Extracellular ATP: an unexpected role as a signaler in plants

ATP and other nucleoside triphosphates not only drive energy-dependent reactions inside cells, but can also function outside the plasma membrane in the extracellular matrix, where they function as agonists that can induce diverse physiological responses without being hydrolyzed. This external role of ATP is well established in animal cells but only recently has it become apparent that extracellular ATP (eATP) can also function as a signaling agent in plants. Recent data have shown that eATP and other nucleotides can induce an increase in the cytosolic Ca(2+) concentration and diverse downstream changes that influence plant growth and defense responses. Ectoapyrase enzymes that regulate the eATP concentration also have an impact on plant growth. These results beg the question of whether there is a receptor that can bind to eATP and transduce this into signaling changes. Answering this will be key to understanding how eATP and ectoapyrases influence plant growth and development.     

Extracellular ATP signaling and homeostasis in plant cells

Extracellular ATP (eATP) is now recognized as an important signaling agent in plant growth and defense response to environmental stimuli. eATP has dual functions in plant cell signaling, which is largely dependent on its concentration in the extracellular matrix (ECM). A lethal level of eATP (extremely low or high) causes cell death, whereas a moderate level of eATP benefits plant growth and development. Ecto-apyrases (Nucleoside Triphosphate-Diphosphohydrolase) help control the eATP concentrations in the ECM, and thus contributing to the mediation of plant growth and defense response upon environmental stress. In this review, we summarize eATP signaling in plants and highlight the correlation between eATP homeostasis control and programmed cell death.     

植物细胞外信号分子eATP研究进展

细胞外ATP(extracellular ATP, eATP)是目前公认的细胞外信号分子, 参与调控多种环境刺激下植物的生长、发育和防御反应。在植物细胞信号转导过程中, eATP具有双重功能, 其作用主要取决于细胞外基质中eATP的浓度。eATP含量过高或者过低都会导致细胞死亡, 适度水平的eATP则有助于植物的生长和发育。细胞外三磷酸腺苷双磷酸酶(Apyrase)严格控制细胞外基质中eATP的水平, 因此有助于调控植物在逆境条件下的生长和防御反应。该文总结了植物中eATP的发现、产生和清除以及受体和信号转导等研究进展, 重点论述eATP在逆境条件下的生理功能, 并对植物eATP的研究方向作了展望。     

Homeostasis of extracellular ATP in uninfected RBCs from a Plasmodium falciparum culture and derived microparticles

Plasmodium falciparum, a dangerous parasitic agent causing malaria, invades human red blood cells (RBCs), causing hemolysis and microvascular obstruction. These and other pathological processes of malaria patients are due to metabolic and structural changes occurring in uninfected RBCs. In addition, infection activates the production of microparticles (MPs).ATP and byproducts are important extracellular ligands modulating purinergic signaling within the intravascular space.Here, we analyzed the contribution of uninfected RBCs and MPs to the regulation of extracellular ATP (eATP) of RBCs, which depends on the balance between ATP release by specific transporters and eATP hydrolysis by ectonucleotidases.RBCs were cultured with P. falciparum for 24–48 h prior to experiments, from which uninfected RBCs and MPs were purified. On-line luminometry was used to quantify the kinetics of ATP release. Luminometry, colorimetry and radioactive methods were used to assess the rate of eATP hydrolysis by ectonucleotidases. Rates of ATP release and eATP hydrolysis were also evaluated in MPs.Uninfected RBCs challenged by different stimuli displayed a strong and transient activation of ATP release, together with an elevated rate of eATP hydrolysis. MPs contained ATP in their lumen, which was released upon vesicle rupture, and were able to hydrolyze eATP.Results suggest that uninfected RBCs and MPs can act as important determinants of eATP regulation of RBCs during malaria.The comparison of eATP homeostasis in infected RBCs, ui-RBCs, and MPs allowed us to speculate on the impact of P. falciparum infection on intravascular purinergic signaling and the control of the vascular caliber by RBCs.     

Extracellular ATP: a potential regulator of plant cell death

Adenosine 5′‐triphosphate (ATP) has been regarded as an intracellular energy currency molecule for many years. In recent decades, it has been determined that ATP is released into the extracellular milieu by animal, plant and microbial cells. In animal cells, this extracellular ATP (eATP) functions as a signalling compound to mediate many cellular processes through its interaction with membrane‐associated receptor proteins. It has also been reported that eATP is a signalling molecule required for the regulation of plant growth, development and responses to environmental stimuli. Recently, the first plant receptor for eATP was identified in Arabidopsis thaliana. Interestingly, some studies have shown that eATP is of particular importance in the control of plant cell death. In this review article, we summarize and discuss the theoretical and experimental advances that have been made with regard to the roles and mechanisms of eATP in plant cell death. We also make an attempt to address some speculative aspects to help develop and expand future research in this area.     

Extracellular ATP and nitric oxide signaling pathways regulate redox-dependent responses associated to root hair growth in etiolated Arabidopsis seedlings

Extracellular ATP (eATP) and nitric oxide (NO) have emerged as crucial players in plant development, stress responses and cell viability. Glutathione (GSH) is an abundant reducing agent with proposed roles in plant growth, development and stress physiology. In a recent publication, we demonstrated that eATP and NO restore hypocotyl elongation of etiolated Arabidopsis seedlings treated with GSH. Here it is reported that exogenous ATP also restores root hair growth suggesting a role for ATP and NO in the regulation of redox balance associated to specific processes of plant morphogenesis. A tentative model integrating redox-, eATP- and NO-signaling pathways during root hair growth in Arabidopsis seedlings is presented.Key words: Arabidopsis thaliana, extracellular ATP signaling, nitric oxide, redox system, root hair     

Extracellular ATP in plants. Visualization, localization, and analysis of physiological significance in growth and signaling

Extracellular ATP (eATP) in animals is well documented and known to play an important role in cellular signaling (e.g. at the nerve synapse). The existence of eATP has been postulated in plants; however, there is no definitive experimental evidence for its presence or an explanation as to how such a polar molecule could exit the plant cell and what physiological role it may play in plant growth and development. The presence of eATP in plants (Medicago truncatula) was detected by constructing a novel reporter; i.e. fusing a cellulose-binding domain peptide to the ATP-requiring enzyme luciferase. Application of this reporter to plant roots allowed visualization of eATP in the presence of the substrate luciferin. Luciferase activity could be detected in the interstitial spaces between plant epidermal cells and predominantly at the regions of actively growing cells. The levels of eATP were closely correlated with regions of active growth and cell expansion. Pharmacological compounds known to alter cytoplasmic calcium levels revealed that ATP release is a calcium-dependent process and may occur through vesicular fusion, an important step in the polar growth of actively growing root hairs. Reactive oxygen species (ROS) activity at the root hair tip is not only essential for root hair growth, but also dependent on the cytoplasmic calcium levels. Whereas application of exogenous ATP and a chitin mixture increased ROS activity in root hairs, no changes were observed in response to adenosine, AMP, ADP, and nonhydrolyzable ATP (betagammameATP). However, application of exogenous potato (Solanum tuberosum) apyrase (ATPase) decreased ROS activity, suggesting that cytoplasmic calcium gradients and ROS activity are closely associated with eATP release.     

Extracellular nucleotides enhance agonist potency at the parathyroid hormone 1 receptor

Parathyroid hormone (PTH) activates the PTH/PTH-related peptide receptor (PTH1R) on osteoblasts and other target cells. Mechanical stimulation of cells, including osteoblasts, causes release of nucleotides such as ATP into the extracellular fluid. In addition to its role as an energy source, ATP serves as an agonist at P2 receptors and an allosteric regulator of many proteins. We investigated the effects of concentrations of extracellular ATP, comparable to those that activate low affinity P2X7 receptors, on PTH1R signaling. Cyclic AMP levels were monitored in real-time using a bioluminescence reporter and β-arrestin recruitment to PTH1R was followed using a complementation-based luminescence assay. ATP markedly enhanced cyclic AMP and β-arrestin signaling as well as downstream activation of CREB. CMP – a nucleotide that lacks a high energy bond and does not activate P2 receptors – mimicked this effect of ATP. Moreover, potentiation was not inhibited by P2 receptor antagonists, including a specific blocker of P2X7. Thus, nucleotide-induced potentiation of signaling pathways was independent of P2 receptor signaling. ATP and CMP reduced the concentration of PTH (1–34) required to produce a half-maximal cyclic AMP or β-arrestin response, with no evident change in maximal receptor activity. Increased potency was similarly apparent with PTH1R agonists PTH (1–14) and PTH-related peptide (1–34). These observations suggest that extracellular nucleotides increase agonist affinity, efficacy or both, and are consistent with modulation of signaling at the level of the receptor or a closely associated protein. Taken together, our findings establish that ATP enhances PTH1R signaling through a heretofore unrecognized allosteric mechanism.     

Stimulation of lipogenesis in rat adipocytes by ATP, a ligand for P2-receptors

The activation of P2-receptors has a wide range of diverse effects in many tissues. Here we show that extracellular ATP stimulates lipogenesis in adipocytes derived from the epididymal fat pads of male Wistar rats. The lipogenic effect of ATP is not susceptible to treatment of adipocytes with adenosine deaminase or an adenosine receptor antagonist. Degradation of ATP in adipocyte suspension by ectonucleotidases is slow and remaining ATP concentrations are sufficient to activate P2-receptors. ATP does not affect basal or insulin stimulated glucose transport, or basal or isoproterenol stimulated lipolysis, respectively. The lipogenic effect of ATP is mimicked by the adenine compounds, ADP, AMP, and beta,gamma-methylene-ATP, but not by other nucleotides (UTP, UDP, CTP, GTP, ITP, and diadenosine tetraphosphate), indicating that extracellular nucleotides stimulate lipogenesis via a P2-receptor. ATP and its receptor may define a signalling system in adipocytes, which regulates fat stores independently from established hormones.     

The progressive ankylosis gene product ANK regulates extracellular ATP levels in primary articular chondrocytes

Introduction

Extracellular ATP (eATP) is released by articular chondrocytes under physiological and pathological conditions. High eATP levels cause pathologic calcification, damage cartilage, and mediate pain. We recently showed that stable over-expression of the progressive ankylosis gene product, ANK, increased chondrocyte eATP levels, but the mechanisms of this effect remained unexplored. The purpose of this work was to further investigate mechanisms of eATP efflux in primary articular chondrocytes and to better define the role of ANK in this process.

Methods

We measured eATP levels using a bioluminescence-based assay in adult porcine articular chondrocyte media with or without a 10 minute exposure to hypotonic stress. siRNAs for known ATP membrane transporters and pharmacologic inhibitors of ATP egress pathways were used to identify participants involved in chondrocyte eATP release.

Results

eATP levels increased after exposure to hypotonic media in a calcium-dependent manner in monolayer and 3-dimensional agarose gel cultures (p < 0.001). A potent transient receptor potential vanilloid 4 (TRPV4) agonist mimicked the effects of hypotonic media. ANK siRNA suppressed basal (p < 0.01) and hypotonically-stressed (p < 0.001) ATP levels. This effect was not mediated by altered extracellular pyrophosphate (ePPi) levels, and was mimicked by the ANK inhibitor, probenecid (p < 0.001). The P2X7/4 receptor inhibitor Brilliant Blue G also suppressed eATP efflux induced by hypotonic media (p < 0.001), while ivermectin, a P2X4 receptor stimulant, increased eATP levels (p < 0.001). Pharmacologic inhibitors of hemichannels, maxianion channels and other volume-sensitive eATP efflux pathways did not suppress eATP levels.

Conclusions

These findings implicate ANK and P2X7/4 receptors in chondrocyte eATP efflux. Understanding the mechanisms of eATP efflux may result in novel therapies for calcium crystal arthritis and osteoarthritis.     

Extracellular ATP is a potent signaling molecule in the activation of the Japanese flounder (Paralichthys olivaceus) innate immune responses

Innate immunity is the first line of defense against pathogen infections. Extracellular ATP (eATP) is one of the most studied danger-associated molecular pattern molecules that can activate host innate immune responses through binding with and activating purinergic receptors on the plasma membrane. The detailed actions of eATP on fish innate immunity, however, remain poorly understood. In this study, we investigated bacterial pathogen-induced ATP release in head kidney cells of the Japanese flounder Paralichthys olivaceus. We also examined the actions of eATP on pro-inflammatory cytokine and immune-related gene expression, the activity of induced NO synthase (iNOS), and the production of reactive oxygen species (ROS) and NO in Japanese flounder immune cells. We demonstrate that ATP is dynamically released from Japanese flounder head kidney cells into the extracellular milieu during immune challenge by formalin-inactivated Edwardsiella tarda and Vibrio anguillarum. In addition, we show that eATP administration results in profound up-regulation of pro-inflammatory cytokine gene expression, iNOS activity, and inflammatory mediator production, including ROS and NO, in Japanese flounder immune cells. Altogether, our findings demonstrate that eATP is a potent signaling molecule for the activation of innate immune responses in fish.     

Extracellular ATP and other nucleotides—ubiquitous triggers of intercellular messenger release

Extracellular nucleotides, and ATP in particular, are cellular signal substances involved in the control of numerous (patho)physiological mechanisms. They provoke nucleotide receptor-mediated mechanisms in select target cells. But nucleotides can considerably expand their range of action. They function as primary messengers in intercellular communication by stimulating the release of other extracellular messenger substances. These in turn activate additional cellular mechanisms through their own receptors. While this applies also to other extracellular messengers, its omnipresence in the vertebrate organism is an outstanding feature of nucleotide signaling. Intercellular messenger substances released by nucleotides include neurotransmitters, hormones, growth factors, a considerable variety of other proteins including enzymes, numerous cytokines, lipid mediators, nitric oxide, and reactive oxygen species. Moreover, nucleotides activate or co-activate growth factor receptors. In the case of hormone release, the initially paracrine or autocrine nucleotide-mediated signal spreads through to the entire organism. The examples highlighted in this commentary suggest that acting as ubiquitous triggers of intercellular messenger release is one of the major functional roles of extracellular nucleotides. While initiation of messenger release by nucleotides has been unraveled in many contexts, it may have been overlooked in others. It can be anticipated that additional nucleotide-driven messenger functions will be uncovered with relevance for both understanding physiology and development of therapy.     

Proteomic Analysis of Extracellular ATP-Regulated Proteins Identifies ATP Synthase β-Subunit as a Novel Plant Cell Death Regulator

Extracellular ATP is an important signal molecule required to cue plant growth and developmental programs, interactions with other organisms, and responses to environmental stimuli. The molecular targets mediating the physiological effects of extracellular ATP in plants have not yet been identified. We developed a well characterized experimental system that depletes Arabidopsis cell suspension culture extracellular ATP via treatment with the cell death-inducing mycotoxin fumonisin B1. This provided a platform for protein profile comparison between extracellular ATP-depleted cells and fumonisin B1-treated cells replenished with exogenous ATP, thus enabling the identification of proteins regulated by extracellular ATP signaling. Using two-dimensional difference in-gel electrophoresis and matrix-assisted laser desorption-time of flight MS analysis of microsomal membrane and total soluble protein fractions, we identified 26 distinct proteins whose gene expression is controlled by the level of extracellular ATP. An additional 48 proteins that responded to fumonisin B1 were unaffected by extracellular ATP levels, confirming that this mycotoxin has physiological effects on Arabidopsis that are independent of its ability to trigger extracellular ATP depletion. Molecular chaperones, cellular redox control enzymes, glycolytic enzymes, and components of the cellular protein degradation machinery were among the extracellular ATP-responsive proteins. A major category of proteins highly regulated by extracellular ATP were components of ATP metabolism enzymes. We selected one of these, the mitochondrial ATP synthase β-subunit, for further analysis using reverse genetics. Plants in which the gene for this protein was knocked out by insertion of a transfer-DNA sequence became resistant to fumonisin B1-induced cell death. Therefore, in addition to its function in mitochondrial oxidative phosphorylation, our study defines a new role for ATP synthase β-subunit as a pro-cell death protein. More significantly, this protein is a novel target for extracellular ATP in its function as a key negative regulator of plant cell death.ATP is a ubiquitous, energy-rich molecule of fundamental importance in living organisms. It is a key substrate and vital cofactor in many biochemical reactions and is thus conserved by all cells. However, in addition to its localization and functions inside cells, ATP is actively secreted to the extracellular matrix where it forms a halo around the external cell surface. The existence of this extracellular ATP (eATP)¹ has been reported in several organisms including bacteria (1), primitive eukaryotes (2), animals (3), and plants (4–6). This eATP is not wasted, but harnessed at the cell surface as a potent signaling molecule enabling cells to communicate with their neighbors and regulate crucial growth and developmental processes.In animals, eATP is a crucial signal molecule in several physiological processes such as neurotransmission (7, 8), regulation of blood pressure (9), enhanced production of reactive oxygen species (ROS) (10), protein translocation (11), and apoptosis (12). Extracellular ATP signal perception at the animal cell surface is mediated by P2X and P2Y receptors, which bind ATP extracellularly and recruit intracellular second messengers (13, 14). P2X receptors are ligand-gated ion channels that provide extracellular Ca²⁺ a corridor for cell entry after binding eATP, facilitating a surge in cytosolic [Ca²⁺] that is essential in activating down-stream signaling. P2Y receptors transduce the eATP signal by marshalling heteromeric G-proteins on the cytosolic face of the plasma membrane and activating appropriate downstream effectors.Although eATP exists in plants, homologous P2X/P2Y receptors for eATP signal perception have not yet been identified, even in plant species with fully sequenced genomes. Notwithstanding the obscurity of plant eATP signal sensors, some of the key downstream messengers recruited by eATP-mediated signaling are known. For example, eATP triggers a surge in cytosolic Ca²⁺ concentration (15–17) and a heightened production of nitric oxide (18–20) and reactive oxygen species (17, 21, 22). Altering eATP levels is attended by activation of plant gene expression (16, 21) and changes in protein abundance (5, 23), indicating that eATP-mediated signaling impacts on plant physiology. Indeed eATP has been demonstrated to regulate plant growth (20, 24–26), gravitropic responses (27), xenobiotic resistance (4), plant-symbiont interactions (28), and plant-pathogen interactions (23, 29). However, the mechanism by which eATP regulates these processes remains unclear, largely because the eATP signal sensors and downstream signal regulatory genes and proteins have not been identified.We previously reported that eATP plays a central regulatory role in plant cell death processes (5). Therefore, an understanding of the signaling components galvanized by eATP in cell death regulation might serve a useful purpose in providing mechanistic detail of how eATP signals in plant physiological processes. We found that eATP-mediated signaling negatively regulates cell death as its removal by application of ATP-degrading enzymes to the apoplast activates plant cell death (5). Remarkably, fumonisin B1 (FB1), a pathogen-derived molecule that activates defense gene expression in Arabidopsis (30), commandeers this eATP-regulated signaling to trigger programmed cell death (5). FB1 is a mycotoxin secreted by fungi in the genus Fusarium and initiates programmed cell death in both animal and plant cells (31, 32). In Arabidopsis, FB1 inaugurates cell death by inactivating eATP-mediated signaling via triggering a drastic collapse in the levels of eATP (5). FB1-induced Arabidopsis programmed cell death is dependent on the plant signaling hormone salicylic acid (33), which is a key regulator of eATP levels (29). Because concurrent application of FB1 and exogenous ATP to remedy the FB1-induced eATP deficit blocks death, FB1 and exogenous ATP treatments can therefore be used as probes to identify the key signal regulators downstream of eATP in cell death control. This is vital for achieving the global objective of elucidating the mechanism of eATP signaling in plant physiology.Gel-based proteomic analyses have been previously applied to successfully identify the novel role of eATP in the regulation of plant defense gene expression and disease resistance (23, 29). We have now employed FB1 and ATP treatments together with two-dimensional difference in-gel electrophoresis (DIGE) and matrix-assisted laser desorption-time of flight MS (MALDI-TOF MS) to identify the changes in Arabidopsis protein profiles associated with a shift from normal to cell death-inception metabolism. Additional reverse genetic analyses enabled us to definitively identify a putative ATP synthase β-subunit as a target for eATP-mediated signaling with an unexpected function in the regulation of plant programmed cell death.     

胞外ATP对壳聚糖诱导的ROS和PAL活性变化的影响

以烟草BY-2悬浮细胞为材料,探讨了胞外ATP对壳聚糖引起的活性氧(reactive oxygen species,ROS)水平和苯丙氨酸解氨酶(phenylalanine ammonia-lyase,PAL)活性变化的影响。结果表明,5~20μg·mL^-1壳聚糖处理导致了烟草悬浮细胞细胞内ROS水平逐渐增加;壳聚糖也导致了PAL活性的增加,其活性在15μg·mL^-1壳聚糖处理下达到峰值,此后有所降低。10~40μmol·L^-1外源ATP处理未引起烟草悬浮细胞内ROS水平和PAL活性的显著变化。细胞外ATP水平则随壳聚糖浓度的增加而逐渐下降。本文进一步分析了细胞外ATP对壳聚糖引起的ROS水平和PAL活性变化的影响。结果显示,外源施加20μmol·L^-1ATP可以有效降低壳聚糖诱导的烟草悬浮细胞ROS水平上升,同时外源ATP也明显减缓了壳聚糖所诱导的PAL活性的上升。上述结果表明,细胞外ATP水平能够影响壳聚糖引起的ROS水平和PAL活性的变化。     

Both the stimulation and inhibition of root hair growth induced by extracellular nucleotides in Arabidopsis are mediated by nitric oxide and reactive oxygen species

Root hairs secrete ATP as they grow, and extracellular ATP and ADP can trigger signaling pathways that regulate plant cell growth. In several plant tissues the level of extracellular nucleotides is limited in part by ectoapyrases (ecto-NTPDases), and the growth of these tissues is strongly influenced by their level of ectoapyrase expression. Both chemical inhibition of ectoapyrase activity and suppression of the expression of two ectoapyrase enzymes by RNAi in Arabidopsis resulted in inhibition of root hair growth. As assayed by a dose-response curve, different concentrations of the poorly hydrolysable nucleotides, ATPγS and ADPβS, could either stimulate (at 7.5–25 μM) or inhibit (at ≥ 150 μM) the growth rate of root hairs in less than an hour. Equal amounts of AMPS, used as a control, had no effect on root hair growth. Root hairs of nia1nia2 mutants, which are suppressed in nitric oxide (NO) production, and of atrbohD/F mutants, which are suppressed in the production of H₂O₂, did not show growth responses to applied nucleotides, indicating that the growth changes induced by these nucleotides in wild-type plants were likely transduced via NO and H₂O₂ signals. Consistent with this interpretation, treatment of root hairs with different concentrations of ATPγS induced different accumulations of NO and H₂O₂ in root hair tips. Two mammalian purinoceptor antagonists also blocked the growth responses induced by extracellular nucleotides, suggesting that they were initiated by a receptor-based mechanism.     

An ATP signalling pathway in plant cells: extracellular ATP triggers programmed cell death in Populus euphratica

We elucidated the extracellular ATP (eATP) signalling cascade active in programmed cell death (PCD) using cell cultures of Populus euphratica. Millimolar amounts of eATP induced a dose‐ and time‐dependent reduction in viability, and the agonist‐treated cells displayed hallmark features of PCD. eATP caused an elevation of cytosolic Ca²⁺ levels, resulting in Ca²⁺ uptake by the mitochondria and subsequent H₂O₂ accumulation. P. euphratica exhibited an increased mitochondrial transmembrane potential, and cytochrome c was released without opening of the permeability transition pore over the period of ATP stimulation. Moreover, the eATP‐induced increase of intracellular ATP, essential for the activation of caspase‐like proteases and subsequent PCD, was found to be related to increased mitochondrial transmembrane potential. NO is implicated as a downstream component of the cytosolic Ca²⁺ concentration but plays a negligible role in eATP‐stimulated cell death. We speculate that ATP binds purinoceptors in the plasma membrane, leading to the induction of downstream intermediate signals, as the proposed sequence of events in PCD signalling was terminated by the animal P2 receptor antagonist suramin.     

Extracellular ATP is a regulator of pathogen defence in plants

In healthy plants extracellular ATP (eATP) regulates the balance between cell viability and death. Here we show an unexpected critical regulatory role of eATP in disease resistance and defensive signalling. In tobacco, enzymatic depletion of eATP or competition with non-hydrolysable ATP analogues induced pathogenesis-related ( PR ) gene expression and enhanced resistance to tobacco mosaic virus and Pseudomonas syringae pv. tabaci . Artificially increasing eATP concentrations triggered a drop in levels of the important defensive signal chemical salicylic acid (SA) and compromised basal resistance to viral and bacterial infection. Inoculating tobacco leaf tissues with bacterial pathogens capable of activating PR gene expression triggered a rapid decline in eATP. Conversely, inoculations with mutant bacteria unable to induce defence gene expression failed to deplete eATP. Furthermore, a collapse in eATP concentration immediately preceded PR gene induction by SA. Our study reveals a previously unsuspected role for eATP as a negative regulator of defensive signal transduction and demonstrates its importance as a key signal integrating defence and cell viability in plants.     

Expression and function of P2 receptors in bone

ATP (adenosine 5'-triphosphate) is one of the most important extracellular regulatory molecules in the skeleton. Extracellular ATP and other nucleotides signal through P2 receptors, a diverse group of receptors that are widely expressed by bone cells. P2 receptors are divided into two subclasses; P2Y G-protein coupled receptors, and P2X ligand-gated ion channels, and there is functional and molecular evidence for the expression of these receptors on both osteoblasts and osteoclasts. In order to activate P2 receptors, nucleotides must be released into the bone microenvironment. ATP is present in mmol concentrations in cells and can be released by cell lysis, cell trauma or physiological mechanisms, possibly through ABC transporters. Following co-activation of P2Y and PTH1 receptors on osteoblasts, there are multiple levels of interaction in downstream signalling that eventually lead to synergistic expression of osteoblastic genes, providing a mechanism for integrating local and systemic regulatory signals in bone particularly with regard to the activation of bone remodelling. Activation of P2Y1 receptors on osteoblasts enhances expression of RANKL leading indirectly to an increase in osteoclast formation and resorption. Expression of P2X7 inducible pores on osteoclast precursor cell membranes allows fusion to form multinucleated osteoclasts and blockade of this receptor inhibits resorption. The capacity of extracellular nucleotides to provide a highly localized and transient signal coupled with the profound effects of P2 receptor activation on osteoblastic and osteoclastic cells and the synergistic interactions with systemic hormones, indicate that nucleotides have a strong influence over bone tissue growth and regeneration.     

Extracellular ATP signaling during differentiation of C2C12 skeletal muscle cells: role in proliferation

Evidence shows that extracellular ATP signals influence myogenesis, regeneration and physiology of skeletal muscle. Present work was aimed at characterizing the extracellular ATP signaling system of skeletal muscle C2C12 cells during differentiation. We show that mechanical and electrical stimulation produces substantial release of ATP from differentiated myotubes, but not from proliferating myoblasts. Extracellular ATP-hydrolyzing activity is low in myoblasts and high in myotubes, consistent with the increased expression of extracellular enzymes during differentiation. Stimulation of cells with extracellular nucleotides produces substantial Ca(2+) transients, whose amplitude and shape changed during differentiation. Consistently, C2C12 cells express several P2X and P2Y receptors, whose level changes along with maturation stages. Supplementation with either ATP or UTP stimulates proliferation of C2C12 myoblasts, whereas excessive doses were cytotoxic. The data indicate that skeletal muscle development is accompanied by major functional changes in extracellular ATP signaling.     

Plant responses to extracellular nucleotides: Cellular processes and biological effects

Higher plants exhibit cellular responsiveness to the exogenous application of purine nucleotides in a manner consistent with a cell–cell signaling function for these molecules. Like animals, plants respond to extracellular ATP, ADP, and stable analogues (e.g., ATPγS and ADPβS) by increasing the cytoplasmic concentration of calcium. Agonist substrate specificity and concentration dependency suggest receptor mediation of these events, and, although the identity of the plant receptor is currently unknown, pharmacological analysis points to the involvement of a plasma membrane-localized calcium channel. Extracellular ATP can also induce the production of reactive oxygen species and stimulate an increase in the mRNA levels of a number of stress- and calcium-regulated genes, suggesting a role for nucleotide-based signaling in plant wound and defense responses. Furthermore, the growth and development of plants can also be altered by the application of external ATP. Recent studies are only beginning to uncover the complexities of plant signaling networks activated in response to extracellular ATP and how these might interact to affect plant physiological processes.