Purinergic Receptor Subtypes Research Articles

Characterization of 11C-GSK1482160 for Targeting the P2X7 Receptor as a Biomarker for Neuroinflammation.

The purinergic receptor subtype 7 (P2X7R) represents a novel molecular target for imaging neuroinflammation via PET. GSK1482160, a potent P2X7R antagonist, has high receptor affinity, high blood-brain barrier penetration, and the ability to be radiolabeled with 11C. We report the initial physical and biologic characterization of this novel ligand. Methods:11C-GSK1482160 was synthesized according to published methods. Cell density studies were performed on human embryonic kidney cell lines expressing human P2X7R (HEK293-hP2X7R) and underwent Western blotting, an immunofluorescence assay, and radioimmunohistochemistry analysis using P2X7R polyclonal antibodies. Receptor density and binding potential were determined by saturation and association-disassociation kinetics, respectively. Peak immune response to lipopolysaccharide treatment in mice was determined in time course studies and analyzed via Iba1 and P2X7R Western blotting and Iba1 immunohistochemistry. Whole-animal biodistribution studies were performed on saline- or lipopolysaccharide-treated mice at 15, 30, and 60 min after radiotracer administration. Dynamic in vivo PET/CT was performed on the mice at 72 h after administration of saline, lipopolysaccharide, or lipopolysaccharide + blocking, and 2-compartment, 5-parameter tracer kinetic modeling of brain regions was performed. Results: P2X7R changed linearly with concentrations or cell numbers. For high-specific-activity 11C-GSK1482160, receptor density and Kd were 1.15 ± 0.12 nM and 3.03 ± 0.10 pmol/mg, respectively, in HEK293-hP2X7R membranes. Association constant kon, dissociation constant koff, and binding potential (kon/koff) in HEK293-hP2X7R cells were 0.2312 ± 0.01542 min-1⋅nM-1, 0.2547 ± 0.0155 min-1, and 1.0277 ± 0.207, respectively. Whole-brain Iba1 expression in lipopolysaccharide-treated mice peaked by 72 h on immunohistochemistry, and Western blot analysis of P2X7R for saline- and lipopolysaccharide-treated brain sections showed a respective 1.8- and 1.7-fold increase in signal enhancement at 72 h. Biodistribution of 11C-GSK1482160 in saline- and lipopolysaccharide-treated mice at 72 h was statistically significant across all tissues studied. In vivo dynamic 11C-GSK1482160 PET/CT of mice at 72 h after administration of saline, lipopolysaccharide, or lipopolysaccharide + blocking showed a 3.2-fold increase and 97% blocking by 30 min. The total distribution volumes for multiple cortical regions and the hippocampus showed statistically significant increases and were blocked by an excess of authentic standard GSK1482160. Conclusion: The current study provides compelling data that support the suitability of 11C-GSK1482160 as a radioligand targeting P2X7R, a biomarker of neuroinflammation.

Role of Purines in Müller Glia

Müller glia, the principal macroglia of the retina, express diverse subtypes of adenosine and metabotropic purinergic (P2Y) receptors. Müller cells of several species, including man, also express ionotropic P2X7 receptors. ATP is liberated from Müller cells after activation of metabotropic glutamate receptors and during osmotic and mechanical induction of membrane stretch; adenosine is released through equilibrative nucleoside transporters. Müller cell-derived purines modulate the neuronal activity and have autocrine effects, for example, induction of glial calcium waves and regulation of the cellular volume. Glial calcium waves induced by neuron-derived ATP mediate functional hyperemia in the retina. Purinergic signaling contributes to the induction of Müller cell gliosis, for example, of cellular proliferation and downregulation of potassium channels, which are important for the homeostatic functions of Müller cells. Purinergic glial calcium waves may also promote the long-range propagation of gliosis and neuronal degeneration across the retinal tissue. The osmotic ATP release is inhibited under pathological conditions. Inhibition of the ATP release may result in osmotic Müller cell swelling and dysregulation of the water transport through the cells; both may contribute to the development of retinal edema. Suppression of the osmotic ATP release and upregulation of the ecto-apyrase (NTPDase1), which facilitate the extracellular degradation of ATP and the formation of adenosine, may protect neurons and photoreceptors from death due to overactivation of P2X receptors. Pharmacological inhibition of P2X7 receptors and stimulation of adenosine receptors may represent clinical approaches to prevent retinal cell death and dysregulated cell proliferation, and to treat retinal edema.

P2X7 Receptor Activation By ATP As Target of Novel Therapies in Acute Myeloid Leukemia

ATP is the key energy molecule as well as an ubiquitous extracellular messenger. Depending on its dose and the engaged purinergic P2 receptor (P2R) subtype, ATP can trigger many different cell responses, including proliferation and cell death. Recent studies have shown that high ATP level exhibits direct cytotoxicity on many tumor cell types. Among the receptors engaged by ATP, P2X7 is the most consistently expressed by tumor cells and its overexpression is related to tumor growth and progression. The P2X7 is an ATP-gated ion channel that, upon sustained stimulation with millimolar ATP concentrations, drives the opening of a non-selective large conductance pore, triggering cell-death signal. We previously demonstrated that ATP is a potent stimulator of normal hematopoietic stem cell compartment while inhibiting acute myeloid leukemia (AML) cells. Based on this observation, we studied AML samples (n=20) collected from the bone marrow or the peripheral blood of leukemic patients at diagnosis before treatment (percentage of circulating blasts >90%). In addition, normal hematopoietic stem cells (HSC) were isolated from leukapheresis products of 5 healthy donors receiving G-CSF.Our data demonstrate that AML cells express high level of P2X7 and that its activation with high dose of ATP reduces blast cell viability while is not effective on normal CD34+ cells. The cytotoxic effect is due to the induction of apoptosis, associated with reduction of mithocondrial membrane potential and activation of caspase cascade. Interestingly, P2X7 is also expressed by leukemic stem/progenitor cells (LSC) and ATP treatment exerts a direct cytotoxicity on different subsets of stem/progenitor cell compartment i.e. CD34- CD38-, CD34+ CD38-, CD34+ CD38+ and CD34- CD38+. Of note, this cytotoxic effect was not observed on HSC subpopulations. Furthermore, we transplanted 1x106 human AML cells into NSG immunodeficient mice followed by intraperitoneal administration of ATP every other day for thirty days post-transplantation. Our results show a 40% inhibition of AML engraftment in ATP-treated mice vs controls.Different P2X7 splice variants have been identified among which only two are functional: P2X7A, which shows both pro-apoptotic and trophic activity and P2X7B, which retains only the growth promoting phenotype. In order to explain ATP different effects on LSCs and normal HSCs, we assumed a different P2X7 isoforms expression on normal and leukemic cells. Preliminary results showed a reduced expression of both P2X7A and P2X7B on normal CD34+ compared to leukemic cells. In particular normal CD34+ express very low level of P2X7A, which is responsible for pore formation after ATP stimulation.Moreover, since P2X7 pore formation facilitates the passage of hydrophilic chemotherapeutic agents, we hypothesized that ATP may potentiate the cytotoxic effect of antineoplastic drugs. Our results showed that ATP potentiates the cytotoxic effect of ARA-C, by significantly reducing cell proliferation and increasing apoptosis of leukemia cell lines.In conclusion, overall survival of adult AML remains poor due to the lack of novel and effective therapies. Novel compounds that have the potential to improve the treatment efficacy with low toxicity are highly warranted. Overall, our results may provide the biological rationale to use P2X7 as a target for novel therapeutical approaches against AML. DisclosuresCavo:Janssen-Cilag, Celgene, Amgen, BMS: Honoraria.

Hetero-oligomerization and Functional Interaction between Purinergic Receptors Expressed in Platelets to Regulate Platelet Shape Change

Adenosine and its precursors, ATP and ADP, exert various physiological effects via binding to purinergic receptors. We previously used co-immunoprecipitation, bioluminescence resonance energy transfer (BRET) and immunoelectron microscopy to demonstrate the hetero-oligomerization of purinergic receptor subtypes. Furthermore, pharmacological studies found significant changes in receptor-mediated signaling in human embryonic kidney (HEK) 293T cells co-transfected with these receptors. These findings suggest that heterodimers of purinergic receptors may have distinct pharmacological profiles, possibly due to dimerization-induced conformational changes, further suggesting that hetero-dimerization may be employed to "fine-tune" purinergic receptor signaling. Adenosine A(2A) receptor (A(2A)R), P2Y1 receptor (P2Y1R) and P2Y12 receptor (P2Y12R) are predominantly expressed on human platelets. ADP activates human platelets by stimulating both P2Y1R and P2Y12R, which act sequentially and in concert to achieve complete platelet aggregation. In contrast, adenosine stimulates Gs-coupled A(2A)R, followed by activativation of adenylate cyclase, leading to an increase in intracellular cAMP levels, which potently inhibits platelet activation. We examined the hetero-oligomerization and functional interactions of A(2A)R, P2Y1R, and P2Y12R. In HEK293T cells triply expressing all three receptors, hetero-oligomerization was observed among the three receptors. Additionally, P2Y1R agonist-evoked Ca(2+) signaling was significantly inhibited by co-treatment with an A(2A)R antagonist in HEK293T cells. In human platelets, we identified endogenous A(2A)R/P2Y1R and A(2A)R/P2Y12R heterodimers. We also observed functional Ca(2+)-signaling-related cross-talk similar to those found in HEK293T cells, and found that they appeared to affect platelet shape. These results collectively suggest that intermolecular signal transduction and specific conformational changes occur among components of the hetero-oligomers formed by these three receptors.

Reply to 'Mechanism for microtsunami-induced intercellular mechanosignalling'

Reply to 'Mechanism for microtsunami-induced intercellular mechanosignalling'

Epigenetic alteration of the purinergic type 7 receptor in salivary epithelial cells

Purinergic receptors, particularly type 7 (P2RX7), are involved in apoptotic cell death. However, the expression and function of P2RX7 are suppressed in HSG cells. In the present study, we explored whether P2RX7 function is regulated by epigenetic alteration of the receptors in two different cell lines, HSG cells derived from human submandibular ducts, and A253 cells, originated from human submandibular carcinoma. We discovered that HSG cells expressed all subtypes of purinergic receptors, excluding P2RX7, at the mRNA level. However, treatment of the cells with 5-Aza-CdR, a DNA demethylating agent, increased the mRNA expression levels of P2RX7 in a time-dependent manner. Furthermore, 5-Aza-CdR completely rescued the calcium response induced by P2RX7 agonist BzATP, a response that was absent in untreated HSG cells. In contrast, A253 cells showed a moderate methylation pattern in the P2RX7 CpG island. Most CG pairs from the first to the 21st were methylated in untreated HSG cells, but 5-Aza-CdR-treatment partially demethylated the methylated CG pairs. We obtained similar results when investigated human tissues; the CG pairs in the P2RX7 CpG islands showed hypermethylation and hypomethylation patterns in human normal and cancer tissues, respectively. Our results suggest that the expression level and function of P2RX7 are regulated by DNA methylation in epithelial cells.

Effects of ATP and NGF on Proliferation and Migration of Neural Precursor Cells.

Purinergic receptors belong to the most ancient neurotransmitter system. While their relevance in neurotransmission is well characterized, it has become clear that they have many other cellular functions. During development, they participate in regulation of proliferation and differentiation of stem cells. Here, we used rat embryonic telencephalon neurosphere cultures to detect purinergic P2 receptor subtype expression and possible synergistic actions of these receptors with NGF. Neurospheres proliferate in the presence of EGF and FGF-2; however, upon depletion of these growth factors, they migrate and differentiate into neurons and glial phenotypes. Expression patterns of P2X and P2Y receptors changed along neural differentiation. Gene expression of P2X2-7 and P2Y1,2,4,6,12,14 receptors was confirmed in undifferentiated and neural-differentiated neurospheres, with an up-regulation of P2X2 and P2X6 subtypes, together with a down-regulation of P2X4, P2X7 and P2Y subtypes upon induction to differentiation. BrdU-labeling and subsequent flow cytometry analysis was used to measure cell proliferation, which was increased by chronic exposure to NGF and increasing concentrations of ATP, in line with the expression levels of PCNA. Furthermore, a synergistic effect on proliferation was observed in conditions of co-incubation with ATP and NGF. While ATP and NGF independently promoted neural migration, no inter-relation between these factors was detected for this cellular process. As conclusion, an unknown synergism of ATP and NGF in proliferation is described. Future efforts may elucidate the underlying mechanisms of the interrelationship of ATP and NGF during neurogenesis.

Altered expression of P2Y2 and P2X7 purinergic receptors in the isolated rat heart mediates ischemia–reperfusion injury

The aim of this study is to analyze the expression of purinergic receptors in the heart after ischemia–reperfusion, and their possible role in ischemia–reperfusion injury. Rat hearts were perfused according to the Langendorff technique and subjected to 30min ischemia followed by 15min reperfusion. Ischemia–reperfusion reduced the gene expression and protein content of purinergic receptors of the P2Y2 subtype, and increased the gene expression and protein content of the P2X7 subtype. Treatment with the agonist of the P2Y2 subtype 2-thio-UTP and with the antagonist of the P2X7 subtype Brilliant Blue improved myocardial function parameters, reduced cell death and increased the myocardial expression of antiapoptotic markers after ischemia–reperfusion. These results suggest that the myocardial expression of the protective P2Y2 subtype of purinergic receptors is reduced, whereas that of the harmful subtype P2X7 subtype is increased during coronary ischemia–reperfusion. This may contribute to myocardial injury in this condition.

The Beneficial Effects of P2X7 Antagonism in Rats with Bile Duct Ligation-induced Cirrhosis.

Splanchnic angiogenesis in liver cirrhosis often leads to complications as gastroesophageal variceal hemorrhage and the treatment efficacy is adversely affected by poor portal-systemic collateral vasoresponsiveness related to nitric oxide (NO). Purinergic receptor subtype P2X7 participates in the modulation of inflammation, angiogenesis, fibrogenesis and vasoresponsiveness, but the relevant influence in cirrhosis is unknown. Common bile duct-ligated (CBDL) or sham-operated Spraque-Dawley rats received brilliant blue G (BBG, a P2X7 antagonist and food additive) or vehicle from the 15th to 28th day after operations, then hemodynamics, mesenteric angiogenesis, portal-systemic shunting, liver fibrosis, and protein expressions of angiogenic and fibrogenic factors were evaluated. The influence of oxidized ATP (oATP, another P2X7 receptor antagonist) on the collateral vasoresponsiveness to arginine vasopressin (AVP) was also surveyed. BBG decreased superior mesenteric artery (SMA) flow, portal-systemic shunting, mesenteric vascular density, and mesenteric protein expressions of vascular endothelial growth factor (VEGF), VEGF receptor 2 (VEGFR2), phospho (p)-VEGFR2, platelet-derived growth factor (PDGF), PDGF receptor beta (PDGFRβ), cyclooxygenase (COX)-1, COX-2, and endothelial NO synthase (eNOS) in CBDL rats. BBG also ameliorated liver fibrosis and down-regulated hepatic interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), PDGF, IL-1β, transforming growth factor-beta (TGF-β), p-extracellular-signal-regulated kinases (ERK), and alpha-smooth muscle actin (α-SMA) expressions in CBDL rats. The collateral vasocontractility to AVP was enhanced by oATP. oATP down-regulated eNOS, inducible NOS (iNOS), VEGF, Akt, p-Akt, and nuclear factor-kappa B (NF-κB) expressions in splenorenal shunt, the most prominent intra-abdominal collateral vessel in rodents. P2X7 antagonism alleviates splanchnic hyperemia, severity of portal-systemic shunting, mesenteric angiogenesis, liver fibrosis, and enhances portal-systemic collateral vasoresponsiveness in cirrhotic rats. P2X7 blockade may be a feasible strategy to control cirrhosis and complications.

Purinergic signaling pathways in endocrine system.

Adenosine-5'-triphosphate is released by neuroendocrine, endocrine, and other cell types and acts as an extracellular agonist for ligand-gated P2X cationic channels and G protein-coupled P2Y receptors in numerous organs and tissues, including the endocrine system. The breakdown of ATP by ectonucleotidases not only terminates its extracellular messenger functions, but also provides a pathway for the generation of two additional agonists: adenosine 5'-diphosphate, acting via some P2Y receptors, and adenosine, a native agonist for G protein-coupled adenosine receptors, also expressed in the endocrine system. This article provides a review of purinergic signaling pathways in the hypothalamic magnocellular neurosecretory cells and neurohypophysis, hypothalamic parvocellular neuroendocrine system, adenohypophysis, and effector glands organized in five axes: hypothalamic-pituitary-gonadal, hypothalamic-pituitary-thyroid, hypothalamic-pituitary-adrenal, hypothalamic-pituitary-growth hormone, and hypothalamic-pituitary-prolactin. We attempted to summarize current knowledge of purinergic receptor subtypes expressed in the endocrine system, including their roles in intracellular signaling, hormone secretion, and other cell functions. We also briefly review the release mechanism for adenosine-5'-triphosphate by neuroendocrine, endocrine and surrounding cells, the enzymes involved in adenosine-5'-triphosphate hydrolysis to adenosine-5'-diphosphate and adenosine, and the relevance of this pathway for sequential activation of receptors and termination of signaling.

Extracellular Adenosine Diphosphate Ribose Mobilizes Intracellular Ca2+ via Purinergic-Dependent Ca2+ Pathways in Rat Pulmonary Artery Smooth Muscle Cells

Background/Aims: Adenosine diphosphate ribose (ADPR), a product of β-NAD⁺ metabolism generated by the multifunctional enzyme CD38, is recognized as a novel signaling molecule. The catalytic site of CD38 orients extracellularly or intracellularly, capable of generating ADPR outside and inside the cells. CD38-dependent pathways have been characterized in pulmonary artery smooth muscle cells (PASMCs); however the physiological function of extracellular ADPR is unclear. Methods: Ca²⁺ mobilizing and proliferative effects of extracellular ADPR were characterized and compared with the ATP-induced responses in rat PASMCs; and the expression of purinergic receptor (P2X and P2Y) subtypes were examined in pulmonary arteries. Results: ADPR elicited concentration-dependent increase in [Ca²⁺]_i with a fast transient and a sustained phase in PASMCs. The sustained phase was abolished by Ca²⁺ removal and inhibited by the non-selective cation channel blocker SKF-96365, but was unaffected by TRPM2 antagonists or nifedipine. The purinergic receptor (P2X) antagonist pyridoxal-phosphate-6-azophenyl-2', 4'-disulfonate inhibited partially the transient and the sustained Ca²⁺ response, while the P2(XY) inhibitor suramin and the phospholipase C inhibitor U73122 abolished the sustained Ca²⁺ influx. The P2Y1 antagonist MRS2179 had no effect on the response. By contrast, ATP and ADP activated Ca²⁺ response exhibited a high and a low affinity component, and the pharmacological profile of ATP-induced Ca²⁺ response was distinctive from that of ADPR. BrdU incorporation assay showed that ADPR caused significant inhibition whereas ATP caused slight stimulation of PASMC proliferation. RT-PCR analysis found that almost all P2X and P2Y subtypes are expressed in PAs. Conclusion: ADPR and ATP activate Ca²⁺ responses through different combinations of multiple purinergic receptor subtypes; and extracellular ADPR may exert an autocrine/paracrine action via purinergic receptors on PASMCs.

The role of nucleotides and purinergic signaling in apoptotic cell clearance - implications for chronic inflammatory diseases.

Billions of cells undergo apoptosis every day in healthy individuals. A prompt removal of dying cells prevents the release of pro-inflammatory intracellular content and progress to secondary necrosis. Thus, inappropriate clearance of apoptotic cells provokes autoimmunity and has been associated with many chronic inflammatory diseases. Recent studies have suggested that extracellular adenosine 5′-triphosphate and related nucleotides play an important role in the apoptotic clearance process. Here, we review the current understanding of nucleotides and purinergic receptors in apoptotic cell clearance and the potential therapeutic targets of purinergic receptor subtypes in inflammatory conditions.

Elevated placental adenosine signaling contributes to the pathogenesis of preeclampsia

Purpose: Dephosphorylation of the myosin II regulatory light chain (MLC) promotes barrier integrity of cellular monolayers through relaxation of the actin cytoskeleton. This study has investigated the influence of adenosine (ADO) on MLC phosphorylation in cultured bovine corneal endothelial cells (BCEC).Methods: MLC phosphorylation was assessed by urea-glycerol gel electrophoresis and immunoblotting. Elevation of cAMP in response to agonists of A2b receptors (subtype of P1 purinergic receptors) was confirmed by phosphorylation of the cAMP response element binding protein (CREB), which was determined by Western blotting. Activation of MAP kinases (i.e. activated ERK1 and ERK2) was assessed by Western blotting to examine their influence on MLC phosphorylation. Transepithelial electrical resistance (TER) of cells grown on porous filters was measured to assess the altered barrier integrity.Results: Exposure to ADO (200 μm; 30 min) and N-ethyl (carboxamido) adenosine (NECA; 50 μm; 30 min), known agonists of A2b receptors, induced phosphorylation of CREB similar to forskolin (FSK, 20 μm; 30 min), a direct activator of adenylate cyclase. Exposure to ADO, NECA, and FSK led to dephosphorylation of MLC by 51, 40, and 47%, respectively. ADO-induced dephosphorylation was dose-dependent with as much as 31% dephosphorylation at 1 μm ADO. CGS-21680, a selective A2a agonist, neither induced MLC dephosphorylation nor CREB phosphorylation. ADO phosphorylated MAP kinases which could be prevented by exposure to the MAP kinase-specific inhibitor, U0126 (10 μM). NECA and FSK also induced ERK1 and ERK2 activation similar to ADO. Exposure to U0126 inhibited MLC phosphorylation under basal conditions by 17%. ADO-induced MLC dephosphorylation was enhanced by a simultaneous exposure to U0126 (25% increase in dephosphorylation). Exposure to ADO caused an increase in TER from 17 to 22 ohms cm2.Conclusions: (1) CREB phosphorylation in response to ADO and NECA, which indicates activation of the cAMP-PKA axis, suggests expression of A2b receptors in BCEC. (2) ERK1 and ERK2, activated by cAMP and A2b receptors, promote MLC phosphorylation. However, the net result of cAMP elevation is MLC dephosphorylation, presumably because the competing pathways involving inactivation of MLCK and/or ROCK are dominant (Rho-associated coiled coil-containing protein kinase or Rho kinase). (3) Consistent with MLC dephosphorylation, exposure to ADO increases TER, which suggests increased barrier integrity.

P2Y2 purinergic receptor activation is essential for efficient hepatocyte proliferation in response to partial hepatectomy.

Extracellular nucleotides via activation of P2 purinergic receptors influence hepatocyte proliferation and liver regeneration in response to 70% partial hepatectomy (PH). Adult hepatocytes express multiple P2Y (G protein-coupled) and P2X (ligand-gated ion channels) purinergic receptor subtypes. However, the identity of key receptor subtype(s) important for efficient hepatocyte proliferation in regenerating livers remains unknown. To evaluate the impact of P2Y2 purinergic receptor-mediated signaling on hepatocyte proliferation in regenerating livers, wild-type (WT) and P2Y2 purinergic receptor knockout (P2Y2-/-) mice were subjected to 70% PH. Liver tissues were analyzed for activation of early events critical for hepatocyte priming and subsequent cell cycle progression. Our findings suggest that early activation of p42/44 ERK MAPK (5 min), early growth response-1 (Egr-1) and activator protein-1 (AP-1) DNA-binding activity (30 min), and subsequent hepatocyte proliferation (24-72 h) in response to 70% PH were impaired in P2Y2-/- mice. Interestingly, early induction of cytokines (TNF-α, IL-6) and cytokine-mediated signaling (NF-κB, STAT-3) were intact in P2Y2-/- remnant livers, uncovering the importance of cytokine-independent and nucleotide-dependent early priming events critical for subsequent hepatocyte proliferation in regenerating livers. Hepatocytes isolated from the WT and P2Y2-/- mice were treated with ATP or ATPγS for 5-120 min and 12-24 h. Extracellular ATP alone, via activation of P2Y2 purinergic receptors, was sufficient to induce ERK phosphorylation, Egr-1 protein expression, and key cyclins and cell cycle progression of hepatocytes in vitro. Collectively, these findings highlight the functional significance of P2Y2 purinergic receptor activation for efficient hepatocyte priming and proliferation in response to PH.

Expression of astrocyte-related receptors in cortical dysplasia with intractable epilepsy.

Epilepsy is one of the major neurologic diseases, and astrocytes play important roles in epileptogenesis. To investigate possible roles of astrocyte-related receptors in patients with intractable epilepsy associated with focal cortical dysplasia (FCD) and other conditions, we examined resected epileptic foci from 31 patients, including 23 with FCD type I, IIa, or IIb, 5 with tuberous sclerosis complex, and 3 with low-grade astrocytoma. Control samples were from 21 autopsied brains of patients without epilepsy or neurologic deficits and 5 patients with pathologic gliosis without epilepsy. Immunohistochemical and immunoblot analyses with antibodies against purinergic receptor subtypes P2RY1, P2RY2, P2RY4, potassium channels Kv4.2 and Kir4.1, and metabotropic receptor subtypes mGluR1 and mGluR5 were performed. Anti-glial fibrillary acidic protein, anti-NeuN, and anti-CD68 immunostaining was used to identify astrocytes, neurons, and microglia, respectively. Most glial fibrillary acidic protein-immunopositive astrocyte cells in the brain samples from patients with epilepsy were P2RY1-, P2RY2-, P2RY4-, Kv4.2-, Kir4.1-, mGluR1-, and mGluR5-positive, whereas samples from controls and pathologic gliosis showed lower expression levels of these astrocyte-related receptors. Our findings suggest that, although these receptors are necessary for astrocyte transmission, formation of the neuron-glia network, and other physiologic functions, overexpression in the brains of patients with intractable epilepsy may be associated with activation of intracellular and glio-neuronal signaling pathways that contribute to epileptogenesis.

Lack of effect of a P2Y6 receptor antagonist on neuropathic pain behavior in mice

Accumulating evidence indicates that various subtypes of purinergic receptors (P2X and P2Y receptor families) play an essential role in the development and the maintenance of neuropathic pain. However, there is only limited data available about the role of P2Y6 receptors in pain processing. Here we detected P2Y6 receptor immunoreactivity in primary afferent neurons of mice and observed an upregulation in response to peripheral nerve injury. However, systemic and intrathecal administration of the P2Y6 receptor antagonist MRS2578 failed to affect the injury-induced neuropathic pain behavior. Our results suggest that P2Y6 receptors, in contrast to other purinergic receptor subtypes, are not critically involved in nerve injury-induced neuropathic pain processing in mice.

P2Y1 receptor-mediated potentiation of inspiratory motor output in neonatal rat in vitro.

PreBötzinger complex inspiratory rhythm-generating networks are excited by metabotropic purinergic receptor subtype 1 (P2Y1R) activation. Despite this, and the fact that inspiratory MNs express P2Y1Rs, the role of P2Y1Rs in modulating motor output is not known for any MN pool. We used rhythmically active brainstem-spinal cord and medullary slice preparations from neonatal rats to investigate the effects of P2Y1R signalling on inspiratory output of phrenic and XII MNs that innervate diaphragm and airway muscles, respectively. MRS2365 (P2Y1R agonist, 0.1 mm) potentiated XII inspiratory burst amplitude by 60 ± 9%; 10-fold higher concentrations potentiated C4 burst amplitude by 25 ± 7%. In whole-cell voltage-clamped XII MNs, MRS2365 evoked small inward currents and potentiated spontaneous EPSCs and inspiratory synaptic currents, but these effects were absent in TTX at resting membrane potential. Voltage ramps revealed a persistent inward current (PIC) that was attenuated by: flufenamic acid (FFA), a blocker of the Ca(2+)-dependent non-selective cation current ICAN; high intracellular concentrations of BAPTA, which buffers Ca(2+) increases necessary for activation of ICAN; and 9-phenanthrol, a selective blocker of TRPM4 channels (candidate for ICAN). Real-time PCR analysis of mRNA extracted from XII punches and laser-microdissected XII MNs revealed the transcript for TRPM4. MRS2365 potentiated the PIC and this potentiation was blocked by FFA, which also blocked the MRS2365 potentiation of glutamate currents. These data suggest that XII MNs are more sensitive to P2Y1R modulation than phrenic MNs and that the P2Y1R potentiation of inspiratory output occurs in part via potentiation of TRPM4-mediated ICAN, which amplifies inspiratory inputs.

P2X receptors in maintenance and differentiation of neural progenitor cells.

P2X receptors in maintenance and differentiation of neural progenitor cells.

Distinct activation profiles in microglia of different ages: A systematic study in isolated embryonic to aged microglial cultures

Microglia have been implicated in disease progression for several age-related brain disorders. However, while microglia’s contribution to the progression of these disorders is accepted, the effect of aging on their endogenous cellular characteristics has received limited attention. In fact, a comprehensive study of how the structure and function of microglia changes as a function of developmental age has yet to be performed. Here, we describe the functional response characteristics of primary microglial cultures prepared from embryonic, neonatal (Neo), 2–3month-old, 6–8month-old, 9–11month-old, and 13–15month-old rats. Microglial morphology, glutamate (GLU) uptake, and release of trophic and inflammatory factors were assessed under basal conditions and in microglia activated with adenosine 5′-triphosphate (ATP) or lipopolysaccharide. We found that microglia from different age groups were both morphologically and functionally distinct. Upon activation by ATP, Neo microglia were the most reactive, upregulating nitric oxide, tumor necrosis factor-α, and brain-derived neurotrophic factor release as well as GLU uptake. This upregulation translated into neurotoxicity in microglia-neuron co-cultures that were not observed with microglia of different developmental ages. Interestingly, 13–15month-old microglia exhibited similar activation profiles to Neo microglia, whereas microglia from younger adults and embryos were activated less by ATP. Our data also identify age-dependent differences in purinergic receptor subtype expression that contribute to the regulation of neuronal survival. Combined, our data demonstrate that microglial activation and purinergic receptor profiles vary non-linearly with developmental age, a potentially important finding for studies examining the role of microglia in neurodegenerative disorders.

Blunted coronary vasodilator response to uridine adenosine tetraphosphate in post-infarct remodeled myocardium is due to reduced P1 receptor activation

We previously demonstrated that uridine adenosine tetraphosphate (Up4A) exerts a potent vasodilator effect in the healthy porcine coronary vasculature. Since the coronary microvascular effects of Up4A after myocardial infarction (MI) are unknown, the present study investigated the response to Up4A in coronary microvessels from post-MI remodeled porcine myocardium, and the involvement of purinergic receptor subtypes. Coronary small arteries (diameter ∼150μm) were dissected from the apex of Sham-operated swine and swine in which MI had been produced 5 weeks earlier by transient (2h) occlusion of the left circumflex coronary artery, and mounted on Mulvany wire myographs. Up4A (10−9–10−5M) produced coronary vasodilation that was reduced in MI as compared to Sham-operated swine. Up4A-induced vasodilation was reduced by P1 blockade with 8-phenyltheophylline in Sham-operated swine and to a lesser extent in MI, while the attenuation by the A2A receptor blocker SCH58261 was similar in Sham-operated and MI swine. Up4A-induced vasodilation remained unaffected by non-selective P2 receptor antagonist PPADS, but was attenuated by selective P2X1 and P2Y1 receptor antagonists MRS2159 and MRS2179, albeit to a similar extent in Sham-operated and MI swine. These responses were paralleled by similar mRNA expression levels of A2A, P2X1 and P2Y1 receptors in MI compared to slaughterhouse control swine. Finally, attenuation of Up4A-induced coronary vasodilation by nitric oxide synthase inhibition was not attenuated in MI as compared to Sham-operated swine. In conclusion, blunted coronary vasodilation in response to Up4A in MI swine is most likely due to reduced activation of P1, rather than P2, receptors and does not involve a loss of NO bioavailability.