Purinergic signalling in neuron-glia interactions

Activity-dependent release of ATP from synapses, axons and glia activates purinergic membrane receptors that modulate intracellular calcium and cyclic AMP. This enables glia to detect neural activity and communicate among other glial cells by releasing ATP through membrane channels and vesicles. Through purinergic signalling, impulse activity regulates glial proliferation, motility, survival, differentiation and myelination, and facilitates interactions between neurons, and vascular and immune system cells. Interactions among purinergic, growth factor and cytokine signalling regulate synaptic strength, development and responses to injury. We review the involvement of ATP and adenosine receptors in neuron-glia signalling, including the release and hydrolysis of ATP, how the receptors signal, the pharmacological tools used to study them, and their functional significance.     

Variations of ATP and its metabolites in the hippocampus of rats subjected to pilocarpine-induced temporal lobe epilepsy

Although purinergic receptor activity has lately been associated with epilepsy, little is known about the exact role of purines in epileptogenesis. We have used a rat model of temporal lobe epilepsy induced by pilocarpine to study the dynamics of purine metabolism in the hippocampus during different times of status epilepticus (SE) and the chronic phase. Concentrations of adenosine 5′-triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), and adenosine in normal and epileptic rat hippocampus were determined by microdialysis in combination with high-performance liquid chromatography (HPLC). Extracellular ATP concentrations did not vary along 4 h of SE onset. However, AMP concentration was elevated during the second hour, whereas ADP and adenosine concentrations augmented during the third and fourth hour following SE. During chronic phase, extracellular ATP, ADP, AMP, and adenosine concentrations decreased, although these levels again increased significantly during spontaneous seizures. These results suggest that the increased turnover of ATP during the acute period is a compensatory mechanism able to reduce the excitatory role of ATP. Increased adenosine levels following 4 h of SE may contribute to block seizures. On the other hand, the reduction of purine levels in the hippocampus of chronic epileptic rats may result from metabolic changes and be part of the mechanisms involved in the onset of spontaneous seizures. This work provides further insights into purinergic signaling during establishment and chronic phase of epilepsy.     

Measurement of purine release with microelectrode biosensors

Purinergic signalling departs from traditional paradigms of neurotransmission in the variety of release mechanisms and routes of production of extracellular ATP and adenosine. Direct real-time measurements of these purinergic agents have been of great value in understanding the functional roles of this signalling system in a number of diverse contexts. Here, we review the methods for measuring purine release, introduce the concept of microelectrode biosensors for ATP and adenosine and explain how these have been used to provide new mechanistic insight in respiratory chemoreception, synaptic physiology, eye development and purine salvage. We finish by considering the association of purine release with pathological conditions and examine the possibilities that biosensors for purines may one day be a standard part of the clinical diagnostic tool chest.     

The effect of metoclopramide on inhibition induced by purine nucleotides, noradrenaline, and theophylline ethylenediamine on intestinal muscle and on peristalsis in vitro.

Metoclopramide (N-(diethylaminoethyl)-2-methoxy-4-amino-5-chlorobenzamide) (Mcp) at concentrations of 0.1 and 1.0 muM partially and significantly reduced the relaxations induced by adenosine 5'-triphosphate (ATP), adenosine diphosphate (ADP), and adenosine, was without effect on theophylline ethylenediamine whilst significantly potentiating noradrenaline on the atropine-pretreated (0.1 muM) taenia coli, rabbit ileum, and rat duodenum. Mcp (1.0 muM) decreased the inhibitory effects of ATP, ADP, and adenosine on peristalsis induced in the isolated guinea-pig ileum by a constant increase in intraluminal pressure, did not affect inhibition due to theophylline ethylenediamine, whilst it potentiated inhibition of peristalsis due to noradrenaline. It is proposed that this effect of Mcp may be a specific antagonistic action on receptors sensitive to the putative purinergic transmitter, ATP and ADP, and may be partly responsible for its observed facilitatatory action on peristalsis.     

Measurement of purine release with microelectrode biosensors

Purinergic signalling departs from traditional paradigms of neurotransmission in the variety of release mechanisms and routes of production of extracellular ATP and adenosine. Direct real-time measurements of these purinergic agents have been of great value in understanding the functional roles of this signalling system in a number of diverse contexts. Here, we review the methods for measuring purine release, introduce the concept of microelectrode biosensors for ATP and adenosine and explain how these have been used to provide new mechanistic insight in respiratory chemoreception, synaptic physiology, eye development and purine salvage. We finish by considering the association of purine release with pathological conditions and examine the possibilities that biosensors for purines may one day be a standard part of the clinical diagnostic tool chest.

     

Ectonucleotidases in Müller glial cells of the rodent retina: Involvement in inhibition of osmotic cell swelling

Extracellular nucleotides mediate glia-to-neuron signalling in the retina and are implicated in the volume regulation of retinal glial (Müller) cells under osmotic stress conditions. We investigated the expression and functional role of ectonucleotidases in Müller cells of the rodent retina by cell-swelling experiments, calcium imaging, and immuno- and enzyme histochemistry. The swelling of Müller cells under hypoosmotic stress was inhibited by activation of an autocrine purinergic signalling cascade. This cascade is initiated by exogenous glutamate and involves the consecutive activation of P2Y₁ and adenosine A1 receptors, the action of ectoadenosine 5′-triphosphate (ATP)ases, and a nucleoside-transporter-mediated release of adenosine. Inhibition of ectoapyrases increased the ATP-evoked calcium responses in Müller cell endfeet. Müller cells were immunoreactive for nucleoside triphosphate diphosphohydrolases (NTPDase)2 (but not NTPDase1), ecto-5′-nucleotidase, P2Y₁, and A1 receptors. Enzyme histochemistry revealed that ATP but not adenosine 5′-diphosphate (ADP) is extracellularly metabolised in retinal slices of NTPDase1 knockout mice. NTPDase1 activity and protein is restricted to blood vessels, whereas activity of alkaline phosphatase is essentially absent at physiological pH. The data suggest that NTPDase2 is the major ATP-degrading ectonucleotidase of the retinal parenchyma. NTPDase2 expressed by Müller cells can be implicated in the regulation of purinergic calcium responses and cellular volume.     

Purinergic signalling: Its unpopular beginning, its acceptance and its exciting future

Adenosine 5'-triphosphate (ATP) was identified in 1970 as the transmitter responsible for non-adrenergic, non-cholinergic neurotransmission in the gut and bladder and the term 'purinergic' was coined. Purinergic cotransmission was proposed in 1976 and ATP is now recognized as a cotransmitter in all nerves in the peripheral and central nervous systems. P1 (adenosine) and P2 (ATP) receptors were distinguished in 1978. Cloning of these receptors in the early 1990s was a turning point in the acceptance of the purinergic signalling hypothesis. There are both short-term purinergic signalling in neurotransmission, neuromodulation and secretion and long-term (trophic) purinergic signalling of cell proliferation, differentiation and death in development and regeneration. Much is known about the mechanisms of ATP release and its breakdown by ectonucleotidases. Recently, there has been emphasis on purinergic pathophysiology, including neurodegenerative and neuropsychiatric disorders. Purinergic therapeutic strategies are being developed for treatment of gut, kidney, bladder, lung, skeletal and reproductive system disorders, pain and cancer.     

ATP synthesis and storage

Since 1929, when it was discovered that ATP is a substrate for muscle contraction, the knowledge about this purine nucleotide has been greatly expanded. Many aspects of cell metabolism revolve around ATP production and consumption. It is important to understand the concepts of glucose and oxygen consumption in aerobic and anaerobic life and to link bioenergetics with the vast amount of reactions occurring within cells. ATP is universally seen as the energy exchange factor that connects anabolism and catabolism but also fuels processes such as motile contraction, phosphorylations, and active transport. It is also a signalling molecule in the purinergic signalling mechanisms. In this review, we will discuss all the main mechanisms of ATP production linked to ADP phosphorylation as well the regulation of these mechanisms during stress conditions and in connection with calcium signalling events. Recent advances regarding ATP storage and its special significance for purinergic signalling will also be reviewed.     

P2 receptors in cardiovascular regulation and disease

The role of ATP as an extracellular signalling molecule is now well established and evidence is accumulating that ATP and other nucleotides (ADP, UTP and UDP) play important roles in cardiovascular physiology and pathophysiology, acting via P2X (ion channel) and P2Y (G protein-coupled) receptors. In this article we consider the dual role of ATP in regulation of vascular tone, released as a cotransmitter from sympathetic nerves or released in the vascular lumen in response to changes in blood flow and hypoxia. Further, purinergic long-term trophic and inflammatory signalling is described in cell proliferation, differentiation, migration and death in angiogenesis, vascular remodelling, restenosis and atherosclerosis. The effects on haemostasis and cardiac regulation is reviewed. The involvement of ATP in vascular diseases such as thrombosis, hypertension and diabetes will also be discussed, as well as various heart conditions. The purinergic system may be of similar importance as the sympathetic and renin-angiotensin-aldosterone systems in cardiovascular regulation and pathophysiology. The extracellular nucleotides and their cardiovascular P2 receptors are now entering the phase of clinical development. An erratum to this article can be found at     

Overexpression of NTPDase2 in gliomas promotes systemic inflammation and pulmonary injury

Gliomas are the most common and devastating type of primary brain tumor. Many non-neoplastic cells, including immune cells, comprise the tumor microenvironment where they create a milieu that appears to dictate cancer development. ATP and the phosphohydrolytic products ADP and adenosine by activating P2 and P1 receptors may participate in these interactions among malignant and immune cells. Purinergic receptor-mediated cell communication is closely regulated by ectonucleotidases, such as by members of the ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) family, which hydrolyze extracellular nucleotides. We have shown that gliomas, unlike astrocytes, exhibit low NTPDase activity. Furthermore, ATP induces glioma cell proliferation and the co-administration of apyrase decreases progression of injected cells in vivo. We have previously shown that NTPDase2 reconstitution dramatically increases tumor growth in vivo. Here we evaluated whether NTPDase2 reconstitution to gliomas modulates systemic inflammatory responses. We observed that NTPDase2 overexpression modulated pro-inflammatory cytokine production and platelet reactivity. Additionally, pathological alterations in the lungs were observed in rats bearing these tumors. Our results suggest that disruption of purinergic signaling via ADP accumulation creates an inflammatory state that may promote tumor spread and dictate clinical progression.     

Nucleotide homeostasis and purinergic nociceptive signaling in rat meninges in migraine-like conditions

Extracellular ATP is suspected to contribute to migraine pain but regulatory mechanisms controlling pro-nociceptive purinergic mechanisms in the meninges remain unknown. We studied the peculiarities of metabolic and signaling pathways of ATP and its downstream metabolites in rat meninges and in cultured trigeminal cells exposed to the migraine mediator calcitonin gene-related peptide (CGRP). Under resting conditions, meningeal ATP and ADP remained at low nanomolar levels, whereas extracellular AMP and adenosine concentrations were one-two orders higher. CGRP increased ATP and ADP levels in meninges and trigeminal cultures and reduced adenosine concentration in trigeminal cells. Degradation rates for exogenous nucleotides remained similar in control and CGRP-treated meninges, indicating that CGRP triggers nucleotide release without affecting nucleotide-inactivating pathways. Lead nitrate-based enzyme histochemistry of whole mount meninges revealed the presence of high ATPase, ADPase, and AMPase activities, primarily localized in the medial meningeal artery. ATP and ADP induced large intracellular Ca²⁺ transients both in neurons and in glial cells whereas AMP and adenosine were ineffective. In trigeminal glia, ATP partially operated via P2X7 receptors. ATP, but not other nucleotides, activated nociceptive spikes in meningeal trigeminal nerve fibers providing a rationale for high degradation rate of pro-nociceptive ATP. Pro-nociceptive effect of ATP in meningeal nerves was reproduced by α,β-meATP operating via P2X3 receptors. Collectively, extracellular ATP, which level is controlled by CGRP, can persistently activate trigeminal nerves in meninges which considered as the origin site of migraine headache. These data are consistent with the purinergic hypothesis of migraine pain and suggest new targets against trigeminal pain.     

Nucleotide- and nucleoside-converting ectoenzymes: Important modulators of purinergic signalling cascade

The involvement of extracellular nucleotides and adenosine in an array of cell-specific responses has long been known and appreciated, but the integrative view of purinergic signalling as a multistep coordinated cascade has emerged recently. Current models of nucleotide turnover include: (i) transient release of nanomolar concentrations of ATP and ADP; (ii) triggering of signalling events via a series of ligand-gated (P2X) and metabotropic (P2Y) receptors; (iii) nucleotide breakdown by membrane-bound and soluble nucleotidases, including the enzymes of ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase) family, ecto-nucleotide pyrophosphatase/phosphodiesterase (E-NPP) family, ecto-5'-nucleotidase/CD73, and alkaline phosphatases; (iv) interaction of the resulting adenosine with own nucleoside-selective receptors; and finally, (v) extracellular adenosine inactivation via adenosine deaminase and purine nucleoside phosphorylase reactions and/or nucleoside uptake by the cells. In contrast to traditional paradigms that focus on purine-inactivating mechanisms, it has now become clear that "classical" intracellular ATP-regenerating enzymes, adenylate kinase, nucleoside diphosphate (NDP) kinase and ATP synthase can also be co-expressed on the cell surface. Furthermore, data on the ability of various cells to retain micromolar ATP levels in their pericellular space, as well as to release other related compounds (adenosine, UTP, dinucleotide polyphosphates and nucleotide sugars) gain another important insight into our understanding of mechanisms regulating a signalling cascade. This review summarizes recent advances in this rapidly evolving field, with particular emphasis on the nucleotide-releasing and purine-converting pathways in the vasculature.     

Purinergic signaling as a new mechanism underlying physical exercise benefits: a narrative review

In the last years, it has become evident that both acute and chronic physical exercise trigger responses/adaptations in the purinergic signaling and these adaptations can be considered one important mechanism related to the exercise benefits for health improvement. Purinergic system is composed of enzymes (ectonucleotidases), receptors (P1 and P2 families), and molecules (ATP, ADP, adenosine) that are able to activate these receptors. These components are widely distributed in almost all cell types, and they respond/act in a specific manner depending on the exercise types and/or intensities as well as the cell type (organ/tissue analyzed). For example, while acute intense exercise can be associated with tissue damage, inflammation, and platelet aggregation, chronic exercise exerts anti-inflammatory and anti-aggregant effects, promoting health and/or treating diseases. All of these effects are dependent on the purinergic signaling. Thus, this review was designed to cover the aspects related to the relationship between physical exercise and purinergic signaling, with emphasis on the modulation of ectonucleotidases and receptors. Here, we discuss the impact of different exercise protocols as well as the differences between acute and chronic effects of exercise on the extracellular signaling exerted by purinergic system components. We also reinforce the concept that purinergic signaling must be understood/considered as a mechanism by which exercise exerts its effects.     

The signaling effects of ATP on melanoma-like skin cancer

Melanoma is a type of skin cancer originated by the malignant transformation of melanocytes. Increasing incidence and mortality require efforts focused on studies and research about this cancer. Its microenvironment is rich in extracellular ATP, but there are no studies evaluating the ectonucleotidases and ATP effects on tumor-derived melanoma cells with known amounts of ATP. This way, the objective of this work was to evaluate the purinergic signaling in the pathophysiology of in vivo melanoma and the in vitro effects of ATP signaling. We found increased and effective extracellular ATP hydrolysis in platelets and a significant decrease of extracellular ATP levels and adenosine hydrolysis. In addition, we cultured PBMCs of melanoma patients and used ATP salt with specific concentrations to evaluate its signaling effects. The enzymatic activity analysis revealed that even with higher ATP doses cells metabolize adenine nucleotides less efficiently, and present low ATP, ADP and AMP hydrolytic activity in CM compared to CT cells. In summary, we showed for the first time important data about the purinergic signaling in the pathophysiology of melanoma and ATP signaling exercising immunosuppressive effects. Therefore, as already shown for other tumors, the purinergic signaling should be considered a potential target for melanoma management and treatment and could offer novel therapeutic prospects.     

Schistosome apyrase SmATPDase1, but not SmATPDase2, hydrolyses exogenous ATP and ADP

Schistosomes are parasitic worms that can live in the bloodstream of their vertebrate hosts for many years. It has been proposed that the worms impinge on host purinergic signalling by degrading proinflammatory molecules like ATP as well as prothrombotic mediators like ADP. This capability may help explain the apparent refractoriness of the worms to both immune elimination and thrombus formation. Three distinct ectoenzymes, expressed at the host-exposed surface of the worm’s tegument, are proposed to be involved in the catabolism of ATP and ADP. These are alkaline phosphatase (SmAP), phosphodiesterase (SmNPP-5), and ATP diphosphohydrolase (SmATPDase1). It has recently been shown that only one of these enzymes—SmATPDase1—actually degrades exogenous ATP and ADP. However, a second ATP diphosphohydrolase homolog (SmATPDase2) is located in the tegument and has been reported to be released by the worms. It is possible that this enzyme too participates in the cleavage of exogenous nucleotide tri- and di-phosphates. To test this hypothesis, we employed RNA interference (RNAi) to suppress the expression of the schistosome SmATPDase1 and SmATPDase2 genes. We find that only SmATPDase1-suppressed parasites are significantly impaired in their ability to degrade exogenously added ATP or ADP. Suppression of SmATPDase2 does not appreciably affect the worms’ ability to catabolize ATP or ADP. Furthermore, we detect no evidence for the secretion or release of an ATP-hydrolyzing activity by cultured parasites. The results confirm the role of tegumental SmATPDase1, but not SmADTPDase2, in the degradation of the exogenous proinflammatory and prothrombotic nucleotides ATP and ADP by live intravascular stages of the parasite.     

Profile of Nucleotide Catabolism and Ectonucleotidase Expression from the Hippocampi of Neonatal Rats After Caffeine Exposure

Nucleotides and nucleosides play an important role in neurodevelopment acting through specific receptors. Ectonucleotidases are the major enzymes involved in controlling the availability of purinergic receptors ligands. ATP is co-released with several neurotransmitters and is the most important source of extracellular adenosine by catabolism exerted by ectonucleotidases. The main ectonucleotidases are named NTPDases (1–8) and 5′-nucleotidase. Adenosine is a powerful modulator of neurotransmitter release. Caffeine blocks adenosine receptor activity as well as adenosine-mediated neuromodulation. Considering the susceptibility of the immature brain to caffeine and the need for correct purinergic signaling during fetal development, we have analyzed the effects of caffeine exposure during gestational and lactational periods on nucleotide degradation and ectonucleotidase expression from the hippocampi of 7-, 14- and 21-days-old rats. Nucleotides hydrolysis was assessed by colorimetric determination of inorganic phosphate released. Ectonucleotidases expression was performed by RT-PCR. ATP and ADP hydrolysis displayed parallel age-dependent decreases in both control and caffeine-treated groups. AMP hydrolysis increased with caffeine treatment in 7-days-old rats (75%); although there was no significant difference in AMP hydrolysis between control (non caffeine-treated) rats and 14- or 21-days caffeine-treated rats. ADP hydrolysis was not affected by caffeine treatment. Caffeine treatment in 7- and 14-days-old rats decreased ATP hydrolysis when compared to the control group (19% and 60% decrease, respectively), but 21-days-treated rats showed an increase in ATP hydrolysis (39%). Expression levels of NTPDase 1 and 5 decreased in hippocampi of caffeine-treated rats. The expression of 5′-nucleotidase was not affected after caffeine exposure. The changes observed in nucleotide hydrolysis and ectonucleotidases expression could promote subtle effects on normal neural development considering the neuromodulatory role of adenosine.     

Transmission of Mechanical Information by Purinergic Signaling

The skeleton constantly interacts and adapts to the physical world. We have previously reported that physiologically relevant mechanical forces lead to small repairable membrane injuries in bone-forming osteoblasts, resulting in release of ATP and stimulation of purinergic (P2) calcium responses in neighboring cells. The goal of this study was to develop a theoretical model describing injury-related ATP and ADP release, their extracellular diffusion and degradation, and purinergic responses in neighboring cells. After validation using experimental data for intracellular free calcium elevations, ATP, and vesicular release after mechanical stimulation of a single osteoblast, the model was scaled to a tissue-level injury to investigate how purinergic signaling communicates information about injuries with varying geometries. We found that total ATP released, peak extracellular ATP concentration, and the ADP-mediated signaling component contributed complementary information regarding the mechanical stimulation event. The total amount of ATP released governed spatial factors, such as the maximal distance from the injury at which purinergic responses were stimulated. The peak ATP concentration reflected the severity of an individual cell injury, allowing to discriminate between minor and severe injuries that released similar amounts of ATP because of differences in injury repair, and determined temporal aspects of the response, such as signal propagation velocity. ADP-mediated signaling became relevant only in larger tissue-level injuries, conveying information about the distance to the injury site and its geometry. Thus, we identified specific features of extracellular ATP and ADP spatiotemporal signals that depend on tissue mechanoresilience and encode the severity, scope, and proximity of the mechanical stimulus.     

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, A2_A and/or A2_B receptor expression, whereas the P2X7 receptor was downregulated. Interestingly, IL-1β secretion in response to nanoparticles is increased by enhanced ATP and ADP hydrolysis, whereas it is decreased by adenosine degradation or selective A2_A or A2_B 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-1β 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.The inflammasome is a major factor of the innate immune system acting as a multiprotein platform to activate caspase-1. We showed recently that nanoparticles of TiO₂ (nano-TiO₂) and SiO₂ (nano-SiO₂) are sensed by the NLRP3 inflammasome to induce the release of mature IL-1β,¹ as observed previously with the environmental irritants asbestos or silica.² Despite the identification and characterisation of numerous sterile or microbial activators, the precise mechanisms mediating NLRP3 inflammasome activation remain to be determined. Here, we investigated whether ATP release and purinergic signalling through ATP, ADP and adenosine may be involved in inflammasome activation by nanoparticles. Intracellular ATP is released after cellular stress and/or activation, and purinergic signalling has been shown to modulate inflammation and immunity.^{3, 4} In the extracellular space, ATP is rapidly hydrolysed in a stepwise manner to ADP, AMP (adenosine monophosphate) and adenosine by ectoenzymes.⁴ Adenosine is then irreversibly hydrolysed to inosine by adenosine deaminase (ADA). Extracellular ATP (eATP) signals through both ATP-gated ion channels P2X and G protein-coupled receptor (GPCR) P2Y membrane receptors, whereas ADP signals through P2Y receptors and adenosine through P1 receptors (or A receptors).⁵ P2Y receptors and A receptors may be coupled to the G_q protein, which activates phospholipase C-beta (PLC-β), to the stimulatory G (G_s) protein, which stimulates adenylate cyclase inducing an increase in cyclic AMP (cAMP) levels, or to the G inhibitory (G_i) protein, which inhibits adenylate cyclase. Extracellular adenosine level is the result of adenosine production from extracellular ATP and ADP, its degradation into inosine and its reuptake by cells. Both ATP and adenosine can be transported outside of the cell via diffusion or active transport, whereas only adenosine can enter the cells through adenosine transporters.⁶ Most cells possess equilibrative and concentrative adenosine transporters (respectively, ENTs and CNTs), which allow adenosine to quickly cross the plasma membrane.⁷ Intracellular adenosine is converted to ATP via phosphorylation steps mediated by adenosine kinase (AK) and AMP kinase (AMPK). The basal physiological level of extracellular adenosine has been estimated to be in the range of 30–200 nM.⁸ ATP-derived adenosine and its subsequent signalling through P1 receptors have beneficial roles in acute disease states.^{4, 9} However, during tissue injury, elevated adenosine levels participate in the progression to chronic diseases by promoting aberrant wound healing leading to fibrosis in different organs including the lungs, liver, skin and kidney. In these conditions the blockade of adenosine signalling is beneficial.^{10, 11, 12, 13, 14, 15, 16} In murine models, ADA-knockout mice present high persistent adenosine levels, which lead to airspace enlargement and fibrosis, cardinal signs of COPD and IPF.^{14, 17, 18}Here we investigate in more detail the critical contribution of purinergic signalling in driving NLRP3 inflammasome activation in response to nanoparticles pointing out the effect of ATP, ADP, as well as adenosine and its receptors. We also identify ATP-derived adenosine as a potential activator of the inflammasome.     

The effects of adenosine and adenine nucleotides on the guinea-pig vas deferens: evidence for existence of purinergic receptors.

The effect of adenosine 5′-triphosphate (ATP) and its congeners on the alpha-adrenergic neuroeffector transmission in the isolated vas deferens of the guinea pig was evaluated. Both intracellular activity and contractile response of the smooth muscle of the vas deferens were recorded by using the sucrose-gap method. Adenosine, adenosine diphosphate (ADP) and adenosine monophosphate (AMP) influenced alpha-adrenergic receptor-mediated excitatory responses by depolarizing the cell membrane potential. ATP, on the other hand, produced action potentials rather than sustained depolarization, and its activity was blocked by theophylline and 2, 2′-pyridylisatogen, an ATP antagonist, but not blocked by either phentolamine or phenoxybenzamine, which inhibit alpha-adrenoreceptor responsiveness caused by norepinephrine or phenylephrine. Furthermore, dipyridamole, an adenosine uptake blocker, potentiated both ATP and adenosine activities. These findings indicate that adenosine and adenine nucleotides may exert their action at an extracellular site. From these results, it may be speculated that alpha adrenoreceptors and purinergic receptors do indeed exist on the smooth muscle of the vas deferens.     

Purinergic signalling in the liver in health and disease

Purinergic signalling is involved in both the physiology and pathophysiology of the liver. Hepatocytes, Kupffer cells, vascular endothelial cells and smooth muscle cells, stellate cells and cholangiocytes all express purinoceptor subtypes activated by adenosine, adenosine 5′-triphosphate, adenosine diphosphate, uridine 5′-triphosphate or UDP. Purinoceptors mediate bile secretion, glycogen and lipid metabolism and indirectly release of insulin. Mechanical stress results in release of ATP from hepatocytes and Kupffer cells and ATP is also released as a cotransmitter with noradrenaline from sympathetic nerves supplying the liver. Ecto-nucleotidases play important roles in the signalling process. Changes in purinergic signalling occur in vascular injury, inflammation, insulin resistance, hepatic fibrosis, cirrhosis, diabetes, hepatitis, liver regeneration following injury or transplantation and cancer. Purinergic therapeutic strategies for the treatment of these pathologies are being explored.