Wound healing and tissue repair are critical processes, and adenosine, released from injured or ischemic tissues, plays an important role in promoting wound healing and tissue repair. Recent studies in genetically manipulated mice demonstrate that adenosine receptors are required for appropriate granulation tissue formation and in adequate wound healing. A2A and A2B adenosine receptors stimulate both of the critical functions in granulation tissue formation (i.e., new matrix production and angiogenesis), and the A1 adenosine receptor (AR) may also contribute to new vessel formation. The effects of adenosine acting on these receptors is both direct and indirect, as AR activation suppresses antiangiogenic factor production by endothelial cells, promotes endothelial cell proliferation, and stimulates angiogenic factor production by endothelial cells and other cells present in the wound. Similarly, adenosine, acting at its receptors, stimulates collagen matrix formation directly. Like many other biological processes, AR-mediated promotion of tissue repair is critical for appropriate wound healing but may also contribute to pathogenic processes. Excessive tissue repair can lead to problems such as scarring and organ fibrosis and adenosine, and its receptors play a role in pathologic fibrosis as well. Here we review the evidence for the involvement of adenosine and its receptors in wound healing, tissue repair and fibrosis.
Adenosine receptors; Wound healing; Fibrosis; Angiogenesis; Neovascularization
Nucleotides within the airway surface liquid (ASL) regulate airway epithelial ion transport rates by Ca2+- and protein kinase C-dependent mechanisms via activation of specific P2Y receptors. Extracellular adenine nucleotides also serve as precursors for adenosine, which promotes cyclic AMP-mediated activation of the cystic fibrosis transmembrane regulator chloride channel via A2b adenosine receptors. A biological role for extracellular ATP in ASL volume homeostasis has been suggested by the demonstration of regulated ATP release from airway epithelia. However, nucleotide hydrolysis at the airway surface makes it difficult to assess the magnitude of ATP release and the relative abundance of adenyl purines and, hence, to define their biological functions. We have combined ASL microsampling and high performance liquid chromatography analysis of fluorescent 1,N6-ethenoadenine derivatives to measure adenyl purines in ASL. We found that adenosine, AMP, and ADP accumulated in high concentrations relative to ATP within the ASL covering polarized primary human normal or cystic fibrosis airway epithelial cells. By using immortalized epithelial cell monolayers that endogenously express a luminal A2b adenosine receptor, we found that basal as well as forskolin-promoted cyclic AMP production was reduced by exogenous adenosine deaminase, suggesting that A2b receptors sense endogenous adenosine within the ASL. The physiological role of adenosine was further established by illustrating that adenosine removal or inhibition of adenosine receptors in primary cultures impaired ASL volume regulation. Our data reveal a complex pattern of nucleotides/nucleosides in ASL under resting conditions and suggest that adenosine may play a key role in regulating ASL volume homeostasis.
Fatty liver is commonly associated with alcohol ingestion and abuse. While the molecular pathogenesis of these fatty changes is well understood, the biochemical and pharmacological mechanisms by which ethanol stimulates these molecular changes remain unknown. During ethanol metabolism, adenosine is generated by the enzyme ecto-5′-nucleotidase, and adenosine production and adenosine receptor activation are known to play critical roles in the development of hepatic fibrosis. We therefore investigated whether adenosine and its receptors play a role in the development of alcohol-induced fatty liver. WT mice fed ethanol on the Lieber-DeCarli diet developed hepatic steatosis, including increased hepatic triglyceride content, while mice lacking ecto-5′-nucleotidase or adenosine A1 or A2B receptors were protected from developing fatty liver. Similar protection was also seen in WT mice treated with either an adenosine A1 or A2B receptor antagonist. Steatotic livers demonstrated increased expression of genes involved in fatty acid synthesis, which was prevented by blockade of adenosine A1 receptors, and decreased expression of genes involved in fatty acid metabolism, which was prevented by blockade of adenosine A2B receptors. In vitro studies supported roles for adenosine A1 receptors in promoting fatty acid synthesis and for A2B receptors in decreasing fatty acid metabolism. These results indicate that adenosine generated by ethanol metabolism plays an important role in ethanol-induced hepatic steatosis via both A1 and A2B receptors and suggest that targeting adenosine receptors may be effective in the prevention of alcohol-induced fatty liver.
Fatty liver is commonly associated with alcohol ingestion and abuse. While the molecular pathogenesis of these fatty changes is well understood, the histochemical and pharmacological mechanisms by which ethanol stimulates these molecular changes remain unknown. During ethanol metabolism, adenosine is generated by the enzyme ecto-5′-nucleotidase, and adenosine production and adenosine receptor activation are known to play critical roles in the development of hepatic fibrosis. We therefore investigated whether adenosine and its receptors play a role in the development of alcohol-induced fatty liver. WT mice fed ethanol on the Lieber-DeCarli diet developed hepatic steatosis, including increased hepatic triglyceride content, while mice lacking ecto-5-nucleotidase or adenosine A1 or A2B receptors were protected from developing fatty liver. Similar protection was also seen in WT mice treated with either an adenosine A1 or A2B receptor antagonist. Steatotic livers demonstrated increased expression of genes involved in fatty acid synthesis, which was prevented by blockade of adenosine A1 receptors, and decreased expression of genes involved in fatty acid metabolism, which was prevented by blockade of adenosine A2B receptors. In vitro studies supported roles for adenosine A1 receptors in promoting fatty acid synthesis and for A2B receptors in decreasing fatty acid metabolism. These results indicate that adenosine generated by ethanol metabolism plays an important role in ethanol-induced hepatic steatosis via both A1 and A2B receptors and suggest that targeting adenosine receptors may be effective in the prevention of alcohol-induced fatty liver.
Adenosine; CD39; CD73; ethanol; extonucleotidase; fibrosis; fructose; hepatic stellate cell; inflammation; insulin resistance; liver; knockout; liver fibrosis; NAFLD; NASH; nucleotide; PPAR; receptor; steatosis; T cell
Adenosine is a potent endogenous regulator of inflammation and tissue repair. Adenosine, which is released from injured and hypoxic tissue or in response to toxins and medications, may induce pulmonary fibrosis in mice, presumably via interaction with a specific adenosine receptor. We therefore determined whether adenosine and its receptors contribute to the pathogenesis of hepatic fibrosis.As in other tissues and cell types, adenosine is released in vitro in response to the fibrogenic stimuli ethanol (40 mg dl−1) and methotrexate (100 nM).Adenosine A2A receptors are expressed on rat and human hepatic stellate cell lines and adenosine A2A receptor occupancy promotes collagen production by these cells. Liver sections from mice treated with the hepatotoxins carbon tetrachloride (CCl4) (0.05 ml in oil, 50 : 50 v : v, subcutaneously) and thioacetamide (100 mg kg−1 in PBS, intraperitoneally) released more adenosine than those from untreated mice when cultured ex vivo.Adenosine A2A receptor-deficient, but not wild-type or A3 receptor-deficient, mice are protected from development of hepatic fibrosis following CCl4 or thioacetamide exposure.Similarly, caffeine (50 mg kg−1 day−1, po), a nonselective adenosine receptor antagonist, and ZM241385 (25 mg kg−1 bid), a more selective antagonist of the adenosine A2A receptor, diminished hepatic fibrosis in wild-type mice exposed to either CCl4 or thioacetamide.These results demonstrate that hepatic adenosine A2A receptors play an active role in the pathogenesis of hepatic fibrosis, and suggest a novel therapeutic target in the treatment and prevention of hepatic cirrhosis.
Purinergic receptor; methylxanthines; hepatic fibrosis; ethanol; methotrexate
Platelets contain three types of granules: alpha granules, dense granules, and lysosomal granules. Each granule contains various growth factors, cytokines, and other physiological substances. Platelets trigger many kinds of biological responses, such as hemostasis, wound healing, and tissue regeneration. This review presents experimental evidence of platelets in accelerating liver regeneration and improving liver fibrosis. The regenerative effect of liver by platelets consists of three mechanisms; i.e., the direct effect on hepatocytes, the cooperative effect with liver sinusoidal endothelial cells, and the collaborative effect with Kupffer cells. Many signal transduction pathways are involved in hepatocyte proliferation. One is activation of Akt and extracellular signal-regulated kinase (ERK)1/2, which are derived from direct stimulation from growth factors in platelets. The other is signal transducer and activator of transcription-3 (STAT3) activation by interleukin (IL)-6 derived from liver sinusoidal endothelial cells and Kupffer cells, which are stimulated by contact with platelets during liver regeneration. Platelets also improve liver fibrosis in rodent models by inactivating hepatic stellate cells to decrease collagen production. The level of intracellular cyclic adenosine monophosphate (cyclic AMP) is increased by adenosine through its receptors on hepatic stellate cells, resulting in inactivation of these cells. Adenosine is produced by the degradation of adenine nucleotides such as adenosine diphosphate (ADP) and adenosine tri-phosphate (ATP), which are stored in abundance within the dense granules of platelets.
platelet; STAT3; Akt; ERK1/2; S1P; adenosine; cyclic AMP
Adenosine, acting through the A2A receptor, promotes tissue matrix production in the skin and the liver and induces the development of dermal fibrosis and cirrhosis in murine models. Since expression of A2A receptors is increased in scleroderma fibroblasts, we examined the mechanisms by which the A2A receptor produces its fibrogenic effects.
The effects of A2A receptor ligation on the expression of the transcription factor, Fli1, a constitutive repressor for the synthesis of matrix proteins, such as collagen, is studied in dermal fibroblasts. Fli1 is also known to repress the transcription of CTGF/CCN2, and the effects of A2A receptor stimulation on CTGF and TGF-β1 expression are also examined.
A2A receptor occupancy suppresses the expression of Fli1 by dermal fibroblasts. A2A receptor activation induces the secretion of CTGF by dermal fibroblasts, and neutralization of CTGF abrogates the A2A receptor-mediated enhancement of collagen type I production. A2AR activation, however, resulted in a decrease in TGF-β1 protein release.
Our results suggest that Fli1 and CTGF are important mediators of the fibrogenic actions of adenosine and the use of small molecules such as adenosine A2A receptor antagonists may be useful in the therapy of dermal fibrosis in diseases such as scleroderma.
Fibrosis; fibroblast; scleroderma
Adenosine is a signaling nucleoside that is produced following tissue injury, particularly injury involving ischemia and hypoxia. The production of extracellular adenosine and its subsequent signaling through adenosine receptors plays an important role in orchestrating injury responses in multiple organs. There are four adenosine receptors that are widely distributed on immune, epithelial, endothelial, neuronal and stromal cells throughout the body. Interestingly, these receptors are subject to altered regulation following injury. Studies in mouse models and human cells and tissues have identified that the production of adenosine and its subsequent signaling through its receptors plays largely beneficial roles in acute disease states, with the exception of brain injury. In contrast, if elevated adenosine levels are sustained beyond the acute injury phase, adenosine responses can become detrimental by activating pathways that promote tissue injury and fibrosis. Understanding when during the course of disease adenosine signaling is beneficial as opposed to detrimental and defining the mechanisms involved will be critical for the advancement of adenosine based therapies for acute and chronic diseases. The purpose of this review is to discuss key observations that define the beneficial and detrimental aspects of adenosine signaling during acute and chronic disease states with an emphasis on cellular processes such as inflammatory cell regulation, vascular barrier function and tissue fibrosis.
adenosine receptors; inflammation; fibrosis; vascular barrier function; CD73; ADORA2B; ADORA2A; ADORA3; ADORA1; acute lung injury; remodeling; anti-inflammatory
Macrophages rely on reverse cholesterol transport mechanisms to rid themselves of excess cholesterol. By reducing accumulation of cholesterol in the artery wall, reverse cholesterol transport slows or prevents development of atherosclerosis. In stable macrophages, efflux mechanisms balance influx mechanisms and accumulating lipids do not overwhelm the cell. Under atherogenic conditions, inflow of cholesterol exceeds outflow and the cell is ultimately transformed into a foam cell, the prototypical cell in the atherosclerotic plaque. Adenosine is an endogenous purine nucleoside released from metabolically active cells by facilitated diffusion and generated extracellularly from adenine nucleotides. Under stress conditions, such as hypoxia, a depressed cellular energy state leads to an acute increase in the extracellular concentration of adenosine. Extracellular adenosine interacts with one or more of a family of G protein coupled receptors (A1, A2A, A2B and A3) to modulate the function of nearly all cells and tissues. Modulation of adenosine signaling participates in regulation of reverse cholesterol transport. Of particular note for the development of atherosclerosis, activation of A2A receptors dramatically inhibits inflammation and protects against tissue injury. Potent anti-atherosclerotic effects ofA2A receptor stimulation include inhibition of macrophage foam celltransformation and upregulation of the reverse cholesterol transport proteins cholesterol 27-hydroxylase and ATP binding cassette transporter (ABC) A1. Thus, A2A receptor agonists may correct or prevent the adverse effects of inflammatory processes on cellular cholesterol homeostasis. This review focuses on the importance of extracellular adenosine acting at specific receptors as a regulatory mechanism to control the formation of foam cells under conditions of lipid loading.
Background and purpose
Adenosine, an endogenous purine nucleoside, is a potent regulator of the inflammatory response and stimulus for fibrosis. We have previously demonstrated that adenosine, acting at the A2A receptor, plays a central role in hepatic fibrosis via direct promotion of collagen production by hepatic stellate cells. As we have previously demonstrated that macrophage A2A receptor function is regulated by interferon-gamma (IFNγ), a noted antifibrotic but pro-inflammatory cytokine, we examined its effect on A2AR-stimulated collagen production in the human hepatic stellate cell line LX-2.
Collagen expression was determined by western blotting and realtime reverse transcription polymerase chain reaction (RT-PCR). Receptor desensitization was assessed by western blotting for membrane associated GRK2. Receptor signaling was determined by western blotting for phosphorylated extracellular signal-related protein kinase (ERK) protein and immunoassay for intracellular cyclic AMP (cAMP). siRNA was used to knock down expression of adenylyl cyclase and signal transducer and activator of transcription (STAT). Adenylyl cyclase expression was assessed by realtime RT-PCR, and STAT expression was assessed by western blotting.
IFNγ diminishes A2A receptor-mediated collagen production at both protein and mRNA levels. IFNγ alters signal transduction at A2A receptors by a STAT1 mediated mechanism involving the suppression of adenylyl cyclase expression.
Conclusions and implications
IFNγ inhibits the function of the adenosine A2A receptor in hepatic stellate cells by downregulating the expression of adenylyl cyclase. This finding explains, at least in part, the protective effect of IFNγ in hepatic fibrosis.
hepatic fibrosis; collagen-1; interferon-gamma; inflammation; adenylyl cyclase; siRNA; hepatic stellate cells
Cancer is a chronic disease and its pathogenesis is well correlated with infection and inflammation. Adenosine is a purine nucleoside, which is produced under metabolic stress like hypoxic conditions. Acute or chronic inflammatory conditions lead to the release of precursor adenine nucleotides (adenosine triphosphate (ATP), adenosien diphosphate (ADP) and adenosine monophosphate (AMP)) from cells, which are extracellularly catabolized into adenosine by extracellular ectonucleotidases, i.e., CD39 or nucleoside triphosphate dephosphorylase (NTPD) and CD73 or 5′-ectonucleotidase. It is now well-known that adenosine is secreted by cancer as well as immune cells during tumor pathogenesis under metabolic stress or hypoxia. Once adenosine is released into the extracellular environment, it exerts various immunomodulatory effects via adenosine receptors (A1, A2A, A2B, and A3) expressed on various immune cells (i.e., macrophages, myeloid-derived suppressor cells (MDSCs), natural killer (NK) cells, dendritic cells (DCs), T cells, regulatory T cell (Tregs), etc.), which play very important roles in the pathogenesis of cancer. This review is intended to summarize the role of inflammation and adenosine in the immunopathogenesis of tumor along with regulation of tumor-specific immune response and its modulation as an adjunct approach to tumor immunotherapy.
Adenosine; Inflammation; Tumor; Cancer; DCs; Macrophages; MDSCs; NK cells; T cells; Tregs
Microglia are activated by pathogen-associated molecular patterns and produce pro-inflammatory cytokines, such as TNF-α, IL-6, and IL-12, and the anti-inflammatory cytokine IL-10. Adenosine is an endogenous purine nucleoside and is a ligand of four G protein-coupled adenosine receptors (ARs), which are the A1AR, A2AAR, A2BAR and A3AR. ARs have been shown to suppress TNF-α production by microglia, but their role in regulating IL-10 production has not been studied. Here, we demonstrate that adenosine augments IL-10 production by activated murine microglia while suppressing the production of pro-inflammatory cytokines. Since the order of potency of selective AR agonists in inducing IL-10 production was 5′-N-ethylcarboxamidoadenosine (NECA) > N6-(3-iodobenzyl)-adenosine-5′-N-methyluronamide (IB-MECA) > 2-chloro-N6-cyclopentyladenosine (CCPA) ≥ 2-p-(2-carboxyethyl)phenethylamino-5′-N-ethyl-carboxamidoadenosine (CGS21680), and the A2BAR antagonist MRS-1754 prevented the effect of NECA, we conclude that the stimulatory effect of adenosine on IL-10 production is mediated by the A2BAR. Mechanistically, adenosine augmented IL-10 mRNA accumulation by a transcriptional process. Using mutant IL-10 promoter constructs we showed that a CREB-binding region in the promoter mediated the augmenting effect of adenosine on IL-10 transcription. Chromatin immunoprecipitation analysis demonstrated that adenosine induced CREB phosphorylation at the IL-10 promoter. Silencing CREB using lentivirally delivered shRNA blocked the enhancing effect of adenosine on IL-10 production confirming a role for CREB in mediating the stimulatory effect of adenosine on IL-10 production. In addition, adenosine augmented IL-10 production by stimulating p38 MAPK. Collectively, our results establish that A2BARs augment IL-10 production by activated murine microglia.
The association of a human genetic deficiency of adenosine deaminase activity with combined immunodeficiency prompted a study of the effects of adenosine and of inhibition of adenosine deaminase activity on human lymphocyte transformation and a detailed study of adenosine metabolism throughout phytohemagglutinin-induced blastogenesis. The adenosine deaminase inhibitor, coformycin, at a concentration that inhibited adenosine deaminase activity more than 95%, or 50 muM adenosine, did not prevent blastogenesis by criteria of morphology or thymidine incorporation into acid-precipitable material. The combination of coformycin and adenosine, however, substantially reduced both the viable cell count and the incorporation of thymidine into DNA in phytohemagglutinin-stimulated lymphocytes. Incubation of lymphocytes with phytohemagglutinin for 72 h produced a 12-fold increase in the rate of deamination and a 6-fold increase in phosphorylation of adenosine by intact lymphocytes. There was no change in the apparent affinity for adenosine with either deamination or phosphorylation. The increased rates of metabolism, apparent as early as 3 h after addition of mitogen, may be due to increased entry of the nucleoside into stimulated lymphocytes. Increased adenosine metabolism was not due to changes in total enzyme activity; after 72 h in culture, the ratios of specific activities in extracts of stimulated to unstimulated lymphocytes were essentially unchanged for adenosine kinase, 0.92, and decreased for adenosine deaminase, 0.44. As much as 38% of the initial lymphocyte adenosine deaminase activity accumulated extracellularly after a 72-h culture with phytohemagglutinin. In phytohemagglutinin-stimulated lymphocytes, the principal route of adenosine metabolism was phosphorylation at less than 5 muM adenosine, and deamination at concentrations greater than 5 muM. In unstimulated lymphocytes, deamination was the principal route of adenosine metabolism over the range of adenosine concentrations studied (0.5-250 muM). These studies demonstrate the dependence of both the unstimulated and stimulated lymphocyte on adenosine and may account for the observed sensitivity of mitogen-stimulated lymphocytes to the toxic effects of exogenously supplied adenosine in the presence of the adenosine deaminase inhibitor coformycin. A single case of immunodeficiency disease has been reported in association with purine nucleoside phosphorylase deficiency. The catabolism of guanosine was also found to be enhanced in stimulated normal lymphocytes; phosphorolysis of guanosine to guanine by intact lymphocytes increased six fold after 72-h culture with phytohemagglutinin. The specific activity of purine nucleoside phosphorylase in extracts, with guanosine as substrate, was essentially the same in stimulated and unstimulated lymphocytes after 72 h of culture.
BACKGROUND AND PURPOSE
Adenosine and inosine accumulate extracellularly during hypoxia/ischaemia in the brain and may act as neuroprotectants. In spinal cord, there is pharmacological evidence for increases in extracellular adenosine during hypoxia, but no direct measurements of purine release. Furthermore, the efflux pathways and origin of extracellular purines are not defined. To characterize hypoxia-evoked purine accumulation, we examined the effect of acute hypoxia on the extracellular levels of adenosine and inosine in isolated spinal cords from rats.
Extracellular adenosine and inosine concentrations were assayed in an in vitro preparation of the isolated spinal cord of the neonatal rat by HPLC.
The extracellular level of inosine was about 10-fold higher than that of adenosine. Acute hypoxia (10 min) caused a temperature-dependent increase in these two purines, which were inhibited by an increase in external Ca2+, but not by several inhibitors of efflux pathways or metabolic enzymes of adenine nucleotides. Inhibitors of adenosine deaminase or the equilibrative nucleoside transporter (ENT) abolished the hypoxia-evoked increase in inosine but not adenosine. The inhibition of glial metabolism abolished the increase of both purines evoked by hypoxia but not by oxygen-glucose deprivation, hypercapnia or an adenosine kinase inhibitor.
CONCLUSIONS AND IMPLICATIONS
Our data suggest that hypoxia releases adenosine itself from intracellular sources. Inosine formed intracellularly may be released through ENTs. During hypoxia, astrocytes appear to play a key role in purine release from neonatal rat spinal cord.
hypoxia; adenosine; inosine; spinal cord; fluoroacetate
Adenosine is produced during cellular hypoxia and apoptosis, resulting in elevated tissue levels at sites of injury. Adenosine is also known to regulate a number of cellular responses to injury, but its role in hepatic stellate cell (HSC) biology and liver fibrosis is poorly understood. We tested the effect of adenosine on the cytosolic Ca2+ concentration, chemotaxis, and upregulation of activation markers in HSCs. We showed that adenosine did not induce an increase in the cytosolic Ca2+ concentration in LX-2 cells and, in addition, inhibited increases in the cytosolic Ca2+ concentration in response to ATP and PDGF. Using a Transwell system, we showed that adenosine strongly inhibited PDGF-induced HSC chemotaxis in a dose-dependent manner. This inhibition was mediated via the A2a receptor, was reversible, was reproduced by forskolin, and was blocked by the adenylate cyclase inhibitor 2,5-dideoxyadenosine. Adenosine also upregulated the production of TGF-β and collagen I mRNA. In conclusion, adenosine reversibly inhibits Ca2+ fluxes and chemotaxis of HSCs and upregulates TGF-β and collagen I mRNA. We propose that adenosine provides 1) a “stop” signal to HSCs when they reach sites of tissue injury with high adenosine concentrations and 2) stimulates transdifferentiation of HSCs by upregulating collagen and TGF-β production.
platelet-derived growth factor; Ca2+; fibrosis
Adenosine is an endogenous purine nucleoside that, following its release into the extracellular space, binds to specific adenosine receptors expressed on the cell surface. Adenosine appears in the extracellular space under metabolically stressful conditions, which are associated with ischemia, inflammation, and cell damage. There are 4 types of adenosine receptors (A1, A2A, A2B and A3) and all adenosine receptors are members of the G protein-coupled family of receptors. Adenosine receptors are expressed on monocytes and macrophages and through these receptors adenosine modulates monocyte and macrophage function. Since monocytes and macrophages are activated by the same danger signals that cause accumulation of extracellular adenosine, adenosine receptors expressed on macrophages represent a sensor system that provide monocytes and macrophages with information about the stressful environment. Adenosine receptors, thus, allow monocytes and macrophages to fine-tune their responses to stressful stimuli. Here, we review the consequences of adenosine receptor activation on monocyte/macrophage function. We will detail the effect of stimulating the various adenosine receptor subtypes on macrophage differentiation/proliferation, phagocytosis, and tissue factor (TF) expression. We will also summarize our knowledge of how adenosine impacts the production of extracellular mediators secreted by monocytes and macrophages in response to toll-like receptor (TLR) ligands and other inflammatory stimuli. Specifically, we will delineate how adenosine affects the production of superoxide, nitric oxide (NO), tumor necrosis factor-α, interleukin (IL)-12, IL-10, and vascular endothelial growth factor (VEGF). A deeper insight into the regulation of monocyte and macrophage function by adenosine receptors should assist in developing new therapies for inflammatory diseases.
Infection; Autoimmunity; Asthma; Sepsis; Arthritis; Colitis
Adenosine concentrations are elevated in the lungs of patients with asthma and chronic obstructive pulmonary disease, where it balances between tissue repair and excessive airway remodeling. We previously demonstrated that the activation of the adenosine A2A receptor promotes epithelial wound closure. However, the mechanism by which adenosine-mediated wound healing occurs after cigarette smoke exposure has not been investigated. The present study investigates whether cigarette smoke exposure alters adenosine-mediated reparative properties via its ability to induce a shift in the oxidant/antioxidant balance. Using an in vitro wounding model, bronchial epithelial cells were exposed to 5% cigarette smoke extract, were wounded, and were then stimulated with either 10 μM adenosine or the specific A2A receptor agonist, 5′-(N-cyclopropyl)–carboxamido–adenosine (CPCA; 10 μM), and assessed for wound closure. In a subset of experiments, bronchial epithelial cells were infected with adenovirus vectors encoding human superoxide dismutase and/or catalase or control vector. In the presence of 5% smoke extract, significant delay was evident in both adenosine-mediated and CPCA-mediated wound closure. However, cells pretreated with N-acetylcysteine (NAC), a nonspecific antioxidant, reversed smoke extract–mediated inhibition. We found that cells overexpressing mitochondrial catalase repealed the smoke extract inhibition of CPCA-stimulated wound closure, whereas superoxide dismutase overexpression exerted no effect. Kinase experiments revealed that smoke extract significantly reduced the A2A-mediated activation of cyclic adenosine monophosphate–dependent protein kinase. However, pretreatment with NAC reversed this effect. In conclusion, our data suggest that cigarette smoke exposure impairs A2A-stimulated wound repair via a reactive oxygen species–dependent mechanism, thereby providing a better understanding of adenosine signaling that may direct the development of pharmacological tools for the treatment of chronic inflammatory lung disorders.
cigarette smoke extract; adenosine; wound closure; oxidants
Background: Newborns have elevated plasma adenosine levels, which may influence their immunological function.
Results: Compared with adults, newborns have elevated plasma 5′-NT and alkaline phosphatase activities and lower adenosine deaminase activity.
Conclusion: Soluble enzymes significantly influence extracellular purine metabolism in blood, and the levels of these enzymes in newborns promote elevated adenosine.
Significance: Higher adenosine generation in newborn blood may promote an anti-inflammatory immunological status.
Extracellular adenosine, a key regulator of physiology and immune cell function that is found at elevated levels in neonatal blood, is generated by phosphohydrolysis of adenine nucleotides released from cells and catabolized by deamination to inosine. Generation of adenosine monophosphate (AMP) in blood is driven by cell-associated enzymes, whereas conversion of AMP to adenosine is largely mediated by soluble enzymes. The identities of the enzymes responsible for these activities in whole blood of neonates have been defined in this study and contrasted to adult blood. We demonstrate that soluble 5′-nucleotidase (5′-NT) and alkaline phosphatase (AP) mediate conversion of AMP to adenosine, whereas soluble adenosine deaminase (ADA) catabolizes adenosine to inosine. Newborn blood plasma demonstrates substantially higher adenosine-generating 5′-NT and AP activity and lower adenosine-metabolizing ADA activity than adult plasma. In addition to a role in soluble purine metabolism, abundant AP expressed on the surface of circulating neonatal neutrophils is the dominant AMPase on these cells. Plasma samples from infant observational cohorts reveal a relative plasma ADA deficiency at birth, followed by a gradual maturation of plasma ADA through infancy. The robust adenosine-generating capacity of neonates appears functionally relevant because supplementation with AMP inhibited whereas selective pharmacologic inhibition of 5′-NT enhanced Toll-like receptor-mediated TNF-α production in neonatal whole blood. Overall, we have characterized previously unrecognized age-dependent expression patterns of plasma purine-metabolizing enzymes that result in elevated plasma concentrations of anti-inflammatory adenosine in newborns. Targeted manipulation of purine-metabolizing enzymes may benefit this vulnerable population.
Adenosine; Adenosine Receptor; ADP; AMP; ATP; Immunology; Infectious Diseases; Innate Immunity; Purine; Purinergic Agonists
Evidence demonstrating that regular ingestion of coffee has salutary effects on patients with chronic liver disease is accumulating rapidly. Specifically, it appears that coffee ingestion can slow the progression of liver fibrosis, preventing cirrhosis and hepatocellular carcinoma (HCC). This should excite clinicians and scientists alike, since these observations, if true, would create effective, testable hypotheses that should lead to improved understanding on fibrosis pathogenesis and thus may generate novel pharmacologic treatments of patients with chronic liver disease.
This review is designed to examine the relevant clinical and epidemiological data in critical fashion and to examine the putative pharmacological effects of coffee relevant to the pathogenesis of liver fibrosis and cirrhosis. We hope that this will inspire relevant critical analyses, especially among “coffee skeptics”. Of note, one major assumption made by this review is that the bulk of the effects of coffee consumption are mediated by caffeine, rather than by other chemical constituents of coffee. Our rationales for this assumption are threefold: first, caffeine’s effects on adenosinergic signaling provide testable hypotheses; second, although there are myriad chemical constituents of coffee, they are present in very low concentrations, and perhaps more importantly, vary greatly between coffee products and production methods (it is important to note that we do not dismiss the “botanical” hypothesis here; rather, we do not emphasize it at present due to the limitations of the studies examined); lastly, some (but not all) observational studies have examined both coffee and non-coffee caffeine consumption and found consistent effects, and when examined, no benefit to decaffeinated coffee has been observed. Further, in the interval since we examined this phenomenon last, further evidence has accumulated supporting caffeine as the effector molecule for coffee’s salutary effects.
adenosine receptor; coffee; liver fibrosis; cirrhosis
Adenosine is generated in response to cellular stress and damage and is elevated in the lungs of patients with chronic lung disease. Adenosine signaling through its cell surface receptors serves as an amplifier of chronic lung disorders, suggesting adenosine-based therapeutics may be beneficial in the treatment of lung diseases such as chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF). Previous studies in mouse models of chronic lung disease demonstrate that the key components of adenosine metabolism and signaling are altered. Changes include an up-regulation of CD73, the major enzyme of adenosine production and down-regulation of adenosine deaminase (ADA), the major enzyme for adenosine metabolism. In addition, adenosine receptors are elevated.
The focus of this study was to utilize tissues from patients with COPD or IPF to examine whether changes in purinergic metabolism and signaling occur in human disease. Results demonstrate that the levels of CD73 and A2BR are elevated in surgical lung biopsies from severe COPD and IPF patients. Immunolocalization assays revealed abundant expression of CD73 and the A2BR in alternatively activated macrophages in both COPD and IPF samples. In addition, mediators that are regulated by the A2BR, such as IL-6, IL-8 and osteopontin were elevated in these samples and activation of the A2BR on cells isolated from the airways of COPD and IPF patients was shown to directly induce the production of these mediators.
These findings suggest that components of adenosine metabolism and signaling are altered in a manner that promotes adenosine production and signaling in the lungs of patients with COPD and IPF, and provide proof of concept information that these disorders may benefit from adenosine-based therapeutics. Furthermore, this study provides the first evidence that A2BR signaling can promote the production of inflammatory and fibrotic mediators in patients with these disorders.
Adenosine is an endogenously released purine nucleoside that signals via four widely expressed G-protein coupled receptors: A1, A2A, A2B, and A3. In the setting of inflammation, the generation and release of adenosine is greatly enhanced. Neutrophils play an important role in host defense against invading pathogens and are the cellular hallmark of acute inflammation. Neutrophils both release adenosine and can respond to it via expression of all four adenosine receptor subtypes. At low concentrations, adenosine can act via the A1 and A3 adenosine receptor subtypes to promote neutrophil chemotaxis and phagocytosis. At higher concentrations, adenosine acts at the lower-affinity A2A and A2B receptors to inhibit neutrophil trafficking and effector functions such as oxidative burst, inflammatory mediator production, and granule release. Modulation of neutrophil function by adenosine is relevant in a broad array of disease models, including ischemia reperfusion injury, sepsis, and non-infectious acute lung injury. This review will summarize relevant research in order to provide a framework for understanding how adenosine directly regulates various elements of neutrophil function.
adenosine; neutrophil; chemotaxis; adhesion; host defense
Coffee and tea contain the stimulants caffeine and theophylline. These compounds act as antagonists of adenosine receptors. Adenosine promotes sleep and its extracellular concentration rises in association with prolonged wakefulness, particularly in the basal forebrain (BF) region involved in activating the cerebral cortex. However, the effect of adenosine on identified BF neurons, especially non-cholinergic neurons, is incompletely understood. Here we used whole-cell patch-clamp recordings in mouse brain slices prepared from two validated transgenic mouse lines with fluorescent proteins expressed in GABAergic or parvalbumin (PV) neurons to determine the effect of adenosine. Whole-cell recordings were made from BF cholinergic neurons and from BF GABAergic and PV neurons with the size (>20 μm) and intrinsic membrane properties (prominent H-currents) corresponding to cortically projecting neurons. A brief (2 min) bath application of adenosine (100 μM) decreased the frequency but not the amplitude of spontaneous excitatory postsynaptic currents (EPSCs) in all groups of BF cholinergic, GABAergic, and PV neurons we recorded. In addition, adenosine decreased the frequency of miniature EPSCs in BF cholinergic neurons. Adenosine had no effect on the frequency of spontaneous inhibitory postsynaptic currents in cholinergic neurons or GABAergic neurons with large H-currents but reduced them in a group of GABAergic neurons with smaller H-currents. All effects of adenosine were blocked by a selective, adenosine A1 receptor antagonist, cyclopentyltheophylline (CPT, 1 μM). Adenosine had no postsynaptic effects. Taken together, our work suggests that adenosine promotes sleep by an A1 receptor-mediated inhibition of glutamatergic inputs to cortically projecting cholinergic and GABA/PV neurons. Conversely, caffeine and theophylline promote attentive wakefulness by inhibiting these A1 receptors in BF thereby promoting the high-frequency oscillations in the cortex required for attention and cognition.
patch-clamp; adenosine A1 receptor; presynaptic modulation; sleep; transgenic mice
The lymphatic system controls tissue homeostasis by draining protein-rich lymph to the vascular system. Lymphangiogenesis, the formation of lymphatic vessels, is a normal event in childhood but promotes tumor spread and metastasis during adulthood. Blocking lymphangiogenesis may therefore be of therapeutic interest. Production of adenosine is enhanced in the tumor environment and contributes to tumor progression through stimulation of angiogenesis. In this study, we determined whether adenosine affects lymphangiogenesis.
Lymphatic endothelial cells (HMVEC-dLy) were cultured in presence of adenosine and their proliferation, migration and tube formation was assessed. Gelatin sponges embedded with the stable analogue of adenosine 2-chloro adenosine were implanted in mice ear and lymphangiogenesis was quantified. Mice were intravenously injected with adenoviruses containing expression vector for 5′-endonucleotidase, which plays a major role in the formation of adenosine.
In vitro, we observed that adenosine decreased the proliferation of lymphatic endothelial cells, their migration and tube formation. However, in vivo, gelatin sponges containing 2-chloro adenosine and implanted in mice ear displayed an elevated level of lymphangiogenesis (2.5-fold, p<0.001). Adenovirus-mediated over-expression of cytosolic 5′-nucleotidase IA stimulated lymphangiogenesis and the recruitment of macrophages in mouse liver. Proliferation of lymphatic endothelial cells was enhanced (2-fold, p<0.001) when incubated in the presence of conditioned medium from murine macrophages.
We have shown that adenosine stimulates lymphangiogenesis in vivo, presumably through a macrophage-mediated mechanism. This observation suggests that blockade of adenosine receptors may help in anti-cancer therapies.
In cell culture, extracellular guanosine increases extracellular adenosine by attenuating the disposition of extracellular adenosine (American Journal of Physiology – Cell Physiology 304: C406–C421, 2013). The goal of this investigation was to determine whether this “guanosine–adenosine mechanism” is operative in an intact organ. Twenty‐seven isolated, perfused mouse kidneys were subjected to metabolic poisons (iodoacetate plus 2,4‐dinitrophenol) to cause energy depletion and thereby stimulate renal adenosine production. Adenosine levels in the renal venous perfusate increased from a baseline of 36 ± 8 to 499 ± 96, 258 ± 50, and 71 ± 13 nmol/L at 15, 30, and 60 min, respectively, after administering metabolic poisons (% of basal; 1366 ± 229, 715 ± 128, and 206 ± 33, respectively). Changes in renal venous levels of guanosine closely mirrored the time course of changes in adenosine: baseline of 15 ± 2 to 157 ± 13, 121 ± 8, and 50 ± 5 nmol/L at 15, 30, and 60 min, respectively (% of basal; 1132 ± 104, 871 ± 59, and 400 ± 51, respectively). Freeze‐clamp experiments in 12 kidneys confirmed that metabolic poisons increased kidney tissue levels of adenosine and guanosine. In eight additional kidneys, we examined the ability of guanosine to reduce the renal clearance of exogenous adenosine; and these experiments revealed that guanosine significantly decreased the renal extraction of adenosine. Because guanosine is metabolized by purine nucleoside phosphorylase (PNPase), in another set of 16 kidneys we examined the effects of 8‐aminoguanine (PNPase inhibitor) on renal venous levels of adenosine and inosine (adenosine metabolite). Kidneys treated with 8‐aminoguanine showed a more robust increase in both adenosine and inosine in response to metabolic poisons. We conclude that in the intact kidney, guanosine regulates adenosine levels.
In cell culture, extracellular guanosine increases extracellular adenosine by attenuating the disposition of extracellular adenosine (American Journal of Physiology – Cell Physiology 304: C406–C421, 2013). The goal of this study was to determine whether the “guanosine–adenosine mechanism” is operative in an intact organ. In isolated, perfused mouse kidneys, inhibition of energy production induced changes in renal venous levels of guanosine that closely mirrored the time course of changes in adenosine, and freeze‐clamp experiments confirmed that metabolic poisons similarly increased kidney tissue levels of adenosine and guanosine. Moreover, exogenous guanosine significantly decreased the renal extraction of exogenous adenosine, and inhibition of purine nucleoside phosphorylase (metabolizes guanosine) augmented the effects of energy depletion on renal levels of both guanosine and adenosine. We conclude that in the intact kidney, guanosine regulates adenosine levels.
8‐Aminoguanine; adenosine; guanosine; inosine; kidney; purine nucleoside phosphorylase
The aim of this study was to investigate the effects of adenine nucleosides and nucleotides on contractility of the smooth muscle of rat prostate gland.Nerve terminals within rat isolated prostatic tissues were electrically field stimulated (60 V, 0.5 ms, 10 Hz, 20 pulses every 60 s). Adenosine 5′-triphosphate (ATP), adenosine 5′-diphosphate (ADP), adenosine 5′-monophosphate (AMP) and adenosine had no effect on baseline smooth muscle tone but concentration-dependently inhibited electrically-evoked contractile responses. The relative order of potency was ATP ≅ AMP ≅ adenosine>ADP.The inhibition by ATP and adenosine of field stimulation-induced contractions in the rat prostate was antagonized by 8-phenyltheophylline (10 μM), but not by suramin (100 μM) and only slightly by reactive blue 2 (5 μM).The adenosine metabolizing enzyme adenosine deaminase (0.1 unit ml−1) inhibited the inhibitory effects of ATP and adenosine. The P2 purinoceptor agonist 2-methylthio ATP (10 nM–0.1 mM), had no effect on field stimulation-induced contractions of the rat prostate.ATP and adenosine did not modify the contractile responses of the rat prostate to exogenously added noradrenaline (10 μM).Inhibitory concentration-response curves to a number of adenosine analogues with differing stabilities and selectivities for the different adenosine receptors yielded a relative rank order of agonist potency of: N6-cyclopentyladenosine (CPA)>N6-cyclohexyladenosine (CHA) ≅ (−)-N6-(2-phenylisopropyl)-adenosine (R-PIA) ≅ 5′-(N-ethylcarboxamido)-adenosine (NECA)>(+)-N6-(2-phenylisopropyl)-adenosine (S-PIA)>2-p-[2-carboxyethyl]phenethyl-amino-5′-N-ethylcarboxamido-adenosine (CGS 21680).These results indicate that adenine nucleoside and nucleotide induced inhibition of electrically-evoked contractions in the rat prostate occurs through activation of adenosine but not ATP receptors. The relative order of potency of adenosine analogues is consistent with activation of receptors of the A1-adenosine receptor subtype. These receptors appear to be prejunctional.
Prostate; adenosine receptors; adenosine 5′-triphosphate; ATP; adenosine; neuromodulation; smooth muscle