The NLRP3 inflammasome is activated by nanoparticles through ATP, ADP and adenosine.

The NLR pyrin domain containing 3 (NLRP3) inflammasome is a major component of the innate immune system, but its mechanism of activation by a wide range of molecules remains largely unknown. Widely used nano-sized inorganic metal oxides such as silica dioxide (nano-SiO2) and titanium dioxide (nano-TiO2) activate the NLRP3 inflammasome in macrophages similarly to silica or asbestos micro-sized particles. By investigating towards the molecular mechanisms of inflammasome activation in response to nanoparticles, we show here that active adenosine triphosphate (ATP) release and subsequent ATP, adenosine diphosphate (ADP) and adenosine receptor signalling are required for inflammasome activation. Nano-SiO2 or nano-TiO2 caused a significant increase in P2Y1, P2Y2, A2A and/or A2B receptor expression, whereas the P2X7 receptor was downregulated. Interestingly, IL-1ß secretion in response to nanoparticles is increased by enhanced ATP and ADP hydrolysis, whereas it is decreased by adenosine degradation or selective A2A or A2B receptor inhibition. Downstream of these receptors, our results show that nanoparticles activate the NLRP3 inflammasome via activation of PLC-InsP3 and/or inhibition of adenylate cyclase (ADCY)-cAMP pathways. Finally, a high dose of adenosine triggers inflammasome activation and IL-1ß secretion through adenosine cellular uptake by nucleotide transporters and by its subsequent transformation in ATP by adenosine kinase. In summary, we show for the first time that extracellular adenosine activates the NLRP3 inflammasome by two ways: by interacting with adenosine receptors at nanomolar/micromolar concentrations and through cellular uptake by equilibrative nucleoside transporters at millimolar concentrations. These findings provide new molecular insights on the mechanisms of NLRP3 inflammasome activation and new therapeutic strategies to control inflammation.

ATP release and purinergic signaling: a common pathway for particle-mediated inflammasome activation.

Deposition of uric acid crystals in joints causes the acute and chronic inflammatory disease known as gout and prolonged airway exposure to silica crystals leads to the development of silicosis, an irreversible fibrotic pulmonary disease. Aluminum salt (Alum) crystals are frequently used as vaccine adjuvant. The mechanisms by which crystals activate innate immunity through the Nlrp3 inflammasome are not well understood. Here, we show that uric acid, silica and Alum crystals trigger the extracellular delivery of endogenous ATP, which just precedes the secretion of mature interleukin-1ß (IL-1ß) by macrophages, both events depending on purinergic receptors and connexin/pannexin channels. Interestingly, not only ATP but also ADP and UTP are involved in IL-1ß production upon these Nlrp3 inflammasome activators through multiple purinergic receptor signaling. These findings support a pivotal role for nucleotides as danger signals and provide a new molecular mechanism to explain how chemically and structurally diverse stimuli can activate the Nlrp3 inflammasome.

Amino acid residues involved in gating identified in the first membrane-spanning domain of the rat P2X(2) receptor.

The first hydrophobic segment of the rat P2X(2) receptor extends from residue Leu(29) to Val(51). In the rat P2X(2) receptor, we mutated amino acids in this segment and adjoining flanking regions (Asp(15) through Thr(60)) individually to cysteine and expressed the constructs in human embryonic kidney cells. Whole-cell recordings were used to measure membrane currents evoked by brief (2-s) applications of ATP (0.3-100 microM). Currents were normal except for Y16C, R34C, Y43C, Y55C, and Q56C (no currents but normal membrane expression by immunohistochemistry), Q37C (small currents), and F44C (normal current but increased sensitivity to ATP, as well as alphabeta-methylene-ATP). We used methanethiosulfonates of positive, negative, or no charge to test the accessibility of the substituted cysteines. D15C, P19C, V23C, V24C, G30C, Q37C, F44C, and V48C were strongly inhibited by neutral, membrane-permeant methanethiosulfonates. Only V48C was also inhibited by positively and negatively charged methanethiosulfonates, consistent with an extracellular position; however, accessibility of V48C was increased by channel opening. V48C could disulfide with I328C, as shown by the large increase in ATP-evoked current caused by reducing agents. The results suggest that Val(48) at the outer end of the first hydrophobic segment takes part in the gating movement of channel opening.

Identification of amino acid residues contributing to the ATP-binding site of a purinergic P2X receptor.

P2X receptor subunits have intracellular N and C termini, two membrane-spanning domains, and an extracellular loop of about 280 amino acids. We expressed the rat P2X(2) receptor in human embryonic kidney cells, and used alanine-scanning mutagenesis on 30 residues with polar side chains conserved among the seven rat P2X receptor subunits. This identified a region proximal to the first transmembrane domain which contained 2 lysine residues that were critical for the action of ATP (Lys(69) and Lys(71)). We substituted cysteines in this region (Asp(57) to Asp(71)) and found that for S65C and I67C ATP-evoked currents were inhibited by methanethiosulfonates. At I67C, the inhibition by negatively charged ethylsulfonate and pentylsulfonate derivatives could be overcome by increasing the ATP concentration, consistent with a reduced affinity of ATP binding. The inhibitory action of the methanethiosulfonates was prevented by pre-exposure to ATP, suggesting occlusion of the binding site. Finally, introduction of negative charges into the receptor by mutagenesis at this position (I67E and I67D) also gave receptors in which the ATP concentration-response curve was right-shifted. The results suggest that residues close to Ile(67) contribute to the ATP-binding site.

Contribution of individual subunits to the multimeric P2X(2) receptor: estimates based on methanethiosulfonate block at T336C.

P2X receptors are membrane proteins that incorporate a cation-selective ion channel that can be opened by the binding of extracellular ATP. They associate as hetero- and homo-multimers of currently unknown stoichiometry. In this study, we have used Xenopus laevis oocytes to express rat P2X(2) receptor subunits, which carry a cysteine mutation at position 336. ATP-induced currents at this mutant receptor subunit were blocked by more than 90% when exposed to [2-(trimethylammonium) ethyl] methanethiosulfonate (MTSET), whereas currents from wild-type subunits were not affected. To compare mutant and wild-type channel expression, we introduced an epitope in their extracellular domains and found for both channels a similar linear relationship between antibody binding and currents induced by ATP. To study the contribution of the individual subunits to the block by MTSET, we coinjected different mixtures of wild-type and mutant-encoding mRNAs. We found that the inhibition by MTSET depended linearly on the proportion of mutant subunits, which was clearly contrary to the hypothesis that a single mutant subunit could act in a dominant fashion. Subsequent concatenation of wild-type and mutant-encoding cDNAs resulted in an inhibition by MTSET that also depended linearly on the number of mutant subunits and was independent of the position of the mutant subunit, as long as only two or three P2X(2) subunits were joined. With four or six subunits joined, however, the inhibition by MTSET became strongly position-dependent. The present results show that a "per-subunit" channel block causes the blocking effects of MTSET and they suggest that not four but maximally three subunits actively participate in the channel formation.

Pore dilation of neuronal P2X receptor channels.

P2X receptors are ligand-gated ion channels activated by the binding of extracellular adenosine 5'-triphosphate (ATP). Brief (< 1 s) applications of ATP to nodose ganglion neurons or to cells transfected with P2X2 or P2X4 receptor cDNAs induce the opening of a channel selectively permeable to small cations within milliseconds. We now show that, during longer ATP application (10-60 s), the channel also becomes permeable to much larger cations such as N-methyl-D-glucamine and the propidium analog YO-PRO-1. This effect is enhanced in P2X2 receptors carrying point mutations in the second transmembrane segment. Progressive dilation of the ion-conducting pathway during prolonged activation reveals a mechanism by which ionotropic receptors may alter neuronal function.

Membrane topology of an ATP-gated ion channel (P2X receptor).

Western blots of Xenopus oocyte membrane preparations showed that the apparent molecular mass of the wild type P2X2 receptor (about 65 kDa) was reduced by pretreatment with endoglycosidase H. Mutagenesis of one or more of three potential asparagines (N182S, N239S, and N298S) followed by Western blots showed that each of the sites was glycosylated in the wild type receptor. Functional channels were formed by receptors lacking any single asparagine, but not by channels mutated in two or three positions. Artificial consensus sequences (N-X-S/T) introduced into the N-terminal region (asparagine at position 9, 16, or 26) were not glycosylated. Asparagines were glycosylated when introduced at the C-terminal end of the first hydrophobic domain (positions 62 and 66) and at the N-terminal end of the second hydrophobic domain (position 324). A protein in which the C terminus of one P2X2 subunit was joined to the N terminus of a second P2X2 subunit (from a concatenated cDNA) had twice the molecular mass of the P2X2 receptor subunit, and formed fully functional channels. The experiments provide direct evidence for the topology originally proposed for the P2X receptor, with intracellular N and C termini, two membrane-spanning domains, and a large extracellular loop.

Identification of amino acid residues contributing to the pore of a P2X receptor.

P2X receptors are ion channels opened by extracellular ATP. The seven subunits currently known are encoded by different genes. It is thought that each subunit has two transmembrane domains, a large extracellular loop, and intracellular N- and C-termini, a topology which is fundamentally different from that of other ligand-gated channels such as nicotinic acetylcholine or glutamate receptors. We used the substituted cysteine accessibility method to identify parts of the molecule that form the ionic pore of the P2X2 receptor. Amino acids preceding and throughout the second hydrophobic domain (316-354) were mutated individually to cysteine, and the DNAs were expressed in HEK293 cells. For three of the 38 residues (I328C, N333C, T336C), currents evoked by ATP were inhibited by extracellular application of methanethiosulfonates of either charge (ethyltrimethylammonium, ethylsulfonate) suggesting that they lie in the outer vestibule of the pore. For two further substitutions (L338C, D349C) only the smaller ethylamine derivative inhibited the current. L338C was accessible to cysteine modification whether or not the channel was opened by ATP, but D349C was inhibited only when ATP was concurrently applied. The results indicate that part of the pore of the P2X receptor is formed by the second hydrophobic domain, and that L338 and D349 are on either side of the channel 'gate'.

The permeabilizing ATP receptor, P2X7. Cloning and expression of a human cDNA.

A cDNA was isolated from a human monocyte library that encodes the P2X7 receptor; the predicted protein is 80% identical to the rat receptor. Whole cell recordings were made from human embryonic kidney cells transfected with the human cDNA and from human macrophages. Brief applications (1-3 s) of ATP and 2', 3'-(4-benzoyl)-benzoyl-ATP elicited cation-selective currents. When compared with the rat P2X7 receptor, these effects required higher concentrations of agonists, were more potentiated by removal of extracellular magnesium ions, and reversed more rapidly on agonist removal. Longer applications of agonists permeabilized the cells, as evidenced by uptake of the propidium dye YO-PRO1, but this was less marked than for cells expressing the rat P2X7 receptor. Expression of chimeric molecules indicated that some of the differences between the rat and human receptor could be reversed by exchanging the intracellular C-terminal domain of the proteins.

The cytolytic P2Z receptor for extracellular ATP identified as a P2X receptor (P2X7).

The P2Z receptor is responsible for adenosine triphosphate (ATP)-dependent lysis of macrophages through the formation of membrane pores permeable to large molecules. Other ATP-gated channels, the P2X receptors, are permeable only to small cations. Here, an ATP receptor, the P2X7 receptor, was cloned from rat brain and exhibited both these properties. This protein is homologous to other P2X receptors but has a unique carboxyl-terminal domain that was required for the lytic actions of ATP. Thus, the P2X7 (or P2Z) receptor is a bifunctional molecule that could function in both fast synaptic transmission and the ATP-mediated lysis of antigen-presenting cells.

Purinoceptors in the Central Nervous System.

New exciting developments on the occurrence and functional role of purinoceptors in mammalian brain were presented at the session "Purinoceptors in the central nervous system" chaired by Flaminio Cattabeni and Tom Dunwiddie at the Purines '96 international conference. The focus of the session were topics of recent interest, including the sources and mechanisms involved in ATP and adenosine release during physiological neurotransmission in hippocampus, the brain expression of the recently cloned P2 receptors, and the role of the various adenosine receptor subtypes in brain protection from neurodegeneration associated with trauma-, ischemia-and excessive excitatory amino acid neurotransmission. New important insights into the mechanisms responsible for the formation and release of adenosine into the extracellular space were provided by data obtained by Dunwiddie and coworkers in hippocampal pyramidal neurons. These data may have functional implications for the role of purines in modulation of synaptic plasticity and long-term potentiation in this brain area, and hence in cognitive functions. Buell provided an updated overview on the cloning, molecular characteristics and brain expression of various ligand-gated P2X purinoceptors; although the functional role of these receptors in mammalian brain still awaits elucidation, their widespread distribution in the nervous system strongly suggests that ATP-mediated events are more prevalent and important in brain than expected. Pedata presented data on the functional interrelationships between adenosine and glutamate in the brain, and also provided evidence for alterations of the reciprocal regulation between these two systems in aged brain, which may have important implications for both ischemia-and trauma-associated neurodegenerative events and senescence-associated cognitive impairment. Finally, von Lubitz provided novel data on the molecular mechanisms likely to be at the basis of the brain protective effects associated with the chronic stimulation of the adenosine A3 receptor, further confirming that this receptor represents a crucial target for the development of new antiischemic and antineurodegenerative therapeutic agents.

A new class of noninactivating K+ channels from aplysia capable of contributing to the resting potential and firing patterns of neurons.

From the nervous system of Aplysia, we have cloned a new class of noninactivating K+ channels (aKv5.1) that are activated at low voltage and are capable of contributing to the resting potential and firing patterns of neurons. Expression of aKv5.1 in Aplysia neuron R15 revealed that aKv5.1 exerts an unusual control over cell excitability; it increased the resting potential by more than 20 mV and abolished the spontaneous bursting activity of the cell. In its ability to suppress the endogenous rhythm of R15, aKv5.1 differs in its actions from transient, inactivating K+ channels such as aKv1.1a, an Aplysia homolog of Shaker. aKv1.1a shortens the duration of the spike and increases the afterpotential, but does not suppress bursting. Thus, by expressing different classes of K+ channels, it is possible to redesign, in specific ways, the signaling capabilities of specific, identified neurons.

Levels of mRNA coding for motoneuron growth-promoting factors are increased in denervated muscle.

Partial denervation of skeletal muscle induces sprouting of axons remaining within the muscle, possibly as a result of increased synthesis by denervated muscle fibers of motoneuron growth-promoting factors. Direct verification of this hypothesis has not been possible because the molecules responsible are not unambiguously characterized. We used Xenopus oocytes as a functional assay for mRNAs coding for secreted growth factors: preparations of mRNA from innervated and denervated neonatal muscle were injected into oocytes. Three days later, oocytes injected with denervated muscle mRNA expressed increased levels of nicotinic acetylcholine receptor and voltage-dependent sodium channels at their membrane. Proteins secreted by the same oocytes were tested for their effects on (i) neurite outgrowth from embryonic chicken ventral spinal cord neurons; (ii) survival in mixed culture of embryonic chicken motoneurons identified using the SC1 antibody; and (iii) survival of embryonic motoneurons purified by panning on SC1 antibody. In all three assays, media conditioned by oocytes injected with mRNA from denervated muscle contained significantly higher levels of biological activity than did those from oocytes injected with innervated muscle mRNA or water. mRNA was prepared from muscle at different times after denervation: a maximal increase was obtained already after 1 day, consistent with an involvement in sprouting. Synthesis of motoneuron growth-promoting factors is thus regulated by denervation in a parallel fashion to that of other key components of the neuromuscular junction.

Both enantiomers of 1-aminocyclopentyl-1,3-dicarboxylate are full agonists of metabotropic glutamate receptors coupled to phospholipase C.

We tested the effects of two enantiomers of a glutamate analogue, (trans)-1-aminocyclopentyl-1,3-dicarboxylate (t-ACPD), in striatal and cerebellar neurons in primary culture, as well as in Xenopus oocytes injected with cerebellar rat RNA. In the presence of MK-801, to avoid N-methyl-D-aspartate receptor activation, and 3 microM tetrodotoxin, both enantiomers [(1R,3S)- and (1S,3R)-t-ACPD] stimulated inositol phosphate (InsP) formation both in striatal neurons after 9-11 days in vitro [EC50, 3.7 +/- 1.1 microM, three experiments, and 33 +/- 7.5 microM, three experiments; maximal stimulatory effects, 252 +/- 15%, 13 experiments, and 269 +/- 15% of basal InsP formation, 14 experiments, for (1R,3S)- and (1S,3R)-t-ACPD, respectively] and in cerebellar granule cells after 9-11 days in vitro [EC50, 50 +/- 18 microM, four experiments, and 307 +/- 92 microM, four experiments; maximal stimulatory effects, 401 +/- 71%, eight experiments, and 423 +/- 75% of basal InsP formation, eight experiments, for (1R,3S)- and (1S,3R)-t-ACPD, respectively]. These effects were not additive, indicating that both enantiomers acted at the same receptor molecule. When we monitored t-ACPD-induced increases in intracellular Ca2+ concentration ([Ca2+]i) with fura-2 ratio-imaging, we found that both enantiomers could elicit similar increase in [Ca2+]i, in the presence of 1 microM MK-801 and 3 microM tetrodotoxin; these effects were also observed in the absence of external Ca2+. Moreover, in Xenopus oocytes injected with adult rat cerebellar RNA, both drugs elicited oscillatory increases of a Ca(2+)-dependent chloride conductance, with similar efficacy, with (1R,3S)-t-ACPD being the more potent isomer. These data are in contradiction to previous reports showing that, in "immature" cerebellar neurons and adult hippocampal slices, (1S,3R)-t-ACPD was either the only active enantiomer or a full agonist of metabotropic receptors, with (1R,3S)-t-ACPD being ineffective or a partial agonist. However, performing these experiments in immature (2-3 days in vitro) striatal or cerebellar neurons, we found that only (1S,3R)-t-ACPD was active in stimulating [Ca2+]i.

Characterization of voltage-dependent calcium channels expressed in Xenopus oocytes injected with mRNA from rat heart.

1. The properties of voltage dependent cardiac Ca channels expressed in Xenopus laevis oocytes after injection of mRNA from rat heart were investigated using the double-microelectrode voltage-clamp technique. 2. Endogenous Ba current (IBa,E) and expressed cardiac Ba current (IBa,C) were studied at various external concentrations of barium (Ba2+). These two entities could be distinguished by their amplitude and their pharmacology. IBa,C was more sensitive to the inorganic Ca channel blocker manganese (Mn2+). The contaminant IBa,E presented properties of voltage dependence identical to IBa,C, but was negligible in the presence of a low external Ba2+ concentration (2 mM). 3. In 2 mM-Ba2+, IBa,C activated at -35 mV, peaked at -14 mV, and reversed at +26 mV. Steady-state inactivation properties, in consideration of the half-inactivation potential of -35 mV, were also typical of L-type Ba currents. However, the decay of IBa,C was very slow (time constant of inactivation near 600 ms). No evidence for the expression of cardiac transient Ca channels (T-type) was found. 4. IBa,C was enhanced after exposure to the 1,4-dihydropyridine (DHP) agonist Bay K 8644. The enhancement of IBa,C was voltage dependent (maximum at -30 +/- 5 mV) and associated with a slowing in current decay. Current-voltage and concentration-response curves obtained for various Ba2+ concentrations revealed an antagonism between external Ba2+ and the 1,4-DHP agonist Bay K 8644. Similar results were found using the (-)Bay K 8644 pure agonist isomer. 5. We conclude that oocytes injected with mRNA from rat heart expressed only the high threshold, long-lasting or L-type Ca channels. The availability of expressed L-type Ca channels for quantitative pharmacological studies using low Ba2+ concentration has been demonstrated.

(trans)-1-amino-cyclopentyl-1,3-dicarboxylate stimulates quisqualate phosphoinositide-coupled receptors but not ionotropic glutamate receptors in striatal neurons and Xenopus oocytes.

The effects of a novel glutamate analogue, (trans)-1-amino-cyclopentyl-1,3-dicarboxylate (ACPD), have been tested in striatal neurons in primary culture and in Xenopus oocytes injected with rat brain RNA. Both systems have been previously shown to contain well characterized metabotropic receptors coupled to phospholipase C (Qp), as well as ionotropic glutamate receptors. In striatal neurons, ACPD stimulated inositol phosphate (InsP) accumulation (EC50 = 9.7 +/- 2.5 microM; maximal effect, 184.7 +/- 11.6% of basal accumulation). This effect of ACPD was likely to be mediated by Qp receptors, because maximal ACPD and quisqualate-induced InsP formation were not additive. In contrast, the effects of ACPD and norepinephrine on InsP formation were additive. ACPD-induced InsP formation was not antagonised by antagonists of muscarinic and alpha 1-adrenergic receptors (1 microM atropine and 0.1 microM prazosin, respectively). In Xenopus oocytes, ACPD and quisqualate induced an oscillatory increase of a Ca2(+)-dependent chloride conductance, which is characteristic of the activation of phospholipase C-coupled receptors in this model. The specificity of ACPD on Qp receptors was demonstrated by testing the effect of this drug on quisqualate/kainate as well as on N-methyl-D-aspartate ionotropic receptors. In striatal neurons, the activation of quisqualate/kainate and N-methyl-D-aspartate receptors was tested by measurement of [3H]-gamma-aminobutyric acid release and by electrophysiological recordings using the patch-clamp technique. At concentrations as high as 1 mM, ACPD was inactive on these inotropic receptors, either as agonist or as antagonist. In conclusion, ACPD appeared to be a highly specific agonist of Qp receptors, with no activity on ionotropic glutamate receptors. It will be a useful drug to study the physiological properties of Qp receptors in vertebrate brains.

Zinc has opposite effects on NMDA and non-NMDA receptors expressed in Xenopus oocytes.

Pharmacological characterization of Zn2+ effects on glutamate ionotropic receptors was investigated in Xenopus oocytes injected with rat brain mRNA, using a double microelectrode, voltage-clamp technique. At low concentration, Zn2+ inhibited NMDA currents (IC50 = 42.9 +/- 1.3 microM) and potentiated both AMPA (EC50 = 30.0 +/- 1.2 microM) and desensitized kainate responses (EC50 = 13.0 +/- 0.1 microM). At higher concentrations, Zn2+ inhibited non-NMDA responses with IC50 values of 1.3 +/- 0.1 mM and 1.2 +/- 0.3 mM for AMPA and kainate, respectively. The potentiation of AMPA or quisqualate currents by Zn2+ was more than 2-fold, whereas that of the kainate current was only close to 30%. This potentiating effect of Zn2+ on AMPA current modified neither the affinity of the agonist for its site nor the current-voltage relationship. In addition, 500 microM Zn2+ differentially affected NMDA and non-NMDA components of the glutamate-induced response. The possible physiological relevance of Zn2+ modulation is discussed.

Electrophysiological expression of endothelin and angiotensin receptors in Xenopus oocytes injected with rat heart mRNA.

Functional endothelin and angiotensin receptors have been expressed in Xenopus oocyte following the microinjection of rat heart mRNA. Under voltage clamp conditions, application of these peptides clearly induced oscillatory Ca2+-activated chloride currents in a dose-dependent manner. In addition, no direct modulation of expressed or native cardiac Ca channels was observed.