Purinergic signalling in neuron-glia interactions

Activity-dependent release of ATP from synapses, axons and glia activates purinergic membrane receptors that modulate intracellular calcium and cyclic AMP. This enables glia to detect neural activity and communicate among other glial cells by releasing ATP through membrane channels and vesicles. Through purinergic signalling, impulse activity regulates glial proliferation, motility, survival, differentiation and myelination, and facilitates interactions between neurons, and vascular and immune system cells. Interactions among purinergic, growth factor and cytokine signalling regulate synaptic strength, development and responses to injury. We review the involvement of ATP and adenosine receptors in neuron-glia signalling, including the release and hydrolysis of ATP, how the receptors signal, the pharmacological tools used to study them, and their functional significance.     

Purinergic signalling in the musculoskeletal system

It is now widely recognised that extracellular nucleotides, signalling via purinergic receptors, participate in numerous biological processes in most tissues. It has become evident that extracellular nucleotides have significant regulatory effects in the musculoskeletal system. In early development, ATP released from motor nerves along with acetylcholine acts as a cotransmitter in neuromuscular transmission; in mature animals, ATP functions as a neuromodulator. Purinergic receptors expressed by skeletal muscle and satellite cells play important pathophysiological roles in their development or repair. In many cell types, expression of purinergic receptors is often dependent on differentiation. For example, sequential expression of P2X5, P2Y₁ and P2X2 receptors occurs during muscle regeneration in the mdx model of muscular dystrophy. In bone and cartilage cells, the functional effects of purinergic signalling appear to be largely negative. ATP stimulates the formation and activation of osteoclasts, the bone-destroying cells. Another role appears to be as a potent local inhibitor of mineralisation. In osteoblasts, the bone-forming cells, ATP acts via P2 receptors to limit bone mineralisation by inhibiting alkaline phosphatase expression and activity. Extracellular ATP additionally exerts significant effects on mineralisation via its hydrolysis product, pyrophosphate. Evidence now suggests that purinergic signalling is potentially important in several bone and joint disorders including osteoporosis, rheumatoid arthritis and cancers. Strategies for future musculoskeletal therapies might involve modulation of purinergic receptor function or of the ecto-nucleotidases responsible for ATP breakdown or ATP transport inhibitors.     

Ectonucleotidases in Müller glial cells of the rodent retina: Involvement in inhibition of osmotic cell swelling

Extracellular nucleotides mediate glia-to-neuron signalling in the retina and are implicated in the volume regulation of retinal glial (Müller) cells under osmotic stress conditions. We investigated the expression and functional role of ectonucleotidases in Müller cells of the rodent retina by cell-swelling experiments, calcium imaging, and immuno- and enzyme histochemistry. The swelling of Müller cells under hypoosmotic stress was inhibited by activation of an autocrine purinergic signalling cascade. This cascade is initiated by exogenous glutamate and involves the consecutive activation of P2Y₁ and adenosine A1 receptors, the action of ectoadenosine 5′-triphosphate (ATP)ases, and a nucleoside-transporter-mediated release of adenosine. Inhibition of ectoapyrases increased the ATP-evoked calcium responses in Müller cell endfeet. Müller cells were immunoreactive for nucleoside triphosphate diphosphohydrolases (NTPDase)2 (but not NTPDase1), ecto-5′-nucleotidase, P2Y₁, and A1 receptors. Enzyme histochemistry revealed that ATP but not adenosine 5′-diphosphate (ADP) is extracellularly metabolised in retinal slices of NTPDase1 knockout mice. NTPDase1 activity and protein is restricted to blood vessels, whereas activity of alkaline phosphatase is essentially absent at physiological pH. The data suggest that NTPDase2 is the major ATP-degrading ectonucleotidase of the retinal parenchyma. NTPDase2 expressed by Müller cells can be implicated in the regulation of purinergic calcium responses and cellular volume.     

Purinergic signaling in inflammatory cells: P2 receptor expression, functional effects, and modulation of inflammatory responses

Extracellular ATP and related nucleotides promote a wide range of pathophysiological responses via activation of cell surface purinergic P2 receptors. Almost every cell type expresses P2 receptors and/or exhibit regulated release of ATP. In this review, we focus on the purinergic receptor distribution in inflammatory cells and their implication in diverse immune responses by providing an overview of the current knowledge in the literature related to purinergic signaling in neutrophils, macrophages, dendritic cells, lymphocytes, eosinophils, and mast cells. The pathophysiological role of purinergic signaling in these cells include among others calcium mobilization, actin polymerization, chemotaxis, release of mediators, cell maturation, cytotoxicity, and cell death. We finally discuss the therapeutic potential of P2 receptor subtype selective drugs in inflammatory conditions.     

Purinergic receptors in gastrointestinal inflammation

Purinergic receptors comprise a family of transmembrane receptors that are activated by extracellular nucleosides and nucleotides. The two major classes of purinergic receptors, P1 and P2, are expressed widely in the gastrointestinal tract as well as immune cells. The purinergic receptors serve a variety of functions from acting as neurotransmitters, to autocoid and paracrine signaling, to cell activation and immune response. Nucleosides and nucleotide agonist of purinergic receptors are released by many cell types in response to specific physiological signals, and their levels are increased during inflammation. In the past decade, the advent of genetic knockout mice and the development of highly potent and selective agonists and antagonists for the purinergic receptors have significantly advanced the understanding of purinergic receptor signaling in health and inflammation. In fact, agonist/antagonists of purinergic receptors are emerging as therapeutic modalities to treat intestinal inflammation. In this article, the distribution of the purinergic receptors in the gastrointestinal tract and their physiological and pathophysiological role in intestinal inflammation will be reviewed.     

P2 receptor-mediated signaling in mast cell biology

Mast cells are widely recognized as effector cells of allergic inflammatory reactions. They contribute to the pathogenesis of different chronic inflammatory diseases, wound healing, fibrosis, thrombosis/fibrinolysis, and anti-tumor immune responses. In this paper, we summarized the role of P2X and P2Y receptors in mast cell activation and effector functions. Mast cells are an abundant source of ATP which is stored in their granules and secreted upon activation. We discuss the contribution of mast cells to the extracellular ATP release and to the maintenance of extracellular nucleotides pool. Recent publications highlight the importance of purinergic signaling for the pathogenesis of chronic airway inflammation. Therefore, the role of ATP and P2 receptors in allergic inflammation with focus on mast cells was analyzed. Finally, ATP functions as mast cell autocrine/paracrine factor and as messenger in intercellular communication between mast cells, nerves, and glia in the central nervous system.     

线粒体损伤相关模式分子与宿主免疫调节

线粒体是真核细胞至关重要的细胞器,参与机体细胞能量代谢和细胞凋亡等多种生物学过程。线粒体还参与机体的天然免疫反应的调节。线粒体不仅可以作为病毒免疫反应的载体,还可以通过产生ROS参与抗菌反应。线粒体受到损伤、刺激后,可释放mtDNA,TFAM,ROS,ATP,心磷脂和甲酰肽等内容物。这些分子可以作为损伤相关模式分子(damage associated molecular patterns, DAMPs)被模式识别受体识别,从而参与宿主的免疫调节。研究表明,线粒体已成为内源性DAMPs的重要来源,在先天性免疫应答以及疾病进展过程中发挥着重要的作用。本文就线粒体来源的损伤相关模式分子在机体免疫调节中的作用进行综述。     

P2 receptors and immunity

Immune cells express receptors for extracellular nucleotides named P2 receptors. P2 receptors transduce signals delivered by nucleotides present in the extracellular environment. Accruing evidence shows that purinergic signalling has a profound effect on multiple immune cell responses such as T lymphocyte proliferation, chemotaxis, cytokine release, phagocytosis, Ag presentation and cytotoxicity. This makes P2 receptors an attractive target for the therapy of immuno-mediated disease and cancer.     

Human submandibular gland (HSG) cell line as a model for studying salivary gland Ca2+ signalling mechanisms

The human submandibular gland cell line (HSG) has been used as a model for studying the molecular mechanisms of salivary cells. The aim of this study was to investigate some aspects of salivary Ca2+ signalling. We focused on the presence and function of specific molecular markers of salivary cells to see whether this cell line retained normal salivary characteristics, despite the neoplastic changes. We detected the M3 acetylcholine receptor and intracellular salivary amylase mRNA with RT-PCR. Carbachol treatment caused a rapid, transient elevation of [Ca2+]i, showing that the cholinergic receptors are functional in HSG cells. Protein kinase C activation by phorbol-esther PMA, prior to carbachol treatment, inhibited the normal Ca2+ signalling pathway in HSG cells. Using selective antagonists, we also identified the dominant muscarinic receptor subtype M3 on HSG cells. We also observed that functional extracellular purinergic receptors were present on HSG cells and coupled to intracellular Ca2+ signalling. Our results suggested that the coupling mechanisms of these receptors remained relatively intact despite the neoplastic transformation. This enables us to use this cell line to model the role of muscarinic and purinergic control of salivary gland function, cell proliferation and differentiation.     

Perspectives of purinergic signaling in stem cell differentiation and tissue regeneration

Replacement of lost or dysfunctional tissues by stem cells has recently raised many investigations on therapeutic applications. Purinergic signaling has been shown to regulate proliferation, differentiation, cell death, and successful engraftment of stem cells originated from diverse origins. Adenosine triphosphate release occurs in a controlled way by exocytosis, transporters, and lysosomes or in large amounts from damaged cells, which is then subsequently degraded into adenosine. Paracrine and autocrine mechanisms induced by immune responses present critical factors for the success of stem cell therapy. While P1 receptors generally exert beneficial effects including anti-inflammatory activity, P2 receptor-mediated actions depend on the subtype of stimulated receptors and localization of tissue repair. Pro-inflammatory actions and excitatory tissue damages mainly result from P2X7 receptor activation, while other purinergic receptor subtypes participate in proliferation and differentiation, thereby providing adequate niches for stem cell engraftment and novel mechanisms for cell therapy and endogenous tissue repair. Therapeutic applications based on regulation of purinergic signaling are foreseen for kidney and heart muscle regeneration, Clara-like cell replacement for pulmonary and bronchial epithelial cells as well as for induction of neurogenesis in case of neurodegenerative diseases.     

Cutting off the power: inhibition of leukemia cell growth by pausing basal ATP release and P2X receptor signaling?

T cells respond to antigen stimulation with the rapid release of cellular ATP, which stimulates an autocrine feedback mechanism that regulates calcium influx through P2X receptors. This autocrine purinergic feedback mechanism plays an essential role in the activation of T cells resulting in cell proliferation and clonal expansion. We recently reported that increases in mitochondrial ATP production drive this stimulation-induced purinergic signaling mechanism but that low-level mitochondrial ATP production fuels basal T cell functions required to maintain vigilance of unstimulated T cells. Here we studied whether defects in these purinergic signaling mechanisms are involved in the unwanted proliferation of leukemia T cells. We found that acute leukemia T cells (Jurkat) possess a larger number and more active mitochondria than their healthy counterparts. Jurkat cells have higher intracellular ATP concentrations and generat more extracellular ATP than unstimulated T cells from healthy donors. As a result, increased purinergic signaling through P2X1 and P2X7 receptors elevates baseline levels of cytosolic Ca²⁺ in Jurkat cells. We found that pharmacological inhibition of this basal purinergic signaling mechanism decreases mitochondrial activity, Ca²⁺ signaling, and cell proliferation. Similar results were seen in the leukemic cell lines THP-1, U-937, and HL-60. Combined treatment with inhibitors of P2X1 or P2X7 receptors and the chemotherapeutic agent 6-mercaptopurine completely blocked Jurkat cell proliferation. Our results demonstrate that increased mitochondrial metabolism promotes autocrine purinergic signaling and uncontrolled proliferation of leukemia cells. These findings suggest that deranged purinergic signaling can result in T cell malignancy and that therapeutic targeting aimed at purinergic signaling is a potential strategy to combat T cell leukemia.     

Mitochondria Regulate Neutrophil Activation by Generating ATP for Autocrine Purinergic Signaling

Polymorphonuclear neutrophils (PMNs) form the first line of defense against invading microorganisms. We have shown previously that ATP release and autocrine purinergic signaling via P2Y₂ receptors are essential for PMN activation. Here we show that mitochondria provide the ATP that initiates PMN activation. Stimulation of formyl peptide receptors increases the mitochondrial membrane potential (Δψm) and triggers a rapid burst of ATP release from PMNs. This burst of ATP release can be blocked by inhibitors of mitochondrial ATP production and requires an initial formyl peptide receptor-induced Ca²⁺ signal that triggers mitochondrial activation. The burst of ATP release generated by the mitochondria fuels a first phase of purinergic signaling that boosts Ca²⁺ signaling, amplifies mitochondrial ATP production, and initiates functional PMN responses. Cells then switch to glycolytic ATP production, which fuels a second round of purinergic signaling that sustains Ca²⁺ signaling via P2X receptor-mediated Ca²⁺ influx and maintains functional PMN responses such as oxidative burst, degranulation, and phagocytosis.     

New I-type lectins of the CD 33-related siglec subgroup identified through genomics

Siglecs are sialic-acid-binding proteins of the Ig superfamily that are involved in cell-cell interactions and signalling. In recent years, several novel siglecs that are highly related to CD33/Siglec-2 have been identified through genomics and functional screens. In addition to their distinct sialic-acid-binding properties, most of these novel siglecs bear tyrosine-based signalling motifs that are typically found in inhibitory receptors of the immune system. The restricted expression patterns of CD33-related siglecs in the haemopoietic and immune systems suggests that they are involved in regulating leucocyte activation during inflammatory and immune responses.     

Role of P2z purinergic receptors in ATP-mediated killing of tumor necrosis factor (TNF)-sensitive and TNF-resistant L929 fibroblasts.

Two closely related cell lines were characterized in their responses to extracellular ATP (ATPo): the fibroblast cell line L929 and a TNF-resistant variant L929/R. Both lines showed ATPo-activated increases in intracellular Ca2+, inward current, and sustained depolarization of the plasma membrane, cell responses compatible with activation of purinergic receptors of the P2y, P2x, or P2z subtype; however, only the L929/R variant was susceptible to ATPo-dependent early permeabilization of the plasma membrane to hydrophilic solutes of M(r) below 900, a response uniquely caused by the activation of P2z receptors. Both cell types were susceptible to the cytotoxic effect of ATPo, but killing of the L929/R variant required much shorter incubations in the presence of this nucleotide. Morphologic examination of ATPo-challenged L929 and L929/R cells showed that cell death occurred by two alternative mechanisms: colloido-osmotic lysis or apoptosis. Occurrence of apoptosis was confirmed by agarose gel analysis of cellular DNA. Although ATPo caused a fast mobilization of intracellular Ca2+, neither colloido-osmotic lysis nor apoptosis were Ca2+ dependent. Our results show that the L929/R variant, but not the L929 parental fibroblast cell line, expresses functional purinergic receptors of the P2z subtype. The presence of P2z receptors confers to L929/R cells enhanced susceptibility to ATPo-mediated cytotoxicity.     

Signalling by extracellular nucleotides in health and disease

Nucleotides are released from all cells through regulated pathways or as a result of plasma membrane damage or cell death. Outside the cell, nucleotides act as signalling molecules triggering multiple responses via specific plasma membrane receptors of the P2 family. In the nervous system, purinergic signalling has a key function in neurotransmission. Outside the nervous system, purinergic signalling is one of the major modulators of basal tissue homeostasis, while its dysregulation contributes to the pathogenesis of various disease, including inflammation and cancer. Pre-clinical and clinical evidence shows that selective P2 agonists or antagonists are effective treatments for many pathologies, thus highlighting the relevance of extracellular nucleotides and P2 receptors as therapeutic targets.     

Purinergic receptor-induced Ca2+ signaling in the neuroepithelium of the vomeronasal organ of larval Xenopus laevis

Purinergic signaling has considerable impact on the functioning of the nervous system, including the special senses. Purinergic receptors are expressed in various cell types in the retina, cochlea, taste buds, and the olfactory epithelium. The activation of these receptors by nucleotides, particularly adenosine-5′-triphosphate (ATP) and its breakdown products, has been shown to tune sensory information coding to control the homeostasis and to regulate the cell turnover in these organs. While the purinergic system of the retina, cochlea, and taste buds has been investigated in numerous studies, the available information about purinergic signaling in the olfactory system is rather limited. Using functional calcium imaging, we identified and characterized the purinergic receptors expressed in the vomeronasal organ of larval Xenopus laevis. ATP-evoked activity in supporting and basal cells was not dependent on extracellular Ca²⁺. Depletion of intracellular Ca²⁺ stores disrupted the responses in both cell types. In addition to ATP, supporting cells responded also to uridine-5′-triphosphate (UTP) and adenosine-5′-O-(3-thiotriphosphate) (ATPγS). The response profile of basal cells was considerably broader. In addition to ATP, they were activated by ADP, 2-MeSATP, 2-MeSADP, ATPγS, UTP, and UDP. Together, our findings suggest that supporting cells express P2Y₂/P2Y₄-like purinergic receptors and that basal cells express multiple P2Y receptors. In contrast, vomeronasal receptor neurons were not sensitive to nucleotides, suggesting that they do not express purinergic receptors. Our data provide the basis for further investigations of the physiological role of purinergic signaling in the vomeronasal organ and the olfactory system in general.     

Regulators and signalling in insect haemocyte immunity

The innate immune system of insects relies on both humoral and cellular immune responses that are mediated via activation of several signalling pathways. Haemocytes are the primary mediators of cell-mediated immunity in insects, including phagocytosis, nodulation, encapsulation and melanization. The last years, research has focused on the mechanisms of microbial recognition and activation of haemocyte intracellular signalling molecules in response to invaders. The powerful tool, RNA interference gene silencing, helped several regulators involved in immune responses, to be identified. In this review, we summarize recent advances in understanding the role(s) of receptors and intracellular signalling molecules involved in immune responses.     

Glial cell-derived glutamate mediates autocrine cell volume regulation in the retina: activation by VEGF

Astroglial cells are a source for gliotransmitters such as glutamate and ATP. We demonstrate here that gliotransmitters have autocrine functions in the regulation of cellular volume. Hypoosmotic stress in the presence of inflammatory mediators or oxidative stress, and during blockade or down-regulation of potassium channels, induces swelling of retinal glial cells. Vascular endothelial growth factor inhibits the osmotic swelling of glial cells in retinal slices or isolated cells. This effect was mediated by a kinase domain region/flk-1 receptor-evoked calcium dependent release of glutamate from glial cells, and subsequent stimulation of glial group I/II metabotropic glutamate receptors. Activation of kinase domain region/flk-1 or glutamate receptors evoked an autocrine swelling-inhibitory purinergic signaling cascade that was calcium-independent. This cascade involved the release of ATP and adenosine, and the activation of purinergic P2Y₁ and adenosine A1 receptors, resulting in the opening of potassium and chloride channels and inhibition of cellular swelling. The glutamatergic-purinergic regulation of the glial cell volume may be functionally important in the homeostasis of the extracellular space volume during intense neuronal activation which is associated with a swelling of neuronal cell structures in the retina. However, glial cell-derived glutamate may also contribute to the swelling of activated neurons since metabolic poisoning of glial cells by iodoacetate inhibits the neuronal cell swelling mediated by activation of ionotropic glutamate receptors.     

ATP- and Gap Junction–dependent Intercellular Calcium Signaling in Osteoblastic Cells

Many cells coordinate their activities by transmitting rises in intracellular calcium from cell to cell. In nonexcitable cells, there are currently two models for intercellular calcium wave propagation, both of which involve release of inositol trisphosphate (IP₃)- sensitive intracellular calcium stores. In one model, IP₃ traverses gap junctions and initiates the release of intracellular calcium stores in neighboring cells. Alternatively, calcium waves may be mediated not by gap junctional communication, but rather by autocrine activity of secreted ATP on P₂ purinergic receptors. We studied mechanically induced calcium waves in two rat osteosarcoma cell lines that differ in the gap junction proteins they express, in their ability to pass microinjected dye from cell to cell, and in their expression of P2Y₂ (P_2U) purinergic receptors. ROS 17/2.8 cells, which express the gap junction protein connexin43 (Cx43), are well dye coupled, and lack P_2U receptors, transmitted slow gap junction-dependent calcium waves that did not require release of intracellular calcium stores. UMR 106-01 cells predominantly express the gap junction protein connexin 45 (Cx45), are poorly dye coupled, and express P_2U receptors; they propagated fast calcium waves that required release of intracellular calcium stores and activation of P_2U purinergic receptors, but not gap junctional communication. ROS/P_2U transfectants and UMR/Cx43 transfectants expressed both types of calcium waves. Gap junction–independent, ATP-dependent intercellular calcium waves were also seen in hamster tracheal epithelia cells. These studies demonstrate that activation of P_2U purinergic receptors can propagate intercellular calcium, and describe a novel Cx43-dependent mechanism for calcium wave propagation that does not require release of intracellular calcium stores by IP₃. These studies suggest that gap junction communication mediated by either Cx43 or Cx45 does not allow passage of IP₃ well enough to elicit release of intracellular calcium stores in neighboring cells.