Purinergic modulation of interleukin-1 beta release from microglial cells stimulated with bacterial endotoxin.

Microglial cells express a peculiar plasma membrane receptor for extracellular ATP, named P2Z/P2X7 purinergic receptor, that triggers massive transmembrane ion fluxes and a reversible permeabilization of the plasma membrane to hydrophylic molecules of up to 900 dalton molecule weight and eventual cell death (Di Virgilio, F. 1995. Immunol. Today, 16:524-528). The physiological role of this newly cloned (Surprenant, A., F. Rassendren, E. Kawashima, R. A. North and G. Buell, 1996. Science (Wash. DC). 272:735-737) cytolytic receptor is unknown. In vitro and in vivo activation of the macrophage and microglial cell P2Z/P2X7 receptor by exogenous ATP causes a large and rapid release of mature IL-1 beta. In the present report we investigated the role of microglial P2Z/P2X7 receptor in IL-1 beta release triggered by LPS. Our data suggest that LPS-dependent IL-1 beta release involves activation of this purinergic receptor as it is inhibited by the selective P2Z/P2X7 blocker oxidized ATP and modulated by ATP-hydrolyzing enzymes such as apyrase or hexokinase. Furthermore, microglial cells release ATP when stimulated with LPS. LPS-dependent release of ATP is also observed in monocyte-derived human macrophages. It is suggested that bacterial endotoxin activates an autocrine/paracrine loop that drives ATP-dependent IL-1 beta secretion.

Spontaneous cell fusion in macrophage cultures expressing high levels of the P2Z/P2X7 receptor.

Mouse and human macrophages express a plasma membrane receptor for extracellular ATP named P2Z/P2X7. This molecule, recently cloned, is endowed with the intriguing property of forming an aqueous pore that allows transmembrane fluxes of hydrophylic molecules of molecular weight below 900. The physiological function of this receptor is unknown. In a previous study we reported experiments suggesting that the P2Z/P2X7 receptor is involved in the formation of macrophage-derived multinucleated giant cells (MGCs; Falzoni, S., M. Munerati, D. Ferrari, S. Spisani, S. Moretti, and F. Di Virgilio. 1995. J. Clin. Invest. 95:1207- 1216). We have selected several clones of mouse J774 macrophages that are characterized by either high or low expression of the P2Z/P2X7 receptor and named these clones P2Zhyper or P2Zhypo, respectively. P2Zhyper, but not P2Zhypo, cells grown to confluence in culture spontaneously fuse to form MGCs. As previously shown for human macrophages, fusion is inhibited by the P2Z/P2X7 blocker oxidized ATP. MGCs die shortly after fusion through a dramatic process of cytoplasmic sepimentation followed by fragmentation. These observations support our previous hypothesis that the P2Z/P2X7 receptor is involved in macrophage fusion.

Modulation of P2X7 receptor functions by polymyxin B: crucial role of the hydrophobic tail of the antibiotic molecule.

BACKGROUND AND PURPOSE: P2X7 is a membrane receptor for extracellular ATP which is highly expressed in dendritic cells, macrophages and microglia where it mediates pro-inflammatory responses. The antibiotic polymyxin B, which binds to and neutralizes the toxic residue of bacterial lipopolysaccharide, greatly amplifies cellular responses mediated by the P2X7 receptor. However, the molecular mechanism involved is so far unknown. EXPERIMENTAL APPROACH: We investigated the effects of polymyxin B and polymyxin B nonapeptide (PMBN) which is the deacylated amino derivative of polymyxin B lacking the N-terminal fatty amino acid 6-methylheptanoic/octanoic-Dab residue, in human macrophages and HEK293 cells stably expressing the human P2X7 receptor (HEK293-hP2X7). Differences between the two antibiotics were assessed by monitoring the following: nucleotide-induced cytoplasmic free Ca2+ concentration changes, plasma membrane permeability changes, lactate dehydrogenase activity, cell morphology changes. Western blot and microscopic analyses of P2X7GFP-expressing cells were also performed. KEY RESULTS: In contrast to polymyxin B, the polymyxin B nonapeptide was unable to potentiate: a) the ATP-induced Ca2+ increase, b) pore formation and consequently ATP-mediated plasma membrane permeabilization; c) ATP-dependent cytotoxicity. Moreover, in contrast to polymyxin B, polymyxin B nonapeptide did not affect aggregation of the P2X7 receptor subunits and it did not potentiate P2X7-dependent cell fusion. CONCLUSIONS AND IMPLICATIONS: The effects of polymyxin B depended on the presence of its N-terminal fatty amino acid 6-methylheptanoic/octanoic-Dab residue as deletion of this residue abolished polymyxin B-dependent modulation of ATP-triggered responses. These findings are important in the search for allosteric modulators of the P2X7 receptor.

P2X(7) receptor and polykarion formation.

Cell fusion is a central phenomenon during the immune response that leads to formation of large elements called multinucleated giant cells (MGCs) of common occurrence at sites of granulomatous inflammation. We have previously reported on the involvement in this event of a novel receptor expressed to high level by mononuclear phagocytes, the purinergic P2X(7) receptor. Herein, we show that blockade of this receptor by a specific monoclonal antibody prevents fusion in vitro. In contrast, cell fusion is stimulated by addition of enzymes that destroy extracellular ATP (i.e., apyrase or hexokinase). Experiments performed with phagocytes selected for high (P2X(7) hyper) or low (P2X(7) hypo) P2X(7) expression show that fusion only occurs between P2X(7) hyper/P2X(7) hyper and not between P2X(7) hyper/P2X(7) hypo or P2X(7) hypo/P2X(7) hypo. During MGCs formation we detected activation of caspase 3, an enzyme that is powerfully stimulated by P2X(7). Finally, we observed that during MGCs formation, the P2X(7) receptor is preferentially localized at sites of cell-to-cell contact. These findings support the hypothesis originally put forward by our group that the P2X(7) receptor participates in multinucleated giant cell formation.

Cytolytic P2X purinoceptors.

Anecdoctal evidence accumulated over almost 20 years has shown that many different cell types are killed by sustained exposure to high concentrations of extracellular ATP. The plasma membrane receptors involved have been pharmacologically characterized and cloned during the last 3 years, and named purinergic P2X. P2X receptors share an intriguing structural relatedness with Caenorhabditis elegans degenerins and mammalian amiloride-sensitive Na channels (ENaCs). Depending on the ATP dose, length of stimulation and receptor subtype, P2X receptor stimulation may cause necrosis or apoptosis. The intracellular pathways activated are poorly known, but the perturbation in intracellular ion homeostasis clearly plays a major role. ICE proteases (caspases) are also triggered, nonetheless their activation is not requested for ATP-dependent cell death. The physiological meaning of P2X receptor-dependent cytotoxicity is not understood, but an involvement in immune-mediated reactions is postulated.

ATP receptors and giant cell formation.

We have investigated the role of the purinergic P2X7 receptor in the formation of multinucleated giant cells in human monocyte/macrophage cultures stimulated with either concanavalin A or phytohemagglutinin. Macrophage fusion can be blocked by a P2X7-selective pharmacological antagonist or by a mAb directed against the extracellular P2X7 domain. Furthermore, macrophage cell clones expressing high P2X7 levels spontaneously fuse in culture, whereas macrophage clones lacking P2X7 are unable to fuse. Our findings suggest that the newly identified purinergic P2X7 receptor plays a central role in the complex chain of events leading to generation of macrophage-derived giant cells.

Evidence for a factor promoting the conversion of VWF from low and intermediate to high molecular mass polymers on the platelet membrane.

On the membrane surface of resting platelets exists a factor capable of promoting the conversion of plasma von Willebrand factor from low and intermediate molecular mass to high molecular mass polymers. The process is accompanied by a parallel increase in the von Willebrand factor activity. This could be a regulatory system for the molecular mass distribution of von Willebrand factor during its lifetime in plasma. Glycoprotein Ib of the platelet membrane appears not to be involved in the process.

P2 purinergic receptors of human eosinophils: characterization and coupling to oxygen radical production.

Extracellular nucleotides elicit multiple responses in eosinophils but no information on expression of purinergic receptors in these cells is available so far. In the present study we show that human eosinophils express the following P2Y and P2X subtypes: P2Y(1), P2Y(2), P2Y(4), P2Y(6), P2Y(11), and P2X(1), P2X(4), P2X(7), whose stimulation results in intracellular Ca(2+) increase and production of large amounts of reactive oxygen intermediates. These events are stimulated or inhibited, respectively, by P2 receptor agonists or antagonists.

ATP-mediated cytotoxicity in microglial cells.

Microglial cells are known to express purinergic receptors for extracellular ATP of both the P2Y and P2X subtypes. Functional studies have shown that both primary mouse microglial cells and the N9 and N13 microglial cell lines express the pore-forming P2Z/P2X7 receptor. Here we identify the presence of this receptor in N9 and N13 cells with a specific polyclonal Ab and show that microglial cells expressing the P2Z/P2X7 receptor are exquisitively sensitive to ATP-mediated cytotoxicity while clones selected for the lack of this receptor are resistant. Transfection of HEK293 cells with P2X7 (but not P2X2) receptor cDNA confers susceptibility to ATP-mediated cytotoxicity. Morphological and biochemical analysis suggests that ATP-dependent cell death in microglial cells occurs by apoptosis. Finally, microglial cells release ATP via a non-lytic mechanism when activated by bacterial endotoxin, thus suggesting the operation of a purinergic autocrine/paracrine loop.

Tenidap enhances P2Z/P2X7 receptor signalling in macrophages.

Tenidap is an anti-inflammatory drug whose mechanism of action is not fully understood. It has been shown to block plasma membrane anion transport and to decrease release of interleukin-1beta, probably via the inhibition of interleukin-1beta converting enzyme. In the present study we showed that: (a) tenidap increases the sensitivity of mouse macrophages to cytotoxic effects mediated by extracellular ATP; (b) tenidap increases lucifer yellow uptake through the macrophage ATP receptor; (c) pretreatment with oxidised ATP, a blocker of the P2Z/P2X7 receptor, inhibits cytotoxicity and lucifer yellow uptake due to the combined effects of ATP and tenidap; (d) macrophages lacking the P2Z/P2X7 receptor are resistant to the synergistic effect of tenidap and ATP. The results suggest that tenidap synergises with extracellular ATP for activation of the P2Z/P2X7 receptor.

Photoexcitation of the methionine-iron bond in iron(III) cytochrome c: bimolecular reaction with NADH.

When iron(III) cytochrome c aqueous solutions containing NADH are irradiated with polychromatic light (wavelength greater than 280 nm), iron(II) cytochrome c and NAD+ in the stoichiometric ratio 2/1 are observed to be the principal reaction products, independently of the presence of oxygen; in addition, a minor process due to direct photodegradation of the nucleotide is observed. The selection of monochromatic 290 nm irradiation light (at which NADH has an absorbance minimum) and an adequate reactant concentration allowed parallel reactions to be minimized and new information to be obtained on the mechanism of the photoredox process. The experimental results are consistent with a reaction mechanism whereby NADH donates one electron to a "reactive intermediate" of the hemoprotein formed from the light-induced methionine-to-iron charge transfer excited state. In this process an NAD. radical is formed which, in deaerated solution, immediately reduces another molecule of the hemoprotein, and is itself oxidized to NAD+. In aerated solution, the NAD. radical rapidly reacts with oxygen to give NAD+ and superoxide O2- anion radical which, in turn, reduces the second iron(III) cytochrome c molecule.

Natural gamma-radiation in the Aeolian volcanic arc.

Pulse-height distributions of gamma-rays, obtained with a field NaI(Tl) scintillation spectrometer in numerous sites of the Lipari and Vulcano islands (Aeolian volcanic arc, Italy), were measured to determine the U, Th and K concentrations of the bedrock and the relative values of the air absorbed dose rate. U is spatially related to both Th and K and the Th/U ratio is on average 3.1-3.5. The magmatic evolution is reflected by the concentration of the three radioelements, as they are more abundant within the more felsic units of the volcanic series. The higher values of U (15.7-20.0 ppm) coincide with higher Th (48.3-65.9 ppm) and K (4.9-6.1%) concentrations associated with rhyolitic rocks of the third cycle (< 50 ky). The air absorbed dose rate varies from 20 to 470 nGy h(-1). The highest values (> 350 nGy h(-1)) are observed on outcrops of rhyolitic obsidian lava flows. The cosmic-ray contribution is also evaluated to estimate the total background radiation dose rate.

Gamma-ray activity in the volcanic islands of the Southern Tyrrhenian Sea.

Field gamma-ray spectrometry was used for the quantitative assessment of U, Th and K of rocks of Stromboli, Salina, Filicuidi and Panarea (Aeolian arc of the Southern Tyrrhenian, Italy). The air absorbed dose rate was calculated from radioelement concentrations. For some rocks the gamma-ray spectra were analysed with the three photo-peak methods and the response matrix method, which converts the pulse height distribution into the true incident gamma-ray energy spectrum. The higher values of U (8.2-9.8 ppm) coincide with higher Th (20.6-27.8 ppm) concentrations associated with rocks of shoshonitic composition. The spatial variation in radioelement concentration reflects the geochemical differences among the rocks. The air absorbed dose rate varies from 25 to 215 nGy h(-1). The highest values correspond to outcrops located in the eastern part of Stromboli, where the annual effective dose equivalent reaches a value of 264 microSv.

Nimodipine inhibits IL-1ß release stimulated by amyloid ß from microglia.

BACKGROUND AND PURPOSE: There is growing evidence that inflammation plays a major role in the pathogenesis of neural damage caused by deposition of amyloid ß (Aß) in the brain. Nimodipine has received attention as a drug that might improve learning and reduce cognitive deficits in Alzheimer's disease, but the mechanism of action is poorly known. In this study, we tested the hypothesis that nimodipine inhibited Aß-stimulated IL-1ß release from microglia. EXPERIMENTAL APPROACH: Cultures of N13 microglia cells or primary mouse microglia were treated with nimodipine, and intracellular accumulation and release of IL-1ß in response to Aß or to the P2 receptor agonists ATP and benzoyl ATP (BzATP) were measured. Accumulation of IL-1ß was measured in vivo after intrahippocampal inoculation of Aß in the absence or presence of nimodipine. The effect of nimodipine on Aß-triggered cytotoxicity was also investigated. KEY RESULTS: We show here that nimodipine dose-dependently inhibited Aß-stimulated IL-1ß synthesis and release from primary microglia and microglia cell lines. Furthermore, nimodipine also inhibited Aß-induced IL-1ßin vivo accumulation at concentrations known to be reached in the CNS. Finally, nimodipine protected microglia from Aß-dependent cytotoxicity. CONCLUSION AND IMPLICATIONS: These data suggest that alleviation of symptoms of Alzheimer's disease following nimodipine administration might be due to an anti-inflammatory effect and point to a novel role for nimodipine as a centrally acting anti-inflammatory drug.

Natural gamma-ray spectrometry as a tool for radiation dose and radon hazard modelling.

We reviewed the calibration procedures of gamma-ray spectrometry with particular emphasis to factors that affect accuracy, detection limits and background radiation in field measurements for dosimetric and radon potential mapping. Gamma-ray spectra were acquired in western Liguria (Italy). The energy windows investigated are centred on the photopeaks of (214)Bi (1.76 MeV), (208)Tl (2.62 MeV) and (40)K (1.46 MeV). The inferred absorbed dose rate and the radon flux are estimated to be lower than 60 nGy h(-1) and 22 Bq m(-2)h(-1), respectively.

Identification of the domain recognized by anti-(ryanodine receptor) antibodies which affect Ca(2+)-induced Ca2+ release.

In the present paper we have defined putative functional domains of the ryanodine receptor Ca2+ channel. cDNA fragments of the skeletal muscle ryanodine receptor were fused in-frame with the Escherichia coli trpe protein and the resulting fusion proteins were evaluated for their ability to react with anti-(ryanodine receptor) antibodies, which are known to block Ca(2+)-dependent activation of the Ca(2+)-release channel. Anti-(ryanodine receptor) antibodies react with epitopes lying within a 245-amino-acid-long polypeptide which is located in a region (residues 4380-4625) encompassing most of myoplasmic loop 2, the predicted transmembrane segment M5 and part of the next lumenal loop (45 residues). Purification of the anti-(ryanodine receptor) antibodies by affinity chromatography led to the isolation of a population of antibodies which was capable of decreasing (by > 30%) the doxorubicin-induced Ca2+ release from isolated terminal cisternae. Polyclonal antibodies raised against a ryanodine receptor fusion encompassing part (198 out of 245 residues) of the immunopositive polypeptide decreased by 2-fold the first-order rate constant of Ca(2+)-induced 45Ca2+ efflux from isolated terminal cisternae. These results suggest strongly that the Ca(2+)-activating domain of the skeletal muscle Ca(2+)-release channel is close to, or associated with, myoplasmic loop 2.

Interaction of erythrocyte transglutaminase with calcium ions.

We have investigated the interaction between calcium ions and erythrocyte transglutaminase and the enzyme activation. The binding involves both high and low affinity sites, but only the former ones are relevant for activation. The binding of calcium and the activation are modified by treatment with NBD-Cl and with PLP suggesting the presence of cysteine and lysine residues at the high affinity binding sites. The interaction of the enzyme with calcium is not calmodulin dependent and is easily detected as a shift in electrophoretic mobility in the presence of SDS.

Mechanism of human sperm activation by extracellular ATP.

We have identified the mechanism whereby extracellular ATP (ATPe) triggers the acrosome reaction in human spermatozoa. This nucleotide opens a ligand-gated ion channel expressed on the sperm plasma membrane. ATPe threshold and 50% effective concentration calculated on the total added ATPe are 0.1 and 2 mM, respectively, corresponding to a free ATP concentration (ATP4-) of 3 and 200 microM, respectively. The ATPe-gated channel is selective for monovalent cations (Na+, choline, and methylglucamine), whereas on the contrary, permeability to Ca2+ is negligible. Isosmolar replacement of extracellular Na+ with sucrose fully blocked ATPe-dependent sperm activation, thus suggesting a mandatory role for Na+ influx. These results show that human sperm express an ATPe-gated Na+ channel that might have an important role in sperm activation before egg fertilization.