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Responses of mouse lymphocytes to extracellular ATP. II. Extracellular ATP causes cell type-dependent lysis and DNA fragmentation
Abstract
Extracellular ATP (ATPo) caused dose-dependent lysis of YAC-1 and P-815 mouse tumor cells. This event, assessed by 51Cr release, was accompanied by sustained depolarization of the plasma membrane potential and Ca2+ influx. Plasma membrane depolarization and Ca2+ influx occurred within a few seconds of ATPo addition to both cell types, whereas 51Cr was released without apparent lag in YAC-1 cells and after 2 h in P-815 cells. Furthermore, a rise in [Ca2+]i was required for ATPo-dependent lysis of YAC-1 but not P-815 cells. In P-815 cells, ATPo caused an early and [Ca2+]i-independent DNA fragmentation that occurred at lower nucleotide concentrations than those required to trigger 51Cr release. Instead in YAC-1 cells very low concentrations of ATPo caused early lysis (ED50 for lysis about 200 microM) accompanied by only barely detectable DNA fragmentation. Previous studies disclosed that lymphokine-activated killer cells are fully resistant to the membrane-perturbing effects of ATPo. We show that lymphokine-activated killer cells also do not undergo DNA fragmentation even in the presence of high ATPo concentrations. This study complements previous observations on the lytic effects of ATPo and shows that this nucleotide can also cause DNA fragmentation, one of the earliest target cell alterations observed during CTL-mediated lysis.
... It has been shown that ecto-ATP can induce death of T cells and macrophages (Canaday et al., 2002;Chvatchko et al., 1996;Lammas et al., 1997;Zanovello et al., 1990). ATP-induced activation of the cytolytic P2X7 purinoreceptor induces calcium flux, formation of large membrane pores, exposure of phosphatidylserine and uptake of propidium iodide, ultimately resulting in cell death. ...
... Furthermore, PS-exposure and cytotoxicity are well-known effects of ATP-induced triggering of the P2X7 receptor (Di Zanovello et al., 1990). ...
... Proposed roles of P2X7 in immunity include the release of IL-1, killing of intracellular pathogens, and cytotoxicity toward immune cells (Canaday et al., 2002;Chvatchko et al., 1996;Lammas et al., 1997;Zanovello et al., 1990). However, millimolar concentrations of exogenous ATP were required to elicit these P2X7-mediated response. ...
Pr Jean KANELLOPOULOS Président du jury Dr Philippe DETERRE Rapporteur Dr François RASSENDREN Rapporteur Pr Catherine SAUTES-FRIDMAN Examinateur Pr David OJCIUS Examinateur
... The early discovery of the P2X7 macropore drove interest in P2X7 as a receptor capable of mediating cell death through disruption of intracellular homeostasis and cytolysis leading to necrosis (Zanovello et al., 1990;Surprenant et al., 1996). However, many reports have highlighted cell death mechanisms associated with P2X7 activation that are typical of an apoptotic pathway. ...
... However, many reports have highlighted cell death mechanisms associated with P2X7 activation that are typical of an apoptotic pathway. These include membrane depolarization, redistribution of phosphatidylserine (PS) to the outer membrane, caspase 3, 8 and 9 activation and membrane blebbing (Zanovello et al., 1990;Ferrari et al., 1997a;Humphreys et al., 2000;Mackenzie et al., 2005). Interestingly, the recent resolution of the rat P2X7 structure has highlighted the presence of a putative phospholipid in a perpendicular position to the transmembrane helices in the middle of the plasma membrane that led McCarthy and colleagues to suggest that P2X7 might possess an intrinsic flippase-like function driving PS redistribution to the outer membrane (McCarthy et al., 2019). ...
P2X7 is a transmembrane receptor expressed in multiple cell types including neurons, dendritic cells, macrophages, monocytes, B and T cells where it can drive a wide range of physiological responses from pain transduction to immune response. Upon activation by its main ligand, extracellular ATP, P2X7 can form a nonselective channel for cations to enter the cell. Prolonged activation of P2X7, via high levels of extracellular ATP over an extended time period can lead to the formation of a macropore, leading to depolarization of the plasma membrane and ultimately to cell death. Thus, dependent on its activation state, P2X7 can either drive cell survival and proliferation, or induce cell death. In cancer, P2X7 has been shown to have a broad range of functions, including playing key roles in the development and spread of tumor cells. It is therefore unsurprising that P2X7 has been reported to be upregulated in several malignancies. Critically, ATP is present at high extracellular concentrations in the tumor microenvironment (TME) compared to levels observed in normal tissues. These high levels of ATP should present a survival challenge for cancer cells, potentially leading to constitutive receptor activation, prolonged macropore formation and ultimately to cell death. Therefore, to deliver the proven advantages for P2X7 in driving tumor survival and metastatic potential, the P2X7 macropore must be tightly controlled while retaining other functions. Studies have shown that commonly expressed P2X7 splice variants, distinct SNPs and post-translational receptor modifications can impair the capacity of P2X7 to open the macropore. These receptor modifications and potentially others may ultimately protect cancer cells from the negative consequences associated with constitutive activation of P2X7. Significantly, the effects of both P2X7 agonists and antagonists in preclinical tumor models of cancer demonstrate the potential for agents modifying P2X7 function, to provide innovative cancer therapies. This review summarizes recent advances in understanding of the structure and functions of P2X7 and how these impact P2X7 roles in cancer progression. We also review potential therapeutic approaches directed against P2X7.
... ATP appears to mediate both apoptosis and necrosis, depending on the target cell type and the concentration of ATP used (Zanovello et al, 1990;Zheng et al, 1991), suggesting the possibility that both P2X and P2Z receptors may be involved (Chvatchko et al, 1996). Apoptosis is a long term process involving gene expression and protein synthesis, which is characterised by cell and chromatin shrinkage and DNA fragmentation, but maintenance of membrane integrity. ...
... Perhaps the conclusions of Blanchard et al, (1995) implicating Ca 2+ /CaM and CaMKII with P2Z activation, could equally be explained by a direct effect of both TFP and KN-62 on the macrophage P2Z receptor. Several recent studies show that ATP stimulates apoptosis in murine lymphocytes and rat thymocytes (Zanovello et al, 1990: Zheng et al, 1991. ATP also induces apoptosis in CLL cells (Section 3.3), which is probably triggered by Ca 2+ influx through the P2Z ion channel, and may involve PLD activation. ...
... 13 Calcium ion influx (rather than potassium efflux) was reported to be required for extracellular ATP's triggering of cell type-dependent lysis. See Zanovello et al., 1990. ATP's triggering of IL-1β in the pathogen-and danger-associated molecular patterns recognition pathway 14 . ...
Pictorial representations such as diagrams and figures are widely used in scientific literature for explanatory and descriptive purposes. The intuitive nature of pictorial representations coupled with texts foster a better understanding of the objects of study. Biological mechanisms and processes can be clearly illustrated and grasped in pictures. I argue that pictorial representations describe biological phenomena by telling stories. I elaborate on the role of narrative structures of pictures in the frontier research using a case study in immunology. I articulate that pictures with an inherent narrative structure are crucial in biological sciences.
... Therefore, there is an increasing and urgent need not just for novel drugs but, more importantly, for novel classes of compounds targeting novel molecular targets. The cloning of the P2X7 receptor (P2X7R, at the time known as P2Z) [1], in the wake of a cogent series of in vitro experiments showing its likely involvement in inflammation [2][3][4][5], spurred great interest and hopes that identification of this novel target would pave the way to new, more effective anti-inflammatory drugs. However, Phase I/II clinical trials launched by major pharmaceutical companies to explore the effect of P2X7R antagonism in several chronic inflammatory diseases (rheumatoid arthritis, inflammatory bowel disease, depression, osteoarthritis, chronic obstructive pulmonary disease, and Crohn's disease) produced rather disappointing results [6][7][8][9], which all together cooled down interest in P2X7R as a suitable target for anti-inflammatory therapy. ...
The discovery of the P2X7 receptor (P2X7R, originally named P2Z) in immune cells, its cloning, and the identification of its role in a multiplicity of immune-mediated diseases raised great hopes for the development of novel and more potent anti-inflammatory medicaments. Unfortunately, such hopes were partially deluded by the unsatisfactory results of most early clinical trials. This failure substantially reduced the interest of the pharmaceutical and biotech industries in the clinical development of P2X7R-targeted therapies. However, recent findings ushered in a second life for the P2X7R in diagnostic medicine. New P2X7R radioligands proved to be very reliable tools for the diagnosis of neuroinflammation in preclinical and clinical studies, and detection and measurement of free P2X7 receptor (or P2X7 subunit) in human blood suggested its potential use as a circulating marker of inflammation. Here we provide a brief review of these novel developments.
... It is not surprising that the P2X7 receptor is involved in the regulation of cell death since one of the recep-tor's domains localized at the C-terminus is similar to the death domain in the tumor necrosis factor receptor 1 (TNF-R1), which is involved in the induction of apoptosis (Zanovello et al., 1990;Chow et al., 1997;Denlinger et al., 2001). Initially, activation of this receptor was thought to lead to necrotic cell death (Di Virgilio et al., 1989). ...
Nucleotides are the most common compounds produced constantly by living organisms. They are involved in most cellular processes like the synthesis of other nucleotides and nucleic acids, generation of energy needed for the maintenance of cells, and molecular signaling. In the 70s sir. Geoffrey Burstock discovered a new class of transmembrane proteins – nucleotide receptors responding to nucleotides and their derivatives. For historical reasons, we distinguish two main classes of nucleotide receptors: P1 – which are G protein-coupled adenosine receptors, and P2 – nucleotide receptors that respond to ATP and its derivatives. Additionally, the P2 receptors family can be divided into two subgroups: P2Y – G protein-coupled receptors and P2X cation channel receptors. This paper focuses mainly on the most researched receptor in the nucleotide receptors family – the P2X7 receptor – presenting it in the background of the nucleotide signaling landscape. Almost thirty years of extensive studies on the receptor contributed to understanding protein structure, splicing variants, and mechanism of action in somatic cells. Despite such a wide spectrum of research, the role of the receptor in cancer progression is still undetermined. In many reports, we can find information about the anti-tumorigenic role of this receptor caused by activation of the cell death mechanism after membrane pore formation. These results, however, contradict other studies in which the same receptor is known to promote cancer development through stimulation of proliferation and activation of pro-survival pathways. Ultimately, all this gathered knowledge points to two faces of the receptor in tumor progression. Therefore, we do provide a comprehensive overview of the topic. Finally, we also try to systemize previous and recent literature about the role of this receptor in somatic and cancer cells and provide access to it in the form of a convenient table.
... In the mid '70 s, Rozengurt and Heppel and Landry and Lehninger showed that eATP causes permeability changes of the plasma membrane and severely upset intracellular ion balance [144,145]. Later, our own laboratory carried out an extensive investigation of the mechanisms responsible for eATP-dependent cell death [146][147][148][149]. Reports on the anticancer activity of eATP have accumulated over the years. ...
Adenosine triphosphate (ATP) is one of the main biochemical components of the tumor microenvironment (TME), where it can promote tumor progression or tumor suppression depending on its concentration and on the specific ecto-nucleotidases and receptors expressed by immune and cancer cells. ATP can be released from cells via both specific and nonspecific pathways. A non-regulated release occurs from dying and damaged cells, whereas active release involves exocytotic granules, plasma membrane-derived microvesicles, specific ATP-binding cassette (ABC) transporters and membrane channels (connexin hemichannels, pannexin 1 (PANX1), calcium homeostasis modulator 1 (CALHM1), volume-regulated anion channels (VRACs) and maxi-anion channels (MACs)). Extracellular ATP acts at P2 purinergic receptors, among which P2X7R is a key mediator of the final ATP-dependent biological effects. Over the years, P2 receptor- or ecto-nucleotidase-targeting for cancer therapy has been proposed and actively investigated, while comparatively fewer studies have explored the suitability of TME ATP as a target. In this review, we briefly summarize the available evidence suggesting that TME ATP has a central role in determining tumor fate and is, therefore, a suitable target for cancer therapy.
... The effects of adenosine on apoptosis of these cells result from interaction with cell surface PI receptors, but the receptor subtypes mediating the effect in each case is unknown, and the second messenger system by which adenosine triggers apoptosis is also unclear (Chow et al., 1997). The ability of extracellular ATP to kill cells is well established, particularly in cells of the immune system (Zanovello et al., 1990;Apasov et al., 1995), and hépatocytes (Nicotera et al., 1986;Zoeteweij et al., 1996). ATP is known to cause cell death by apoptosis and necrosis, and two members of the P2X family have been implicated. ...
The aim of this thesis is to investigate the possibility that extracellular nucleotides, and their receptors (P2 receptors) are involved in regulating developmental processes during chick embryogenesis. In the first chapter, the classification, structure, and signal transduction mechanisms of P2 receptors are described. The evidence that suggests that this type of signalling has the potential to be important developmentally is also discussed. In the second chapter, in-situ hybridisation is used to describe the developmentally regulated pattern of expression of cP2Y1, a G protein-coupled P2 receptor, in multiple tissue systems during chick embryogenesis. This is the first description of P2 receptor subtype expression during embryonic development of the chick, and only the second time that expression of a specific P2 receptor has been described during the embryonic development of an organism. The following two chapters are devoted to investigating the function of cP2Y1, in one of the regions in which it is expressed, the developing limb. In chapter 3 monitoring of intracellular calcium is used to characterise the response of early limb cells to extracellular nucleotides. This technique is used to show that in addition to cP2Y1, at least one other P2 receptor subtype is expressed in early limb cells. Monitoring of intracellular calcium, and RNase protection is also used to show that within 24 hours of culturing, expression of cP2Y1 is lost from limb cells. Monitoring of intracellular calcium was then used as an assay to demonstrate that expression of functional cP2Y1 could be rescued in these cells by transfection with an expression vector carrying the cP2Y1 cDNA. In the fourth chapter, the loss of cP2Y1 in-vitro is exploited, using a gain-of- function approach, to investigate a function for cP2Y1 in limb development. Early limb bud cells were transfected as described and characterised above, and an in vitro assay of cartilage formation was used to provide evidence that cP2Y1 may have an important role to play in regulating the formation of the limb skeleton. In the final chapter immunohistochemistry is used to show that two members of the P2X, ligand-gated ion channel family of P2 receptors are expressed during chick skeletal muscle development, and in the embryonic chick nervous system.
... The results are also in agreement with Nijweide et al., (1995) Virgilio et al., 1989, Zanovello et al., 1990& Zheng et al., 1991. Cell death caused by ATP involves both necrosis and apoptosis, with two P2 purinoceptors implicated: P2X? (the receptor for ATP^ ), and possibly P2Xi which has sequence homology to genes expressed by thymocytes during the onset of apoptosis (Chow et al., 1997). ...
Osteoclasts are multinucleated cells responsible for the resporption of both the organic and inorganic components of bone. The molecular mechanisms by which these cells are activated to resorb bone are still poorly understood. Previous work has shown that mature rat osteoclasts in short term cultures are extremely sensitive to small shifts in extracellular pH (H+ out) and are strongly stimulated in acidified conditions (pH 6.8 - 7.0). The aim of the work presented in this thesis was to further investigate the actions of [H+ out] on osteoclast function and to study the interactions of ([H+ out]) with other stimulators of resorption. Experiments with mature rat osteoclasts indicated that the acid-activation effect does not abate over time but may become even more pronounced. Conversely, osteoclast formation in long-term mouse marrow cultures was inhibited at low pH and stimulated in more alkaline conditions; however, after formation in marrow cultures, the mature mouse osteoclasts exhibited the same acid-activation characteristics as freshly isolated mature rat osteoclasts. Experiments with cultured mouse calarial bones showed similar effects: osteoclastic resorption was strongly activated below pH 7.0, but acidified conditions resulted in a reduction in the number of osteoclasts visible in bones. Acid-stimulated resorption in calvaria occurred with HCO3- rather than CO2 acidosis and was blocked by inhibitors of prostaglandin and leukotriene synthesis. In contrast, prostaglandin inhibitors stimulated pit formation by cultured rat osteoclasts. I also found that resorption pit formation by isolated chick osteoclasts was very sensitive to small changes in although maximum pH sensitivity occurs over a more alkaline pH range than is the case for rodent osteoclasts. The data from these diverse systems provide strong support for the critical role of acid-base balance in modulating osteoclast function, despite apparent differences in the role of prostaglandins. The results show how pH can be manipulated to optimise resorption assays, and emphasise the importance of controlling this key variable. I also discovered that extracellular ATP, now recognised as an important signalling molecule in many tissues, stimulated both the activation and formation of rodent osteoclasts. There was a marked synergy between the stimulatory effects of [H+ out] and low dose ATP on the resorptive activity of rat osteoclasts. Acid-activated resorption was blocked by apyrase (an ecto-ATPase) and by suramin (an ATP antagonist). Thus, extracellular ATP and low pH both appear to be necessary for osteoclast activation. The findings suggest an important new mechanism for the local control of osteoclast function which may be relevant to, for example, the bone loss associated with inflammation.
... These numerous cascades illustrate the diversity and complexity of P2RX7 signaling, with implications for cellular processes such as growth, proliferation, differentiation, metabolism, migration, invasion, autophagy and also cell death. Mechanisms of P2RX7-dependent cell death have been well documented and are numerous, including apoptosis (Zanovello et al., 1990) as well as several unique processes: aponecrosis (MacKenzie et al., 2001), necroptosis (Cullen et al., 2015), pyroptosis (de Gassart and Martinon, 2015), pseudo-apoptosis (Roger and Pelegrin, 2011), and autosis (Draganov et al., 2015). Autophagic cell death has also been identified . ...
The P2RX7 receptor is a unique member of a family of extracellular ATP (eATP)-gated ion channels expressed in immune cells, where its activation triggers the inflammatory cascade. Therefore, P2RX7 has been long investigated as a target in the treatment of infectious and inflammatory diseases. Subsequently, P2RX7 signaling has been documented in other physiological and pathological processes including pain, CNS and psychiatric disorders and cancer. As a result, a range of P2RX7 antagonists have been developed and trialed. Interestingly, the recent crystallization of mammalian and chicken receptors revealed that most widely-used antagonists may bind a unique allosteric site. The availability of crystal structures allows rational design of improved antagonists and modeling of binding sites of the known or presumed inhibitors. However, several unanswered questions limit the cogent development of P2RX7 therapies. Firstly, this receptor functions as an ion channel, but its chronic stimulation by high eATP causes opening of the non-selective large pore (LP), which can trigger cell death. Not only the molecular mechanism of LP opening is still not fully understood but its function(s) are also unclear. Furthermore, how can tumor cells take advantage of P2RX7 for growth and spread and yet survive overexpression of potentially cytotoxic LP in the eATP-rich environment? The recent discovery of the feedback loop, wherein the LP-evoked release of active MMP-2 triggers the receptor cleavage, provided one explanation. Another mechanism might be that of cancer cells expressing a structurally altered P2RX7 receptor, devoid of the LP function. Exploiting such mechanisms should lead to the development of new, less toxic anticancer treatments. Notably, targeted inhibition of P2RX7 is crucial as its global blockade reduces the immune and inflammatory responses, which have important anti-tumor effects in some types of malignancies. Therefore, another novel approach is the synthesis of tissue/cell specific P2RX7 antagonists. Progress has been aided by the development of p2rx7 knockout mice and new conditional knock-in and knock-out models are being created. In this review, we seek to summarize the recent advances in our understanding of molecular mechanisms of receptor activation and inhibition, which cause its re-emergence as an important therapeutic target. We also highlight the key difficulties affecting this development.
... Among apoptotic-related alterations P2X7 pore opening associates with cell morphological changes like cell blebbing and shrinkage, nuclear fragmentation and chromatin condensation [53][54][55][56] [Fig. 1]. ...
P2X7 receptor is an ion channel activated by extracellular adenosine trisphosphate (eATP) that attracted increasing attention for its role in immune reactions, neurobiology and oncology. As receptor for an extracellular ligand, P2X7 activates a series of intracellular signalling pathways mainly via alterations of the ion permeability, but also through formation of a large unselective pore and direct interaction with other proteins. Here we wish to give an overview on the main biochemical paths initiated by P2X7 activation by revising recent and established literature on P2X7-triggered signalling cascades leading to cell death, inflammatory and immune response activation, proliferation and metabolism modulation. We will focus on the well-known P2X7 inflammasome/NF-kB and pro-apoptotic networks but also cover P2X7-activated emerging autophagic, pyroptotic and proliferative-oncogenic pathways, like beclin-1/LC3-II, caspase-11, Akt and VEGF axes.
... Addition of extracellular ATP at concentrations of 10 to 100 lM have been reported to be required for induction of PCD. 36,37 The concentrations of extracellular ATP detected in bulk cellfree supernatants from CD40-stimulated Müller cells were lower than these concentrations. However, compared with single administration of extracellular ATP, cell death after repeated exposure to ATP requires lower concentrations of the nucleotide. ...
Purpose
Cluster of differentiation 40 (CD40) is required for retinal capillary degeneration in diabetic mice, a process mediated by the retinal endothelial cells (REC) death. However, CD40 activates prosurvival signals in endothelial cells. The purpose of this study was to identify a mechanism by which CD40 triggers programmed cell death (PCD) of RECs and address this paradox.
Methods
Human RECs and Müller cells were incubated with CD154 and L-N6-(1-Iminoethyl)lysine (L-Nil, nitric oxide synthase 2 inhibitor), α-lipoic acid (inhibitor of oxidative stress), anti-Fas ligand antibody, or A-438079 (P2X7 adenosine triphosphate [ATP] receptor inhibitor). Programmed cell death was analyzed by fluorescence-activated cell sorting (FACS) or Hoechst/propidium iodide staining. Release of ATP was measured using a luciferase-based assay. Mice were made diabetic with streptozotocin. Expression of P2X7 was assessed by FACS, quantitative PCR, or immunohistochemistry.
Results
Ligation of CD40 in primary RECs did not induce PCD. In contrast, in the presence of primary CD40⁺ Müller cells, CD40 stimulation caused PCD of RECs that was not impaired by L-Nil, α-lipoic acid, or anti-Fas ligand antibody. We found CD40 did not trigger TNF-α or IL-1β secretion. Primary Müller cells released extracellular ATP in response to CD40 ligation. Inhibition of P2X7 (A-438079) impaired PCD of RECs; CD40 upregulated P2X7 in RECs, making them susceptible to ATP/P2X7–mediated PCD. Diabetic mice upregulated P2X7 in the retina and RECs in a CD40-dependent manner.
Conclusions
Cluster of differentiation 40 induces PCD of RECs through a dual mechanism: ATP release by Müller cells and P2X7 upregulation in RECs. These findings are likely of in vivo relevance since CD40 upregulates P2X7 in RECs in diabetic mice and CD40 is known to be required for retinal capillary degeneration.
... ADP was also recognized as a substrate, indicating that this enzyme is an authentic nucleoside triphosphate diphosphohydrolase as described in other cells [2,13,20]. Trypanosoma brucei brucei and Leishmania amazonensis are pathogens which cannot synthesize purines de novo [6,51]; hence, it has been postulated that these ecto-ATPases in protozoa parasites play a role in the salvage of purines from the host cells [6,43] L. donovani might sequentially dephosphorylate ATP to adenosine: ATP ? ADP ? ...
In this work, we have described the expression of ecto-ATPDase on the external surface of Leishmania donovani. This enzyme has the ability to hydrolyze extracellular ATP. There is a low level of ATP hydrolysis in the absence of divalent cation 2.5 ± 0.51 nM Pi 10(7) cells/h which shows the divalent cation-dependent activity of this enzyme in the intact parasite. However, MgCl2 stimulated the ATP hydrolysis to a greater extent compared with CaCl2 and ZnCl2. This activity was also observed when replaced by MnCl2. The Mg-dependent ecto-ATPase activity was 46.58 ± 6.248 nM Pi 10(7) cells/h. The apparent K m for ATP was 5.76 mM. Since Leishmania also possesses acid phosphatase activity and to discard the possibility that the observed ATP hydrolysis was due to acid phosphatase, the effect of pH was examined. In the pH range 6.0-9.0, in which the cells were viable, the phosphatase activity decreased while ATPase activity increased. To show that the observed ATP hydrolysis was not due to phosphatase or nucleotidase activity, certain inhibitors for these enzymes were tested. Vandate and NaF inhibited the phosphatase activity; Ammonium molybdate inhibited 5'-nucleotidase activity, but these inhibitors did not inhibit the observed ATP hydrolysis. However, when ADP was used as a substrate, there was no inhibition of ATP hydrolysis showing the possibility of ATP diphosphohydrolase activity. To confirm that this Mg-dependent ATPase activity is an ecto-ATPase activity, we used an impermeable inhibitor, 4,4'-diisothiocyanostilbene 2,-2'-disulfonic acid, as well as suramin, an antagonist of P2-purinoceptors and inhibitor of some ecto-ATPases. These two reagents inhibited the Mg(2+)-dependent ATPase activity in a dose-dependent manner. The presence of L. donovani E-NTPDase activity was demonstrated using antibodies against NTPDase by Western blotting and flow cytometry. The presence of Mg(2+)-dependent ATP diphosphohydrolase activity on the surface of L. donovani modulates the nucleotide concentration and protects the parasite from the lytic effects of the nucleotides mainly ATP. Ecto-ATPDase from L. donovani may be further characterized as a good antigen and as a target for immunodiagnosis and drug development, respectively.
... Based on the above observations, these investigators suggested that lymphocyte-induced tumor destruction may use eATP as a mediator in a lytic pathway (14,41,42). Direct evidence for this proposal, although quite limited, is nevertheless compelling. ...
Extracellular adenosine triphosphate (eATP) has been suggested to play a role in lymphocyte-induced tumor destruction. We now provide evidence that a protein responsible for ATP synthesis in mitochondria may also play a physiologic role in major histocompatibility complex-independent, lymphocyte-mediated cytotoxicity. A 51.5-kD protein (p51.5) bearing structural and immunologic characteristics of the beta subunit of H+ transporting ATP synthase (E.C. 3.6.1.34, beta-H+ATPase, published molecular mass of 51.6 kD) was detected on the plasma membrane of three different human tumor cell lines studied. NH2-terminal amino acid sequence analysis of purified p51.5 from K562 tumor cells revealed 100% homology of 16 residues identified in the first 21 positions to the known sequence of human mitochondrial beta-H+ ATPase. Antibody directed against a 21-mer peptide in the ATP binding region of beta-H+ ATPase (anti-beta) reacted with only one band on Western blots of whole tumor extracts and tumor membrane extracts suggesting that the antiserum reacts with a single species of protein. Anti-beta reacted with the cell membranes of tumor cells as determined by fluorescence-activated flow cytometry and immunoprecipitated a 51.5-kD protein from surface-labeled neoplastic cells (but not human erythrocytes and lymphocytes). Purified p51.5 bound to human lymphocytes and inhibited natural killer (NK) cell-mediated cytotoxicity. Furthermore, anti-beta treatment of the K562 and A549 tumor cell lines inhibited NK (by > 95%) and interleukin 2-activated killer (LAK) cell (by 75%) cytotoxicity, respectively. Soluble p51.5 upon binding to lymphocytes retained its reactivity to anti-beta suggesting that the ATP binding domain and the lymphocyte-receptor binding domain reside in distinct regions of the ligand. These results suggest that beta-H+ ATPase or a nearly identical molecule is an important ligand in the effector phase (rather than the recognition phase) of a cytolytic pathway used by naive NK and LAK cells.
... A second and unique response is the sustained membrane depolarization and increase in cytosolic-free calcium by the opening of a nonselective transmembrane pore which is permeable to hydrophilic molecules of up to 900 D. This increase in membrane permeability finally results in the induction of cell death. In many cases, ATP-induced cytotoxicity is mediated by classical alterations of apoptosis, including membrane blebbing, nuclear condensation, and DNA fragmentation (9,18,58). In addition, in lipopolysaccharide (LPS) 1 -primed macrophages and microglial cells, it has been observed that stimulation with ATP induces the rapid release of the The Journal of Cell Biology, Volume 139, 1997 1636 proinflammatory cytokine interleukin (IL)-1  (19,22). ...
Cells of the macrophage lineage express a peculiar surface receptor for extracellular ATP, designated P2Z/P2X7 purinergic receptor, that induces pore formation and collapse of the plasma membrane potential. Although the function of the P2Z receptor is largely unknown, accumulating evidence implicates its role in cell signaling and immune reactions. Here, we investigated the effect of P2Z receptor ligation on the activation of NF-κB, a transcription factor controlling cytokine expression and apoptosis. Exposure of microglial cells to ATP but not other nucleotides resulted in potent NF-κB activation. This effect was specifically mediated by the P2Z receptor, because selective receptor antagonists prevented NF-κB activation. NF-κB activation required reactive oxygen intermediates and proteases of the caspase family, because it was abolished by antioxidants and specific protease inhibitors. The subunit composition of the ATP-induced NF- κB–DNA complex was rather unusual. Whereas exposure to LPS-induced prototypical NF-κB p50 homo- and p65 (RelA)/p50 heterodimers, ATP stimulation resulted in the sole appearance of a p65 homodimer. This is the first demonstration that a certain stimulus activates a particular NF-κB subunit. Because different NF-κB complexes exhibit distinct transcriptional and DNA-binding activities, ATP may control the expression of a subset of NF-κB target genes distinct from those activated by classical proinflammatory mediators.
... 6 It was originally put forward that P2X 7 R might participate in shedding of CD23 and CD62L or killing by apoptosis. 4,20,21 We have previously observed that transfection of P2X 7 R, the most widely diffused P2X receptor subtype in immune cells, confers a substantial proliferative advantage when transfected into human B lymphoblastoid cells that lack it constitutively. 14 The P2X 7 R transfectants become able to grow in the absence of serum, show an elevated resting [Ca ϩϩ ] i , respond to ATP, and secrete large amounts of this nucleotide into the culture supernatant. ...
Human leukocytes express a receptor for extracellular nucleotides, named P2X7R, that in lymphocytes can either mediate cell death or proliferation, depending on the level of activation. The authors have investigated P2X7R expression and function in 21 patients affected by B-cell chronic lymphocytic leukemia, 13 with an evolutive and 8 with an indolent variant of the disease. Resting cytoplasmic Ca++ concentration was significantly higher in lymphocytes from patients with the evolutive compared with indolent variant. Furthermore, in the former, P2X7R stimulation triggered a Ca++ influx significantly larger. Higher Ca++ influx correlated with an increased P2X7R expression in the lymphocytes from patients with the evolutive form. Finally, incubation in the presence of extracellular adenosine triphosphate decreased spontaneous proliferation of lymphocytes from patients affected with the evolutive variant but had no effects on lymphocytes from patients with the indolent form. These results suggest that expression and function of P2X7R may correlate with the severity of B-cell chronic lymphocytic leukemia.
... However it has been convincingly shown that also in this case, the final triggering event is the K + decrease secondary to the increase in cyto-plasmic Ca 2+ ([Ca 2+ ]i) [20]. Changes in the intracellular K + concentration might also be responsible of the strong proapoptotic activity of P2X7 [151], since, as mentioned above, cleavage of caspase-3 and -9 is triggered by low K + . The large P2X7R-dependent increases in [Ca 2+ ]i sets in motion several Ca 2+ -dependent intracellular pathways. ...
Until recently, P2X receptors have not received much attention in the context of immunology and inflammation. While this is justified to a certain extent for P2X1, P2X2, P2X3, P2X5 and P2X6, which still await identification of a convincing role in the pathophysiology of immune cells, it is clearly not any more the case for P2X4 and even more so for P2X7, a molecule that has achieved the status of an essential, non-redundant, immunomodulatory receptor. In this review I will highlight the most important inflammatory responses participated by P2X receptors.
... Unlike apoptosis, in which cells form membrane blebs that are phagocytized in a noninflammatory manner, pyroptotic cells swell and burst. This releases intracellular damageassociated molecular patterns, such as histones [6] and ATP [7], that are thought to induce the inflammatory cascade in surrounding cells and tissues. Importantly, the caspase-1 substrate(s) that mediate pyroptosis are unknown, however this process is independent of IL-1β and IL-18 [8], as neither is sufficient or required for pyroptotic stimulation. ...
... For example, human monocytes primed by bacterial endotoxin/ lipopolysaccharide (LPS) respond to extracellular ATP with the caspase1 dependent proteolytic maturation and externalization of IL1-β (Hogquist KA et al 1991;Perregaux D et al 1994;. Apoptosis is another well-known consequence of P2X7 receptor activation in various types of leukocytes (Zanovello et al. 1990;Zheng et al. 1991). ...
... This is reflected by either dramatic growth of current amplitude, by sustained tail currents, or by both (Chessell et al., 1997;Chessell et al., 2001). This occurrence often results in cell lysis (Zanovello et al., 1990;Zheng et al., 1991), resultant lactate dehydrogenase (LDH) release and is the reason why the P2X7 receptor had previously been referred to as the "cell death receptor". ...
P2X7 receptors belong to a family of membrane bound ion channels which are activated by extracellular ATP, resulting in the opening of a non-selective cation channel. After prolonged or repeated exposure to agonist, functional and cellular changes can occur, including the formation of a large pore, cell lysis and the release of mature, biologically active interleukin-1β. It is this diversity of functions that underlies the significance of this receptor in pain processing.
P2X7 receptors are expressed on microglia, which when activated, release a host of mediators which contribute to central sensitisation, a phenomenon associated with neuropathic pain. The role of P2X7 receptors in the activation of microglia is less well established and is the main subject of this thesis.
Before considering the interaction between P2X7 receptors and microglia, the first aim was to establish whether P2X7 receptors played a role in a pathological process known to be associated with microglial activation. An additional aim was to establish whether the site of action was in the central nervous system (CNS), where microglia are located. These aims were accomplished using a surgery-based rat model of neuropathic pain, the chronic constriction injury (CCI) model, and by comparing the effects of different P2X7 receptor antagonists when dosed peripherally or directly into the spinal cord. The results indicated that P2X7 receptor antagonists produced efficacy in the CCI model via a mechanism located in the CNS.
To further investigate the association between P2X7 receptors and microglia, a different experimental paradigm was explored. Chronically dosed morphine is known to activate microglia, the consequence of which is thought to underlie morphine tolerance and reduced morphine analgesia. By administering a P2X7 receptor antagonist to CCI-operated rats treated with chronic morphine, the interaction between the P2X7 receptor and morphine tolerance and analgesia was explored. The results showed that P2X7 receptor antagonism delayed morphine tolerance and increased the efficacy of low doses of morphine, suggesting an association between P2X7 receptors and microglia.
It was intended to confirm the interaction between a P2X7 receptor antagonist and morphine in another neuropathic pain model, namely varicella zoster virus-induced neuropathy. However due to a lack of reproducibility, this model was not used for pharmacological studies.
Having demonstrated an association between P2X7 receptor antagonist and morphine in a chronic pain setting, studies were initiated to explore whether this interaction occurred in other morphine-related behaviours. The effect on body weight, motor coordination and single dosed morphine-induced analgesia was assessed in rats co-administered with P2X7 receptor antagonist and morphine. Results demonstrated that the blockade of P2X7 receptors enhanced morphine acute dose-induced analgesia, but had no influence on motor-impairment and body weight.
The final part of the thesis used immunohistochemical and molecular techniques to confirm that microglia played a role in established allodynia induced by CCI-surgery and that P2X7 receptors directly influenced microglia-activation.
In conclusion, the data in this thesis has illustrated an association between centrally activated P2X7 receptors and microglia, as well as an association between the P2X7 receptor and morphine-induced tolerance and analgesia. It is possible that co-administration of a P2X7 receptor antagonist with morphine could reduce the effective dose of morphine clinically, thereby reducing the side effects of this commonly used analgesic.
... P2X7 is historically known for its ability of ATP to induce cell death through apoptosis (Zanovello et al., 1990;Chow et al., 1997;Ferrari et al., 1999). A role in cell proliferation would seem to be at odds with this, but evidence suggests that in certain cell types, activation of P2X7 is more likely involved with proliferation than cell death. ...
The P2X7 receptor is a trimeric ATP-gated cation channel found predominantly, but not exclusively, on immune cells. P2X7 activation results in a number of downstream events, including the release of proinflammatory mediators and cell death and proliferation. As such, P2X7 plays important roles in various inflammatory, immune, neurologic and musculoskeletal disorders. This review focuses on the use of P2X7 antagonists in rodent models of neurologic disease and injury, inflammation, and musculoskeletal and other disorders. The cloning and characterization of human, rat, mouse, guinea pig, dog, and Rhesus macaque P2X7, as well as recent observations regarding the gating and permeability of P2X7, are discussed. Furthermore, this review discusses polymorphic and splice variants of P2X7, as well as the generation and use of P2X7 knockout mice. Recent evidence for emerging signaling pathways downstream of P2X7 activation and the growing list of negative and positive modulators of P2X7 activation and expression are also described. In addition, the use of P2X7 antagonists in numerous rodent models of disease is extensively summarized. Finally, the use of P2X7 antagonists in clinical trials in humans and future directions exploring P2X7 as a therapeutic target are described.
... While P1 and P2Y subtypes are G-protein-coupled metabotropic receptors, P2X receptors are resembled as homo-or hetero-trimeric ligand-gated ion channels from seven possible subunits. The ion channels formed by P2X1-P2X7 subunits are permeable to Na + , K + and Ca 2+ ions, while at high agonist concentrations P2X7 receptor (P2X7R) subtypes assemble cation ion channels that are capable of pore forming, allowing the unselective flow of compounds with molecular masses of 700Da besides the uncontrolled entry of ions, including Ca 2+ , into the cell which may induce intrinsic cell death programs [1,2,3]. Moreover, the P2X7R has an intracellular domain that couples receptor activation to intracellular signaling events and is classically involved with apoptosis [4,5]. ...
Novel developmental functions have been attributed to the P2X7 receptor (P2X7R) including proliferation stimulation and neural differentiation. Mouse embryonic stem cells (ESC), induced with retinoic acid to neural differentiation, closely assemble processes occurring during neuroectodermal development of the early embryo.
P2X7R expression together with the pluripotency marker Oct-4 was highest in undifferentiated ESC. In undifferentiated cells, the P2X7R agonist Bz-ATP accelerated cell cycle entry, which was blocked by the specific P2X7R inhibitor KN-62. ESC induced to neural differentiation with retinoic acid, reduced Oct-4 and P2X7R expression. P2X7R receptor-promoted intracellular calcium fluxes were obtained at lower Bz-ATP ligand concentrations in undifferentiated and in neural-differentiated cells compared to other studies. The presence of KN-62 led to increased number of cells expressing SSEA-1, Dcx and β3-tubulin, as well as the number of SSEA-1 and β3-tubulin-double-positive cells confirming that onset of neuroectodermal differentiation and neuronal fate determination depends on suppression of P2X7R activity. Moreover, an increase in the number of Ki-67 positive cells in conditions of P2X7R inhibition indicates rescue of progenitors into the cell cycle, augmenting the number of neuroblasts and consequently neurogenesis.
In embryonic cells, P2X7R expression and activity is upregulated, maintaining proliferation, while upon induction to neural differentiation P2X7 receptor expression and activity needs to be suppressed.
... In native cells, ATP elicits different effects over a wide concentration range. For instance, ATP increases the cell membrane permeability in T lymphocytes, but does not cause lysis at concentrations below 100 µM (El-Moatassim et al. 1989), whereas in the millimolar range ATP induces cytolysis and the subsequent death of T lymphocytes (Di Virgilio et al. 1989;Filippini et al. 1990;Zanovello et al. 1990). Furthermore, immunomodulatory effects, such as the inhibition of human natural killer cell reactivity (Schmidt et al. 1984) and the inhibition of macrophagemediated cytotoxicity (Cameron, 1984), have been observed to occur at < 100 µM ATP, whereas at least 500 µM ATP was necessary for ATP-induced killing of mycobacteria by human macrophages (Lammas et al. 1997). ...
• The effect of the agonist ATP on whole cell currents of Xenopus oocytes expressing either the wild-type human P2X7 receptor (hP2X7), an N-terminally hexahistidyl-tagged hP2X7 receptor (His-hP2X7), or a truncated His-hP2X7 receptor (His-hP2X7ΔC) lacking the C-terminal 156 amino acids was investigated using the two-microelectrode voltage clamp technique. • The activation time course of the wild-type hP2X7 receptor can be described as the sum of an exponentially growing and an additional almost linearly activating current component. • The amplitude of the exponentially activating current component of the wild-type hP2X7 receptor displayed a biphasic dependence on the agonist concentration, which could be best approximated by a model of two equal high-sensitivity and two equal low-sensitivity non-cooperative activation sites with apparent dissociation constants of about 4 and 200 mfree ATP4-, respectively. • The linearly activating current was monophasically dependent on the agonist concentration with an apparent dissociation constant of about 200 m. • The contribution of the low-sensitivity sites to current kinetics was reduced or almost abolished in oocytes expressing His-hP2X7 or His-hP2X7ΔC. • Our data indicate that the hP2X7 receptor possesses at least two types of activation sites, which differ in ATP4- sensitivity by a factor of 50. The degree of occupation of these two sites influences both activation and deactivation kinetics. Both N- and C-terminal domains appear to be important determinants of the current elicited by activation of the sites with low ATP sensitivity, but not for that mediated by the highly ATP-sensitive sites.
p>The initial work aimed to characterise monocyte-derived dendritic cells (Mo-DCs) from normal and asthmatic subjects to identify inherited differences with possible relevance for allergic asthma. It was found that Mo-DCs from asthmatic subjects expressed significantly lower level of CD23 at their surface and produced more IL-6 than Mo-DCs from normal subjects.
A primary screen was then developed to address the hypothesis. The primary screen aimed to identify individual agents found at elevated levels in the asthmatic lung, referred to as allergic mediators, that significantly change the phenotype or function of Mo-DCs. Further in-depth investigation of their effects on Mo-DCs and possible relevance in allergic asthma would then be conducted. The effects of allergic mediators on Mo-DCs from both normal and asthmatic subjects were studied. The experimental approach taken showed that the Mo-DC phenotype and function was significantly changed in response to TNF-α (the positive control), IFN-γ, IL-3, IL-5 and IgE, but not in response to MMIP-1α, histamine, PGD<sub>2</sub> and IL-13. Four areas of particular interest were identified. 1) Phenotypically mature IFN-γ treated Mo-DCs failed to significantly enhance T cell proliferation. 2) CD23 disappeared from the surface of Mo-DCs in response to TNF-α, IFN-γ and IgE. 3) IL-3 and IL-5 frequently induced a mature Mo-DC phenotype. 4) Mo-DCs from normal and asthmatic subjects responded differently to allergic mediators.
The work presented in this thesis supports a role for DCs in allergic asthma. Evidence also suggested that DCs from asthmatic subjects are more prone to initiate or perpetuate the chronic allergic inflammation seen in asthmatic airways.</p
For many years the P2X7 receptor (P2X7R) was considered the prototypic cytolytic receptor due to its ability to cause dramatic changes in plasma membrane permeability, eventually leading to cell death. However, later studies revealed that controlled P2X7R activation has beneficial effects on cell metabolism and nowadays our perception of the physiological role of this receptor has radically changed. Some of the biochemical pathways underlying the trophic effect of the P2X7R are being unveiled, thus disclosing an unanticipated role of P2X7Rs in mitochondrial and glycolytic metabolism. We provide here an update of the effects of the P2X7R on cell energy metabolism.
The role of the target cell in its own death mediated by cytotoxic T lymphocytes (CTL) has been controversial. The ability of the pore-forming granule components of CTL to induce target cell death directly has been taken to suggest an essentially passive role for the target. This view of CTL-mediated killing ascribes to the target the single role of providing an antigenic stimulus to the CTL; this signal results in the vectoral degranulation and secretion of pore-forming elements onto the target. On the other hand, by a number of criteria, target cell death triggered by CTL appears fundamentally different from death resulting from membrane damage and osmotic lysis. CTL-triggered target cell death involves primary internal lesions of the target cell that reflect a physiological cell death process. Orderly nuclear disintegration, including lamin phosphorylation and solubilization, chromatin condensation, and genome digestion, are among the earliest events, preceding the loss of plasma membrane integrity. We have tested directly the involvement of the target cell in its own death by examining whether we could isolate mutants of target cells that have retained the ability to be recognized by and provide an antigenic stimulus to CTL while having lost the capacity to respond by dying. Here, we describe one such mutant, BW87. We have used this CTL-resistant mutant to analyze the mechanisms of CTL-triggered target cell death under a variety of conditions. The identification of a mutable target cell element essential for the cell death response to CTL provides genetic evidence that target cell death reflects an active cell suicide process similar to other physiological cell deaths.
In human and rodent macrophages, activation of the P2X7 nucleotide receptor stimulates interleukin-1β processing and release, apoptosis, and killing of intracellular Mycobacterium tuberculosis. Signaling pathways downstream of this ionotropic ATP receptor are poorly understood. Here we describe the rapid activation of the stress-activated protein kinase (SAPK)/JNK pathway in BAC1 murine macrophages stimulated by extracellular ATP. Brief exposure of the cells to ATP (10–30 min) was sufficient to trigger a rapid accumulation of activated SAPK that was then sustained for >120 min. Several observations indicated that the P2X7 receptor mediated this effect. 1) ATP and 3′-O-(4-benzoyl)benzoyl-ATP were the only agonistic nucleotides. 2) The effect was inhibited by oxidized ATP and the isoquinoline KN-62, two known P2X7 receptor antagonists. 3) ATP-induced SAPK activation could be recapitulated in P2X7 receptor-transfected HEK293 cells, but not in wild-type HEK293 cells. Because P2X7 receptor stimulation can rapidly activate caspase family proteases that have been implicated in the induction of the SAPK pathway, we investigated whether ATP-dependent SAPK activation involved such proteases. Brief exposure of BAC1 macrophages to extracellular ATP induced DNA fragmentation, α-fodrin breakdown, and elevated levels of caspase-3-type activity. Asp-Glu-Val-Asp-cho, a caspase-3 inhibitor, inhibited ATP-induced DNA fragmentation and α-fodrin proteolysis, but had no effect on ATP-induced SAPK activation. Tyr-Val-Ala-Asp-chloromethyl ketone, a caspase-1 inhibitor, prevented ATP-induced release of processed interleukin-1β, but not ATP-dependent SAPK activity. We conclude that activation of ionotropic P2X7 nucleotide receptors triggers a strong activation of SAPK via a pathway independent of caspase-1- or caspase-3-like proteases.
ATP is released in the body from several cells under various physiological and pathological conditions. A number of authors have postulated a role for extracellular ATP (ATP o ) as a neurotransmitter, a secretagogue or an inflammatory mediator. Here, we propose an additional role for ATP o , as a cytotoxic factor, and discuss in vitro experiments showing that this nucleotide causes cell death by two mechanisms: colloido-osmotic lysis and apoptosis.
The present study shows that human mononuclear phagocytes express a P2Z- like purinergic membrane receptor activity. Extracellular adenosine triphosphate (ATP) induces the formation of nonselective membrane pores in human mononuclear phagocytes that allow the entry of otherwise membrane impermeant fluorescent dyes (YO-PRO-1 or Lucifer yellow) into the cytoplasm of these cells. The percentage of mononuclear phagocytes that was permeabilized by ATP increased as monocytes matured into macrophages. Their response to ATP was inhibited by Mg2+ and oxidized ATP. Benzoylbenzoic-ATP (BzBzATP) was approximately 60% as effective as ATP and adenosine-5 -O-(thiophosphate) (ATP gamma S) was less than 20% as effective as ATP in permeabilizing human macrophages to YO-PRO-1 or Lucifer Yellow. Thus, the human P2Z-like receptor differs from its murine counterpart because BzBzATP, ATP, and ATP gamma S are equally efficacious in permeabilizing murine macrophage-like J774 cells to these dyes. UTP, GTP, and CTP were ineffective in permeabilizing human or murine macrophages to YO-PRO-1. Taken together, these data indicate that human monocyte-derived macrophages express a P2Z-like activity that is pharmacologically distinct from that expressed by their murine counterparts and that expression of these receptors is developmentally regulated in human mononuclear phagocytes.
The present study shows that extracellular adenosine triphosphate (ATP) has the capacity to mediate dose-dependent lysis of the monocytic leukemia cell line THP-1. The lysis, assessed by 51Cr release, was found to be selective for ATP, because adenosine diphosphate (ADP) or other nucleotides were less effective in their ability to lyse the cells. The amount of 51Cr released was particularly enhanced by the stimulation of the cells with 1,000 U/mL of interferon gamma (IFN- gamma) for 3 days, and the sensitivity was time and dose dependent. Analysis of the mechanism of lysis indicated that the fully ionized form, ATP4-, mediated the lysis, because the addition of cation chelators or the absence of the divalent cations, Ca2+ and Mg2+, in the culture medium of a 6-hour 51Cr release assay increased the percent specific lysis. Therefore, the ATP receptors on THP-1 cells were classified as P2Z purinoceptors. Moreover, it is shown here that the Ca2+/calmodulin complex plays a role in the regulation of the lysis by extracellular ATP of THP-1 cells, because antagonists of this complex, such as trifluoperazine or KN-62, were found to inhibit the ATP- mediated cell lysis.
Approximately 30-40% of patients with Parkinson's disease (PD) exhibit cognitive impairments. However, there are currently no clinically effective drugs for the treatment of cognitive impairment in patients with PD. Previous studies have suggested that mitochondrial dysfunction such as decreased adenosine triphosphate (ATP) production triggers dopaminergic neurodegeneration in patients with PD and that mitochondria represent a potential target for the development of novel treatments for preventing PD. Therefore, in the present study, we investigated the cognition-enhancing effects of ethyl pyruvate (EP) and 1-(3,4-dimethoxyphenethyl)-4-(3-phenylpropyl) piperazine dihydrochloride (SA4503) in mice with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinsonism. PD model mice were generated via treatment with MPTP (25 mg/kg, i.p.) once a day for 5 consecutive days. Twenty-four hours after the final injection of MPTP, mice were intraperitoneally injected with EP (25, 50, 100 mg/kg) or SA4503 (1 mg/kg) once a day for 4 weeks. Chronic administration of EP (100 mg/kg i.p.) or SA4503 (1 mg/kg, i.p.) improved both motor deficits and cognitive impairments in MPTP-treated mice. Furthermore, treatment with EP or SA4503 attenuated decreases in the levels of ATP and tyrosine hydroxylase (TH) in the substantia nigra pars compacta (SNpc)/ventral tegmental area (VTA), striatum, and hippocampal CA1 region. Administration of EP or SA4503 protected the dopaminergic neurons from MPTP-induce toxicity and restored the dopamine levels in the striatum. Elevated 4-hydroxy-2-nonenal- (4-HNE-) and nitrotyrosine-reactive protein levels induced by MPTP-treatment were suppressed by EP or SA4503 treatment in the SNpc-VTA, striatum, and hippocampal CA1 region. These observations suggest that EP and SA4503 attenuate cognitive impairments and motor dysfunction in mice with MPTP-induced PD.
Adenosine triphosphate (ATP) accumulates at sites of tissue injury and inflammation. Effects of extracellular ATP are mediated by plasma membrane receptors named P2 receptors (P2Rs). The P2R most involved in inflammation and immunity is the P2X7 receptor (P2X7R), expressed by virtually all cells of innate and adaptive immunity. P2X7R mediates NLRP3 inflammasome activation, cytokine and chemokine release, T lymphocyte survival and differentiation, transcription factor activation, and cell death. Ten human P2RX7 gene splice variants and several SNPs that produce complex haplotypes are known. The P2X7R is a potent stimulant of inflammation and immunity and a promoter of cancer cell growth. This makes P2X7R an appealing target for anti-inflammatory and anti-cancer therapy. However, an in-depth knowledge of its structure and of the associated signal transduction mechanisms is needed for an effective therapeutic development.
In 1967 the late Zanvil A. Cohn reported that adenine nucleotides had striking effects on the morphology and pinocytic activity of mouse macrophages [1]. These early studies were followed by the demonstration by Sung et al. that extracellular ATP (ATPe) caused plasma membrane depolarization, increased intracellular Ca2+ ([Ca2+]i), and inhibition of Fc receptor-mediated phagocytosis [2]. These intriguing effects remained unexplained until the work by Steinberg and Silverstein, who started a thorough investigation of the mechanism of macrophage responses to extracellular nucleotides, ATP in particular [3,4].
Purinergic signalling plays a crucial role in immunity and autoimmunity. Among purinergic receptors, the P2X7 receptor (P2X7R) has an undisputed role as it is expressed to high level by immune cells, triggers cytokine release and modulates immune cell differentiation. In this review, we focus on evidence supporting a possible role of the P2X7R in the pathogenesis of systemic lupus erythematosus (SLE).
P2X receptors are ligand-gated ion channels that activate within milliseconds of agonist binding, causing rapid cellular depolarization and excitation. This makes them ideally suited to mediate the rapid neurotransmitter functions of adenosine 5′-triphosphate (ATP). In the periphery the roles of ATP as an excitatory cotransmitter from sympathetic and parasympathetic nerves have been extensively characterized (Burnstock and Kennedy, 1986; Burnstock, 1990). More recently ATP was also identified as a fast excitatory neurotransmitter in the brain (Edwards et al., 1992). In this section we will discuss the biophysical and pharmacological properties of the P2X receptors that underlie these actions.
Natural killer (NK) cells are CD3−, T-cell receptor (TCR)−, large granular lymphocytes (LGL) that express spontaneous lytic activity against tumor cells, virally infected cells, and perhaps certain hematopoietic progenitor cells (Trinchieri, 1989). Understanding the cellular and molecular mechanisms by which NK cells recognize and destroy target cells has become an area of considerable interest. Previous studies (Henney, 1973; Herberman et al., 1986) with NK cells have proposed to divide the process of NK killing into four identifiable stages, consisting of: (1) target cell binding (adhesion), (2) effector cell activation (recognition/ signal transduction), (3) delivery of the lethal signal to the target (lethal hit), and (4) effector cell detachment and recycling. Ca2+ plays a central role in the killing process, yet only recently has it been possible to delineate more clearly the site(s) of Ca2+ requirements in the lytic mechanism. Early studies involving cytotoxic T lymphocyte (CTL) models and more recent studies with NK cells demonstrated that Ca2+ was required at a point in the lytic process distal to Mg2+-dependent target cell adhesion but proximal to target cell disintegration (Roder and Haliotis, 1980; Quan et al., 1982; Martz et al., 1983; Berke, 1989). Recent evidence supported a role for Ca2+ in the activation of a stimulus-secretion response by killer cells. It also suggested that Ca2+ can be a potent toxic agent if allowed to accumulate at high concentrations in target cells. Therefore, Ca2+ appears to be required not only to activate killer cell function but must also enter the killer cell to activate additional processes related to stimulus-secretion coupling. Analysis of the lethal hit by which killer lymphocytes mediate target cell damage has focused on CTL and their interactions with specific target cells (Roder and Haliotis, 1980; Quan et al., 1982). It suggested, but did not prove, that during “programming for lysis” the killer cells may deposit (or secrete) materials onto the target cell that mediate the lytic signals. The hypothesis that material(s) were transferred from a killer cell to a target cell was a requisite for this target cell lysis model (Henkart and Henkart, 1982). Collectively, numerous data support this hypothesis as a general phenomenon common to numerous types of killer cells. The observation that NK cells and in vitro- activated CTL contain intracytoplasmic azurophilic granules suggested that these granules may be released during the Ca2+-dependent stage of killing and may contain materials capable of mediating target cell lysis.
There is excellent evidence that T cells are required for control of most virus infections and that cloned cytotoxic T lymphocytes (CTL) are sufficient for such control (reviewed in Martz and Howell, 1989; see also Moskophidis et al., 1989). In three cases that have been tested, control of one virus fails to control a second virus in a mixed infection (Lukacher et al., 1984; Maclntyre et al., 1985; Scherle et al., 1992). This evidence for control by a bystander-sparing effector action is consistent with control by the contact-dependent lytic action of CTL. It speaks against a nonspecific CTL effector mechanism such as release of inter-ferons or other antireplicating cytokines, or activation of phagocytes by cytokines released by CTL and other cells, at least to the extent that the actions of such mediators would be “long-range,” spilling into the compartment infected with bystander virus.
Platelets, erythrocytes and mast cells have been a focus of interest in purinergic receptor biology for many years. These cells have been invaluable for the development of new pharmacological agonists and antagonists (notably platelets), the molecular cloning of members of the P2Y family, the dissection of the signal transduction pathways (especially erythrocytes), and the identification recently of the elusive permeabilizing ATP receptor (mast cells). Platelets, erythrocytes and mast cells not only express P2 receptors but also release ATP, thus setting the stage for an autocrine/paracrine loop that might have a key role in the control of local blood flow and tissue responses to noxious agents.
The nature of the so-called lethal hit that causes target cells (TC) to die after a relatively short encounter with cytotoxic T lyphocytes (CTL) is of great interest not only to immunologists. This problem is also very challenging to those who realize that CTL-TC interactions present an interesting model system in which contacts between two different cells-(CTL) and antigen (Ag)-bearing (TC)-result in dramatic consequences for both participants: the CTL is activated due to the crosslinking of the T-cell receptor (TCR) with the Ag. whereas the TC eventually dies (Goldstein, 1987).
Although the phenomenon of lymphocyte-mediated cytotoxicity is well known, the underlying mechanisms are at present a hotly debated issue because the molecular participants involved still elude definition (Berke, 1991; Krahenbuhl and Tschopp, 1991). Although perforin is a well-characterized cytocidal mediator, much evidence indicates that other molecules are implicated (Trenn et al., 1987). In the last three years, we have explored the possibility that extracellular ATP (ATPo) might constitute a mediator of lymphocyte-mediated cytotoxicity (Di Virgilio et al., 1989; Zanovello et al., 1990; Di Virgilio et al., 1990)
Despite decades of work and much real progress, the mechanisms by which cytotoxic T lymphocytes (CTL) damage target cells remain largely obscure. In addition, the question has been raised whether cytolysis is the primary antiviral effector mechanism employed by CTL in vivo (Martz and Howell, 1989). Greater progress has been made in understanding the mechanisms by which CTL adhere to targets. Many of the molecules involved in recognition and adhesion have been identified, although the mechanism of adhesion-strengthening, the recognition-controlled rapid increase in avidity of adhesion molecules for target ligands, remains to be elucidated.
The P2X7 plasma membrane receptor is an intriguing molecule that is endowed with the ability to kill cells, as well as to activate many responses and even stimulate proliferation. Here, the authors give an overview on the multiplicity and complexity of P2X7-mediated responses, discussing recent information on this receptor. Particular attention has been paid to early and late signs of apoptosis and necrosis linked to activation of the receptor and to the emerging field of P2X7 function in carcinogenesis.
This chapter discusses the three properties of intravenously administered adenosine triphosphate (ATP) that contribute to its efficacy and are expected to significantly affect the outcomes of patients with advanced cancer. The three properties of ATP discussed in the chapter are cytolytic activities of ATP, the induction of resistance by ATP, and anticachexia effects of ATP. The cytolytic activities of ATP are attributed to both purinergic receptors and non- receptor-mediated mechanisms. The induction of resistance by ATP induces resistance to chemo- and radiation therapy in normal tissues. The third proven activity of ATP (anticachexia effects)—its ability to effectively expand the liver, red blood cells, and blood plasma ATP pools—is expected to significantly contribute to a favorable outcome in the treatment of advanced cachectic cancers, especially in older patients, by delivering purines to purine-depleted peripheral sites. A large amount of data establishes that on aging and in cachectic disease models, a significant depletion of purines in vivo can be easily noted. The anticachexia effects of ATP translate into significant improvements in the quality of life parameters in the advanced disease patients. The anticachexia effects of ATP and its effectiveness in delivering purines to purine-depleted peripheral sites act to protect the host from the otherwise devastating effects of high-dose cytotoxic and/or radiation therapy.
Twenty years ago Gomperts and Cockcroft were the first to describe ATP-dependent permeability increases in a cell type involved in the immune response (mast cells), hypothesizing that “a possible mechanism would involve channel formation by the aggregation of transmembrane monomeric units…” (Cockcroft and Gomperts 1979). Ever since, similar responses to ATP have been described in many other immune and inflammatory cells (356-1|Table 1), leading many authors to propose that expression of the “ATP permeabilizing receptor” is a feature of cells involved in host defense. In mast cells, as well as in other immune cells, the active form was found to be ATP in its fully dissociated form. Accordingly, this hypothetical receptor was named the ATP4-receptor, later to become P2Z when Gordon (1986) carefully defined the properties of the P2 receptor of mast cells and lymphocytes, and found that this receptor did not entirely fit the P2X/P2Y subclassification originally proposed by Burnstock and Kennedy (1985). The functional “signature” of the P2Z receptor (reversible permeabilization of the plasma membrane) was so peculiar that many doubted the actual “receptor” nature of P2Z, reckoning that ATP might cause in immune cell types a nonspecific perturbation of the plasma membrane that in turn led to permeability transition. However, even before the cloning of the actual molecule responsible for ATP-dependent permeabilization, evidence supporting the receptor nature of P2z was compelling as:
1.
Other nucleosides or nucleotides could not mimic this effect, even at high concentrations (Cockcroftand Gomperts 1980; Steinberg et al. 1987).
2.
It was possible to select from ATP-sensitive lines cell clones fully resistant to ATP (Steinberg and Silverstein 1987; Murgia et al. 1992).
3.
Periodate-oxidized ATP (oATP) was shown to completely block permeabilization, and another ATP analog, 2-methylthio-9-β-l-ribofuranosyladenine 5′-triphosphate (2-MeS-L-ATP) was 50% inhibitory (Murgia et al. 1993; Tatham et al. 1988).
Table 1
Cells expressing the P2X7/P2Z receptor
Cell type
Method
References
Rat mast cells
Functional responses
Cockcroft and Gomperts (1979)
Mouse T and B lymphocytes
Functional responses (?)
Di Virgilio et al. (1989); Filippini et al. (1990a,b); Chused et al. (1996)
Human B lymphocytes
Functional responses (?), Abs, RT-PCR
Wiley and Dubyak (1989); Ferrari et al. (1994); Bretschneider et al.(1995); O.R. Baricordi et al. (in preparation)
Human T lymphocytes
Functional responses (?); RT-PCR
Baricordi et al. (1996); O.R. Baricordi et al. (in preparation)
Mouse macrophages
Functional responses, cloning, Abs, in-situ hybridization, RT-PCR
Steinberg et al. (1987);Surprenant et al. (1996); Chiozzi et al. (1997)
Human macrophages
Functional responses.cloning, Abs, RT-PCR
Hickman et al. (1994);Falzoni et al. (1995);Dubyak et al. (1996); Rassendren et al. (1997)
Mouse/rat microglial cells
Functional responses.Abs, in situ hybridization
Ferrari et al. (1996,1997a);Collo et al. (1997)
Human Langerhans cells
Functional responses
Girolomoni et al. (1993)
Human dendritic cells
Abs
Buell et al. (1998)
Mouse dendritic cells
Functional responses, Abs
Mutini et al. (1999)
Human fibroblasts
Functional responses, Abs, RT-PCR
Solini et al. (1999)
Porcine and bovine endothelial cells
Functional responses.RT-PCR
Von Albertini et al. (1998)
Rat retina neurons
Functional responses, Abs, RT-PCR
Brandle et al. (1998)
Rat mesangial cells
Functional responses, Northern
Schulze-Lohoff et al. (1998)
CHO cells
Functional responses.RT-PCR
Michel et al. (1998)
Rat supraoptic neurons
RT-PCR
Shibuya et al. (1999)
Rat salivary gland
RT-PCR
Turner et al. (1998)
Rat submandibular glands
Functional responses RT-PCR
Alzola et al. (1998)
Rat parotid acinar cells
Functional responses.RT-PCR
Tenneti et al. (1998)
Rat hepatic arteries
RT-PCR
Phillips et al. (1998)
Human saphenous vein smooth muscle
Functional responses, RT-PCR
Cario-Toumaniantz et al. (1998)
Receptors for extracellular nucleotides are widely expressed by mammalian cells. They mediate a large array of responses ranging from growth stimulation to apoptosis, from chemotaxis to cell differentiation and from nociception to cytokine release, as well as neurotransmission. Pharma industry is involved in the development and clinical testing of drugs selectively targeting the different P1 nucleoside and P2 nucleotide receptor subtypes. As described in detail in the present review, P2 receptors are expressed by all tumours, in some cases to a very high level. Activation or inhibition of selected P2 receptor subtypes brings about cancer cell death or growth inhibition. The field has been largely neglected by current research in oncology, yet the evidence presented in this review, most of which is based on in vitro studies, although with a limited amount from in vivo experiments and human studies, warrants further efforts to explore the therapeutic potential of purinoceptor targeting in cancer.
Besides their well-established roles as short-term neurotransmitters, nucleotides and nucleosides are also potent regulators of cell growth and differentiation. Compelling evidence obtained on neural, immune, cardiovascular and respiratory system cells, as well as on many other cell types, suggests that these compounds may act as physiological regulators of the phenotype, proliferation and apoptosis of target cells, with a potential in both development and maturation, and in tissue repair and remodeling after trauma and ischemia. Studies by several authors have also made clear that the trophic actions of nucleotides and nucleosides may be either direct or mediated by modulation of synthesis and release of secondary trophic substances (e.g., polypeptidic growth factors and cytokines). In many instances, the involvement of specific P1 or P2 receptors has been established, which discloses the possibility of regulating the local production of neurotrophins, pleiotrophis, cytokines and other trophic agents via selective purinoceptor ligands. The pharmacological modulation of these receptors may therefore have a therapeutic potential in various diseases, ranging from cancer and neurodegenerative disorders, to prevention of recurrent stroke and promotion of wound and gastric ulcer healing. Drug Dev. Res. 39:393–406, 1996. © 1997 Wiley-Liss, Inc.
Incubation of isolated rat liver nuclei with ATP, NAD⁺, and submicromolar Ca²⁺ concentrations resulted in extensive DNA hydrolysis. Half-maximal activity occurred with 200 nM Ca²⁺, and saturation of the process was observed with 1 µM Ca²⁺. ATP stimulated a calmodulin-dependent nuclear Caa+ uptake system which apparently mediated endonuclease activation. Ca²⁺-activated DNA fragmentation was inhibited by the inhibitor of poly(ADP-ribose) synthetase, 3-aminobenzamide, and was associated with poly(ADP-ribosyl)ation of nuclear protein. The characteristics of this endonuclease activity indicate that it may be responsible for the Ca²⁺-dependent fragmentation of DNA involved in programmed cell death (apoptosis) and in certain forms of chemically induced cell killing.
Addition of 0.4-25 microM extracellular ATP results in transient, dose-dependent increases in cytosolic free calcium measured in Ehrlich ascites tumor cells. In cells incubated with 1 mM extracellular Ca2+, ATP induces a triphasic Ca2+ transient: an initial rapid increase (2-3 s), a second, slower phase of increase (60-90 s), and, finally, a gradual return to near resting [Ca2+]i (4-5 min). Several findings demonstrate that the initial, rapid phase of Ca2+ transient results from a mobilization of Ca2+ from a non-mitochondrial intracellular store, while the second, slow phase of increase is produced by enhanced influx of Ca2+ across the plasma membrane. Successive additions of extracellular ATP can elicit repetitive Ca2+ transients if the initially added ATP is removed either through the action of native ecto-ATPase activity or exogenous hexokinase. Other adenine nucleotides, including non-hydrolyzable ATP analogs, neither alter cytosolic [Ca2+] nor antagonize the ATP-induced effects. Conversely, other nucleotide triphosphates (ITP, UTP, and GTP) induce Ca2+ transients which are identical to those produced by ATP. A variety of experimental results indicate that these actions of ATP and other nucleotide triphosphates are not due to a generalized increase in plasma membrane permeability. The results suggest that, in these transformed cells, ATP may act in a manner similar to other Ca2+ mobilizing hormones and growth factors.
In addition to its important role in intracellular metabolic pathways, ATP appears to function as a neurotransmitter in mammalian neurones. The extracellular effects of ATP are not restricted to neurones. We describe the effects of ATP on transmembrane fluxes of monovalent and divalent cations and on phagocytosis in the J774 mouse macrophage cell line and in mouse macrophages elicited by intraperitoneal injection of thioglycollate broth. Of all nucleotides tested, only ATP is capable of depolarizing the macrophage plasma membrane potential, promoting Na+ influx and K+ efflux, effecting an increase in intracellular free Ca2+, and inhibiting phagocytosis. Nonhydrolyzable ATP analogs had no effect on membrane permeability or phagocytosis. The effect mediated by ATP is not accompanied by an increase in membrane permeability to nucleotides, indicating that the action of ATP is restricted to the external surface of macrophages.
A Lyt-2+, trinitrophenyl-specific, lymphotoxin-secreting, cytotoxic T-cell line, PCl 55, mediates the digestion of target cell DNA into discretely sized fragments. This phenomenon manifests itself within 30 min after effector cell encounter as measured by the release of 3H counts from target cells prelabeled with [3H]deoxythymidine and occurs even at very low effector to target cell ratios (0.25:1). A Lyt-1+, ovalbumin-specific, lymphotoxin-secreting T-helper cell clone, 5.9.24, is also able to mediate fragmentation of target cell DNA over a time course essentially indistinguishable from the cytotoxic T lymphocyte-mediated hit. Cell-free lymphotoxin-containing supernatants also cause release of DNA from targets, although they require a longer time course, on the order of 24 hr. In contrast, lysis of cells by antibody plus complement or Triton X-100 does not result in DNA release even after extended periods of incubation (24 hr). All three treatments that result in the release of DNA from cells cause fragmentation of that DNA into discretely sized pieces that are multiples of 200 base pairs. The results thus suggest that cytotoxic T cells, lymphotoxin-secreting helper clones with cytolytic activity, and lymphotoxin all effect target cell destruction by means of a similar mechanism and that observed differences in time course and the absence of target cell specificity in killing mediated by lymphotoxin may simply reflect differences in the mode of toxin delivery.
The intracellularly trappable fluorescent Ca2+ indicator quin-2 was used to measure free cytosolic Ca2+, [Ca2+]i, in the two highly dedifferentiated tumor cell lines, Ehrlich and Yoshida ascites carcinomas. It was found that these carcinoma cells can trap quin-2 similarly to normal cells, but [Ca2+]i was apparently significantly lower than in any normal cell tested previously with this method. By using a new lipid-soluble heavy metal chelator TPEN (N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine), which crosses artificial and natural membranes, it was found that endogenous heavy metals are responsible for partially quenching quin-2 fluorescence trapped inside the cells. Although the quenching of intracellular quin-2 fluorescence is quantitatively more relevant in these ascites carcinomas, TPEN was effective also in normal cells like lymphocytes and granulocytes. Both in the normal and especially in the malignant cell lines [Ca2+]i can be grossly underestimated at low intracellular quin-2 concentrations. Endogenous heavy metal quenching is thus a potential source of artifact when [Ca2+]i is measured with quin-2. When corrected for quin-2 fluorescence quenching by intracellular heavy metals, [Ca2+]i and basic regulatory mechanisms of [Ca2+]i homeostasis in Ehrlich and Yoshida carcinomas are similar to those of nontransformed cells.
Cyclic AMP powerfully inhibits the fMet-Leu-Phe-dependent respiratory burst and exocytosis of azurophilic and specific granules without affecting Ca2+ release from intracellular stores. The elevation of [Ca2+]i induced by fMet-Leu-Phe is short-lived in cyclic AMP-treated cells and similar to that of untreated cells stimulated in the absence of external Ca2+. Nevertheless, in these latter cells fMet-Leu-Phe induces metabolic activation. We therefore suggest that the inhibitory action of cyclic AMP on neutrophil responses is not due to its effects on [Ca2+]i homoeostasis.
We have previously demonstrated that exogenous ATP can give medullary thymocytes the calcium message required for the induction of their blastogenesis. In the present study, using the highly sensitive calcium indicator Indo-1, we have measured the effect of exogenous nucleotides on the cytosolic-free calcium concentration [Ca2+]i of thymocytes, and determined inositol phosphate (IP) formation in the same cells, in parallel assays. The results were compared to those obtained with the mitogenic lectin concanavalin A (ConA) in similar experiments. They show that ATP does not mobilize calcium from its internal stores but stimulates its influx from the extracellular medium. Nevertheless, these data do not rule out the possibility that the nucleotide acts through specific P2 purinergic sites.
We observed that lymphokine-activated T lymphocytes, obtained in short- and long-term cultures following stimulation with recombinant interleukin-2 (rIL-2), are resistant to cell-mediated cytotoxicity. In particular, lymphokine-activated killer (LAK) cells do not undergo self-lysis or lysis by alloreactive cytotoxic T lymphocytes (CTL), in line with recent reports concerning CTL clones. Similar findings were further confirmed in a lectin-dependent cell cytotoxicity assay. LAK cell lysis resistance was not due to an inability to recognize itself, since inactivated LAK cells used as cold competitors inhibited tumor cell lysis in a dose-dependent manner. In contrast, the addition on Day 0 of irradiated LAK cells or alloreactive CTL, as well as a CTL clone having LAK-like activity to rIL-2-stimulated cultures abrogated or strongly reduced LAK cell generation. Therefore, LAK cell precursors were most likely susceptible to the lytic activity of differentiated cytotoxic cells, as no inhibition was detected when cell to cell contact was prevented by using a diffusible chamber culture system. These findings, on the whole, suggest that the emergence of the lysis-resistant phenotype is most likely the result of a selective process occurring in vitro that leads to the elimination of lysis-susceptible lymphocytes present in culture.
The presence of micromolar concentrations (5 to 100 microM) of adenosine 5'-triphosphate (ATP) in cytotoxicity assays of human natural killer (NK) cells with K562 targets resulted in marked inhibition of NK activity. NK activity of peripheral blood mononuclear cells (PBL) and of purified NK cells (i.e., large granular lymphocytes (LGL] were similarly sensitive to inhibition by ATP. The inhibitory activity was specific to ATP and was not demonstrated with GTP, UTP, CTP, or with other adenosine compounds. This inhibitory activity resulted from an effect of ATP on the effector cells and was not dependent on serum components. ATP did not inhibit binding of the LGL to target cells, and therefore the inhibition by ATP is presumed to be related to some post-recognition metabolic events. Some physiologic role in regulation of NK activity by ATP seems possible, because nonspecific phosphatases (bacterial alkaline phosphatase or human alkaline phosphatase) stimulated NK activity and partially reversed the ATP-induced inhibition of reactivity.
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