Benzoyl ATP Research Articles

Purinergic agonists increase [Ca2+]i in rat conjunctival goblet cells through ryanodine receptor type 3.

ATP and benzoylbenzoyl-ATP (BzATP) increase free cytosolic Ca2+ concentration ([Ca2+]i) in conjunctival goblet cells (CGCs) resulting in mucin secretion. The purpose of this study was to investigate the source of the Ca2+i mobilized by ATP and BzATP. First-passage cultured rat CGCs were incubated with Fura-2/AM, and [Ca2+]i was measured under several conditions with ATP and BzATP stimulation. The following conditions were used: 1) preincubation with the Ca2+ chelator EGTA, 2) preincubation with the SERCA inhibitor thapsigargin (10-6 M), which depletes ER Ca2+ stores, 3) preincubation with phospholipase C (PLC) or protein kinase A (PKA) inhibitor, or 4) preincubation with the voltage-gated calcium channel antagonist nifedipine (10-5 M) and the ryanodine receptor (RyR) antagonist dantrolene (10-5 M). Immunofluorescence microscopy (IF) and quantitative reverse transcription polymerase chain reaction (RT-qPCR) were used to investigate RyR presence in rat and human CGCs. ATP-stimulated peak [Ca2+]i was significantly lower after chelating Ca2+i with 2 mM EGTA in Ca2+-free buffer. The peak [Ca2+]i increase in CGCs preincubated with thapsigargin, the PKA inhibitor H89, nifedipine, and dantrolene, but not the PLC inhibitor, was reduced for ATP at 10-5 M and BzATP at 10-4 M. Incubating CGCs with dantrolene alone decreased [Ca2+]i and induced CGC cell death at a high concentration. RyR3 was detected in rat and human CGCs with IF and RT-qPCR. We conclude that ATP- and BzATP-induced Ca2+i increases originate from the ER and that RyR3 may be an essential regulator of CGC [Ca2+]i. This study contributes to the understanding of diseases arising from defective Ca2+ signaling in nonexcitable cells.NEW & NOTEWORTHY ATP and benzoylbenzoyl-ATP (BzATP) induce mucin secretion through an increase in free cytosolic calcium concentration ([Ca2+]i) in conjunctival goblet cells (CGCs). The mechanisms through which ATP and BzATP increase [Ca2+]i in CGCs are unclear. Ryanodine receptors (RyRs) are fundamental in [Ca2+]i regulation in excitable cells. Herein, we find that ATP and BzATP increase [Ca2+]i through the activation of protein kinase A, voltage-gated calcium channels, and RyRs, and that RyRs are crucial for nonexcitable CGCs' Ca2+i homeostasis.

Involvement of P2X7R-mediated microglia polarization and neuroinflammation in the response to electroacupuncture on post-stroke memory impairment

PurposePost-stroke cognitive impairment (PSCI) is a common complication of ischemic stroke episodes. Memory impairment is an important component of the poststroke cognitive syndrome. Microglial activation plays a critical role in stroke-induced neuroinflammation. Previous studies have reported that electroacupuncture (EA) provides neuroprotective effects by reducing the expression levels of the Purinergic receptor P2X ligand-gated ion channel 7 (P2X7) and inhibiting neuroinflammation in rat model of ischemic stroke. Further understanding of the role and connections between P2X7R and microglial activation in EA-induced anti-inflammatory can reveal novel targets for post-stroke memory impairment treatment. MethodsA Middle cerebral artery occlusion and reperfusion (MCAO/R) model was established. We used 2’(3’)-O-(4-benzoyl) benzoyl ATP (BzATP) as a P2X7R agonist. Following MCAO/R injury, the rats underwent EA therapy at the Baihui (DU20) and Shenting (DU24) acupoints for seven consecutive days. The Barnes maze test was used to evaluate memory function. Following intervention, a T2 weighted images (T2WI) scan was performed to identify changes in cerebral infarction volume in MCAO/R rats. The levels of Interleukin-1β (IL-1β), Interleukin-6 (IL-6) and Interleukin-4 (IL-4), Interleukin-10 (IL-10) in the peri-infarct hippocampal were examined by ELISA. Immunofluorescence was employed to evaluate Iba-1+ / P2X7R+, Iba-1+/ iNOS+ and Iba-1+/ Arg-1+ cell populations in the peri-infarct hippocampal DG area. The protein expression of P2X7R, Nuclear factor E2-related factor 2 (Nrf2), Recombinant nlr family, pyrin domain containing protein 3 (NLRP3), Inducible nitric oxide synthase (iNOS) and Arginase-1 (Arg-1) in the peri-infarct hippocampal were investigated using western blot assays. Besides, we also measured the levels of reactive oxygen species (ROS), superoxide dismutase (SOD) and malondialdehyde (MDA). ResultsWe found EA treatment reduced inflammation and oxidative stress, which is consistent with a decrease in P2X7R expression and improved learning and memory functions. In contrast, we found BzATP enhanced inflammation and oxidative stress. Moreover, our results showed EA treatment up-regulated Nrf2, down-regulated NLRP3, and promoted microglia M2 polarization. Finally, EA-mediated positive effects were reversed by intracerebroventricular injection of BzATP, which is consistent with an increase in P2X7R expression. ConclusionEA ameliorates memory impairment in a rat model of ischemic stroke by reducing inflammation and ROS through the inhibition of P2X7R expression. In turn, this mechanism regulates Nrf2 and NLRP3 expression, suggesting EA is beneficial for ischemic stroke treatment using P2X7R as target.

Coordinated Transcriptional Waves Define the Inflammatory Response of Primary Microglial Culture.

The primary role of microglia is to maintain homeostasis by effectively responding to various disturbances. Activation of transcriptional programs determines the microglia's response to external stimuli. In this study, we stimulated murine neonatal microglial cells with benzoyl ATP (bzATP) and lipopolysaccharide (LPS), and monitored their ability to release pro-inflammatory cytokines. When cells are exposed to bzATP, a purinergic receptor agonist, a short-lived wave of transcriptional changes, occurs. However, only combining bzATP and LPS led to a sustainable and robust response. The transcriptional profile is dominated by induced cytokines (e.g., IL-1α and IL-1β), chemokines, and their membrane receptors. Several abundant long noncoding RNAs (lncRNAs) are induced by bzATP/LPS, including Ptgs2os2, Bc1, and Morrbid, that function in inflammation and cytokine production. Analyzing the observed changes through TNF (Tumor necrosis factor) and NF-κB (nuclear factor kappa light chain enhancer of activated B cells) pathways confirmed that neonatal glial cells exhibit a distinctive expression program in which inflammatory-related genes are upregulated by orders of magnitude. The observed capacity of the microglial culture to activate a robust inflammatory response is useful for studying neurons under stress, brain injury, and aging. We propose the use of a primary neonatal microglia culture as a responsive in vitro model for testing drugs that may interact with inflammatory signaling and the lncRNA regulatory network.

Prevention of M2 polarization and temporal limitation of differentiation in monocytes by extracellular ATP

BackgroundElevated levels of extracellular adenosine triphosphate (ATP) modulate immunologic pathways and are considered to be a danger signal in inflammation, lung fibrosis and cancer. Macrophages can be classified into two main types: M1 macrophages are classically activated, pro-inflammatory macrophages, whereas M2 macrophages are alternatively activated, pro-fibrotic macrophages. In this study, we examined the effect of ATP on differentiation of native human monocytes into these macrophage subtypes. We characterized M1 and M2 like macrophages by their release of Interleukin-1beta (IL-1β) and Chemokine (C–C motif) ligand 18 (CCL18), respectively.ResultsMonocytes were stimulated with ATP or the P2X7 receptor agonist Benzoylbenzoyl-ATP (Bz-ATP), and the production of various cytokines was analyzed, with a particular focus on CCL18 and IL-1β, along with the expression of different purinergic receptors. Over a 72 h period of cell culture, monocytes spontaneously differentiated to M2 like macrophages, as indicated by an increased release of CCL18. Immediate stimulation of monocytes with ATP resulted in a dose-dependent reduction in CCL18 release, but had no effect on the concentration of IL-1β. In contrast, delayed stimulation with ATP had no effect on either CCL18 or IL-1β release. Similar results were observed in a model of inflammation using lipopolysaccharide-stimulated human monocytes. Stimulation with the P2X7 receptor agonist Bz-ATP mimicked the effect of ATP on M2-macrophage differentiation, indicating that P2X7 is involved in ATP-induced inhibition of CCL18 release. Indeed, P2X7 was downregulated during spontaneous M2 differentiation, which may partially explain the ineffectiveness of late ATP stimulation of monocytes. However, pre-incubation of monocytes with PPADS, Suramin (unselective P2X- and P2Y-receptor blockers) and KN62 (P2X7-antagonist) failed to reverse the reduction of CCL18 by ATP.ConclusionsATP prevents spontaneous differentiation of monocytes into M2-like macrophages in a dose- and time-dependent manner. These effects were not mediated by P2X and P2Y receptors.

P2X7R promotes migration and invasion of Lewis lung cancer cells by activating the AKT signaling pathway

To explore the role of P2X7 receptor (P2X7R) in migration and invasion of mouse Lewis lung cancer (LLC) cells and examine the tumorigenic ability of LLC cells in P2X7R-knockout mice. RT-PCR was used to examine P2X7R mRNA expression in LLC cells. LLC cells were treated with ATP (as a P2X7R agonist) or 2'- 3'- O- (4-benzoyl- benzoyl)-ATP (BzATP) (a P2X7R agonist) with or without pretreatment with P2X7R antagonist oxATP or A438079. The changes in migration and invasive abilities of the cells were evaluated using wound healing assay and Transwell assay; Western blotting was performed to determine the activation level of the key proteins in the AKT signaling pathway. The effects of BzATP, A438079, and LY294002 (a inhibitor of the PI3K/AKT pathway) on migration and invasion of LLC cells were also examined. In wild-type (WT) and P2X7R knockout (P2X7-/-) C57BL/6 mice, the growth of subcutaneous LLC cell xenografts were observed by measuring tumor volume and weight. P2X7R expression was detected in LLC cells. Treatment with P2X7R agonist significantly enhanced migration and invasion abilities of LLC cells, and this effect was inhibited by application of P2X7R antagonists (P < 0.001). Western blotting showed that BzATP treatment of LLC cells significantly increased the expression level of p-AKT protein, which was obviously lowered by treatment with P2X7R antagonist (P < 0.01). P2X7R antagonist strongly inhibited BzATP-induced enhancement of LLC cell migration and invasion (P < 0.001). In the tumor- bearing mice, the tumor volume and weight were significantly lower in P2X7-/- mice than in WT mice (P < 0.05). P2X7R promotes migration and invasion of LLC cells by activating the AKT signaling pathway, and LLC cells show lowered tumorigenic capacity in P2X7-/- mice.

BzATP Activates Satellite Glial Cells and Increases the Excitability of Dorsal Root Ganglia Neurons In Vivo.

The purinergic system plays an important role in pain transmission. Recent studies have suggested that activation of P2-purinergic receptors (P2Rs) may be involved in neuron-satellite glial cell (SGC) interactions in the dorsal root ganglia (DRG), but the details remain unclear. In DRG, P2X7R is selectively expressed in SGCs, which closely surround neurons, and is highly sensitive to 3’-O-(4-Benzoyl) benzoyl-ATP (BzATP). Using calcium imaging in intact mice to survey a large number of DRG neurons and SGCs, we examined how intra-ganglionic purinergic signaling initiated by BzATP affects neuronal activities in vivo. We developed GFAP-GCaMP6s and Pirt-GCaMP6s mice to express the genetically encoded calcium indicator GGCaM6s in SGCs and DRG neurons, respectively. The application of BzATP to the ganglion induced concentration-dependent activation of SGCs in GFAP-GCaMP6s mice. In Pirt-GCaMP6s mice, BzATP initially activated more large-size neurons than small-size ones. Both glial and neuronal responses to BzATP were blocked by A438079, a P2X7R-selective antagonist. Moreover, blockers to pannexin1 channels (probenecid) and P2X3R (A317491) also reduced the actions of BzATP, suggesting that P2X7R stimulation may induce the opening of pannexin1 channels, leading to paracrine ATP release, which could further excite neurons by acting on P2X3Rs. Importantly, BzATP increased the responses of small-size DRG neurons and wide-dynamic range spinal neurons to subsequent peripheral stimuli. Our findings suggest that intra-ganglionic purinergic signaling initiated by P2X7R activation could trigger SGC-neuron interaction in vivo and increase DRG neuron excitability.

The P2X7 purinergic receptor in intervertebral disc degeneration.

Mechanisms involved in the development of intervertebral disc (IVD) degeneration are only partially known, thus making the implementation of effective therapies very difficult. In this study, we investigated P2X7 purinergic receptor (P2X7R), NLRP3 inflammasome, and interleukin (IL)‐1β expression in IVD specimens at different stages of disease progression, and during the in vitro dedifferentiation process of the primary cells derived thereof. We found that P2X7R, NLRP3, and IL‐1β expression was higher in the IVD samples at a more advanced stage of degeneration and in the expanded IVD cells in culture which partially recapitulated the in vivo degeneration process. In IVD cells, the P2X7R showed a striking nuclear localization, while NLRP3 was mainly cytoplasmic. Stimulation with the semiselective P2X7R agonist benzoyl ATP together with lipopolysaccharide treatment triggered P2X7R transfer to the cytoplasm and P2X7R/NLRP3 colocalization. Taken together, these findings support pathophysiological evidence that the degenerated disc is a highly inflamed microenvironment and highlight the P2X7R/NLRP3 axis as a suitable therapeutic target. The immunohistochemical analysis and the assessment of subcellular localization revealed a substantial expression of P2X7R also in normal disc tissue. This gives us the opportunity to contribute to the few studies performed in natively expressed human P2X7R so far, and to understand the possible physiological ATP‐mediated P2X7R homeostasis signaling. Therefore, collectively, our findings may offer a new perspective and pave the way for the exploration of a role of P2X7R‐mediated purinergic signaling in IVD metabolism that goes beyond its involvement in inflammation.

Monocyte-derived and M1 macrophages from ankylosing spondylitis patients released higher TNF-\u03b1 and expressed more IL1B in response to BzATP than macrophages from healthy subjects

Macrophages participate in the pathogenesis of ankylosing spondylitis (AS) by producing inflammatory cytokines. Extracellular adenosine triphosphate (eATP), released during cell stress, acts through purinergic receptors (P2XR and P2YR) and induces inflammatory responses. We investigated the effect of 2ʹ(3ʹ)-O-(4-benzoyl benzoyl) ATP (BzATP) (a prototypic agonist of P2X7R) on the production of inflammatory cytokines in both monocyte-generated (M2-like) and M1 macrophages from patients and controls. Macrophages were differentiated from isolated periphery-monocytes (n = 14 in each group) by macrophage colony-stimulating factor (M-CSF). Using LPS and IFN-γ, macrophages were skewed toward M1 type and were treated with BzATP. Gene expression and protein release of IL-1β, IL-23, and TNF-α were evaluated by real-time PCR and ELISA methods respectively before and after treatment. BzATP significantly increased the protein release of TNF-α and the expression of TNFA and IL1B in monocyte-generated macrophages. Besides, BzATP treatment significantly upregulated IL1B expression, reduced TNFA and IL23A expression, and TNF-α release in M1 macrophages from both groups. Monocyte-generated and M1 macrophages from AS patients released higher TNF-α and expressed more IL1B in response to the same concentration of BzATP treatment respectively. Based on our results, AS macrophages were more sensitive to BzATP treatment and responded more intensively. Besides, the diverse effects of BzATP on monocyte-derived and M1 macrophages in our study may represent the differed inflammatory properties of these two groups of macrophages in response to eATP in the body.

IRE1-mTOR-PERK Axis Coordinates Autophagy and ER Stress-Apoptosis Induced by P2X7-Mediated Ca2+ Influx in Osteoarthritis

Moderate-intensity exercise can help delay the development of osteoarthritis (OA). Previous studies have shown that the purinergic receptor P2X ligand gated ion channel 7 (P2X7) is involved in OA development and progression. To investigate the effect of exercise on P2X7 activation and downstream signaling in OA, we used the anterior cruciate ligament transection (ACLT)-induced OA rat model and primary chondrocyte culture system. Our in vivo experiments confirmed that treadmill exercise increased P2X7 expression and that this effect was more pronounced at the later time points. Furthermore, P2X7 activation induced endoplasmic reticulum (ER) stress and increased the expression levels of ER stress markers, such as 78 kDa glucose-regulated protein (GRP78), protein kinase R-like endoplasmic reticulum kinase (PERK), inositol-requiring enzyme-1 (IRE1), and activating transcription factor 6 (ATF6). At the early time points, IRE1 and PERK were activated, and mTOR was inhibited. At the later time points, mTOR was activated, mediating PERK to promote ER stress-apoptosis, whereas IRE1 and autophagy were inhibited. To confirm our observations in vitro, we treated primary chondrocytes with the P2X7 agonist benzoylbenzoyl-ATP (Bz-ATP). Our results confirmed that P2X7-mediated Ca2+ influx activated IRE1-mediated autophagic flux and induced PERK-mediated ER stress-apoptosis. To further investigate the role of P2X7 in OA, we injected mTOR antagonist rapamycin or P2X7 antagonist A740003 into the knee joints of ACLT rats. Our results demonstrated that mTOR inhibition induced autophagy, decreased apoptosis, and reduced cartilage loss. However, injection of mTOR agonist MHY1485 or Bz-ATP had the opposite effect. In summary, our results indicated that during the early stages of moderate-intensity exercise, P2X7 was activated and autophagic flux was increased, delaying OA development. At the later stages, P2X7 became over-activated, and the number of apoptotic cells increased, promoting OA development. We propose that the IRE1-mTOR-PERK signaling axis was involved in the regulation of autophagy inhibition and the induction of apoptosis. Our findings provide novel insights into the positive and preventative effects of exercise on OA, suggesting that the intensity and duration of exercise play a critical role. We also demonstrated that on a molecular level, P2X7 and its downstream pathways could be potential therapeutic targets for OA.

Electroacupuncture may alleviate neuropathic pain via suppressing P2X7R expression.

Neuropathic pain is a severe problem that is difficult to treat clinically. Reducing abnormal remodeling of dendritic spines/synapses and increasing the anti-inflammatory effects in the spinal cord dorsal horn are potential methods to treat this disease. Previous studies have reported that electroacupuncture (EA) could increase the pain threshold after peripheral nerve injury. However, the underlying mechanism is unclear. P2X7 receptors (P2X7R) mediate the activation of microglia and participate in the occurrence and development of neuropathic pain. We hypothesized that the effects of EA on relieving pain may be related to the downregulation of the P2X7R. Spinal nerve ligation (SNL) rats were used as a model in this experiment, and 2'(3')-O-(4-benzoyl)benzoyl ATP (BzATP) was used as a P2X7R agonist. We found that EA treatment decreased dendritic spine density, inhibited synaptic reconstruction and reduced inflammatory response, which is consistent with the decrease in P2X7R expression as well as the improved neurobehavioral performance. In contrast to the beneficial effects of EA, BzATP enhanced abnormal remodeling of dendritic spines/synapses and inflammation. Furthermore, the EA-mediated positive effects were reversed by BzATP, which is consistent with the increased P2X7R expression. These findings indicated that EA improves neuropathic pain by reducing abnormal dendritic spine/synaptic reconstruction and inflammation via suppressing P2X7R expression.

Flow cytometry: An accurate tool for screening P2RX7 modulators

AbstractThe P2X purinergic receptor 7 (P2RX7) is a poorly selective ATP‐gated ion channel. Although P2RX7 binds ATP with relatively low affinity, prolonged activation can lead to nonselective membrane pore formation. Indeed, brief exposure to ATP triggers a rapid Ca2+ influx, whereas prolonged exposure to high ATP concentrations results in the passage of larger organic molecules. P2RX7 is involved in the physiopathology of a number of diseases and has notably emerged as a potential therapeutic target in inflammation, neuropathic pain, Alzheimer's disease, and cancer—prompting growing interest in the synthesis of novel P2RX7 modulators and the development of reliable, stringent screening methods. In the present study, we developed methods based on conventional flow cytometry, imaging flow cytometry and spectral flow cytometry and used them to measure P2RX7's activity upon activation by 3'‐O‐(4‐benzoyl)benzoyl ATP. We also demonstrated the use of the highly sensitive DNA‐intercalating dye TO‐PRO‐3 to determine P2RX7's large pore activity. The simultaneous quantification of calcium influx (Fluo‐3 AM), large pore opening (TO‐PRO‐3), and viability (propidium iodide) is a very efficient method for low‐ to medium‐throughput screening of P2RX7 modulators. Agonist and antagonist potencies can be accurately evaluated. Spectral cytometry notably enabled us to assay several biological activities while correcting for the intrinsic fluorescence of the screened compounds—otherwise a well‐known limitation of fluorescence‐based screening. Hence, spectral cytometry appears to be a useful, novel tool for drug candidate screening.

Role of Conserved Residues and F322 in the Extracellular Vestibule of the Rat P2X7 Receptor in Its Expression, Function and Dye Uptake Ability.

Activation of the P2X7 receptor results in the opening of a large pore that plays a role in immune responses, apoptosis, and many other physiological and pathological processes. Here, we investigated the role of conserved and unique residues in the extracellular vestibule connecting the agonist-binding domain with the transmembrane domain of rat P2X7 receptor. We found that all residues that are conserved among the P2X receptor subtypes respond to alanine mutagenesis with an inhibition (Y51, Q52, and G323) or a significant decrease (K49, G326, K327, and F328) of 2′,3′-O-(benzoyl-4-benzoyl)-ATP (BzATP)-induced current and permeability to ethidium bromide, while the nonconserved residue (F322), which is also present in P2X4 receptor, responds with a 10-fold higher sensitivity to BzATP, much slower deactivation kinetics, and a higher propensity to form the large dye-permeable pore. We examined the membrane expression of conserved mutants and found that Y51, Q52, G323, and F328 play a role in the trafficking of the receptor to the plasma membrane, while K49 controls receptor responsiveness to agonists. Finally, we studied the importance of the physicochemical properties of these residues and observed that the K49R, F322Y, F322W, and F322L mutants significantly reversed the receptor function, indicating that positively charged and large hydrophobic residues are important at positions 49 and 322, respectively. These results show that clusters of conserved residues above the transmembrane domain 1 (K49–Y51–Q52) and transmembrane domain 2 (G326–K327–F328) are important for receptor structure, membrane expression, and channel gating and that the nonconserved residue (F322) at the top of the extracellular vestibule is involved in hydrophobic inter-subunit interaction which stabilizes the closed state of the P2X7 receptor channel.

Neuron-derived factors negatively modulate ryanodine receptor-mediated calcium release in cultured mouse astrocytes

Changes in intracellular Ca2+ concentration ([Ca2+]i) produced by ryanodine receptor (RyR) agonist, caffeine (caf), and ionotropic agonists: N-methyl-d-aspartate (NMDA) receptor (NMDAR) agonist, NMDA and P2X7 receptor (P2X7R) agonist, 3′-O-(4-benzoyl)benzoyl adenosine 5′-triphosphate (BzATP) were measured in cultured mouse cortical astrocytes loaded with the fluorescent calcium indicator Fluo3-AM in a confocal laser scanning microscope. In mouse astrocytes cultured in standard medium (SM), treatment with caf increased [Ca2+]i, with a peak response occurring about 10 min after stimulus application. Peak responses to NMDA or BzATP were observed about <1 min and 4.5 min post stimulus, respectively. Co-treatment with NMDA or BzATP did not alter the peak response to caf in astrocytes cultured in SM, the absence of the effects being most likely due to asynchrony between the response to caf, NMDA and BzATP. Incubation of astrocytes with neuron-condition medium (NCM) for 24 h totally abolished the caf-evoked [Ca2+]i increase. In NCM-treated astrocytes, peak of [Ca2+]i rise evoked by NMDA was delayed to about 3.5 min, and that induced by BzATP occurred about three minutes earlier than in SM. The results show that neurons secrete factors that negatively modulate RyR-mediated Ca2+-induced Ca2+ release (CICR) in astrocytes and alter the time course of Ca2+ responses to ionotropic stimuli.

P2X7 receptors exert a permissive effect on the activation of presynaptic AMPA receptors in rat trigeminal caudal nucleus glutamatergic nerve terminals

BackgroundPurine receptors play roles in peripheral and central sensitization and are associated with migraine headache. We investigated the possibility that ATP plays a permissive role in the activation of AMPA receptors thus inducing Glu release from nerve terminals isolated from the rat trigeminal caudal nucleus (TCN).MethodsNerve endings isolated from the rat TCN were loaded with [3H]D-aspartic acid ([3H]D-ASP), layered into thermostated superfusion chambers, and perfused continuously with physiological medium, alone or with various test drugs. Radioactivity was measured to assess [3H]D-ASP release under different experimental conditions.ResultsSynaptosomal [3H]D-ASP spontaneous release was stimulated by ATP and to an even greater extent by the ATP analogue benzoylbenzoylATP (BzATP). The stimulation of [3H]D-ASP basal release by the purinergic agonists was prevented by the selective P2X7 receptor antagonist A438079. AMPA had no effect on basal [3H]D-ASP release, but the release observed when synaptosomes were exposed to AMPA plus a purinoceptor agonist exceeded that observed with ATP or BzATP alone. The selective AMPA receptor antagonist NBQX blocked this “excess” release. Co-exposure to AMPA and BzATP, each at a concentration with no release-stimulating effects, evoked a significant increase in [3H]D-ASP basal release, which was prevented by exposure to a selective AMPA antagonist.ConclusionsP2X7 receptors expressed on glutamatergic nerve terminals in the rat TCN can mediate Glu release directly and indirectly by facilitating the activation of presynaptic AMPA receptors. The high level of glial ATP that occurs during chronic pain states can promote widespread release of Glu as well as can increase the function of AMPA receptors. In this manner, ATP contributes to the AMPA receptor activation involved in the onset and maintenance of the central sensitization associated with chronic pain.

Abstract P5-05-15: Activation of osteocytes connexin43 hemichannels suppresses breast cancer bone metastasis, a potential therapeutic target

Abstract Bone is a fertile place for metastasized breast cancer cell (BCC), however, mechanical loading is reported to inhibit BCC growth. Osteocytes, representing over 95% of bone cells, are mechanosensitive; they coordinate bone remodeling and modulate the extracellular environment. Mechanical loading activates osteocityc connexin 43 (Cx43) hemichannels, which mediates communication between the intracellular and extracellular space with message of molecules below 1 kDa. We have shown that ATP released by osteocytic hemichannels through the activation of BCC P2X7 receptor, inhibits BCC growth and migration. Here, we evaluate if activation of Cx43 hemichannels in osteocytes plays a critical role in inhibiting breast cancer bone metastasis. Our lab develops a monoclonal antibody, MHC2, which binds to extracellular domain of Cx43 and increases the activity of osteocytic hemichannels. This antibody was immune-reactive in HeLa cells transfected with Cx43 and osteocytes cell line MLO-Y4. It also labeled mouse osteocytes in situ. MHC2 antibody enhanced the activity of Cx43 hemichannels in cells and in vivo osteocytes and promoted the ATP release from osteocytic MLO-Y4 cells. Moreover, conditioned media collected from MHC2-treated osteocytes reduced BCC migration in vitro. The weekly injection of MHC2 reduced the breast cancer growth in bone in WT mice, but not in specific osteocyte Cx43 knockout mice. MHC2 antibody increase levels of lymphocytes T cytotoxic (CD3+/CD8+) and helper (CD3+/CD4+) in tumor and draining lymph nodes. Moreover, the weekly injection of MHC2, in intracardiac injected tumor cells model, in both immunocompetent and immune-compromised mice increased survival rate and reduced tumor spreading. As is known, osteocytic Cx43 hemichannels are permeable to ATP. We showed that P2X7 specific agonist benzoyl ATP reduced in vitro breast cancer cell migration, increased cytoplasmic free Ca2+ and induced formation of tubulin bundles, an indicator of cytoskeleton reorganization. The effect of benzoyl ATP required activation of CamKII in breast cancer cells. Our results suggest that Cx43 hemichannels are likely a potential therapeutic target in treating breast cancer growth and metastasizing to the bone, probably through the enhancement of the extracellular ATP level and the activation of P2X7/CamKII axis. Citation Format: Manuel A. Riquelme, Sumin Gu, Jeffery B. Chavez, Zhiqiang An, Jean X. Jiang. Activation of osteocytes connexin43 hemichannels suppresses breast cancer bone metastasis, a potential therapeutic target [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-05-15.

P2X7 Receptor Expression in Patients With Serositis Related to Systemic Lupus Erythematosus.

Introduction: P2X7R is an extracellular ATP-gated receptor involved in inflammatory and autoimmune processes mainly acting through NLPR3-inflammasome activation and IL-1β release, also implicated in lymphocyte proliferation and cellular apoptosis. Several observations from animal models and patients’ studies highlight a possible link between P2X7R-NLRP3 axis and Systemic Lupus Erythematosus (SLE) pathogenesis. The P2X7R-inflammasome axis in addition to the direct production of IL-1β and IL-18, indirectly mediates the release of other cytokines implicated in the pathogenesis of SLE, such as IL-6. The aim of this study was to investigate the role of P2X7R and NLRP3-inflammasome in SLE.Methods: Forty-eight SLE patients, 16 with (SLE-S) and 32 without (SLE-NS) history of serositis, and 20 healthy control (HC) subjects were enrolled. Demographic, clinical, and therapeutic data were collected. IL-1β and IL-6 plasma levels were evaluated by ELISA. Peripheral blood mononuclear cells (PBMCs) were isolated from venous blood by Ficoll gradient sedimentation and employed as follows: (1) evaluation of P2X7R and NLRP3 expression by RT-PCR; (2) determination of P2X7R activity as Benzoyl ATP (BzATP)-induced [Ca2+]i increments using Fura-2-AM fluorescent probe; (3) isolation of monocytes/macrophages and assessment of in vitro IL-1β and IL-6 release following stimulation with lipopolysaccharide (LPS) and BzATP, either separately or in combination.Results: Plasma IL-1β levels were unmodified in SLE respect to HC whereas IL-6 levels were higher in SLE than in HC, resulting significantly increased in SLE-S. Macrophages isolated from SLE patients released lower quantities of IL-1β after stimulation with BzATP, whereas IL-6 release was significantly augmented in SLE-NS respect to both HC and SLE-S after all types of stimulation. The [Ca2+]i increase following BzATP stimulation was significantly lower in PBMCs from SLE patients than in PBMCs from HC. RT-PCR showed significantly reduced P2X7R and significantly augmented NLRP3 expression in PBMCs from SLE patients.Conclusion: Our data indicate reduced P2X7R expression and function in SLE patients compared with HC and, conversely, increased IL-6 signaling. The possible consequences of reduced P2X7R, mainly on cytokines network deregulation and lymphocyte proliferation, will be further investigated as well as the role of IL-6 as a possible therapeutic target especially in lupus serositis.

The P2X7 Receptor Is Shed Into Circulation: Correlation With C-Reactive Protein Levels

The P2X7 receptor (P2X7R) is a key pro-inflammatory plasma membrane receptor responsible for NLRP3 inflammasome activation and IL-1β release. Various inflammatory plasma membrane receptors (e.g., IL-1 type I receptor, TNF type I and II receptors, IL-2 receptor) are shed under different pathophysiological conditions. In the present study, we show that the full length P2X7R is released into circulation in patients as well as in healthy subjects. Blood levels of shed P2X7R (sP2X7R) correlate to those of the inflammatory marker C reactive protein (CRP). Blood sP2X7R ranged from 16.74 to 82.17 ng/L, mean ± SE 40.97 ± 3.82 (n = 26) in healthy subjects, from 33.1 to 484.0 ng/L, mean ± SE 114.78 ± 12.22 (n = 45) in patients with CRP <3 mg/L, and from 63.65 to 1092.3 ng/L, mean ± SE 204.2 ± 30.94 (n = 42) in patients with CRP >3 mg/L. sP2X7R in plasma was largely associated to microvesicles/microparticles. Peripheral blood monocytes from healthy subjects released sP2X7R upon stimulation with the semi-selective P2X7R agonist benzoyl ATP. These data show that the P2X7R can be released into circulation, and that its blood levels increase in various disease conditions.

Activation of P2X7 Receptors in Peritoneal and Meningeal Mast Cells Detected by Uptake of Organic Dyes: Possible Purinergic Triggers of Neuroinflammation in Meninges.

Extracellular ATP activates inflammasome and triggers the release of multiple cytokines in various immune cells, a process primarily mediated by P2X7 receptors. However, the expression and functional properties of P2X7 receptors in native mast cells in tissues such as meninges where migraine pain originates from have not been explored. Here we report a novel model of murine cultured meningeal mast cells and using these, as well as easily accessible peritoneal mast cells, studied the mechanisms of ATP-mediated mast cell activation. We show that ATP induced a time and dose-dependent activation of peritoneal mast cells as analyzed by the uptake of organic dye YO-PRO1 as well as 4,6-diamidino-2-phenylindole (DAPI). Both YO-PRO1 and DAPI uptake in mast cells was mediated by the P2X7 subtype of ATP receptors as demonstrated by the inhibitory effect of P2X7 antagonist A839977. Consistent with this, significant YO-PRO1 uptake was promoted by the P2X7 agonist 2′,3′-O-(benzoyl-4-benzoyl)-ATP (BzATP). Extracellular ATP-induced degranulation of native and cultured meningeal mast cells was shown with Toluidine Blue staining. Taken together, these data demonstrate the important contribution of P2X7 receptors to ATP-driven activation of mast cells, suggesting these purinergic mechanisms as potential triggers of neuroinflammation and pain sensitization in migraine.

Modulation of P2X7 purinergic receptor activity by extracellular Zn2+ in cultured mouse hippocampal astroglia

The P2X7R protein, a P2 type purinergic receptor functioning as a non-selective cation channel, is expressed in different cell types of the central nervous system in several regions of the brain. The activation of the P2X7R protein by ATP modulates excitatory neurotransmission and contributes to microglial activation, apoptosis and neuron-glia communication. Zinc is an essential micronutrient that is highly concentrated in the synaptic vesicles of glutamatergic hippocampal neurons where free zinc ions released into the synaptic cleft alter glutamatergic signal transmission. Changes in both P2X7R-mediated signaling and brain zinc homeostasis have been implicated in the pathogenesis of mood disorders. Here, we tested the hypothesis that extracellular zinc regulates P2X7R activity in the hippocampus. We observed that P2X7R is expressed in both neurons and glial cells in primary mouse hippocampal neuron-glia culture. Propidium iodide (PI) uptake through large pores formed by pannexins and P2X7R was dose-dependently inhibited by extracellular zinc ions. Calcium influx mediated by P2X7R in glial cells was also reduced by free zinc ions. Interestingly, no calcium influx was detected in response to ATP or 3′-O-(4-Benzoyl) benzoyl ATP (BzATP) in neurons despite the expression of P2X7R at the plasma membrane. Our results show that free zinc ions can modulate hippocampal glial purinergic signaling, and changes in the activity of P2X7R may contribute to the development of depression-like behaviors associated with zinc deficiency.

Purinergic Vasotoxicity: Role of the Pore/Oxidant/KATP Channel/Ca2+ Pathway in P2X7-Induced Cell Death in Retinal Capillaries.

P2X7 receptor/channels in the retinal microvasculature not only regulate vasomotor activity, but can also trigger cells in the capillaries to die. While it is known that this purinergic vasotoxicity is dependent on the transmembrane pores that form during P2X7 activation, events linking pore formation with cell death remain uncertain. To better understand this pathophysiological process, we used YO-PRO-1 uptake, dichlorofluorescein fluorescence, perforated-patch recordings, fura-2 imaging and trypan blue dye exclusion to assess the effects of the P2X7 agonist, benzoylbenzoyl-ATP (BzATP), on pore formation, oxidant production, ion channel activation, [Ca2+]i and cell viability. Experiments demonstrated that exposure of retinal microvessels to BzATP increases capillary cell oxidants via a mechanism dependent on pore formation and the enzyme, NADPH oxidase. Indicative that oxidation plays a key role in purinergic vasotoxicity, an inhibitor of this enzyme completely prevented BzATP-induced death. We further discovered that vasotoxicity was boosted 4-fold by a pathway involving the oxidation-driven activation of hyperpolarizing KATP channels and the resulting increase in calcium influx. Our findings revealed that the previously unappreciated pore/oxidant/KATP channel/Ca2+ pathway accounts for 75% of the capillary cell death triggered by sustained activation of P2X7 receptor/channels. Elucidation of this pathway is of potential therapeutic importance since purinergic vasotoxicity may play a role in sight-threatening disorders such as diabetic retinopathy.