Exocytosis of ATP From Astrocytes Modulates Phasic and Tonic Inhibition in the Neocortex

Communication between neuronal and glial cells is important for many brain functions. Astrocytes can modulate synaptic strength via Ca²⁺-stimulated release of various gliotransmitters, including glutamate and ATP. A physiological role of ATP release from astrocytes was suggested by its contribution to glial Ca²⁺-waves and purinergic modulation of neuronal activity and sleep homeostasis. The mechanisms underlying release of gliotransmitters remain uncertain, and exocytosis is the most intriguing and debated pathway. We investigated release of ATP from acutely dissociated cortical astrocytes using “sniff-cell” approach and demonstrated that release is vesicular in nature and can be triggered by elevation of intracellular Ca²⁺ via metabotropic and ionotropic receptors or direct UV-uncaging. The exocytosis of ATP from neocortical astrocytes occurred in the millisecond time scale contrasting with much slower nonvesicular release of gliotransmitters via Best1 and TREK-1 channels, reported recently in hippocampus. Furthermore, we discovered that elevation of cytosolic Ca²⁺ in cortical astrocytes triggered the release of ATP that directly activated quantal purinergic currents in the pyramidal neurons. The glia-driven burst of purinergic currents in neurons was followed by significant attenuation of both synaptic and tonic inhibition. The Ca²⁺-entry through the neuronal P2X purinoreceptors led to phosphorylation-dependent down-regulation of GABAA receptors. The negative purinergic modulation of postsynaptic GABA receptors was accompanied by small presynaptic enhancement of GABA release. Glia-driven purinergic modulation of inhibitory transmission was not observed in neurons when astrocytes expressed dn-SNARE to impair exocytosis. The astrocyte-driven purinergic currents and glia-driven modulation of GABA receptors were significantly reduced in the P2X4 KO mice. Our data provide a key evidence to support the physiological importance of exocytosis of ATP from astrocytes in the neocortex.     

Extracellular ATP regulates cell death of lymphocytes and monocytes induced by membrane-bound lipoproteins of Mycoplasma fermentans and Mycoplasma salivarium

The cytotoxicities of lipoproteins of Mycoplasma fermentans and Mycoplasma salivarium to a lymphocytic cell line, MOLT-4, and a monocytic cell line, HL-60, was upregulated by ATP added extracellularly in a dose-dependent manner. These lipoproteins induced ATP release and plasma membrane permeability increase in these cell lines. In addition, periodate-oxidized ATP, an antagonist for P2X purinergic receptors, suppressed the cytotoxicity of the lipoproteins, suggesting the possibility that P2X receptors for ATP play crucial roles in the cytotoxicity. Activation of caspase-3 induced by the lipoproteins, which was assessed by the cleavage of the synthetic substrate DEVD-pNA and the endogenous substrate poly(ADP-ribose) polymerase, was also upregulated and downregulated by extracellular ATP and periodate-oxidized ATP, respectively. On the basis of these results, this study suggests that mycoplasmal lipoproteins induce the permeability increase in lymphocytes and monocytes, by which ATP is released, and the ATP regulates the cytotoxicities of the lipoproteins to the cells, possibly by interaction with ATP receptors such as P2X purinergic receptors.     

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.     

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.     

ATP induces intracellular calcium increases and actin cytoskeleton disaggregation via P2x receptors

The consequences of purinoceptor activation on calcium signalling, inositol phosphate metabolism, protein secretion and the actin cytoskeleton were demonstrated in the WRK-1 cell line. Extracellular ATP was used as a secretagogue to induce a rise in intracellular Ca(2+) concentration ([Ca(2+)](i)), acting via P2x purinergic receptors, which causes actin skeleton disaggregation and protein secretion. ATP bound specifically to purinergic receptors, with Ki of 0.8 microM. The magnitude order for binding of different nucleotides was alpha beta-Met-ATP >or= dATPalphaS > ATP >or= ADP > UTP > AMP > suramin. No increase in inositol phosphates (IPs) was observed after ATP application suggesting that the purinergic sites in WRK-1 cells are not of a P2y type. ATP (1-100 microM) caused a concentration-dependent increase in [Ca(2+)](i)(EC(50)= 30 microM). The responses were reproducible without any desensitization over several applications. The response to ATP was abolished when extracellular calcium ([Ca(2+)](e)) was reduced to 100 nM. A non-specific purinergic antagonist, suramin, reversibly inhibited the ATP-response suggesting that ATP is able to bind to P2x purinergic sites to trigger Ca(2+) entry and increase of [Ca(2+)](i). ATP induced a concentration-dependent disaggregation of actin and exocytotic release of proteins both, which were dependent upon [Ca(2+)](e). Similarly, alpha,beta-Met-ATP, a potent P2x agonist also stimulated Ca(2+) mobilization, actin network destructuration, and protein release. In the isolated rat neurohypophysial nerve terminals, ATP was shown to act as a physiological stimulus for vasopressin release via Ca(2+) entry through a P2x receptor [6]. Here, we show that in these nerve terminals, ATP is also able to induce actin disaggregation by a Ca(2+) dependent mechanism. Thus, actin cytoskeleton alterations induced by ATP through activation of P2x receptors could be a prelude to exocytosis.     

Dual roles of P2 purinergic receptors in insulin-stimulated leptin production and lipolysis in differentiated rat white adipocytes

ATP is co-localized with norepinephrine at the sympathetic nerve terminals and may be released simultaneously upon neuronal stimulation, which results in activation of purinergic receptors. To examine whether leptin synthesis and lipolysis are influenced by P2 purinergic receptor activation, the effects of ATP and other nucleotides on leptin secretion and glycerol release have been investigated in differentiated rat white adipocytes. Firstly, insulin-induced leptin secretion was inhibited by nucleotide treatment with the following efficacy order: 3'-O-(4-benzoyl)benzoyl ATP (BzATP) > ATP > UTP. Secondly, treatment of adipocytes with ATP increased both intracellular Ca(2+) concentration and cAMP content. Intracellular calcium concentration was increased by ATP and UTP, but not BzATP, an effect attributed to phospholipase C-coupled P2Y(2). On the other hand, cAMP was generated by treatment with BzATP and ATPgammaS, but not UTP, indicating functional expression of adenylyl cyclase-coupled P2Y(11) receptors in white adipocytes. Thirdly, lipolysis was significantly activated by BzATP and ATP, which correlated with the characteristics of the P2Y(11) subtype. Taken together, the data presented here suggest that white adipocytes express at least two different types of P2Y receptors and that activation of P2Y(11) receptor might be involved in inhibition of leptin production and stimulation of lipolysis, suggesting that purinergic transmission can play an important role in white adipocyte physiology.     

Regulation by P2 agonists of the intracellular calcium concentration in epithelial cells freshly isolated from rat trachea.

Epithelial cells were isolated from rat trachea by incubation of the organ in a calcium-free medium. The intracellular concentration of calcium ([Ca(2+)](i)) was measured with the calcium-sensitive fluorescent dye fura2. In resting conditions, the cells maintained a low [Ca(2+)](i) in spite of the presence of millimolar concentration of calcium in the incubation medium. These cells had retained intracellular stores of calcium which were emptied after exposure of the cells to thapsigargin, an inhibitor of intracellular calcium ATPases. Substance P (125 nM) transiently increased 2.5-fold the [Ca(2+)](i). ATP (1 mM) doubled the [Ca(2+)](i) after a few seconds and further induced a sustained increase of the [Ca(2+)](i). Coomassie blue fully blocked the response to ATP and extracellular magnesium only inhibited the delayed response to ATP. Among purinergic analogs, only benzoyl-ATP (Bz-ATP), an agonist on P2X ionotropic purinergic receptors, reproduced the response to ATP. UTP and 2-methylthioATP (two agonists on P2Y metabotropic purinergic receptors) transiently increased the [Ca(2+)](i). Thapsigargin, ATP and Bz-ATP increased the uptake of extracellular calcium. RT-PCR analysis revealed that two metabotropic receptors (P2Y(1) and P2Y(2)) and two ionotropic receptors (P2X(4) and P2X(7)) were expressed by the cells present in the suspension. It is concluded that purinergic agonists can modulate the response of rat tracheal epithelial cells by several mechanisms. The activation of metabotropic receptors should mobilize intracellular IP(3)-sensitive calcium pools. The activation of the ionotropic receptors should not only open a non-specific cation channel leading to the entry of calcium but should also induce the formation of pores in cells expressing the P2X(7) receptors, which could be deleterious to these cells.     

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.     

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.     

Purinergic receptor signaling regulates N-cadherin expression in primary astrocyte cultures

Extracellular ATP exerts both short-term and long-term effects in the CNS by stimulating cell-surface purinergic receptors. Here we have examined the effect of purinergic receptor activation on N-cadherin expression, a calcium-dependent cell adhesion molecule involved in many processes, including glia-glia and axon-glia interactions. When primary cultures of rat cortical astrocytes were treated with ATP, N-cadherin protein expression increased in a time- and concentration-dependent manner. In addition, ATP treatment caused an increase in N-cadherin immunoreactivity in both the cytoplasm and on the cell surface membrane. Interestingly, experiments with cycloheximide revealed that relocalization of N-cadherin to the cell surface membrane were independent of protein synthesis. The ATP-induced increase in N-cadherin protein expression was blocked by reactive blue 2 and 8-(p-sulfophenyl)-theophylline, suggesting involvement of both P2 and P1 purinergic receptors, respectively. In addition, N-cadherin expression was partially blocked when signaling from purinergic receptors to extracellular signal regulated protein kinase or Akt was inhibited by 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene or wortmannin, respectively. By using an in vitro model of traumatic CNS injury, we found that N-cadherin expression was increased when astrocytes were subjected to rapid and reversible mechanical strain. The findings presented here demonstrate a role for extracellular ATP, purinergic receptors and protein kinase signaling in regulating N-cadherin expression and suggest a role for this mechanism in cell-cell interactions.     

Effect of P2X purinergic receptors in tumor progression and as a potential target for anti-tumor therapy

The development of tumors is a complex pathological process involving multiple factors, multiple steps, and multiple genes. Their prevention and treatment have always been a difficult problem at present. A large number of studies have proved that the tumor microenvironment plays an important role in the progression of tumors. The tumor microenvironment is the place where tumor cells depend for survival, and it plays an important role in regulating the growth, proliferation, apoptosis, migration, and invasion of tumor cells. P2X purinergic receptors, which depend on the ATP ion channel, can be activated by ATP in the tumor microenvironment, and by mediating tumor cells and related cells (such as immune cells) in the tumor microenvironment. They play an important regulatory role on the effects of the skeleton, membrane fluidity, and intracellular molecular metabolism of tumor cells. Therefore, here, we outlined the biological characteristics of P2X purinergic receptors, described the effect of tumor microenvironment on tumor progression, and discussed the effect of ATP on tumor. Moreover, we explored the role of P2X purinergic receptors in the development of tumors and anti-tumor therapy. These data indicate that P2X purinergic receptors may be used as another potential pharmacological target for tumor prevention and treatment.

     

mTOR and differential activation of mitochondria orchestrate neutrophil chemotaxis

Neutrophils use chemotaxis to locate invading bacteria. Adenosine triphosphate (ATP) release and autocrine purinergic signaling via P2Y2 receptors at the front and A2a receptors at the back of cells regulate chemotaxis. Here, we examined the intracellular mechanisms that control these opposing signaling mechanisms. We found that mitochondria deliver ATP that stimulates P2Y2 receptors in response to chemotactic cues, and that P2Y2 receptors promote mTOR signaling, which augments mitochondrial activity near the front of cells. Blocking mTOR signaling with rapamycin or PP242 or mitochondrial ATP production (e.g., with CCCP) reduced mitochondrial Ca²⁺ uptake and membrane potential, and impaired cellular ATP release and neutrophil chemotaxis. Autocrine stimulation of A2a receptors causes cyclic adenosine monophosphate accumulation at the back of cells, which inhibits mTOR signaling and mitochondrial activity, resulting in uropod retraction. We conclude that mitochondrial, purinergic, and mTOR signaling regulates neutrophil chemotaxis and may be a pharmacological target in inflammatory diseases.     

P2X7-mediated ATP secretion is accompanied by depletion of cytosolic ATP

Purinergic Signalling - ATP and its metabolites are important extracellular signal transmitters acting on purinergic P2 and P1 receptors. Most cells can actively secrete ATP in response to a...     

Purinergic receptors in the splanchnic circulation

There is considerable evidence that purines are vasoactive molecules involved in the regulation of blood flow. Adenosine is a well known vasodilator that also acts as a modulator of the response to other vasoactive substances. Adenosine exerts its effects by interacting with adenosine receptors. These are metabotropic G-protein coupled receptors and include four subtypes, A(1), A(2A), A(2B) and A(3). Adenosine triphosphate (ATP) is a co-transmitter in vascular neuroeffector junctions and is known to activate two distinct types of P2 receptors, P2X (ionotropic) and P2Y (metabotropic). ATP can exert either vasoconstrictive or vasorelaxant effects, depending on the P2 receptor subtype involved. Splanchnic vascular beds are of particular interest, as they receive a large fraction of the cardiac output. This review focus on purinergic receptors role in the splanchnic vasomotor control. Here, we give an overview on the distribution and diversity of effects of purinergic receptors in splanchnic vessels. Pre- and post-junctional receptormediated responses are summarized. Attention is also given to the interactions between purinergic receptors and other receptors in the splanchnic circulation.     

Extracellular ATP induces fast and transient non-selective cationic currents and cytosolic Ca2+ changes in human umbilical artery smooth muscle cells

Ionotropic purinergic receptors (P2X) are expressed in endothelial and smooth muscle cells of blood vessels. ATP acting on smooth muscle P2X receptors is able to induce vasoconstriction in different kind of vessels. However, to our knowledge, there are no reports that directly show the activity of these purinergic receptors in native human vascular smooth muscle cells. In this work, we describe for the first time an ATP-induced current in freshly isolated human umbilical artery (HUA) smooth muscle cells. The current was measured by patch-clamp technique in whole-cell condition on cells clamped at -50 mV. At 100 μM of ATP the current showed a rapid activation and desensitization, and was carried by both Na(+) and Ca(2+). The current was completely blocked by suramin (300 μM) and partially blocked by 100 μM of Zn(2+) without affecting the kinetic of desensitization. All these properties suggest that the ATP-induced ionic currents are mediated through P2X(1)-like receptors. Moreover, we show that ATP transiently increased cytosolic Ca(2+) in "in situ" smooth muscle cells of intact HUA segments and that this response is dependent of extracellular and intracellular Ca(2+). These data expand the knowledge of purinergic receptors properties in vascular smooth muscle cells and the probable role of ATP as a paracrine modulator of contractile tone in a human artery which is fundamental for feto-placental blood flow.     

Inhibition of Ca2+ signaling and glucagon secretion in mouse pancreatic alpha-cells by extracellular ATP and purinergic receptors

Glucagon secreted from pancreatic alpha-cells plays a critical role in glycemia, mainly by hepatic glucose mobilization. In diabetic patients, an impaired control of glucagon release can worsen glucose homeostasis. Despite its importance, the mechanisms that regulate its secretion are still poorly understood. Since alpha-cells are particularly sensitive to neural and paracrine factors, in this report we studied the role of purinergic receptors and extracellular ATP, which can be released from nerve terminals and beta-cell secretory granules. Using immunocytochemistry, we identified in alpha-cells the P2 receptor subtype P2Y1, as well as the P1 receptors A1 and A2A. In contrast, only P2Y1 and A1 receptors were localized in beta-cells. To analyze the role of purinergic receptors in alpha-cell function, we studied their participation in Ca2+ signaling. At low glucose concentrations, mouse alpha-cells exhibited the characteristic oscillatory Ca2+ signals that lead to secretion. Application of ATP (1-10 microM) abolished these oscillations or reduced their frequency in alpha-cells within intact islets and isolated in culture. ATPgammaS, a nonhydrolyzable ATP derivative, indicated that the ATP effect was mainly direct rather than through ATP-hydrolytic products. Additionally, adenosine (1-10 microM) was also found to reduce Ca2+ signals. ATP-mediated inhibition of Ca2+ signaling was accompanied by a decrease in glucagon release from intact islets in contrast to the adenosine effect. Using pharmacological agonists, we found that only P2Y1 and A2A were likely involved in the inhibitory effect on Ca2+ signaling. All these findings indicate that extracellular ATP and purinergic stimulation are effective regulators of the alpha-cell function.     

Roles of purinergic P2X receptors as pacemaking channels and modulators of calcium-mobilizing pathway in pituitary gonadotrophs

Anterior pituitary cells release ATP and express several subtypes of purinergic P2 receptors, but their biophysical properties and roles in spontaneous and receptor-controlled electrical activity have not been characterized. Here we focused on extracellular ATP actions in gonadotrophs from embryonic, neonatal, and adult rats. In cells from all three age groups, the Ca2+-mobilizing agonist GnRH induced oscillatory, hyperpolarizing, nondesensitizing, and slow deactivating currents. In contrast, ATP induced nonoscillatory, depolarizing, slowly desensitizing, and rapidly deactivating current, indicating that these cells express cation-conducting P2X channels but not Ca2+-mobilizing P2Y receptors. The amplitudes of P2X current response and the rates of receptor desensitization were dependent on ATP concentration. The biophysical and pharmacological properties of P2X currents were consistent with the expression of P2X2 subtype of channels in these cells. ATP-induced rapid depolarization of gonadotrophs lead to initiation of firing in quiescent cells, an increase in the frequency of action potentials in spontaneously active cells, and a transient stimulation of LH release. ATP also influenced GnRH-induced current and membrane potential oscillations and LH release in an extracellular Ca2+-dependent manner. These inositol 1,4,5-triphosphate-dependent oscillations were facilitated, slowed, or stopped, depending of ATP concentration, the time of its application, and the level of Ca2+ content in intracellular stores. These results indicate that, in gonadotrophs, P2X receptors could operate as pacemaking channels and modulators of GnRH-controlled electrical activity and secretion.     

Cell-autonomous regulation of hematopoietic stem cell cycling activity by ATP

Extracellular nucleotides regulate many cellular functions through activation of purinergic receptors in the plasma membrane. Here, we show that in hematopoietic stem cell (HSC), ATP is stored in vesicles and released in a calcium-sensitive manner. HSC expresses ATP responsive P2X receptors and in vitro pharmacological P2X antagonism restrained hematopoietic progenitors proliferation, but not myeloid differentiation. In mice suffering from chronic inflammation, HSCs were significantly expanded and their cycling activity was sensitive to treatment with the P2X antagonist periodate-oxidized 2,3-dialdehyde ATP. Our results indicate that ATP acts as an autocrine stimulus in regulating HSCs pool size.