Extracellular ATP is increased by release of ATP-loaded microparticles triggered by nutrient deprivation

Rationale: Caloric restriction improves the efficacy of anti-cancer therapy. This effect is largely dependent on the increase of the extracellular ATP concentration in the tumor microenvironment (TME). Pathways for ATP release triggered by nutrient deprivation are largely unknown. Methods: The extracellular ATP (eATP) concentration was in vivo measured in the tumor microenvironment of B16F10-inoculated C57Bl/6 mice with the pmeLuc probe. Alternatively, the pmeLuc-TG-mouse was used. Caloric restriction was in vivo induced with hydroxycitrate (HC). B16F10 melanoma cells or CT26 colon carcinoma cells were in vitro exposed to serum starvation to mimic nutrient deprivation. Energy metabolism was monitored by Seahorse. Microparticle release was measured by ultracentrifugation and by Nanosight. Results: Nutrient deprivation increases eATP release despite the dramatic inhibition of intracellular energy synthesis. Under these conditions oxidative phosphorylation was dramatically impaired, mitochondria fragmented and glycolysis and lactic acid release were enhanced. Nutrient deprivation stimulated a P2X7-dependent release of ATP-loaded, mitochondria-containing, microparticles as well as of naked mitochondria. Conclusions: Nutrient deprivation promotes a striking accumulation of eATP paralleled by a large release of ATP-laden microparticles and of naked mitochondria. This is likely to be a main mechanism driving the accumulation of eATP into the TME

P2X7 expression modulates mitochondrial metabolism

L’espessione del recettore P2X7 modula il metabolismo mitocondriale. Il recettore P2X7 è principalmente conosciuto per la sua abilità nel causare morte cellular dovuta ad una prolunata attivazione data dall’ATP, tramite un aumento della permeabilizzazione della membrane plasmatica. Al contrario una brave attivazione causa una modificazioni della concentrazione intracellulare di cationi che si associa a differenti processi fisiologici come induzione della cascata infiammatoria, proliferazione e soppravivemza cellulare. Negli ultimi anni si è cercato di comprendere meglio i meccanismi tramite i quali il recettore P2X7 supporta il metabolismo energetico delle cellule. Il nostro laboratorio ha in precedenza dimostrato come il recettore P2X7 ha un effetto trofico sul metabolismo energetico cellulare tramite l’aumento del potenziale mitocondriale di membrane e la sintesi di ATP. Al contrario stimolazione farmacologica del recettore purinergico causa frammentazione mitocondriale e collasso del potenziale di membrane mitocondriale. Questi dati portano in luce l’importante ruolo del P2X7 nel modulare il metabolismo mitocondriale. Nel presente studio dimostraimo come il recettore P2X7 è presente a livello dei mitocondri e in seguito a attivazione si abbia un suo aumento in questi siti. Inoltre delezione genetica del recettore P2X7 compromette la respirazione mitocondriale, il potenziale di membrane e l’abilita di produrre ROS. Questo stato cellulare de-energizzato provoca un impatto negativo sulle diverse funzioni cellulari come la migrazione. Queste osservazioni dimostrano come il P2X7 gioca un ruolo centrale nell’omeostasi energetica cellulare e nei processi che la coinvolgono.P2X7 expression modulates mitochondrial metabolism. The P2X7 receptor is a trimeric ATP-gated cation channel best known for its ability to cause plasma membrane permeabilization and cell death after prolonged exposure to extracellular ATP. However, recent data show that its brief activation triggers rapid inward cation currents and intracellular signalling pathways associated with a multiplicity of physiological processes such as induction of the inflammatory cascade, cell proliferation and survival. Recently, there has been an increased effort to understand the mechanism by which P2X7 supports energy-requiring cell functions. We previously showed that basal P2X7 expression has a trophic effect on cellular energetics as it increases mitochondrial potential and ATP synthesis, while on the contrary pharmacological P2X7 stimulation causes mitochondrial potential collapse and fragmentation. These findings point to major role for P2X7 in the modulation of mitochondrial metabolism. In the present study we show that P2X7 localizes to the mitochondria especially following activation. Furthermore P2X7 genetic deletion severely impairs mitochondrial respiration, mitochondrial membrane potential and ability to produce ROS. Decreased energy generation impacts negatively on key cell functions such as migration. These observations demonstrate the central role played by P2X7 in the modulation of cellular energy homeostasis and energy-requiring processes

Microglia P2X4 receptors as pharmacological targets for demyelinating diseases

Pharmacological activation of the P2X4 receptor expressed by brain microglia may provide a novel avenue to promote remyelination and improve clinical symptoms in experimental autoimmune encephalomyelitis and potentially in multiple sclerosis

Extracellular nucleotides and nucleosides as signalling molecules

Extracellular nucleotides, mainly ATP, but also ADP, UTP, UDP and UDP-sugars, adenosine, and adenine base participate in the “purinergic signalling” pathway, an ubiquitous system of cell-to-cell communication. Fundamental pathophysiological processes such as tissue homeostasis, wound healing, neurodegeneration, immunity, inflammation and cancer are modulated by purinergic signalling. Nucleotides can be released from cells via unspecific or specific mechanisms. A non-regulated nucleotide release can occur from damaged or dying cells, whereas exocytotic granules, plasma membrane-derived microvesicles, membrane channels (connexins, pannexins, calcium homeostasis modulator (CALHM) channels and P2X7 receptor) or specific ATP binding cassette (ABC) transporters are involved in the controlled release. Four families of specific receptors, i.e. nucleotide P2X and P2Y receptors, adenosine P1 receptors, and the adenine-selective P0 receptor, and several ecto- nucleotidases are essential components of the “purinergic signalling” pathway. Thanks to the activity of ecto-nucleotidases, ATP (and possibly other nucleotides) are degraded into additional messenger molecules with specific action. The final biological effects depend on the type and amount of released nucleotides, their modification by ecto-nucleotidases, and their possible cellular re-uptake. Overall, these processes confer a remarkable level of selectivity and plasticity to purinergic signalling that makes this network one of the most relevant extracellular messenger systems in higher organisms

Extracellular ATP: A Feasible Target for Cancer Therapy

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 P2X7 Receptor in Infection and Inflammation

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

A rationale for targeting the P2X7 receptor in Coronavirus disease 19 (Covid-19)

Severe pneumonia which shares several of the features of acute respiratory distress syndrome (ARDS) is the main cause of morbidity and mortality in Coronavirus disease 19 (Covid-19) for which as of now there is no effective treatment. ARDS is caused and sustained by an uncontrolled inflammatory activation characterized by a massive release of cytokines (cytokine storm), diffuse lung edema, inflammatory cell infiltraton and disseminated coagulation. Macrophage and T lymphocyte dysfunction plays a central role in this syndrome. In several experimental in vitro and in vivo models, many of these pathophysiological changes are triggered by stimulation of the P2X7 receptor. We hypothesize that this receptor might be an ideal candidate to target in Covid-19-associated severe pneumonia

Amyloid β-dependent mitochondrial toxicity in mouse microglia requires P2X7 receptor expression and is prevented by nimodipine

Previous data from our laboratory show that expression of the P2X7 receptor (P2X7R) is needed for amyloid β (Aβ)-stimulated microglia activation and IL-1β release in vitro and in vivo. We also showed that Aβ-dependent stimulation is inhibited by the dihydropyridine nimodipine at an intracellular site distal to the P2X7R. In the present study, we used the N13 microglia cell line and mouse primary microglia from wt and P2rx7-deleted mice to test the effect of nimodipine on amyloid β (Aβ)-dependent NLRP3 inflammasome expression and function, and on mitochondrial energy metabolism. Our data show that in microglia Aβ causes P2X7R-dependent a) NFκB activation; b) NLRP3 inflammasome expression and function; c) mitochondria toxicity; and these changes are fully inhibited by nimodipine. Our study shows that nimodipine is a powerful blocker of cell damage caused by monomeric and oligomeric Aβ, points to the mitochondria as a crucial target, and underlines the permissive role of the P2X7R

The P2X7 Receptor Is Shed Into Circulation: Correlation With C-Reactive Protein Levels

The P2X7 receptor (P2X7R) is a key pro-inflammatory plasma membrane receptor responsible for NLRP3 inflammasome activation and IL-1β release. Various inflammatory plasma membrane receptors (e.g., IL-1 type I receptor, TNF type I and II receptors, IL-2 receptor) are shed under different pathophysiological conditions. In the present study, we show that the full length P2X7R is released into circulation in patients as well as in healthy subjects. Blood levels of shed P2X7R (sP2X7R) correlate to those of the inflammatory marker C reactive protein (CRP). Blood sP2X7R ranged from 16.74 to 82.17 ng/L, mean ± SE 40.97 ± 3.82 (n = 26) in healthy subjects, from 33.1 to 484.0 ng/L, mean ± SE 114.78 ± 12.22 (n = 45) in patients with CRP 3 mg/L. sP2X7R in plasma was largely associated to microvesicles/microparticles. Peripheral blood monocytes from healthy subjects released sP2X7R upon stimulation with the semi-selective P2X7R agonist benzoyl ATP. These data show that the P2X7R can be released into circulation, and that its blood levels increase in various disease conditions