Sustained calcium entry through P2X nucleotide receptor channels in human airway epithelial cells

Purinergic receptor stimulation has potential therapeutic effects for cystic fibrosis (CF). Thus, we explored roles for P2Y and P2X receptors in stably increasing [Ca(2+)](i) in human CF (IB3-1) and non-CF (16HBE14o(-)) airway epithelial cells. Cytosolic Ca(2+) was measured by fluorospectrometry using the fluorescent dye Fura-2/AM. Expression of P2X receptor (P2XR) subtypes was assessed by immunoblotting and biotinylation. In IB3-1 cells, ATP and other P2Y agonists caused only a transient increase in [Ca(2+)](i) derived from intracellular stores in a Na(+)-rich environment. In contrast, ATP induced an increase in [Ca(2+)](i) that had transient and sustained components in a Na(+)-free medium; the sustained plateau was potentiated by zinc or increasing extracellular pH. Benzoyl-benzoyl-ATP, a P2XR-selective agonist, increased [Ca(2+)](i) only in Na(+)-free medium, suggesting competition between Na(+) and Ca(2+) through P2XRs. Biochemical evidence showed that the P2X(4) receptor is the major subtype shared by these airway epithelial cells. A role for store-operated Ca(2+) channels, voltage-dependent Ca(2+) channels, or Na(+)/Ca(2+) exchanger in the ATP-induced sustained Ca(2+) signal was ruled out. In conclusion, these data show that epithelial P2X(4) receptors serve as ATP-gated calcium entry channels that induce a sustained increase in [Ca(2+)](i). In airway epithelia, a P2XR-mediated Ca(2+) signal may have therapeutic benefit for CF.     

Trimeric architecture of homomeric P2X2 and heteromeric P2X1+2 receptor subtypes

Of the three major classes of ligand-gated ion channels, nicotinic receptors and ionotropic glutamate receptors are known to be organized as pentamers and tetramers, respectively. The architecture of the third class, P2X receptors, is under debate, although evidence for a trimeric assembly is accumulating. Here we provide biochemical evidence that in addition to the rapidly desensitising P2X1 and P2X3 receptors, the slowly desensitising subtypes P2X2, P2X4, and P2X5 are trimers of identical subunits. Similar (heteromeric) P2X subunits also formed trimers, as shown for co-expressed P2X1 and P2X2 subunits, which assembled efficiently to a P2X1+2 receptor that was exported to the plasma membrane. In contrast, P2X6 subunits, which are incapable of forming functional homomeric channels in Xenopus oocytes, were retained in the ER as apparent tetramers and high molecular mass aggregates. Altogether, we conclude from these data that a trimeric architecture is the structural hallmark of functional homomeric and heteromeric P2X receptors.     

Mouse Leydig cells express multiple P2X receptor subunits

ATP acts on cellular membranes by interacting with P2X (ionotropic) and P2Y (metabotropic) receptors. Seven homomeric P2X receptors (P2X₁–P2X₇) and seven heteromeric receptors (P2X_1/2, P2X_1/4, P2X_1/5, P2X_2/3, P2X_2/6, P2X_4/6, P2X_4/7) have been described. ATP treatment of Leydig cells leads to an increase in [Ca²⁺]_i and testosterone secretion, supporting the hypothesis that Ca²⁺ signaling through purinergic receptors contributes to the process of testosterone secretion in these cells. Mouse Leydig cells have P2X receptors with a pharmacological and biophysical profile resembling P2X₂. In this work, we describe the presence of several P2X receptor subunits in mouse Leydig cells. Western blot experiments showed the presence of P2X₂, P2X₄, P2X₆, and P2X₇ subunits. These results were confirmed by immunofluorescence. Functional results support the hypothesis that heteromeric receptors are present in these cells since 0.5 μM ivermectin induced an increase (131.2 ± 5.9%) and 3 μM ivermectin a decrease (64.2 ± 4.8%) in the whole-cell currents evoked by ATP. These results indicate the presence of functional P2X₄ subunits. P2X₇ receptors were also present, but they were non-functional under the present conditions because dye uptake experiments with Lucifer yellow and ethidium bromide were negative. We conclude that a heteromeric channel, possibly P2X_2/4/6, is present in Leydig cells, but with an electrophysiological and pharmacological phenotype characteristic of the P2X₂ subunit.     

Extracellular zinc and ATP-gated P2X receptor calcium entry channels: New zinc receptors as physiological sensors and therapeutic targets

In this review, we focus on two attributes of P2X receptor channel function, one essential and one novel. First, we propose that P2X receptors are extracellular sensors as well as receptors and ion channels. In particular, the large extracellular domain (that comprises 70% of the molecular mass of the receptor channel protein) lends itself to be a cellular sensor. Moreover, its exquisite sensitivity to extracellular pH, ionic strength, and multiple ligands evokes the function of a sensor. Second, we propose that P2X receptors are extracellular zinc receptors as well as receptors for nucleotides. We provide novel data in multiple publications and illustrative data in this invited review to suggest that zinc triggers ATP-independent activation of P2X receptor channel function. In this light, P2X receptors are the cellular site of integration between autocrine and paracrine zinc signaling and autocrine and paracrine purinergic signaling. P2X receptors may sense changes in these ligands as well as in extracellular pH and ionic strength and transduce these sensations via calcium and/or sodium entry and changes in membrane potential.     

P2X7 receptor deletion attenuates oxidative stress and liver damage in sepsis

Purinergic Signalling - Sepsis is a severe disease characterized by an uncontrolled systemic inflammation and consequent organ dysfunction generated in response to an infection. Extracellular ATP...     

Suppression of discoidin domain receptor 1 by RNA interference attenuates lung inflammation

Discoidin domain receptor 1 (DDR1) is a receptor tyrosine kinase whose ligand is collagen. Recently, we have reported the association of DDR1 in the cytokine production of human leukocytes in in vitro and in vivo expression in idiopathic pulmonary fibrosis. However, its role in in vivo inflammation has not been fully elucidated. Small interference RNA (siRNA) can induce specific suppression of in vitro and in vivo gene expression. In this study, using a bleomycin-induced pulmonary fibrosis mouse model, we administered siRNA against DDR1 transnasally and evaluated histological changes, cytokine expression, and signaling molecule activation in the lungs. Histologically, siRNA against DDR1 successfully reduced in vivo DDR1 expression and attenuated bleomycin-induced infiltration of inflammatory cells. Furthermore, it significantly reduced inflammatory cell counts and concentrations of cytokines such as MCP-1, MIP-1alpha, and MIP-2 in bronchoalveolar lavage fluid. Subsequently, bleomycin-induced up-regulation of TGF-beta in bronchoalveolar lavage fluid was significantly inhibited, and collagen deposition in the lungs was reduced. Furthermore, siRNA against DDR1 significantly inhibited bleomycin-induced P38 MAPK activation in the lungs. Considered together, we propose that DDR1 contributes to the development of bleomycin-induced pulmonary inflammation and fibrosis.     

Gain-of-function of P2X7 receptor gene variants in multiple sclerosis

We have previously shown that P2X7 receptor blockade prevents ATP excitotoxicity in oligodendrocytes and ameliorates chronic experimental autoimmune encephalomyelitis. Here, we have explored the putative association of functionally relevant single nucleotide polymorphisms of the P2X7 receptor gene with multiple sclerosis. We found that T allele of rs17525809 polymorphism, which yields an Ala-76 to Val change in the extracellular domain, is more frequent in multiple sclerosis patients than in controls. Importantly, P2X7 variants with Val show a gain-of-function consisting in higher calcium permeability, larger electrophysiological responses and higher ethidium uptake, and enhance the effect of the also gain-of-function His-155 to Tyr substitution (rs208294) in the haplotype formed by these two variants. These findings may contribute to define the genetic background predisposing for multiple sclerosis and its pathophysiology.     

Estrogen attenuates P2X7-R-mediated apoptosis of uterine cervical cells by blocking calcium influx

Estrogen blocks apoptosis of human ectocervical epithelial cells by modulating P2X7/Ca2+ influx. The effect involves decreased Ca2+-influx and cytosolic-calcium-increase via ATP-activated P2X7 pores. This mechanism may have physiological significance in the human cervix, in-vivo, and the results suggest a physiological role for estrogen in the cervix as an anti-apoptotic factor.     

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

The P2 purinergic receptor family implicated in many physiological processes, including neurotransmission, mechanical adaptation and inflammation, consists 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 mechanotransduction. This study thus provides important insights into the biophysical mechanisms underlying ATP-dependent Ca²⁺ response signatures, which are important in mediating bone mechanoadaptation.     

Absence of the P2X7 receptor alters leukocyte function and attenuates an inflammatory response

When challenged with extracellular ATP, leukocytes respond and activate processes attributed to the P2X(7) receptor (P2X(7)R), an unusual ligand-gated ion channel. To prove P2X(7)R involvement, blood samples from P2X(7)R-deficient mice were characterized. Monocytes and lymphocytes associated with wild-type blood responded to ATP and underwent volume/shape changes and shed L-selectin. In contrast, leukocytes from P2X(7)R-deficient animals demonstrated no change in physical properties or L-selectin expression following ATP challenge. Blood stimulated with LPS or ATP individually generated minimal quantities of the leaderless polypeptide IL-1 beta, but sequential treatment of wild-type, but not P2X(7)R-deficient, blood with LPS and ATP yielded large amounts of cell-free cytokine. Based on these differences, wild-type and P2X(7)R-deficient animals were compared following induction of monoclonal anti-collagen-induced arthritis. Ab-treated wild-type animals subsequently challenged with LPS developed inflamed, swollen paws; their joint cartilage demonstrated lesions, loss of proteoglycan content, and the presence of collagen degradation products. P2X(7)R-deficient animals subjected to the same challenge were markedly less affected; both the incidence and severity of disease were reduced. These data indicate that ATP does act via the P2X(7)R to affect leukocyte function and that the P2X(7)R can serve as an important component of an in vivo inflammatory response.     

Novel P2X7 receptor antagonists

The synthesis and pharmacological evaluation of a new series of potent P2X(7) receptor antagonists is disclosed. The compounds inhibit BzATP-mediated pore formation in THP-1 cells. The distribution of the P2X(7) receptor in inflammatory cells, most notably the macrophage, mast cell and lymphocyte, suggests that P2X(7) antagonists have a significant role to play in the treatment of inflammatory disease.     

N-terminal tagging of human P2X7 receptor disturbs calcium influx and dye uptake

The P2X7 receptor is a frequently studied member of the purinergic receptor family signalling via channel opening and membrane pore formation. Fluorescent imaging is an important molecular method for studying cellular receptor expression and localization. Fusion of receptors to fluorescent proteins might cause major functional changes and requires careful functional evaluation such as has been done for the rat P2X7 receptor. This study examines fusion constructs of the human P2X7 receptor. We assessed surface expression, channel opening with calcium influx, and pore formation using YO-PRO-1 dye uptake in response to BzATP stimulation in transfected cells. We found that tagging at the N-terminal of the human P2X7 receptor with the enhanced green fluorescent protein (eGFP) disturbed channel opening and pore formation despite intact surface expression. A triple hemagglutinin (3HA) fused to the N-terminal also disrupted pore formation but not channel opening showing that even a small tag alters the normal function of the receptor. Together, this suggests that in contrast to what has been observed for the rat P2X7 receptor, the human P2X7 receptor contains N-terminal motifs important for signalling that prevent the construction of a functionally active fusion protein.     

Acidosis attenuates P2X purinergic vasoconstriction in skeletal muscle arteries

Vasoconstriction via alpha(2)-receptors is known to be sensitive to acidic pH, but little is known about the pH sensitivity of P2X receptors. ATP is a cotransmitter released with norepinephrine from the sympathetic nerves and causes vasoconstriction via P2X purinergic receptors on vascular smooth muscle. We hypothesized that reductions in pH would attenuate P2X-mediated vasoconstriction in iliofemoral artery rings. Twenty-five rats were killed, and the iliac and femoral arteries were dissected out and placed in modified Krebs-Henseleit buffer. The arteries were cut into 2-mm sections and mounted in an organ tissue bath. Tension (g) was measured during a potassium chloride and norepinephrine challenge (maximal tension). The arteries were then exposed to alpha,beta-methylene ATP (10(-7)-10(-3) M; n = 13) or phenylephrine (10(-7)-10(-4) M; n = 6) with a tissue bath pH of 7.8, 7.4, and 7.0. Dose-response curves were fit with nonlinear regression analysis to calculate the EC(50) and slope. The peak tension with alpha,beta-methylene ATP was lower during pH 7.0 (1.37 +/- 0.09 g) compared with pH 7.8 (1.90 +/- 0.12 g). EC(50) was highest with pH 7.4 (-5.38 +/- 0.18 log M alpha,beta-methylene ATP) and lowest with pH 7.0 (-4.9 +/- 0.10 log M alpha,beta-methylene ATP). The slopes of the dose-response curves were not different. Pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) abolished contraction caused by the addition of alpha,beta-methylene ATP (n = 6). There was no effect of pH on phenylephrine dose-response curves. These data indicate that the vasoconstrictor response to alpha,beta-methylene ATP is sensitive to pH and that lower pH attenuates the response of P2X purinergic receptors.     

P2X7 receptor and macrophage function

Macrophages are unique innate immune cells that play an integral role in the defense of the host by virtue of their ability to recognize, engulf, and kill pathogens while sending out danger signals via cytokines to recruit and activate inflammatory cells. It is becoming increasingly clear that purinergic signaling events are essential components of the macrophage response to pathogen challenges and disorders such as sepsis may be, at least in part, regulated by these important sensors. The activation of the P2X₇ receptor is a powerful event in the regulation of the caspase-1 inflammasome. We provide evidence that the inflammasome activation requires “priming” of macrophages prior to ATP activation of the P2X₇R. Inhibition of the inflammasome activation by the tyrosine kinase inhibitor, AG126, suggests regulation by phosphorylation. Finally, the P2X₇R may also be activated by other elements of the host response such as the antimicrobial peptide LL-37, which adds a new, physiologically relevant agonist to the P2X₇R pathway. Therapeutic approaches to inflammation and sepsis will certainly be enhanced by an increased understanding of how purinergic receptors modulate the inflammasomes.     

Radioligands targeting purinergic P2X7 receptor

The purinergic P2X7 receptor (P2X7R) is an adenosine triphosphate (ATP) ligand-gated cationic channel receptor. P2X7R is closely associated with various inflammatory, immune, cancer, neurological, musculoskeletal and cardiovascular disorders. P2X7R is an interesting therapeutic target as well as molecular imaging target. This brief digest highlights the radioligands targeting P2X7R recently developed in drug discovery and molecular imaging agent development.     

P2X7受体与炎性细胞因子

P2X7受体是嘌呤受体中功能独特的一个亚型,为ATP控制的离子通道,在单核细胞、巨噬细胞、中性粒细胞中高表达,被ATP激活后导致K＋外流和Ca^2＋内流、非选择性膜孔形成,启动一系列信号途径如炎症小体NALP3的活化,丝裂原蛋白激酶途径激活NF-κB增强炎性细胞因子转录,ROS和氮介质的产生,介导IL-1β、IL-6、IL-18、TNF-α、MIP-2、CCL2、HMGB1等多种炎性细胞因子的释放,参与炎症的发生发展,与真菌感染及阿尔茨海默病、类风湿性关节炎、哮喘等炎症性疾病密切相关.     

Isoform specificity of P2X2 purinergic receptor C-terminus binding to tubulin

Adenosine triphosphate (ATP) and other nucleotides can be released in the central and peripheral nervous systems and act as neurotransmitters/neuromodulators. They can activate G-protein coupled receptors and ligand-gated ion channels, which are present throughout the central nervous system (CNS). P2X2 is one of seven known ion channels gated by ATP, and is characterized by having two transmembrane domains, a large extracellular loop and intracellular N- and C-termini. Recently, work from several laboratories has shown that neurotransmitter receptors can interact with other proteins thereby changing their functional attributes. More specifically, it was demonstrated that P2X2 binds beta-tubulin. Our goal was to investigate this interaction, by comparing P2X2 with a naturally occurring splicing variant named P2X2b. These isoforms differ in their C-terminal regions which contain the proposed beta-tubulin-binding domain. Indeed we were able to demonstrate that only the long variant P2X2 binds beta-tubulin I in various biochemical assays. In addition, this interaction can be direct since it also occurred when the P2X2 C-terminus was exposed to purified brain tubulin. When expressed in heterologous cells, P2X2 interacted with beta-tubulin I while present on the outer membrane, as demonstrated by biotinylation of surface proteins. Therefore, the present data strongly support a functional interaction between an ATP-gated channel and the cytoskeleton. Moreover, we show a biochemical difference between the splicing alternatives that might account for novel distinct functional roles.     

克隆的P2受体亚型的药理学研究进展

细胞外嘌呤（腺苷,ADP,ATP）及嘧啶（UDP,UTP）为重要的信使分子,通过细胞表面P2受体介导产生不同的生物效应,P2嘌吟受体的概念于1978年被提出,随后根据药理学特征又被分为P2X及P2X嘌呤受体,90年代,采用分子生物学手段,一系列配体门控的P2X受体及G蛋白耦联的P2Y受体被克隆及功能表达,迄今为止,已有七型P2X受体亚型（P2X1－7）及六型P2Y受体亚型被克隆（P2Y1,2,4,6,11,12）,各型具有不同的分子结构,药理学特征及组织分布,本文还讨论了目前可用于区分各亚型激动剂及拮抗剂。