Extracellular ATP functions as an endogenous external metabolite regulating plant cell viability

ATP is a vital molecule used by living organisms as a universal source of energy required to drive the cogwheels of intracellular biochemical reactions necessary for growth and development. Animal cells release ATP to the extracellular milieu, where it functions as the primary signaling cue at the epicenter of a diverse range of physiological processes. Although recent findings revealed that intact plant tissues release ATP as well, there is no clearly defined physiological function of extracellular ATP in plants. Here, we show that extracellular ATP is essential for maintaining plant cell viability. Its removal by the cell-impermeant traps glucose-hexokinase and apyrase triggered death in both cell cultures and whole plants. Competitive exclusion of extracellular ATP from its binding sites by treatment with beta,gamma-methyleneadenosine 5'-triphosphate, a nonhydrolyzable analog of ATP, also resulted in death. The death response was observed in Arabidopsis thaliana, maize (Zea mays), bean (Phaseolus vulgaris), and tobacco (Nicotiana tabacum). Significantly, we discovered that fumonisin B1 (FB1) treatment of Arabidopsis triggered the depletion of extracellular ATP that preceded cell death and that exogenous ATP rescues Arabidopsis from FB1-induced death. These observations suggest that extracellular ATP suppresses a default death pathway in plants and that some forms of pathogen-induced cell death are mediated by the depletion of extracellular ATP.     

Purinergic signaling and kinase activation for survival in pulmonary oxidative stress and disease

Stimulus-induced release of endogenous ATP into the extracellular milieu has been shown to occur in a variety of cells, tissues, and organs. Extracellular ATP can propagate signals via P2 receptors that are essential for growth and survival of cells. Abundance of P2 receptors, their multiple isoforms, and their ubiquitous distribution indicate that they transmit vital signals. Pulmonary epithelium and endothelium are rich in both P2X and P2Y receptors. ATP release from lung tissue and cells occurs upon stimulation both in vivo and in vitro. Extracellular ATP can activate signaling cascades composed of protein kinases including extracellular signal-regulated kinase (ERK) and phosphatidylinositol-3-kinase (PI3K). Here we summarize progress related to release of endogenous ATP and nucleotide signaling in pulmonary tissues upon exposure to oxidant stress. Hypoxic, hyperoxic, and ozone exposures cause a rapid increase of extracellular ATP in primary pulmonary endothelial and epithelial cells. Extracellular ATP is critical for survival of these cells in high oxygen and ozone concentrations. The released ATP, upon binding to its specific receptors, triggers ERK and PI3K signaling and renders cells resistant to these stresses. Impairment of ATP release and transmission of such signals could limit cellular survival under oxidative stress. This may further contribute to disease pathogenesis or exacerbation.     

Pharmacological dissection of the cellular mechanisms associated to the spontaneous and the mechanically stimulated ATP release by mesentery endothelial cells: roles of thrombin and TRPV

Endothelial cells participate in extracellular ATP release elicited by mechanosensors. To characterize the dynamic interactions between mechanical and chemical factors that modulate ATP secretion by the endothelium, we assessed and compared the mechanisms participating in the spontaneous (basal) and mechanically stimulated secretion using primary cultures of rat mesentery endothelial cells. ATP/metabolites were determined in the cell media prior to (basal) and after cell media displacement or a picospritzer buffer puff used as mechanical stimuli. Mechanical stimulation increased extracellular ATP that peaked within 1 min, and decayed to basal values in 10 min. Interruption of the vesicular transport route consistently blocked the spontaneous ATP secretion. Cells maintained in media lacking external Ca²⁺ elicited a spontaneous rise of extracellular ATP and adenosine, but failed to elicit a further extracellular ATP secretion following mechanical stimulation. 2-APB, a TRPV agonist, increased the spontaneous ATP secretion, but reduced the mechanical stimulation-induced nucleotide release. Pannexin1 or connexin blockers and gadolinium, a Piezo1 blocker, reduced the mechanically induced ATP release without altering spontaneous nucleotide levels. Moreover, thrombin or related agonists increased extracellular ATP secretion elicited by mechanical stimulation, without modifying spontaneous release. In sum, present results allow inferring that the spontaneous, extracellular nucleotide secretion is essentially mediated by ATP containing vesicles, while the mechanically induced secretion occurs essentially by connexin or pannexin1 hemichannel ATP transport, a finding fully supported by results from Panx1^?/? rodents. Only the latter component is modulated by thrombin and related receptor agonists, highlighting a novel endothelium-smooth muscle signaling role of this anticoagulant.     

ATP is stored in lysosomes of greater epithelial ridge supporting cells in newborn rat cochleae

Adenosine triphosphate (ATP), which plays a crucial role in both developing and mature cochleae, is released from greater epithelial ridge (GER) supporting cells of the rat cochlea, but the organelles in which ATP is stored have not yet been identified. Thus, we studied the organelles involved in ATP storage and suggest that lysosomes provide this function. GER supporting cells of newborn rats were isolated, purified, and cultured, and labeled vesicles within the supporting cells were identified via confocal microscopy and transmission electron microscopy (TEM). ATP release from GER supporting cells after glycyl-L -phenylalanine-β-naphthylamide (GPN) treatment was measured. The specifically labeled organelles observed by confocal microscopy and TEM were lysosomes, and GPN treatment enhanced ATP luminescence in the extracellular fluid of the supporting cells. The release of ATP from supporting cells was affected by changes in intra- and extracellular Ca²⁺ concentrations. In addition, changes in the intracellular Ca²⁺ caused by inhibiting the phospholipase signaling pathway affected the release of ATP from supporting cells. We demonstrated that ATP is stored in the lysosomes of GER supporting cells within newborn rat cochleae and that ATP release from GER supporting cells may be Ca²⁺-dependent.     

Characterization of ATP receptor which mediates norepinephrine release in PC12 cells.

PC12 cells, a rat pheochromocytoma cell line, has been reported to release norepinephrine in response to extracellular ATP in the presence of extracellular Ca2+. The potency order of ATP analogues was adenosine 5'-O-(3-thiotriphosphate) greater than ATP greater than adenosine 5'-O-(1-thiotriphosphate) = 2-methylthioadenosine 5'-triphosphate (MeSATP) greater than 2'- and 3'-O-(4-benzoyl-benzoyl)ATP (BzATP) greater than ADP greater than 5-adenylylimidodiphosphate. Adenosine 5'-O-(2-thiodiphosphate), beta, gamma-methyleneadenosine 5'-triphosphate, AMP and adenosine were inactive. The ATP action in the absence of extracellular Ca2+, suggests a small but appreciable contribution of intracellular Ca2+ mobilization, for norepinephrine release. However, for some ATP derivatives, like BzATP, almost no contribution of the phospholipase C-Ca2+ pathway is suggested, based on their low activity in inositol phosphates production. To identify the ATP-receptor protein, PC12 cell membranes were photoaffinity-labeled with [32P]BzATP. SDS-PAGE analysis showed that a 53-kDa protein labeling was inhibited by ATP and its derivatives, as well as by P2-antagonists, suramin and reactive blue 2, which inhibit the nucleotide-induced norepinephrine release. The inhibitory activity of the nucleotides was, in parallel with their potency, to induce norepinephrine release. Despite their inability to release norepinephrine, GTP and GTP gamma S inhibited the BzATP labeling, suggesting the participation of a putative G protein in the ATP-receptor-mediated actions. We suggest that the 53-kDa protein on the PC12 cell surface is an ATP receptor, which mediates the norepinephrine release, depending, mainly, on extracellular Ca2+ gating.     

Extracellular ATP as a signaling molecule for epithelial cells

The charge of this invited review is to present a convincing case for the fact that cells release their ATP for physiological reasons. Many of our "purinergic" colleagues as well as ourselves have experienced resistance to this concept, because it is teleologically counter-intuitive. This review serves to integrate the three main tenets of extracellular ATP signaling: ATP release from cells, ATP receptors on cells, and ATP receptor-driven signaling within cells to affect cell or tissue physiology. First principles will be discussed in the Introduction concerning extracellular ATP signaling. All possible cellular mechanisms of ATP release will then be presented. Use of nucleotide and nucleoside scavengers as well as broad-specificity purinergic receptor antagonists will be presented as a method of detecting endogenous ATP release affecting a biological endpoint. Innovative methods of detecting released ATP by adapting luciferase detection reagents or by using "biosensors" will be presented.Because our laboratory has been primarily interested in epithelial cell physiology and pathophysiology for several years, the role of extracellular ATP in regulation of epithelial cell function will be the focus of this review. For ATP release to be physiologically relevant, receptors for ATP are required at the cell surface. The families of P2Y G protein-coupled receptors and ATP-gated P2X receptor channels will be introduced. Particular attention will be paid to P2X receptor channels that mediate the fast actions of extracellular ATP signaling, much like neurotransmitter-gated channels versus metabotropic heptahelical neurotransmitter receptors that couple to G proteins. Finally, fascinating biological paradigms in which extracellular ATP signaling has been implicated will be highlighted. It is the goal of this review to convert and attract new scientists into the exploding field of extracellular nucleotide signaling and to convince the reader that extracellular ATP is indeed a signaling molecule.     

Kinetics and mechanism of ATP-dependent IL-1 beta release from microglial cells

Endotoxin-dependent release of IL-1 beta from mouse microglial cells is a very inefficient process, as it is slow and leads to accumulation of a modest amount of extracellular cytokine. Furthermore, secreted IL-1 beta is mostly in the procytokine unprocessed form. Addition of extracellular ATP to LPS-primed microglia caused a burst of release of a large amount of processed IL-1 beta. ATP had no effect on the accumulation of intracellular pro-IL-1 beta in the absence of LPS. In LPS-treated cells, ATP slightly increased the synthesis of pro-IL-1 beta. Optimal ATP concentration for IL-1 beta secretion was between 3 and 5 mM, but significant release could be observed at concentrations as low as 1 mM. At all ATP concentrations IL-1 beta release could be inhibited by increasing the extracellular K+ concentration. ATP-dependent IL-1 beta release was also inhibited by 90 and 60% by the caspase inhibitors YVAD and DEVD, respectively. Accordingly, in ATP-stimulated microglia, the p20 proteolytic fragment derived from activation of the IL-1-beta-converting enzyme could be detected by immunoblot analysis. These experiments show that in mouse microglial cells extracellular ATP triggers fast maturation and release of intracellularly accumulated IL-beta by activating the IL-1-beta-converting enzyme/caspase 1.     

Store-operated Ca2+ entry suppresses distention-induced ATP release from the urothelium

Epithelial cells in the urinary bladder (urothelium) trigger sensory signals in micturition by releasing ATP in response to distention of the bladder wall. Our previous study revealed the distinct roles of extracellular Ca(2+) and the Ca(2+) stores in the endoplasmic reticulum (ER) in urothelial ATP release. In the present study, we investigated the regulation of urothelial ATP release by Ca(2+) influx from the extracellular space and Ca(2+) release from the ER using a distention assay of the mouse bladder wall in a small Ussing chamber. Stimulation of Ca(2+) release from the ER in the mucosal side of the bladder induced significant ATP release without distention. Blockade of the inositol 1,4,5-triphosphate receptor reduced distention-induced ATP release, suggesting that Ca(2+) release from the ER is essential for the induction of urothelial ATP release. On the other hand, blockade of store-operated Ca(2+) entry (SOCE) from the extracellular space significantly enhanced distention-induced ATP release. Thus Ca(2+) release from the ER causes urothelial ATP release and depletion of Ca(2+) stores in the ER, which in turn causes the depletion-inducing SOCE to suppress the amount of urothelial ATP released.     

The relationship between constitutive ATP release and its extracellular metabolism in isolated rat kidney glomeruli.

ATP and adenosine are important extracellular regulators of glomerular functions. In this study, ATP release from glomeruli suspension and its extracellular metabolism were investigated. Basal extraglomerular ATP concentration (1nM) increased several fold during inhibition of ecto-ATPase activity, reflecting the basal ATP release rate. Mechanical perturbation increased the amounts of ATP released from glomeruli. ATP added to glomeruli was almost completely degraded within 20 minutes. In that time, AMP was the main product of extracellular ATP metabolism. Significant accumulation of AMP was observed after 5 min (194 +/-16 microM) and 20 min (271 +/-11 microM), whereas at the same time concentration of adenosine was only 10 muM. A competitive inhibitor of ecto-5-nucleotidase alpha-beta-methylene-ADP (AOPCP), decreased extraglomerular ATP and adenosine concentration by 80% and 50%, respectively. Similarly, AMP (100 microM) also markedly reduced extraglomerular ATP accumulation, whereas IMP, its deamination product, was not effective. P1, P5-diadenosine pentaphosphate (Ap5A) - an inhibitor of ecto-adenylate kinase prevented significantly the disappearance of ATP from extraglomerular media caused by AMP. These findings demonstrate that the decrease in extracellular ATP concentration observed after addition of AOPCP or AMP is caused by the presence of ecto-adenylate kinase activity in the glomeruli. The enzyme catalyses reversible reaction 2ADP<->ATP+AMP, and a rise in the AMP concentration can lead to fall in ATP level. The present study provides evidence the extraglomerular accumulation of ATP reflects both release of ATP from glomeruli cells and its metabolism by ecto-enzymes. Our data suggest that AMP, produced from ATP in the Bowman's capsular space, might plays a dual role as a substrate for ecto-adenylate kinase and ecto-nucleotidase reactions being responsible for the regulation of intracapsular ATP and adenosine concentration. We conclude that AMP degrading and converting ecto-enzymes effectively determine the balance between ATP and adenosine concentration and thus the activation of P2 and/or adenosine receptors.     

Endotoxin activation of macrophages does not induce ATP release and autocrine stimulation of P2 nucleotide receptors

Receptors for extracellular nucleotides (P2, or purinergic receptors) have previously been implicated in the transduction of endotoxin signaling in macrophages. The most compelling evidence has been the observation that inhibitors of ionotropic nucleotide (P2X) receptors, including periodate-oxidized ATP (oATP), attenuate a subset of endotoxin-induced effects such as activation of NF-kappaB and up-regulation of inducible NO synthase. We investigated whether endotoxin induces ATP release from a murine macrophage cell line (BAC1.2F5) using sensitive on-line assays for extracellular ATP. These cells constitutively released ATP, producing steady-state extracellular concentrations of approximately 1 nM when assayed as monolayers of 10(6) adherent cells bathed in 1 ml of medium. However, the macrophages did not release additional ATP during either acute or prolonged endotoxin stimulation. In addition, cellular ecto-ATPase activities were measured following prolonged endotoxin activation and were found not to be significantly altered. Although oATP treatment significantly attenuated the endotoxin-induced production of NO, this inhibitory effect was not reproduced when the cells were coincubated with apyrase, a highly effective ATP scavenger. These results indicate that activation of macrophages by endotoxin does not induce autocrine stimulation of P2 nucleotide receptors by endogenous ATP released to extracellular compartments. Moreover, the data suggest that the ability of oATP to interfere with endotoxin signaling is due to its interaction with molecular species other than ATP-binding P2 receptors.     

Pharmacological sensitivity of ATP release triggered by photoliberation of inositol-1,4,5-trisphosphate and zero extracellular calcium in brain endothelial cells

Recently, ATP has gained much interest as an extracellular messenger involved in the communication of calcium signals between cells. The mechanism of ATP release is, however, still a matter of debate. In the present study we investigated the possible contribution of connexin hemichannels or ion channels in the release of ATP in GP8, a rat brain endothelial cell line. Release of ATP was triggered by photoactivation of InsP(3) or by reducing the extracellular calcium concentration. Both trigger protocols induced ATP release significantly above baseline. InsP(3)-triggered ATP release was completely blocked by alpha-glycyrrhetinic acid (alpha-GA), the connexin mimetic peptides gap 26 and 27, and the trivalent ions gadolinium and lanthanum. ATP release triggered by zero calcium was, in addition to these substances, also blocked by flufenamic acid (FFA), niflumic acid, and NPPB. Gap 27 selectively blocked zero calcium-triggered ATP release in connexin-43 transfected HeLa cells, while having no effect in wild-type and connexin-32 transfected cells. Of all the agents used, only alpha-GA, FFA and NPPB significantly reduced gap junctional coupling. In conclusion, InsP(3) and zero calcium-triggered ATP release show major similarities but also some differences in their sensitivity to the agents applied. It is suggested that both stimuli trigger ATP release through the same mechanism, which is connexin-dependent, permeable in both directions, potently blocked by connexin mimetic peptides, and consistent with the opening of connexin hemichannels.     

KCl -Permeabilized Pancreatic Islets: An Experimental Model to Explore the Messenger Role of ATP in the Mechanism of Insulin Secretion

Our previous work has demonstrated that islet depolarization with KCl opens connexin36 hemichannels in β-cells of mouse pancreatic islets allowing the exchange of small metabolites with the extracellular medium. In this study, the opening of these hemichannels has been further characterized in rat islets and INS–1 cells. Taking advantage of hemicannels’opening, the uptake of extracellular ATP and its effect on insulin release were investigated. 70 mM KCl stimulated light emission by luciferin in dispersed rat islets cells transduced with the fire-fly luciferase gene: it was suppressed by 20 mM glucose and 50 μM mefloquine, a specific connexin36 inhibitor. Extracellular ATP was taken up or released by islets depolarized with 70 mM KCl at 5 mM glucose, depending on the external ATP concentration. 1 mM ATP restored the loss of ATP induced by the depolarization itself. ATP concentrations above 5 mM increased islet ATP content and the ATP/ADP ratio. No ATP uptake occurred in non-depolarized or KCl-depolarized islets simultaneously incubated with 50 μM mefloquine or 20 mM glucose. Extracellular ATP potentiated the secretory response induced by 70 mM KCl at 5 mM glucose in perifused rat islets: 5 mM ATP triggered a second phase of insulin release after the initial peak triggered by KCl-depolarization itself; at 10 mM, it increased both the initial, KCl-dependent, peak and stimulated a greater second phase of secretion than at 5 mM. These stimulatory effects of extracellular ATP were almost completely suppressed by 50 μM mefloquine. The magnitude of the second phase of insulin release due to 5 mM extracellular ATP was decreased by addition of 5 mM ADP (extracellular ATP/ADP ratio = 1). ATP acts independently of K_ATP channels closure and its intracellular concentration and its ATP/ADP ratio seems to regulate the magnitude of both the first (triggering) and second (amplifying) phases of glucose-induced insulin secretion.     

细胞外ATP与肿瘤免疫

ATP是最重要的胞内代谢产物之一,也是一种重要的信号分子。研究发现,某些凋亡刺激能诱导肿瘤细胞内ATP释放到细胞外,这种释放到细胞外的ATP能促进吞噬细胞对凋亡细胞的吞噬,由此激发特异性抗肿瘤免疫杀伤效应,提示细胞外ATP在肿瘤免疫治疗中的潜在应用价值。本文就细胞外ATP在肿瘤免疫中的研究进展作一综述。     

Bioluminescence detection of ATP release mechanisms in epithelia

Autocrine and paracrine release of and extracellular signalingby ATP is a ubiquitous cell biological and physiological process. Despite this knowledge, the mechanisms and physiological roles ofcellular ATP release are unknown. We tested the hypothesis thatepithelia release ATP under basal and stimulated conditions by using anewly designed and highly sensitive assay for bioluminescence detectionof ATP released from polarized epithelial monolayers. Thisbioluminescence assay measures ATP released from cystic fibrosis (CF)and non-CF human epithelial monolayers in a reduced serum mediumthrough catalysis of the luciferase-luciferin reaction, yielding aphoton of light collected by a luminometer. This novel assay measuresATP released into the apical or basolateral medium surroundingepithelia. Of relevance to CF, CF epithelia fail to release ATP acrossthe apical membrane under basal conditions. Moreover, hypotonicity isan extracellular signal that stimulates ATP release into bothcompartments of non-CF epithelia in a reversible manner; the responseto hypotonicity is also lost in CF epithelia. The bioluminescencedetection assay for ATP released from epithelia and other cells will beuseful in the study of extracellular nucleotide signaling inphysiological and pathophysiological paradigms. Taken together, theseresults suggest that extracellular ATP may be a constant regulator ofepithelial cell function under basal conditions and an autocrineregulator of cell volume under hypotonic conditions, two functions thatmay be lost in CF and contribute to CF pathophysiology.

     

Induction of extracellular ATP mediates increase in intracellular thioredoxin in RAW264.7 cells exposed to low-dose γ-rays

We previously showed that low doses (0.25-0.5 Gy) of γ-rays elevated thioredoxin (Trx-1) in various organs of mice after whole-body irradiation. Also, it is reported that extracellular ATP, which is released in response to various stresses, regulates the expression of intracellular antioxidants through activation of P2 receptors. We have recently found that low-dose γ-rays induce ATP release from the exposed cells. However, it is not yet clear whether the radiation-induced extracellular ATP modulates the cellular redox balance. Here, we investigated whether γ-ray irradiation-induced release of extracellular ATP contributes to the induction of the cellular antioxidant Trx-1, using mouse macrophage-like RAW264.7 cells. Irradiation with γ-rays or exogenously added ATP increased the expression of Trx-1, and in both cases the increase was blocked by pretreatment with an ectonucleotidase, apyrase. Then, the involvement of ATP-dependent reactive oxygen species (ROS) generation in the increase in antioxidant capacity was examined. ATP stimulation promoted the generation of intracellular ROS and also increased Trx-1 expression. The increase in Trx-1 expression was significantly suppressed by pretreatment of the cells with antioxidants. In conclusion, the γ-ray irradiation-induced release of extracellular ATP may, at least in part, contribute to the production of ROS via purinergic signaling, leading to promotion of intracellular antioxidants as an adaptive response to an oxidative stress.     

Brefeldin A block of integrin-dependent mechanosensitive ATP release from Xenopus oocytes reveals a novel mechanism of mechanotransduction

Many animal cells release ATP into the extracellular medium, and often this release is mechanosensitive. However, the mechanisms underlying this release are not well understood. Using the luciferin-luciferase bioluminescent assay we demonstrate that a Xenopus oocyte releases ATP at a basal rate approximately 0.01 fmol/s, and gentle mechanical stimulation can increase this to 50 fmol/s. Brefeldin A, nocodazole, and progesterone-induced- maturation block basal and mechanosensitive ATP release. These treatments share the common feature of disrupting the Golgi complex and vesicle trafficking to the cell surface and thereby block protein secretion and membrane protein insertion. We propose that ATP release occurs when protein transport vesicles enriched in ATP fuse with the plasma membrane. Collagenase, integrin-binding peptides, and cytochalasin D also block ATP release, indicating that extracellular, membrane and cytoskeletal elements are involved in the release process. Elevation of intracellular Ca(2+) does not evoke ATP release but potentiates mechanosensitive ATP release. Our study indicates a novel mechanism of mechanotransduction that would allow cells to regulate membrane trafficking and protein transport/secretion in response to mechanical loading.     

Cystic fibrosis transmembrane conductance regulator facilitates ATP release by stimulating a separate ATP release channel for autocrine control of cell volume regulation

These studies provide evidence that cystic fibrosis transmembrane conductance regulator (CFTR) potentiates and accelerates regulatory volume decrease (RVD) following hypotonic challenge by an autocrine mechanism involving ATP release and signaling. In wild-type CFTR-expressing cells, CFTR augments constitutive ATP release and enhances ATP release stimulated by hypotonic challenge. CFTR itself does not appear to conduct ATP. Instead, ATP is released by a separate channel, whose activity is potentiated by CFTR. Blockade of ATP release by ion channel blocking drugs, gadolinium chloride (Gd(3+)) and 4,4'-diisothiocyanatostilbene-2,2'disulfonic acid (DIDS), attenuated the effects of CFTR on acceleration and potentiation of RVD. These results support a key role for extracellular ATP and autocrine and paracrine purinergic signaling in the regulation of membrane ion permeability and suggest that CFTR potentiates ATP release by stimulating a separate ATP channel to strengthen autocrine control of cell volume regulation.     

Prostaglandin E2 is a Potential Mediator of Extracellular ATP Action in Osteoblast-Like Cells

Extracellular ATP dose dependently stimulated ⁴⁵Ca²⁺ influx even in the presence of nifedipine, a Ca²⁺ antagonist that inhibits voltage-dependent Ca²⁺ channel, in osteoblast-like MC3T3-E1 cells. ATP stimulated arachidonic acid release and the synthesis of prostaglandin E₂ (PGE₂). However, the ATP-induced arachidonic acid release was significantly reduced by chelating extracellular Ca²⁺ with EGTA. On the other hand, ATP induced DNA synthesis of these cells in a dose-dependent manner in the range between 1μM and 1 mM. The pretreatment with indomethacin, a cyclooxygenase inhibitor, suppressed both ATP-induced PGE₂ synthesis and DNA synthesis in these cells. The inhibitory effect by 50μM indomethacin on the DNA synthesis was reversed by adding 10μM PGE₂. These results strongly suggest that extracellular ATP stimulates Ca²⁺ influx resulting in the release of arachidonic acid in osteoblast-like cells and that extracellular ATP-induced proliferative effect is mediated, at least in part, by ATP-stimulated PGE₂ synthesis.     

Activation of pannexin 1 channels by ATP through P2Y receptors and by cytoplasmic calcium

The ability for long-range communication through intercellular calcium waves is inherent to cells of many tissues. A dual propagation mode for these waves includes passage of IP3 through gap junctions as well as an extracellular pathway involving ATP. The wave can be regenerative and include ATP-induced ATP release via an unknown mechanism. Here, we show that pannexin 1 channels can be activated by extracellular ATP acting through purinergic receptors of the P2Y group as well as by cytoplasmic calcium. Based on its properties, including ATP permeability, pannexin 1 may be involved in both initiation and propagation of calcium waves.     

Mechanisms of ATP release by human trabecular meshwork cells, the enabling step in purinergic regulation of aqueous humor outflow

Our guiding hypothesis is that ecto-enzymatic conversion of extracellular ATP to adenosine activates A(1) adenosine receptors, reducing resistance to aqueous humor outflow and intraocular pressure. The initial step in this purinergic regulation is ATP release from outflow-pathway cells by mechanisms unknown. We measured similar ATP release from human explant-derived primary trabecular meshwork (TM) cells (HTM) and a human TM cell line (TM5). Responses to 21 inhibitors indicated that pannexin-1 (PX1) and connexin (Cx) hemichannels and P2X(7) receptors (P2RX(7) ) were comparably important in modulating ATP release induced by hypotonic swelling, whereas vesicular release was insignificant. Consistent with prior studies of PX1 activity in certain other cells, ATP release was lowered by the reducing agent dithiothreitol. Overexpressing PX1 in HEK293T cells promoted, while partial knockdown (KD) in both HEK293T and TM5 cells inhibited hypotonicity-activated ATP release. Additionally, KD reduced the pharmacologically defined contribution of PX1 and enhanced those of Cx and P2RX(7) . ATP release was also triggered by raising intracellular Ca(2+) activity with ionomycin after a prolonged lag time and was unaffected by the PX1 blocker probenecid, but nearly abolished by P2RX(7) antagonists. We conclude that swelling-stimulated ATP release from human TM cells is physiologically mediated by PX1 and Cx hemichannels and P2X(7) receptors, but not by vesicular release. PX1 appears not to be stimulated by intracellular Ca(2+) in TM cells, but can be modulated by oxidation-reduction state. The P2RX(7) -dependent component of swelling-activated release may be mediated by PX1 hemichannels or reflect apoptotic magnification of ATP release, either through itself and/or hemichannels.