Extracellular Adenosine Triphosphate Concentration Research Articles

PIEZO1 promotes ATP release from periodontal ligament cells following compression force.

Orthodontic mechanical force on the periodontal ligament induces extracellular adenosine triphosphate (ATP) release. However, mechanosensitive molecules have not been confirmed functionally in periodontal ligament cells. In the present study, we examined the roles of mechanosensitive PIEZO channels in the mechanically stimulated release of ATP in human periodontal ligament fibroblasts (HPdLFs). To examine PIEZO expression in HPdLFs, we performed reverse transcription-quantitative polymerase chain reaction, fluorescent immunostaining, and Ca2+ imaging. ATP concentrations were measured in culture medium after applications of the PIEZO1 agonist Yoda1 and compression force in a newly developed in vitro weight-loaded cell model (IVWLC) using balance weights and a 48-well plate. The mechanosensitive channel inhibitor GsMTx4 and the ATP-releasing route inhibitors clodronic acid, meclofenamic acid, and probenecid were used. To suppress PIEZO1 expression, short interference RNA (siRNA) treatment of the PIEZO1 gene was performed. PIEZO1 mRNA was expressed more abundantly than PIEZO2 mRNA in HPdLFs. HPdLF cell bodies were immunoreactive to anti-PIEZO1 antibody. Yoda1 increased intracellular Ca2+ and extracellular ATP concentrations in a dose-dependent manner. ATP release was inhibited by GsMTx4 and inhibitors of ATP release routes. In the IVWLC, HPdLFs released ATP in response to compression force but not in response to hypoxic stimulation that was simultaneously applied to cells. Mechanically stimulated ATP release was inhibited by GsMTx4, inhibitors of ATP-releasing routes and siRNA treatment of PIEZO1. PIEZO1 on the cell membranes of HPdLFs is activated by compression force and then induces ATP release via intracellular Ca2+-dependent exocytosis and ATP-permeable channels.

Impairment of endothelial Pannexin1-TRPV4 signaling and flow-induced vasodilation in obesity-induced pulmonary hypertension

Obesity is a highly prevalent and debilitating disease that significantly contributes to the development of other pathologies, such as pulmonary hypertension (PH). However, the mechanisms triggering obesity-induced PH (OiPH) are currently unknown. Endothelial cell (EC) Ca 2+ signals dilate small pulmonary arteries (PAs) and maintain a low resting pulmonary arterial pressure (PAP). Impairment of endothelial Ca 2+ signaling mechanisms is a crucial contributor to elevated PAP in PH. Although PAs are a “high-flow” vascular bed, flow-induced endothelial Ca 2+ signaling mechanisms have not been investigated in OiPH. In this regard, we recently showed that Ca 2+ influx through endothelial transient receptor potential vanilloid 4 (TRPV4 EC ) ion channels dilates PAs and lowers resting PAP (Daneva et al., PNAS, 2021). Moreover, flow/shear stress (F/SS) activates the efflux of adenosine triphosphate (ATP) through endothelial Pannexin 1 (Panx1 EC ) in PAs. Panx1 EC -effluxed ATP, in turn, stimulates endothelial purinergic P2Y2 receptor (P2Y2R EC ) to increase TRPV4 EC channel activity (Daneva et al., eLife, 2021). Therefore, I hypothesized that impaired Panx1 EC -P2Y2R EC -TRPV4 EC signaling axis reduces flow-induced dilation of PAs and elevates PAP in OiPH. Right ventricular systolic pressure, an indicator of PAP, was significantly higher in mice exposed to a high-fat diet (HFD, 60%, 24 weeks) compared to normal chow-fed mice, confirming OiPH in these mice. F/SS (4–15 dynes/cm 2 )-induced dilation was drastically reduced in small PAs (~ 50 μm), cannulated at both ends and pressurized to 15 mm Hg, from HFD mice, possibly indicating impaired flow-induced endothelial signaling in OiPH. Confocal Ca 2+ imaging studies in fluo-4-loaded PAs showed that the baseline and agonist (GSK1016790A)-induced activity of TRPV4 EC sparklets (unitary Ca 2+ influx signals through TRPV4 EC channels) is reduced in HFD mice compared to normal mice, confirming a decrease in TRPV4 EC channel activity in OiPH. F/SS (4–15 dynes/cm 2 ) resulted in increased activity of TRPV4 EC sparklets in PAs from normal mice, but not in PAs from HFD mice, suggesting that flow-induced TRPV4 EC activity is reduced in OiPH. Bioluminescence measurements of extracellular ATP concentration showed lower baseline ATP levels in PAs from HFD mice than normal mice. Importantly, F/SS (4 – 15 dynes/cm 2 ) increased ATP efflux in PAs from normal mice, but not in PAs from HFD mice, indicating that flow-Panx1 EC signaling is also impaired in OiPH. Together, these data support the idea that flow-induced Panx1 EC -TRPV4 EC signaling is impaired in OiPH. Targeting the individual elements of this pathway may provide novel therapies in OiPH. Future studies will assess the signaling linkages of the flow-Panx1 EC -P2Y2R EC -TRPV4 EC signaling axis in OiPH. R01 HL142808, R01 HL146914, R01 HL157407 to Dr. S.K. Sonkusare; APS Postdoctoral Fellowship Award to Dr. Z. Daneva This is the full abstract presented at the American Physiology Summit 2023 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.

Endothelial Pannexin1‐purinergic signaling pulmonary hypertension

Endothelial cell (EC) Ca2+ signals dilate small pulmonary arteries (PAs) and maintain a low resting pulmonary arterial pressure (PAP). Impairment of endothelial Ca2+ signaling mechanisms is a crucial contributor to elevated PAP in pulmonary hypertension (PH). Studies in systemic arteries show that flow/shear stress activates endothelial Ca2+ signals to cause vasodilation. Although PAs are a “high‐flow” vascular bed, flow‐induced endothelial Ca2+ signaling mechanisms have not been investigated in PH. In this regard, we recently showed that Ca2+ influx through endothelial transient receptor potential vanilloid 4 (TRPV4EC) ion channels dilates PAs and lowers resting PAP (Daneva et al., PNAS, 2021). Moreover, flow/shear stress activates the efflux of adenosine triphosphate (ATP) through endothelial Pannexin 1 (Panx1EC) in PAs. Panx1EC‐effluxed ATP, in turn, stimulates endothelial purinergic P2Y2 receptor (P2Y2REC) to increase TRPV4EC channels activity (Daneva et al., eLife, 2021). I hypothesized that impaired Panx1EC—P2Y2REC—TRPV4EC signaling axis reduces flow‐induced dilation of PAs and elevates PAP in PH. Confocal Ca2+ imaging studies in fluo‐4‐loaded en face PAs (~ 50 mm) showed that the baseline and agonist (GSK1016790A or GSK101)‐induced activity of TRPV4EC sparklets (individual Ca2+ influx signals through TRPV4EC channels) is reduced in the mice exposed to Sugen 5416 + chronic hypoxia (Su+CH; 3 weeks; 10% O2) compared to the normoxic control mice, indicating a decrease in TRPV4EC channel activity in PH. The expression of TRPV4 channels at the mRNA level, however, was not altered in Su+CH mice. The activation of TRPV4EC sparklets by exogenous ATP (1 mM) was also reduced in PAs from Su+CH mice, suggesting an impairment of ATP‐P2Y2REC‐TRPV4EC channel signaling axis in PH. Accordingly, ATP‐induced, endothelium‐dependent dilation was absent in PAs from Su+CH mice. We previously showed that basal ATP efflux in PAs predominantly occurs through endothelial Panx1, and flow/shear stress increases ATP efflux entirely through the activation of endothelial Panx1. Bioluminescence measurements of extracellular ATP concentration showed lower baseline ATP levels in PAs from Su+CH mice than control mice, suggesting a decrease in ATP efflux in PH. Importantly, flow/shear stress (4 – 15 dynes/cm2) increased the efflux of ATP from normal mice, but not in PAs from Su+CH mice, indicating that flow/shear stress‐Panx1EC signaling is also impaired in PH. Together, these data support the idea that flow‐Panx1EC and P2Y2REC‐TRPV4EC signaling are impaired in PH. Targeting individual elements of this pathway may provide novel therapeutic options in PH and other pulmonary vascular disorders characterized by endothelial dysfunction. Future studies will assess the individual signaling linkages of the flow‐Panx1EC‐P2Y2REC‐TRPV4EC signaling axis in PH.

Exploitation of Elevated Extracellular ATP to Specifically Direct Antibody to Tumor Microenvironment

The extracellular adenosine triphosphate (ATP) concentration is highly elevated in the tumor microenvironment (TME) and remains tightly regulated in normal tissues. Using phage display technology, we establish a method to identify an antibody that can bind to an antigen only in the presence of ATP. Crystallography analysis reveals that ATP bound in between the antibody-antigen interface serves as a switch for antigen binding. In a transgenic mouse model overexpressing the antigen systemically, the ATP switch antibody binds to the antigen in tumors with minimal binding in normal tissues and plasma and inhibits tumor growth. Thus, we demonstrate that elevated extracellular ATP concentration can be exploited to specifically target the TME, giving therapeutic antibodies the ability to overcome on-target off-tumor toxicity.

Aptamer-Based Atp-Responsive Delivery Systems for Diagnosis and Treatment of Cancer

In recent years, many stimuli-triggered drug delivery platforms have been designed to deliver drugs accurately to specific sites and reduce their side effects, improving “on-demand” therapeutic efficacy. Adenosine-5'-triphosphate (ATP)-responsive drug delivery methods are examples of these systems that use ATP molecules as a trigger for delivery of therapeutic agents. Since intra- and extra-cellular ATP concentrations are significantly different from each other (1-10 mM and <0.4 mM, respectively), the use of ATP can be a practical method for regulating drug release. Aptamers possess unique properties including, ligand-specific response, short sequence (~ 20-80 bases) and easy functionalization. Thus, their combination with ATP-responsive systems results in more accurate drug delivery systems and greater control of drug release. A wide range of nanoparticle frameworks, such as polymeric nanogels, liposomes, metallic nanoparticles, protein, or DNA nano-assemblies, have been employed in the fabrication of nanocarriers. In this review, we describe several ATP-responsive drug delivery systems based on the various carriers and discuss the challenges and strengths of each method.

Mechanism of action of secondary metabolites from marine-derived Streptoymces on bacterial isolates by membrane permeability

This study was designed to further investigate the potential mechanism of action of our previously characterized and identified marine-derived Streptomyces extracts (ESC003 and ESC012) on selected bacterial isolates from our culture bank. Time-kill kinetics, protein and lipid leakages assay, cell membrane permeability, phosphate and potassium ions efflux assay, extracellular adenosine triphosphate (ATP) concentration and membrane potential (MP) were all carried out using the marine-derived Streptomyces extract to determine and understand the probable mode of action at which the extract inhibit or kill bacterial cell. The MIC of ESC003 and ESC012 ranged from 0.16 to 6.25 mg/mL while the MBC ranged from 1.25 to >10 mg/mL. On the time-kill kinetics, a reduction in mean viable cell amount was detected at respective time studied. For the impermeability of the bacterial isolates, the relative electric conductivity increased with increase in concentration and time interval of exposure. As regards the protein leakage, lipid leakage, 260 nm absorbing materials leakage, phosphate and potassium ions efflux; considerable amount of these products were leaked with increase in concentration and time of exposure to the bacterial isolates. The extracellular ATP concentration from respective bacterial isolates increased appreciably with increased concentration of exposure with a simultaneous decrease in membrane potential. Findings from this study revealed that the Streptomyces extracts revealed a significant breakthrough against susceptible bacterial isolates via the permeability of the bacterial cell membrane, and thus resulted in the outflow of ATP, electrolytes, DNA materials, and proteins. These changes lead to disruption, and eventually cell death, which were proportional to a concurrent decrease in the viability of the bacterial cell.

P0968THE INFLUENCE OF ALPHA KLOTHO ON NPP1 ACTIVITY IN CULTURED RAT PODOCYTES

Abstract Background and Aims Kidney is the principal contributor to circulating αKlotho (αKL), and simultaneously, constitutes the major organ where circulating αKlotho is taken up from the circulation. Klotho is mainly expressed in proximal renal tubule and in distal tubular epithelium. In the kidney soluble αKlotho acts from the urinary luminal side as an autocrine or paracrine enzyme to regulate transporters and ion channels. Recently, it has been demonstrated that the glomerulus is another source of αKlotho production. NPP1 is the best characterized nucleotide pyrophosphatases/phosphodiesterases (NPP) family member, encoded by the ENPP1 gene, and is a type II transmembrane glycoprotein that forms disulfide-bonded homodimers in the plasma membrane and in the mineral-depositing matrix vesicles. The major function of NPP1 is generation of PPi from adenosine triphosphate (ATP). Recently, we demonstrated that extracellular nucleotides stimulate an increase in glomerular albumin permeability, in part due to glomerular NO production and actin reorganization in podocytes . Furthermore, both αKlotho and NPP1 regulate insulin-dependent signaling. Thus, a key aim of this study is to fully examine the role of αKlotho on NPP1 function and its role in the protection of glomerular filtration barrier and podocyte function in diabetes. Method We measured glomerular permeability to albumin (Palb) in a single isolated rat glomerulus based on the video-microscopy method and permeability to albumin across the podocyte monolayer. Expression of αKlotho and NPP1 was confirmed in podocytes using Western blotting and immunofluorescence. Ecto-nucleotide pyrophosphatase/phosphodiesterase activity in podocytes was assessed using p-nitrophenyl-5′-thymidine monophosphate (p-Nph-5′-TMP) as a substrate. The ATP concentration was measured using luciferin-luciferase reaction. Results Since NPP1 and Klotho regulate insulin-dependent signaling we investigated the role of Klotho on NPP1 dimerization and NPPs activity. Podocyte treatment withαKlotho (0.5 nM, 30 min) in non-reducing conditions (maleimide, 100 mM) increased the disulfide bond formation of NPP1 (Fig. 1A). The percentage of dimerized (260 kDa) NPP1 increased approximately by 45%. This effect was abolished in the presence of ATP (100 µM, 30 min). As a positive control, we used hydrogen peroxide (100 μM, 5 min) which induces oxidative disulfide bond formation (Fig. 1A). We also demonstrated time-dependent decrease of NPPs activity in the presence of αKL (Fig. 1B). Klotho also induces an increase of extracellular ATP concentration by 76% (from 3.19±0.01 to 5.62±0.43, n=6, P&amp;lt;0.05). Moreover, we confirmed the role of soluble αKlotho in ATP-evoked increases of glomerular and podocyte albumin permeability. Conclusion Summarizing, we have clearly demonstrated an interaction between αKL and NPP1 in podocytes. Moreover, we observed the protective role of αKlotho in podocyte permeability to albumin.

A202 DYSREGULATED ENDOPLASMIC RETICULUM STRESS PATHWAYS IN COLON-DERIVED ENTEROIDS FROM INFLAMMATORY BOWEL DISEASE PATIENTS DRIVE DC MATURATION LEADING TO A PRO-INFLAMMATORY PHENOTYPE

Abstract Background Intestinal epithelial cells (IECs) rely on danger signals such as adenosine triphosphate (ATP) and endoplasmic reticulum (ER) stress to form an appropriate, coordinated immune response. Moreover, ER stress in IECs contributes to the pathogenesis of inflammatory bowel disease (IBD), and an increase in extracellular ATP concentrations is a risk factor for Crohn’s disease (CD). Aims We hypothesized that cells exposed to ER stress or ATP modulate innate immune responses, creating a pro-inflammatory environment that drives dendritic cell (DC) maturation. Methods Caco-2 cells and human colon-derived enteroid monolayers were exposed to ATP or the ER stress inducer thapsigargin, stimulated with E. coli FliC flagellin, and ER stress markers CHOP, GRP78, and XBP1 s/u were measured via qPCR and western blot, and cytokine release was measured by ELISA. Next, monocyte-derived dendritic cells (moDCs) were cultured in Caco-2 or enteroid conditioned supernatants and their activation status measured via flow cytometry, and cytokine analysis was performed using Luminex platform. We also assessed ER stress markers, TLR5 expression, and cytokine expression differences between IBD and healthy controls (HC). Results We found that ER stress amplified FliC-induced IL-8 and decreased CCL20 in Caco-2 cells. Moreover, in IBD subjects, we found an increase FliC-induced IL-8 response, and decreased TNFa and CCL20. moDCs cultured with conditioned media from Caco-2 or enteroid monolayers showed a proinflammatroy phenotype, with an increase in CD80, CD86, MHCII, and a decrease in CD103. Moreover, moDCs cultured in stressed Caco-2 supernatants increased release of IL-6, TNFa, and IL-12p70, and decreased IL-10, suggesting potential to induce inflammatory Th1 and/or Th17 cells.. DC activation correlated with the amount of FliC-induced IL-8. Interestingly, there were distinct differences in cytokine expression and basal ER stress between IBD and HC enteroid monolayers, suggesting a dysregulated ER stress pathway in IBD-derived enteroids. Conclusions ER stress in Caco-2 cells and colon-derived enteroid monolayers enhances FliC-induced TLR5 responses, leading to a pro-inflammatory environment that drives DC maturation, which may link epithelial ER stress and immune cell activation in IBD. Furthermore, the cytokine and ER stress pathway differences between IBD and HC-derived enteroids suggests that prolonged periods of stress in IBD patients may rewire the IEC stem cell compartment, further perpetuating inflammation and disease. Funding Agencies CCC, CIHR

Examining Local Cell-to-Cell Signalling in the Kidney Using ATP Biosensing.

Cell-to-cell communication is an essential process for the efficient function of cells and tissues. Central to this is the purinergic transmission of purines, with ligands such as adenosine triphosphate (ATP). Altered cell-to-cell communication, and in particular changes in the paracrine release of extracellular ATP, plays crucial roles in pathophysiological conditions, such as diabetes. ATP biosensing provides a reliable, real-time measurement of local extracellular ATP concentrations. This allows the detection of altered ATP release, which underlies the progression of inflammation and fibrosis and is a potential therapeutic target. Here we describe in a step-by-step basis how to utilize sensitive microelectrode biosensors to detect low, real-time concentrations of ATP, in vitro.

Neurocognitive exercise program improves the balance performance in children with ADHD: A follow-up study

That adenosine 5′ triphosphate (ATP) functions as an extracellular signaling molecule has been established since the 1970s. Ubiquitous throughout the body as the principal molecular store of intracellular energy, ATP has a short extracellular half-life and is difficult to measure directly. Extracellular ATP concentrations are dependent both on the rate of cellular release and of enzymatic degradation. Some findings from in vitro studies suggest that extracellular ATP concentrations increase during high levels of neuronal activity and seizure-like events in hippocampal slices. Pharmacological studies suggest that antagonism of ATP-sensitive purinergic receptors can suppress the severity of seizures and block epileptogenesis. Directly measuring extracellular ATP concentrations in the brain, however, has a number of specific challenges, notably, the rapid hydrolysis of ATP and huge gradient between intracellular and extracellular compartments. Two studies using microdialysis found no change in extracellular ATP in the hippocampus of rats during experimentally-induced status epilepticus. One of which demonstrated that ATP increased measurably, only in the presence of ectoATPase inhibitors, with the other study demonstrating increases only during later spontaneous seizures. Current evidence is mixed and seems highly dependent on the model used and method of detection. More sensitive methods of detection with higher spatial resolution, which induce less tissue disruption will be necessary to provide evidence for or against the hypothesis of seizure-induced elevations in extracellular ATP. Here we describe the current hypothesis for ATP release during seizures and its role in epileptogenesis, describe the technical challenges involved and critically examine the current evidence.

Pharmacological blockade of the CD39/CD73 pathway but not adenosine receptors augments disease in a humanized mouse model of graft-versus-host disease.

Allogeneic hematopoietic stem cell transplantation is a curative therapy for a number of hematological malignancies, but is limited by the development of graft-versus-host disease (GVHD). CD39 and CD73 form an ectoenzymatic pathway that hydrolyzes extracellular adenosine 5'-triphosphate (ATP) to adenosine, which respectively exacerbate or alleviate disease in allogeneic mouse models of GVHD. The current study aimed to explore the role of the CD39/CD73 pathway and adenosine receptor (AR) blockade in a humanized mouse model of GVHD. Immunodeficient nonobese diabetic-severe combined immunodeficiency-IL-2 receptor γnull mice were injected with human peripheral blood mononuclear cells, and subsequently injected with the CD39/CD73 antagonist αβ-methylene-ADP (APCP) (50mgkg-1 ) or saline for 7days, or the AR antagonist caffeine (10mgkg-1 ) or saline for 14days. Mice predominantly engrafted human CD4+ and CD8+ T cells, with smaller proportions of human regulatory T cells, invariant natural killer T cells, monocytes and dendritic cells. Neither APCP nor caffeine altered engraftment of these human leukocyte subsets. APCP (CD39/CD73 blockade) augmented GVHD as shown through increased weight loss and worsened liver histology, including increased leukocyte and human T-cell infiltration, and increased apoptosis. This treatment also increased serum human IL-2 concentrations and decreased the frequency of human CD39- CD73- CD4+ T cells. In contrast, caffeine (AR blockade) did not alter GVHD severity or human serum cytokine concentrations (IL-2, IL-6, IL-10 or tumor necrosis factor-α). In conclusion, blockade of CD39/CD73 but not ARs augments disease in a humanized mouse model of GVHD. These results indicate that CD39/CD73 blockade maintains sufficient extracellular ATP concentrations to promote GVHD in this model.

Centrifugation-induced release of ATP from red blood cells.

Centrifugation is the primary preparation step for isolating red blood cells (RBCs) from whole blood, including for use in studies focused on transduction of adenosine triphosphate (ATP), an important vasodilatory signaling molecule. Despite the wide use of centrifugation, little work has focused on how the centrifugation itself affects release of ATP from RBCs prior to subsequent experimentation. Here we report that both the centrifugation force and duration have a pronounced impact on the concentration of ATP present in the packed RBCs following centrifugation. Multiple subsequent centrifugations yield extracellular ATP concentrations comparable to the amount released during the initial centrifugation, suggesting this effect is cumulative. Pairwise measurements of hemoglobin and ATP suggest the presence of ATP is primarily due to an increase in centrifugation-induced hemolysis. These results indicate that common centrifugation parameters, within the ranges explored here, can release ATP in quantities comparable to the low end of the range of values measured in typical ATP transduction experiments, potentially complicating experimental interpretation of those results.

Behavior and stability of adenosine triphosphate (ATP) during chlorine disinfection

Adenosine triphosphate (ATP) analysis is a cultivation-independent alternative method for the determination of bacterial viability in both chlorinated and non-chlorinated water. Here we investigated the behavior and stability of ATP during chlorination in detail. Different sodium hypochlorite doses (0–22.4 mg-Cl2 L−1; 5 min exposure) were applied to an Escherichia coli pure culture suspended in filtered river water. We observed decreasing intracellular ATP with increasing chlorine concentrations, but extracellular ATP concentrations only increased when the chlorine dose exceeded 0.35 mg L−1. The release of ATP from chlorine-damaged bacteria coincided with severe membrane damage detected with flow cytometry (FCM). The stability of extracellular ATP was subsequently studied in different water matrixes, and we found that extracellular ATP was stable in sterile deionized water and also in chlorinated water until extremely high chlorine doses (≤11.2 mg-Cl2 L−1; 5 min exposure). In contrast, ATP decreased relatively slowly (k = 0.145 h−1) in 0.1 μm filtered river water, presumably due to degradation by either extracellular enzymes or the fraction of bacteria that were able to pass through the filter. Extracellular ATP decreased considerably faster (k = 0.368 h−1) during batch growth of a river water bacterial community. A series of growth potential tests showed that extracellular ATP molecules were utilized as a phosphorus source during bacteria proliferation. From the combined data we conclude that ATP released from bacteria at high chlorine doses could promote bacteria regrowth, contributing to biological instability in drinking water distribution systems.

Variations of ATP and its metabolites in the hippocampus of rats subjected to pilocarpine-induced temporal lobe epilepsy.

Although purinergic receptor activity has lately been associated with epilepsy, little is known about the exact role of purines in epileptogenesis. We have used a rat model of temporal lobe epilepsy induced by pilocarpine to study the dynamics of purine metabolism in the hippocampus during different times of status epilepticus (SE) and the chronic phase. Concentrations of adenosine 5'-triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), and adenosine in normal and epileptic rat hippocampus were determined by microdialysis in combination with high-performance liquid chromatography (HPLC). Extracellular ATP concentrations did not vary along 4h of SE onset. However, AMP concentration was elevated during the second hour, whereas ADP and adenosine concentrations augmented during the third and fourth hour following SE. During chronic phase, extracellular ATP, ADP, AMP, and adenosine concentrations decreased, although these levels again increased significantly during spontaneous seizures. These results suggest that the increased turnover of ATP during the acute period is a compensatory mechanism able to reduce the excitatory role of ATP. Increased adenosine levels following 4h of SE may contribute to block seizures. On the other hand, the reduction of purine levels in the hippocampus of chronic epileptic rats may result from metabolic changes and be part of the mechanisms involved in the onset of spontaneous seizures. This work provides further insights into purinergic signaling during establishment and chronic phase of epilepsy.

P2X7 IS AN ARCHAIC SCAVENGER RECEPTOR RECOGNIZING APOPTOTIC NEUROBLASTS IN EARLY HUMAN NEUROGENESIS

The expression and function of P2X7 receptors in adult CNS have been widely studied, however, the roles of these purinergic receptors in human neural development has largely focused on the effects of receptor activation. Previous studies of embryonic and adult rodent neural precursors suggest adenosine triphosphate (ATP), the physiological agonist for P2X receptors, can act as a potent modifier of proliferation, migration and differentiation, mediated via intracellular calcium ([Ca 2+ ] i ) signaling. The P2X7 receptor has a ubiquitous distribution in the body but is most abundant on macrophages and microglia where its activation by ATP leads to secretion of proinflammatory cytokines. However, extracellular ATP concentrations in the CNS are usually at sub-micromolar levels suggesting that ATP-induced activation of the P2X7 receptor will not occur under physiological circumstances in the CNS. Another possible role for P2X7 receptors has been suggested by recent work on macrophages and neural precursor cells. In these studies the P2X7 receptor was shown to act as a scavenger receptor i.e. a receptor present on a phagocytotic cell which detects molecules present on the surface of apoptotic cells and facilitates phagocytosis of the apoptotic cell. In a recent study of human neural precursor cells (hNPCs) and neuroblasts isolated from human fetal telencephalons at 16-19 WG, our group showed that both P2X7R high /DCX low hNPCs and P2X7R high /DCX high neuroblasts were capable of phagocytic engulfment of a range of targets including latex beads, apoptotic ReN cells and apoptotic neuroblasts. We found that these neuroblasts and their precursor cells expressed functional P2X7 receptors on their cell surface. Although expression of P2X7 is widespread in the cells of the neuroblast, it is those DCX + neuroblasts with the highest expression of P2X7 which are actively phagocytic towards an autologous apoptotic neighbour or other phagocytic targets, including latex beads and apoptotic ReNcells. Pre-incubation of P2X7 high neuroblasts with ATP or oxidized ATP inhibited phagocytosis of targets by these cells. Moreover siRNA knockdown of P2X7R also inhibited phagocytosis of the apoptotic targets. This review considers this major new role for the P2X7 receptor in early human neurogenesis.

ATP-P2X7 Receptor Modulates Axon Initial Segment Composition and Function in Physiological Conditions and Brain Injury

Axon properties, including action potential initiation and modulation, depend on both AIS integrity and the regulation of ion channel expression in the AIS. Alteration of the axon initial segment (AIS) has been implicated in neurodegenerative, psychiatric, and brain trauma diseases, thus identification of the physiological mechanisms that regulate the AIS is required to understand and circumvent AIS alterations in pathological conditions. Here, we show that the purinergic P2X7 receptor and its agonist, adenosine triphosphate (ATP), modulate both structural proteins and ion channel density at the AIS in cultured neurons and brain slices. In cultured hippocampal neurons, an increment of extracellular ATP concentration or P2X7-green fluorescent protein (GFP) expression reduced the density of ankyrin G and voltage-gated sodium channels at the AIS. This effect is mediated by P2X7-regulated calcium influx and calpain activation, and impaired by P2X7 inhibition with Brilliant Blue G (BBG), or P2X7 suppression. Electrophysiological studies in brain slices showed that P2X7-GFP transfection decreased both sodium current amplitude and intrinsic neuronal excitability, while P2X7 inhibition had the opposite effect. Finally, inhibition of P2X7 with BBG prevented AIS disruption after ischemia/reperfusion in rats. In conclusion, our study demonstrates an involvement of P2X7 receptors in the regulation of AIS mediated neuronal excitability in physiological and pathological conditions.

Pathological potential of astroglial purinergic receptors.

Acute brain injury and neurodegenerative disorders may result in astroglial activation. Astrocytes are able to determine the progression and outcome of these neuropathologies in a beneficial or detrimental way. Nucleotides, e.g. adenosine 5'-triphosphate (ATP), released after acute or chronic neuronal injury, are important mediators of glial activation and astrogliosis.Acute injury may cause significant changes in ATP balance, resulting in (1) a decline of intracellular ATP levels and (2) an increase in extracellular ATP concentrations via efflux from the intracellular space. The released ATP may have trophic effects, but can also act as a proinflammatory mediator or cytotoxic factor, inducing necrosis/apoptosis as a universal "danger" signal. Furthermore, ATP, primarily released from astrocytes, is a means of communication between neurons, glial cells, and intracerebral blood vessels.Astrocytes express a heterogeneous battery of purinergic ionotropic and metabotropic receptors (P2XRs and P2YRs, respectively) to respond to extracellular nucleotides.In this chapter, we summarize the contemporary knowledge on the pathological potential of P2Rs in relation to changes of astrocytic functions, determined by distinct molecular signaling cascades, in a variety of diseases. We discuss specific aspects of reactive astrogliosis, with respect to the involvement of prominent receptor subtypes, such as the P2X7 and P2Y1/2Rs. Examples of purinergic signaling of microglia, oligodendrocytes, and blood vessels under pathophysiological conditions will also be presented.The understanding of the pathological potential of purinergic signaling in "controlling and fine-tuning" of astrocytic responses is important for identifying possible therapeutic principles to treat acute and chronic central nervous system diseases.

Natural compounds with P2X7 receptor-modulating properties.

The adenosine 5'-triphosphate (ATP)-gated P2X7 receptor is a membrane-bound, non-selective cation channel, expressed in a variety of cell types. The P2X7 senses high extracellular ATP concentrations and seems to be implicated in a wide range of cellular functions as well as pathophysiological processes, including immune responses and inflammation, release of gliotransmitters and cytokines, cancer cell growth or development of neurodegenerative diseases. In the present study, we identified natural compounds and analogues that can block or sensitize the ATP (1 mM)-induced Ca(2+) response using a HEK293 cell line stably expressing human P2X7 and fluorometric imaging plate reader technology. For instance, teniposide potently blocked the human P2X7 at sub-miromolar concentrations, but not human P2X4 or rat P2X2. A marked block of ATP-induced Ca(2+) entry and Yo-Pro-1 uptake was also observed in human A375 melanoma cells and mouse microglial cells, both expressing P2X7. On the other hand, agelasine (AGL) and garcinolic acid (GA) facilitated the P2X7 response to ATP in all three cell populations. GA also enhanced the YO-PRO-1 uptake, whereas AGL did not affect the ATP-stimulated intracellular accumulation of this dye. According to the pathophysiological role of P2X7 in various diseases, selective modulators may have potential for further development, e.g. as neuroprotective or antineoplastic drugs.

Detecting adenosine triphosphate in the pericellular space

Release of adenosine triphosphate (ATP) into the extracellular space occurs in response to a multiplicity of physiological and pathological stimuli in virtually all cells and tissues. A role for extracellular ATP has been identified in processes as different as neurotransmission, endocrine and exocrine secretion, smooth muscle contraction, bone metabolism, cell proliferation, immunity and inflammation. However, ATP measurement in the extracellular space has proved a daunting task until recently. To tackle this challenge, some years ago, we designed and engineered a novel luciferase probe targeted to and expressed on the outer aspect of the plasma membrane. This novel probe was constructed by appending to firefly luciferase the N-terminal leader sequence and the C-terminal glycophosphatidylinositol anchor of the folate receptor. This chimeric protein, named plasma membrane luciferase, is targeted and localized to the outer side of the plasma membrane. With this probe, we have generated stably transfected HEK293 cell clones that act as an in vitro and in vivo sensor of the extracellular ATP concentration in several disease conditions, such as experimentally induced tumours and inflammation.

Abstract 2262: Contribution of autophagy in anticancer immune response induced by chemotherapy

Abstract Antineoplastic chemotherapies are particularly efficient when they elicit immunogenic cell death, thus provoking an anticancer immune response. We demonstrate that autophagy, which is often disabled in cancer, is dispensable for chemotherapy-induced cell death but required for its immunogenicity. In response to chemotherapy, autophagy-competent, but not autophagy-deficient, cancers attracted dendritic cells and T lymphocytes into the tumor bed. Suppression of autophagy inhibited the release of adenosine triphosphate (ATP) from dying tumor cells. Conversely, inhibition of extracellular ATP-degrading enzymes increased pericellular ATP in autophagy-deficient tumors, re-established the recruitment of immune cells, and restored chemotherapeutic responses, but only in immunocompetent hosts. Thus, autophagy is essential for the release of immunogenic ATP from dying cells, and increased extracellular ATP concentrations improve the efficacy of antineoplastic chemotherapies when autophagy is disabled. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2262. doi:1538-7445.AM2012-2262