Roles of Cytokines in the Temporal Changes of Microglial Membrane Currents and Neuronal Excitability and Synaptic Efficacy in ATP-Induced Cortical Injury Model

Cytokines are important neuroinflammatory modulators in neurodegenerative brain disorders including traumatic brain injury (TBI) and stroke. However, their temporal effects on the physiological properties of microglia and neurons during the recovery period have been unclear. Here, using an ATP-induced cortical injury model, we characterized selective effects of ATP injection compared to needle-control. In the damaged region, the fluorescent intensity of CX3CR1-GFP (+) cells, as well as the cell density, was increased and the maturation of newborn BrdU (+) cells continued until 28 day-post-injection (dpi) of ATP. The excitability and synaptic E/I balance of neurons and the inward and outward membrane currents of microglia were increased at 3 dpi, when expressions of tumor necrosis factor (TNF)-α/interleukin (IL)-1β and IL-10/IL-4 were also enhanced. These changes of both cells at 3 dpi were mostly decayed at 7 dpi and were suppressed by any of IL-10, IL-4, suramin (P2 receptor inhibitor) and 4-AP (K+ channel blocker). Acute ATP application alone induced only small effects from both naïve neurons and microglial cells in brain slice. However, TNF-α alone effectively increased the excitability of naïve neurons, which was blocked by suramin or 4-AP. TNF-α and IL-1β increased and decreased membrane currents of naïve microglia, respectively. Our results suggest that ATP and TNF-α dominantly induce the physiological activities of 3 dpi neurons and microglia, and IL-10 effectively suppresses such changes of both activated cells in K+ channel- and P2 receptor-dependent manner, while IL-4 suppresses neurons preferentially.

Expression of purinergic receptors on microglia in the animal model of choroidal neovascularisation

To investigate the effect of P2 receptor on microglia and its inhibitor PPADS on choroidal neovascularization. Forty CX3CR1GFP/+ mice were randomly divided into 8 groups. In addition to the normal group, the rest of groups were receiving laser treatment. The retina and choroid from the second, third, fourth and fifth group of mice were taken in the 1, 4, 7, 14 days after laser treatment. The mice in the sixth and seventh group received intravitreal injection of 2 µl PPADS or PBS respectively immediately after laser treatment. The mice in the eighth group received topical application of PPADS once per day of three days. The mice in sixth, seventh and eighth group received AF and FFA examination on the fourth day after laser treatment. Immunofluorescence histochemical staining and real-time quantitative PCR were used to evaluate P2 expression and its effect on choroidal neovascularization. After laser treatment, activated microglia can express P2 receptors (P2X4, P2X7, P2Y2 and P2Y12). The expression of P2 increased on the first day after laser damage, peaked on the fourth day (tP2X4 = 6.05, tP2X7 = 2.95, tP2Y2 = 3.67, tP2Y12 = 5.98, all P < 0.01), and then decreased. After PPADS inhibition, compared with the PBS injection group, the mRNA of P2X4, P2X7, P2Y2 and P2Y12 were decreased significantly in the PPADS injection group (tP2X4 = 5.54, tP2X7 = 9.82, tP2Y2 = 3.86, tP2Y12 = 7.91, all P < 0.01) and the PPADS topical application group (tP2X4 = 3.24, tP2X7 = 5.89, tP2Y2 = 6.75, tP2Y12 = 4.97, all P < 0.01). Compared with the PBS injection group, not only the activity of microglia cells but also the leakage of CNV decreased significantly (P < 0.01) in the PPADS injection group and the PPADS topical application group. But between two PPADS groups, the leakage of CNV had no difference (P = 0.864). After laser induced CNV, activated microglia can express P2 receptors. The P2 receptor inhibitor, PPADS, can significantly affect the function of microglia and inhibit the formation of choroidal neovascularization.

Purinergic P2 Receptors: Novel Mediators of Mechanotransduction

Mechanosensing and mechanotransduction are vital processes in mechanobiology and play critical roles in regulating cellular behavior and fate. There is increasing evidence that purinergic P2 receptors, members of the purinergic family, play a crucial role in cellular mechanotransduction. Thus, information on the specific mechanism of P2 receptor-mediated mechanotransduction would be valuable. In this review, we focus on purinergic P2 receptor signaling pathways and describe in detail the interaction of P2 receptors with other mechanosensitive molecules, including transient receptor potential channels, integrins, caveolae-associated proteins and hemichannels. In addition, we review the activation of purinergic P2 receptors and the role of various P2 receptors in the regulation of various pathophysiological processes induced by mechanical stimuli.

High-affinity P2Y2 and low-affinity P2X7 receptor interaction modulates ATP-mediated calcium signaling in murine osteoblasts

1AbstractP2 purinergic receptor family implicated in many physiological processes, including neurotransmission, mechanical adaptation and inflammation, consist of ATP-gated non-specific cation channels P2XRs and G-protein coupled receptors P2YRs. Different cells, including bone forming osteoblasts, express multiple P2 receptors; however, how P2X and P2Y receptors interact in generating cellular responses to various doses of [ATP] remains poorly understood. Using primary bone marrow and compact bone derived osteoblasts and BMP2-expressing C2C12 osteoblastic cells, we demonstrated conserved features in the P2-mediated Ca2+ responses to ATP, including a transition of Ca2+ response signatures from transient at low [ATP] to oscillatory at moderate [ATP], and back to transient at high [ATP], and a non-monotonic changes in the response magnitudes which exhibited two troughs at 10−4 and 10−2 M [ATP]. We identified P2Y2 and P2X7 receptors as predominantly contributing to these responses, and constructed a mathematical model of P2Y2R-induced inositol trisphosphate (IP3) mediated Ca2+ release coupled to a Markov model of P2X7R dynamics to study this system. Model predictions were validated using parental and CRISPR/Cas9-generated P2Y2 and P2Y7 knockouts in osteoblastic C2C12-BMP cells. Activation of P2Y2 by progressively increasing [ATP] induced a transition from transient to oscillatory to transient Ca2+ responses due to the biphasic nature of IP3Rs and the interaction of SERCA pumps with IP3Rs. At high [ATP], activation of P2X7R modulated the response magnitudes through an interplay between the biphasic nature of IP3Rs and the desensitization kinetics of P2X7Rs. Moreover, we found that P2Y2 activity may alter the kinetics of P2X7 towards favouring naïve state activation. Finally, we demonstrated the functional consequences of lacking P2Y2 or P2X7 in osteoblast mechanitransduction. This study thus provides important insights into the biophysical mechanisms underlying ATP-dependent Ca2+ response signatures, which are important in mediating bone mechanoadaptation.2Author SummaryATP-sensitive purinergic receptors comprise a network of cell-surface receptors that activate upon ATP binding, allowing them to transmit information in a tissue- and context-dependent manner. In bone, mechanically-stimulated osteoblasts release ATP that stimulates low- and high-affinity P2 receptors in neighboring cellular populations, inducing appropriate physiological responses. P2 receptor signaling is characterized by elevations in intracellular calcium levels. When simultaneously stimulated by their common ligand, ATP, the contribution of each P2 receptor subtype gives rise to a complex calcium response, exhibiting oscillatory characteristics and biphasic dose-dependent behaviours. Here we used experimental and computational modeling approaches to determine the underlying dynamics of ATP-mediated calcium signaling in osteoblasts. The latter was done by developing a mathematical model that was comprised of a subset of low-(P2X7) and high-(P2Y2) affinity P2 receptors, reflecting the conserved P2 expression observed across different osteoblast models. We demonstrated that this model recapitulates experimental recordings of ATP-induced calcium signaling in osteoblasts and describes the dynamic interplay between P2Y2 and P2X7 receptors in the P2 receptor network.

Extracellular ATP Mediates Cancer Cell Migration and Invasion Through Increased Expression of Cyclooxygenase 2

The tumor microenvironment plays a major role in the ability of the tumor cells to undergo metastasis. A major player of tumors gaining metastatic property is the inflammatory protein, cyclooxygenase 2 (COX-2). Several tumors show upregulation of this protein, which has been implicated in mediating metastasis in various cancer types such as of colon, breast and lung. In this report, we show that the concentration of extracellular ATP (eATP) is increased in response to cell death mediated by chemotherapeutic agents such as doxorubicin. By using three different cell-lines—HeLa (cervical), IMR-32 (neuronal) and MCF-7 (breast)—we show that this eATP goes on to act on purinergic (P2) receptors. Among the various P2 receptors expressed in these cells we identified P2X7, in IMR-32 and MCF-7 cells, and P2Y12, in HeLa cells, as important in modulating cell migration and invasion. Downstream of the P2 receptor activation, both p42/44 mitogen-activated protein kinase (MAPK) and the p38 MAPK are activated in these cells. These result in an increase in the expression of COX-2 mRNA and protein. We also observe an increase in the activity of matrix metalloproteinase 2 (MMP-2) enzyme in these cells. Blocking the P2 receptors not only blocks migration and invasion, but also COX-2 synthesis and MMP-2 activity. Our results show the link between purinergic receptors and COX-2 expression. Increased levels of ATP in the tumor microenvironment, therefore, leads to increased COX-2 expression, which, in turn, affords migratory and invasive properties to the tumor. This provides P2 receptor-based anti-inflammatory drugs (PBAIDs) a potential opportunity to be explored as cancer therapeutics.

NTPDase8 protects mice from intestinal inflammation by limiting P2Y6 receptor activation: identification of a new pathway of inflammation for the potential treatment of IBD

ObjectiveNucleotides are danger signals that activate inflammatory responses via binding P2 receptors. The nucleoside triphosphate diphosphohydrolase-8 (NTPDase8) is an ectonucleotidase that hydrolyses P2 receptor ligands. We investigated the role of NTPDase8 in intestinal inflammation.DesignWe generated NTPDase8-deficient (Entpd8–/–) mice to define the role of NTPDase8 in the dextran sodium sulfate (DSS) colitis model. To assess inflammation, colons were collected and analysed by histopathology, reverse transcriptase-quantitative real-time PCR (RT-qPCR) and immunohistochemistry. P2 receptor expression was analysed by RT-qPCR on primary intestinal epithelium and NTPDase8 activity by histochemistry. The role of intestinal P2Y6 receptors was assessed by bone marrow transplantation experiments and with a P2Y6 receptor antagonist.ResultsNTPDase8 is the dominant enzyme responsible for the hydrolysis of nucleotides in the lumen of the colon. Compared with wild-type (WT) control mice, the colon of Entpd8–/– mice treated with DSS displayed significantly more histological damage, immune cell infiltration, apoptosis and increased expression of several proinflammatory cytokines. P2Y6 was the dominant P2Y receptor expressed at the mRNA level by the colonic epithelia. Irradiated P2ry6–/– mice transplanted with WT bone marrow were fully protected from DSS-induced intestinal inflammation. In agreement, the daily intrarectal injection of a P2Y6 antagonist protected mice from DSS-induced intestinal inflammation in a dose-dependent manner. Finally, human intestinal epithelial cells express NTPDase8 and P2Y6 similarly as in mice.ConclusionNTPDase8 protects the intestine from inflammation most probably by limiting the activation of P2Y6 receptors in colonic epithelial cells. This may provide a novel therapeutic strategy for the treatment of inflammatory bowel disease.

P2 Receptors in Cardiac Myocyte Pathophysiology and Mechanotransduction

ATP is a major energy source in the mammalian cells, but it is an extracellular chemical messenger acting on P2 purinergic receptors. A line of evidence has shown that ATP is released from many different types of cells including neurons, endothelial cells, and muscle cells. In this review, we described the distribution of P2 receptor subtypes in the cardiac cells and their physiological and pathological roles in the heart. So far, the effects of external application of ATP or its analogues, and those of UTP on cardiac contractility and rhythm have been reported. In addition, specific genetic alterations and pharmacological agonists and antagonists have been adopted to discover specific roles of P2 receptor subtypes including P2X4-, P2X7-, P2Y2- and P2Y6-receptors in cardiac cells under physiological and pathological conditions. Accumulated data suggest that P2X4 receptors may play a beneficial role in cardiac muscle function, and that P2Y2- and P2Y6-receptors can induce cardiac fibrosis. Recent evidence further demonstrates P2Y1 receptor and P2X4 receptor as important mechanical signaling molecules to alter membrane potential and Ca2+ signaling in atrial myocytes and their uneven expression profile between right and left atrium.

P2Y2 and P2X4 Receptors Mediate Ca2+ Mobilization in DH82 Canine Macrophage Cells

Purinergic receptors of the P2 subclass are commonly found in human and rodent macrophages where they can be activated by adenosine 5′-triphosphate (ATP) or uridine 5′-triphosphate (UTP) to mediate Ca2+ mobilization, resulting in downstream signalling to promote inflammation and pain. However, little is understood regarding these receptors in canine macrophages. To establish a macrophage model of canine P2 receptor signalling, the expression of these receptors in the DH82 canine macrophage cell line was determined by reverse transcription polymerase chain reaction (RT-PCR) and immunocytochemistry. P2 receptor function in DH82 cells was pharmacologically characterised using nucleotide-induced measurements of Fura-2 AM-bound intracellular Ca2+. RT-PCR revealed predominant expression of P2X4 receptors, while immunocytochemistry confirmed predominant expression of P2Y2 receptors, with low levels of P2X4 receptor expression. ATP and UTP induced robust Ca2+ responses in the absence or presence of extracellular Ca2+. ATP-induced responses were only partially inhibited by the P2X4 receptor antagonists, 2′,3′-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP), paroxetine and 5-BDBD, but were strongly potentiated by ivermectin. UTP-induced responses were near completely inhibited by the P2Y2 receptor antagonists, suramin and AR-C118925. P2Y2 receptor-mediated Ca2+ mobilization was inhibited by U-73122 and 2-aminoethoxydiphenyl borate (2-APB), indicating P2Y2 receptor coupling to the phospholipase C and inositol triphosphate signal transduction pathway. Together this data demonstrates, for the first time, the expression of functional P2 receptors in DH82 canine macrophage cells and identifies a potential cell model for studying macrophage-mediated purinergic signalling in inflammation and pain in dogs.