate the functional response of G protein-coupled receptors. In this work we studied whether the human histamine H3 receptor of 445 amino acids (hH3R445) experiences heterologous desensitization mediated by PKC activation. Bioinformatic analysis indicated the presence of Serine and Threonine residues susceptible of PKC-mediated phosphorylation on the third intracellular loop and the carboxyl terminus of the hH3R445. In CHO-K1 cells stably transfected with the hH3R445 direct PKC activation by phorbol 12-myristate 13-acetate (TPA, 200nM) abolished H3R-mediated inhibition of forskolin-stimulated cAMP accumulation. Activation of endogenous purinergic receptors by ATP (adenosine 5'-triphosphate, 10μM) increased the free calcium intracellular concentration ([Ca(2+)]i) confirming their coupling to phospholipase C stimulation. Incubation with ATP also abolished H3R-mediated inhibition of forskolin-induced cAMP accumulation, and this effect was prevented by the PKC inhibitors Ro-31-8220 and Gö-6976. Pre-incubation with TPA or ATP reduced H3R-mediated stimulation of [(35)S]-GTPγS binding to membranes from CHO-K1-hH3R445 cells by 39.7 and 54.2%, respectively, with no change in the agonist potency, and the effect was prevented by either Ro-31-8220 or Gö-6976. Exposure to ATP or TPA also resulted in the loss of cell surface H3Rs (-30.4 and -45.1%) as evaluated by [(3)H]-NMHA binding to intact cells. These results indicate that the hH3R445 undergoes heterologous desensitization upon activation of receptors coupled to PKC stimulation.

Activation of purinergic receptors (P2) in the renal medulla promotes endothelin-dependent natriuresis in male rats.

Renal endothelin-1 (ET-1) and purinergic signaling systems regulate Na(+) reabsorption in the renal medulla. A link between the renal ET-1 and purinergic systems was demonstrated in vitro, however, the in vivo interaction between these systems has not been defined. To test whether renal medullary activation of purinergic (P2) receptors promotes ET-dependent natriuresis, we determined the effect of increased medullary NaCl loading on Na(+) excretion and inner medullary ET-1 mRNA expression in anesthetized adult male Sprague-Dawley rats in the presence and absence of purinergic receptor antagonism. Isosmotic saline (NaCl; 284 mosmol/kgH2O) was infused into the medullary interstitium (500 μl/h) during a 30-min baseline urine collection period, followed by isosmotic or hyperosmotic saline (1,800 mosmol/kgH2O) for two further 30-min urine collection periods. Na(+) excretion was significantly increased during intramedullary infusion of hyperosmotic saline. Compared with isosmotic saline, hyperosmotic saline infused into the renal medulla caused significant increases in inner medullary ET-1 mRNA expression. Renal intramedullary infusion of the P2 receptor antagonist suramin inhibited the increase in Na(+) excretion and inner medullary ET-1 mRNA expression induced by NaCl loading in the renal medulla. Activation of medullary P2Y2/4 receptors by infusion of UTP increased urinary Na(+) excretion. Combined ETA and ETB receptor blockade abolished the natriuretic response to intramedullary infusion of UTP. These data demonstrate that activation of medullary P2 receptors promotes ET-dependent natriuresis in male rats, suggesting that the renal ET-1 and purinergic signaling systems interact to efficiently facilitate excretion of a NaCl load.

Pannexin1 Channels Are Required for Chemokine-Mediated Migration of CD4+ T Lymphocytes: Role in Inflammation and Experimental Autoimmune Encephalomyelitis.

Pannexin1 (Panx1) channels are large high conductance channels found in all vertebrates that can be activated under several physiological and pathological conditions. Our published data indicate that HIV infection results in the extended opening of Panx1 channels (5-60 min), allowing for the secretion of ATP through the channel pore with subsequent activation of purinergic receptors, which facilitates HIV entry and replication. In this article, we demonstrate that chemokines, which bind CCR5 and CXCR4, especially SDF-1α/CXCL12, result in a transient opening (peak at 5 min) of Panx1 channels found on CD4(+) T lymphocytes, which induces ATP secretion, focal adhesion kinase phosphorylation, cell polarization, and subsequent migration. Increased migration of immune cells is key for the pathogenesis of several inflammatory diseases including multiple sclerosis (MS). In this study, we show that genetic deletion of Panx1 reduces the number of the CD4(+) T lymphocytes migrating into the spinal cord of mice subjected to experimental autoimmune encephalomyelitis, an animal model of MS. Our results indicate that opening of Panx1 channels in response to chemokines is required for CD4(+) T lymphocyte migration, and we propose that targeting Panx1 channels could provide new potential therapeutic approaches to decrease the devastating effects of MS and other inflammatory diseases.

Abstract 340: Recombinant Soluble Apyrase Inhibits Vascular Smooth Muscle Cell Migration

Background: Purinergic receptor activation by extracellular nucleotides is involved in thrombosis and neointimal hyperplasia that accompany atherosclerosis and postangioplasty restenosis. Human apyrases [ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDases)] are membrane bound enzymes that hydrolyze extracellular nucleotides, thereby inhibiting purinergic receptor activation. CD39, the first identified human apyrase, is constitutively expressed on endothelial cell (EC) and vascular smooth muscle cell (VSMC) surfaces. APT102, a recombinant soluble form of CD39L3, has been shown to reduce platelet activation through its ADPase activity, but its effects on VSMC and EC function are yet to be established. We tested the hypothesis that APT102 will inhibit migration of VSMCs and ECs. Methods: We studied cell migration using a modified Boyden chamber assay in which 5x10 4 cells suspended in 0.2% FBS/DMEMF12 were added to the upper chamber of transwells separated from the lower chamber medium by a microporous membrane through which VSMCs and ECs can migrate. APT102 (100 nM) or vehicle control was added to the upper chamber; lower chamber contained 2.5% FBS/DMEMF12 and either ATP (10 μM) or vehicle control. Transwells were incubated at 37 0 C for 6 h, after which cells that migrated through pores and adhered to the lower chamber side of the membrane were fixed, stained and counted. Results: ATP (10μM) significantly enhanced migration of both VSMCs and ECs. APT102 significantly inhibited VSMC migration and completely abrogated the pro-migratory effect of ATP. In contrast, APT102 had no inhibitory effect on EC migration, either spontaneous or ATP-enhanced. Conclusion: APT102 inhibits VSMC but not EC migration. These results suggest that pharmacological targeting of extracellular nucleotides may provide a safe and effective therapeutic strategy to inhibit neointimal hyperplasia and restenosis after angioplasty, without delaying endothelial cell recovery, which is a significant limitation of drug-eluting stents. Further studies are needed to clarify the mechanism(s) underlying the differential effect of extracellular nucleotide degradation by APT102 on VSMC and EC migration.

Retinal glial responses to optic nerve crush are attenuated in Bax-deficient mice and modulated by purinergic signaling pathways.

BackgroundRetinal ganglion cell (RGC) soma death is a consequence of optic nerve damage, including in optic neuropathies like glaucoma. The activation of the innate immune network in the retina after nerve damage has been linked to RGC pathology. Since the eye is immune privileged, innate immune functions are the responsibility of the glia, specifically the microglia, astrocytes, and Müller cells that populate the retina. Glial activation, leading to the production of inflammatory cytokines, is a hallmark feature of retinal injury resulting from optic nerve damage and purported to elicit secondary degeneration of RGC somas.MethodsA mouse model of optic nerve crush (ONC) was used to study retinal glial activation responses. RGC apoptosis was blocked using Bax-deficient mice. Glial activation responses were monitored by quantitative PCR and immunofluorescent labeling in retinal sections of activation markers. ATP signaling pathways were interrogated using P2X receptor agonists and antagonists and Pannexin 1 (Panx1)-deficient mice with RGC-specific deletion.ResultsONC induced activation of both macroglia and microglia in the retina, and both these responses were dramatically muted if RGC death was blocked by deletion of the Bax gene. Macroglial, but not microglial, activation was modulated by purinergic receptor activation. Release of ATP after optic nerve damage was not mediated by PANX1 channels in RGCs.ConclusionsRGC death in response to ONC plays a principal stimulatory role in the retinal glial activation response.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-016-0558-y) contains supplementary material, which is available to authorized users.