High Concentrations Of Adenosine Research Articles

Adenosine, energy metabolism and sleep homeostasis

Adenosine is directly linked to the energy metabolism of cells. In the central nervous system (CNS) an increase in neuronal activity enhances energy consumption as well as extracellular adenosine concentrations. In most brain areas high extracellular adenosine concentrations, through A1 adenosine receptors, decrease neuronal activity and thus the need for energy. Adenosine may be a final common pathway for various sleep factors. We have identified a relatively specific area, the basal forebrain (BF), which appears to be central in the regulation/execution of recovery sleep after sleep deprivation (SD), or prolonged wakefulness. Adenosine concentration increases in this area during SD, and this increase induces sleep while prevention of the increase during SD abolishes recovery sleep. The increase in adenosine is associated with local changes in energy metabolism as indicated by increases in levels of pyruvate and lactate and increased phosphorylation of AMP-activated protein kinase. The increases in adenosine and sleep are associated with intact cholinergic system since specific lesion of the BF cholinergic cells abolishes both. Whether adenosine during SD is produced by the cholinergic neurons or astrocytes associated with them remains to be explored. An interesting, but so far unexplored question regards the relationship between the local, cortical regulation of sleep homeostasis and the global regulation of the state of sleep as executed by lower brain mechanisms, including the BF. The increase in adenosine concentration during SD also in cortical areas suggests that adenosine may have a role in the local regulation of sleep homeostasis. The core of sleep need is probably related to primitive functions of life, like energy metabolism. It can be noted that this assumption in no way excludes the possibility that later in evolution additional functions may have developed, e.g., related to complex neuronal network functions like memory and learning.

ADENOSINE-MEDIATED ATTENUATION OF THE INNATE IMMUNE RESPONSE FOR ONLY THOSE WHO NEED IT? THE TAILORED POTENTIAL OF PENTOXIFYLLINE

Sepsis, the systemic inflammatory response syndrome that occurs during an infection, is associated with considerable morbidity and mortality in intensive care units. The initial proinflammatory response can be overwhelming, leading to collateral tissue damage in the host, suggesting that the innate immune response, rather than the nature of the pathogen itself, determines the outcome of a septic patient. During the past decade, a wide variety of studies focused on blockade of proinflammatory mediators, e.g., IL-1 receptor antagonists, anti-TNF antibodies, and soluble TNF receptors; none, however, improved mortality rates (1). Possibly, dampening of the innate immune response, rather than blocking it, will yield better results. Following the initial insult and immune response, sepsis induces a pronounced and sustained immune paralysis, resulting in a higher susceptibility to secondary infections accounting for increased mortality rates in this phase of the disease. Recently, it was proposed that the next major advance in sepsis treatment could be not the attenuation, but the preservation of the immune response (2). These observations illustrate that apart from source control and antibiotic therapy, adjuvant immunomodulating therapies are of interest and may contain future therapeutic potential. In this issue of Shock, Kreth et al. (3) describe a potential role for pentoxifylline (PTX) as an immunomodulating agent able to attenuate the inflammatory response during sepsis. Pentoxifylline is a xanthine derivative that exerts its anti-inflammatory effects through phosphodiesterase inhibition. Pentoxifylline has been intensively studied in cardiovascular diseases, where it has demonstrated to decrease TNF-α levels associated with an improved left ventricular pump function in patients with advanced heart failure (4). The mode of action of PTX is, however, still not fully known. Several theories have emerged, including modulation of (a) the translocation of calcium ions, (b) inhibition of phosphodiesterase, thereby increasing intracellular cyclic AMP (cAMP) concentrations, (c) interaction with the adenosine receptor or inhibition of adenosine uptake, and (d) interaction with prostaglandin metabolism (5). Kreth et al. suggest that the anti-inflammatory properties of PTX are mediated by adenosine, more specifically via the adenosine A2A receptor (A2AR) pathway. Adenosine is able to increase the cAMP concentration through A2AR and A2BR agonism, leading to its anti-inflammatory effects. Possibly, in the presence of PTX, the adenosine-induced increase in cAMP is not diminished because cAMP is not being hydrolyzed to ADP in the presence of PTX. Next, a sufficient amount of cAMP will be available to initiate protective signaling pathways explaining the synergistic effects described by Kreth et al. Unfortunately, the authors did not use specific adenosine receptor antagonists to support this hypothesis. Although the work of Kreth et al. is of interest, several limitations concerning the clinical relevance of this study are present. First, the authors state that knowledge of circulating adenosine levels is needed to predict whether a patient will be a possible responder to PTX therapy. However, only 50% of the septic patients in their study showed "adequately elevated" adenosine levels. Adenosine was first measured in septic patients by Martin et al. (6) in 2000, showing that a high plasma adenosine concentration has a prognostic value for the outcome of septic patients, but is variable in time. Calibration of the severity of illness is therefore of the utmost importance in determining the phase of disease, which is an extremely difficult task. Second, the reliable determination of extracellular adenosine concentrations is cumbersome in daily practice because of 2 reasons: adenosine has an extremely short half-life due to rapid cellular uptake and intracellular degradation, and the endothelium acts as an active metabolic barrier for adenosine. In addition, circulating adenosine concentrations do not represent the concentration in the interstitial compartments (7). Also, various techniques are used to measure adenosine concentrations that make comparisons of these studies difficult and hazardous. Standardization of techniques to measure the endogenous adenosine concentrations in humans in vivo is needed. In conclusion, Kreth et al. promote the concept of "tailored therapies" for sepsis patients, in this case using adenosine levels to predict the efficacy of PTX treatment. Considering the difficulties to determine in which phase of this disease the patient is currently in, the difficulties associated with the measurements of adenosine in daily practice, the fact that only circulating concentrations (and not interstitial concentrations) can be measured, and the lack of clinical evidence that PTX treatment will actually improve the outcome of septic patients, this will not take place in the future. Nevertheless, the concept of tailored therapy is relevant, deserves further attention, and may result in better future treatment options with fewer adverse effects.

Study of peripheral tolerance in ADA SCID patients after different treatments (143.36)

Abstract Adenosine deaminase (ADA)-SCID is characterized by increased purine metabolites and severe combined immunodeficiency. Autoimmune manifestations have been observed in milder forms or after enzyme replacement therapy (ERT), and recently in patients following gene therapy (GT). nTregs have a full enzymatic machinery to generate and sustain high concentrations of extracellular adenosine and may be involved in the pathogenesis of autoimmunity. We analyzed the frequency of CD4+CD25+FOXP3+CD127- in the peripheral blood, the methylation profile of the FoxP3 gene and the suppressive function of nTreg in ADA-SCID patients following ERT, GT or bone marrow transplant (BMT) as compared to controls. We found that the proportion and function of nTregs after GT mirrored that of controls, while nTreg from the ERT group showed a reduced frequency and impaired function. We also investigated the maturation state of B cells in the peripheral blood of ADA-SCID patients. After ERT and early after GT, patients displayed an increased proportion of transitional B cells (CD24highCD38high) and of cells with an incomplete BCR (CD21-CD35-) as compared to BMT and healthy controls. At later follow up patients treated with GT showed a tendency to normalize the phenotype in parallel to an increased frequency of gene corrected cells. Collectively, these data provide a first indication of predisposition to autoimmunity in ADA-SCID and the role of different treatments in the maintenance of peripheral tolerance.

Generation and Accumulation of Immunosuppressive Adenosine by Human CD4+CD25highFOXP3+ Regulatory T Cells

Naturally occurring regulatory T cells (nTreg) are crucial for maintaining tolerance to self and thus preventing autoimmune diseases and allograft rejections. In cancer, Treg down-regulate antitumor responses by several distinct mechanisms. This study analyzes the role the adenosinergic pathway plays in suppressive activities of human nTreg. Human CD4(+)CD25(high)FOXP3(+) Treg overexpress CD39 and CD73, ectonucleotidases sequentially converting ATP into AMP and adenosine, which then binds to A(2a) receptors on effector T cells, suppressing their functions. CD4(+)CD39(+) and CD4(+)CD25(high) T cells express low levels of adenosine deaminase (ADA), the enzyme responsible for adenosine breakdown, and of CD26, a surface-bound glycoprotein associated with ADA. In contrast, T effector cells are enriched in CD26/ADA but express low levels of CD39 and CD73. Inhibitors of ectonucleotidase activity (e.g. ARL67156) and antagonists of the A(2a) receptor (e.g. ZM241385) blocked Treg-mediated immunosuppression. The inhibition of ADA activity on effector T cells enhanced Treg-mediated immunosuppression. Thus, human nTreg characterized by the presence of CD39 and the low expression of CD26/ADA are responsible for the generation of adenosine, which plays a major role in Treg-mediated immunosuppression. The data suggest that the adenosinergic pathway represents a potential therapeutic target for regulation of immunosuppression in a broad variety of human diseases.

Adenosine Inhibits Chemotaxis and Induces Hepatocyte-Specific Genes in Bone Marrow Mesenchymal Stem Cells

Bone marrow-derived mesenchymal stem cells (MSCs) have therapeutic potential in liver injury, but the signals responsible for MSC localization to sites of injury and initiation of differentiation are not known. Adenosine concentration is increased at sites of cellular injury and inflammation, and adenosine is known to signal a variety of cellular changes. We hypothesized that local elevations in the concentration of adenosine at sites of tissue injury regulate MSC homing and differentiation. Here we demonstrate that adenosine does not induce MSC chemotaxis but dramatically inhibits MSC chemotaxis in response to the chemoattractant hepatocyte growth factor (HGF). Inhibition of HGF-induced chemotaxis by adenosine requires the A2a receptor and is mediated via up-regulation of the cyclic adenosine monophosphate (AMP)/protein kinase A pathway. This results in inhibition of cytosolic calcium signaling and down-regulation of HGF-induced Rac1. Because of the important role of Rac1 in the formation of actin stress fibers, we examined the effect of adenosine on stress fiber formation and found that adenosine inhibits HGF-induced stress fiber formation. In addition, we found that adenosine induces the expression of some key endodermal and hepatocyte-specific genes in mouse and human MSCs in vitro. We propose that the inhibition of MSC chemotaxis at sites of high adenosine concentration results in localization of MSCs to areas of cellular injury and death in the liver. We speculate that adenosine might initiate the process of differentiation of MSCs into hepatocyte-like cells.

A2A Adenosine Receptor May Allow Expansion of T Cells Lacking Effector Functions in Extracellular Adenosine-Rich Microenvironments

Immunosuppressive signaling via the A2A adenosine receptor (A2AR) provokes a mechanism that protects inflamed tissues from excessive damage by immune cells. This mechanism is desirable not only for preventing uncontrolled tissue destruction by overactive immune responses, but also for protecting tumor tissues from antitumor immune responses. In aforementioned circumstances, T cell priming may occur in an environment containing high concentrations of extracellular adenosine. To examine qualitative changes in T cells activated in the presence of adenosine, we asked whether different functional responses of T cells are equally susceptible to A2AR agonists. In this study, we demonstrate that A2AR signaling during T cell activation strongly inhibited development of cytotoxicity and cytokine-producing activity in T cells, whereas the inhibition of T cell proliferation was only marginal. Both CD8(+) and CD4(+) T cells proliferated well in the presence of A2AR agonists, but their IFN-gamma-producing activities were susceptible to inhibition by cAMP-elevating A2AR. Importantly, the impaired effector functions were maintained in T cells even after removal of the A2AR agonist, reflecting T cell memory of the immunoregulatory effect of adenosine. Thus, although the adenosine-rich environment may allow for the expansion of T cells, the functional activation of T cells may be critically impaired. This physiological mechanism could explain the inefficiency of antitumor T cells in the tumor microenvironment.

Erythrocytic Adenosine Monophosphate as an Alternative Purine Source in Plasmodium falciparum

Plasmodium falciparum is a purine auxotroph, salvaging purines from erythrocytes for synthesis of RNA and DNA. Hypoxanthine is the key precursor for purine metabolism in Plasmodium. Inhibition of hypoxanthine-forming reactions in both erythrocytes and parasites is lethal to cultured P. falciparum. We observed that high concentrations of adenosine can rescue cultured parasites from purine nucleoside phosphorylase and adenosine deaminase blockade but not when erythrocyte adenosine kinase is also inhibited. P. falciparum lacks adenosine kinase but can salvage AMP synthesized in the erythrocyte cytoplasm to provide purines when both human and Plasmodium purine nucleoside phosphorylases and adenosine deaminases are inhibited. Transport studies in Xenopus laevis oocytes expressing the P. falciparum nucleoside transporter PfNT1 established that this transporter does not transport AMP. These metabolic patterns establish the existence of a novel nucleoside monophosphate transport pathway in P. falciparum.

Adenosine A1-A2A receptor heteromers: new targets for caffeine in the brain

The contribution of blockade of adenosine A1 and A2A receptor to the psychostimulant effects of caffeine is still a matter of debate. When analyzing motor activity in rats, acutely administered caffeine shows a profile of a non-selective adenosine receptor antagonist, although with preferential A1 receptor antagonism. On the other hand, tolerance to the effects of A1 receptor blockade seems to be mostly responsible for the tolerance to the motor-activating effects of caffeine, while the residual motor-activating effects of caffeine in tolerant individuals seem to involve A2A receptor blockade. These behavioral studies correlate with in vivo microdialysis experiments that suggest that A1 receptor-mediated modulation of striatal glutamate release is involved in the psychostimulant effects of caffeine. Experiments in transfected cells demonstrate the ability of A1 receptors to heteromerize with A2A receptors and the A1-A2A receptor heteromer can be biochemically identified in the striatum, in striatal glutamatergic terminals. The striatal A1-A2A receptor heteromer provides a "concentration-dependent switch" mechanism by which low and high concentrations of synaptic adenosine produce the opposite effects on glutamate release. The analysis of the function of A1-A2A receptor heteromers during chronic treatment with caffeine gives new clues about the well-known phenomenon of tolerance to the psychostimulant effects of caffeine.

The Role of Adenosine in Rheumatoid Arthritis

In this review the hypothesis is developed, that high concentrations of adenosine are anti-inflammatory and therefore beneficial in inflammatory disease like rheumatoid arthritis. The anti-inflammatory effect of adenosine is mediated mainly via specific adenosine receptors on immune cells that are involved in the disease process. Unfortunately, at least three mechanisms support a decrease in the local concentration of adenosine at the site of inflammation: 1) A decrease in sympathetic innervation, which leads to decreased local release of the cotransmitter ATP at the site of inflammation and therefore to a decreased level of its degradation product adenosine. 2) High activities of the adenosine degrading enzyme adenosine deaminase, which leads to increased degradation of adenosine to inosine and therefore decreases the concentration of adenosine 3) Lower activity of the ATP degrading enzyme 5-Ecto-nucleotidase, which leads to a slower degradation of ATP to adenosine, also resulting in lower local levels of adenosine present. Therefore, it has to be considered that the optimal treatment for rheumatoid arthritis has to include drugs that specifically target adenosine receptors or the adenosine metabolism to increase local adenosine concentrations. In this respect the mechanism of action of low-dose methotrexate therapy is discussed, which is thought to involve the inhibition of adenosine degrading enzymes rather than antiproliferative effects. Keywords: Adenosine, neuroimmunology, rheumatoid arthritis, sympathetic nervous system

Adenosine A2 Receptor Activation Attenuates Afferent Arteriolar Autoregulation During Adenosine Receptor Saturation in Rats

Adenosine is an important paracrine agent regulating renal hemodynamics via adenosine A1 and A2 receptors. To determine the interactions between adenosine A1 and A2 receptors and the possible role of adenosine as a modulator of afferent arteriolar autoregulatory responses, videomicroscopic measurements of afferent arteriolar dimensions were performed at different perfusion pressures (from 100 to 125 and 150 mm Hg) using the isolated-blood-perfused rat juxtamedullary nephron preparation. Single afferent arterioles were visualized and superfused with low or high concentrations of adenosine, either alone or with the adenosine A1 receptor antagonist 8-noradamantan-3-yl-1,3-dipropylxanthine (10 micromol/L) or the adenosine A2 receptor antagonist dimethyl-1-propargylxanthine (10 micromol/L). Adenosine (20 micromol/L) decreased afferent arteriolar diameter by -9.0+/-0.9%, and this effect was enhanced by dimethyl-1-propargylxanthine (10 micromol/L) to -16.1+/-1.2%. However, autoregulatory capability was maintained. Adenosine-induced vasoconstriction was prevented by 8-noradamantan-3-yl-1,3-dipropylxanthine (10 micromol/L) with diameter increasing by 9.6+/-1.2%. Adenosine receptor saturation with a high concentration of adenosine (120 micromol/L) or blocking A1 receptors with 8-noradamantan-3-yl-1,3-dipropylxanthine in the presence of adenosine resulted in marked vasodilation and marked impairment of autoregulatory responses to increases in perfusion pressure (-1.5+/-1.1% and -3.5+/-0.9%). However, afferent arteriolar autoregulatory responses to elevations in perfusion pressure were restored after blockade of A2 receptors alone or in combination with A1 receptor blockade. During treatment with dimethyl-1-propargylxanthine in the presence of adenosine receptor saturation (120 micromol/L), afferent arteriolar autoregulatory responses were intact (-16.5+/-1.6% and -26.4+/-2.1%). These results indicate that the interactions between adenosine A1 and A2 receptors exert important modulatory influences on afferent arteriolar tone and autoregulatory capability. Activation of A2 receptors abrogates the counteracting influences of A1 receptors leading to marked vasodilation and decreased afferent arteriolar autoregulatory efficiency.

Effect of Adenosine on the Growth of Human T-Lymphocyte Leukemia Cell Line MOLT-4

Adenosine has been observed to suppress the growth of MOLT-4 human leukemia cells in vitro. Changes in the cell cycle, especially increased percentage of cells in S phase, prolonged generation time, and induction of apoptosis at higher adenosine concentrations have been found to be responsible for the growth suppression. Dipyridamole, a drug inhibiting the cellular uptake of adenosine, reversed partially but significantly the adenosine-induced growth suppression. It follows from these results that the action of adenosine on the MOLT-4 cells comprises its cellular uptake and intracellular operation. These findings present new data on anticancer efficacy of adenosine.

Adenosine inhibits cytosolic calcium signals and chemotaxis in hepatic stellate cells

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 A(2a) 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-beta and collagen I mRNA. In conclusion, adenosine reversibly inhibits Ca2+ fluxes and chemotaxis of HSCs and upregulates TGF-beta 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-beta production.

Heterodimeric adenosine receptors: a device to regulate neurotransmitter release.

Since 1990 it has been known that dimers are the basic functional form of nearly all G-protein-coupled receptors (GPCRs) and that homo- and heterodimerization may play a key role in correct receptor maturation and trafficking to the plasma membrane. Nevertheless, homo- and heterodimerization of GPCR has become a matter of debate especially in the search for the precise physiological meaning of this phenomenon. This article focuses on how heterodimerization of adenosine A1 and A2A receptors, which are coupled to apparently opposite signalling pathways, allows adenosine to exert a fine-tuning modulation of striatal glutamatergic neurotransmission, providing a switch mechanism by which low and high concentrations of adenosine inhibit and stimulate, respectively, glutamate release.

Adenosine-Mediated Inhibition of the Cytotoxic Activity and Cytokine Production by Activated Natural Killer Cells

Adenosine is an important signaling molecule that regulates multiple physiologic processes and exerts major anti-inflammatory actions. Tumors have high concentrations of adenosine, which could inhibit the function of tumor-infiltrating lymphoid cells. We investigated the ability of adenosine and its stable analogue 2-chloroadenosine (CADO) to inhibit cytokine production and cytotoxic activity of lymphokine-activated killer (LAK) cells and determined whether both these effects are initiated via a common pathway. CADO strongly inhibited cytotoxic activity of LAK cells and attenuated the production of IFN-gamma, granulocyte macrophage colony-stimulating factor, tumor necrosis factor alpha, and macrophage inflammatory protein-1alpha by LAK cells stimulated by cross-linking of the Ly49D receptor. These inhibitory effects were associated with the ability of CADO to stimulate cyclic AMP (cAMP) production and activate protein kinase A (PKA). Using cAMP analogues with different affinities for the A and B sites of the regulatory subunits of PKA types I and II, we found that activation of PKA I, but not PKA II, mimicked the inhibitory effects of CADO on LAK cell cytotoxic activity and cytokine production. Inhibitors of the PKA catalytic subunits (H89 and PKI(14-22) peptide) failed to abrogate the inhibitory effects of CADO whereas Rp-8-Br-cAMPS, an antagonist of the RI subunit, blocked the inhibitory effects of CADO. We conclude that the inhibitory effects of adenosine are probably mediated via cAMP-dependent activation of the RI subunits of PKA I but are independent of the catalytic activity of PKA. Tumor-produced adenosine could be a potent tumor microenvironmental factor inhibiting the functional activity of tumor-infiltrating immune cells.

The Adenosine System Selectively Inhibits TLR-Mediated TNF-α Production in the Human Newborn

Human newborns are susceptible to microbial infection and mount poor vaccine responses, yet the mechanisms underlying their susceptibility are incompletely defined. We have previously reported that despite normal basal expression of TLRs and associated signaling intermediates, human neonatal cord blood monocytes demonstrate severe impairment in TNF-alpha production in response to triacylated (TLR 2/1) and diacylated (TLR 2/6) bacterial lipopeptides (BLPs). We now demonstrate that in marked contrast, BLP-induced synthesis of IL-6, a cytokine with anti-inflammatory and Th2-polarizing properties, is actually greater in neonates than adults. Remarkably, newborn blood plasma confers substantially reduced BLP-induced monocyte synthesis of TNF-alpha, while preserving IL-6 synthesis, reflecting the presence in neonatal blood plasma of a soluble, low molecular mass inhibitory factor (<10 kDa) that we identify as adenosine, an endogenous purine metabolite with immunomodulatory properties. The neonatal adenosine system also inhibits TNF-alpha production in response to whole microbial particles known to express TLR2 agonist activity, including Listeria monocytogenes, Escherichia coli (that express BLPs), and zymosan particles. Selective inhibition of neonatal TNF-alpha production is due to the distinct neonatal adenosine system, including relatively high adenosine concentrations in neonatal blood plasma and heightened sensitivity of neonatal mononuclear cells to adenosine A3 receptor-mediated accumulation of cAMP, a second messenger that inhibits TLR-mediated TNF-alpha synthesis but preserves IL-6 production. We conclude that the distinct adenosine system of newborns polarizes TLR-mediated cytokine production during the perinatal period and may thereby modulate their innate and adaptive immune responses.

The Cotranscribed Salmonella enterica sv. Typhi tsx and impX Genes Encode Opposing Nucleoside-Specific Import and Export Proteins

The Salmonella enterica tsx gene encodes a nucleoside-specific outer membrane channel. The Tsx porin is essential for the prototrophic growth of S. enterica sv. Typhi in the absence of nucleosides. RT-PCR analysis shows that the tsx gene is cotranscribed with an open reading frame unique to S. enterica, impX (STY0450), which encodes an inner membrane protein 108 amino acids in length, which is predicted to have only two transmembrane alpha-helices. Fusions of the lacZ gene to both tsx and impX reveal that the transcription of both genes is induced in the presence of adenosine. A null mutation in the S. Typhi impX gene suppresses the induced auxotrophy for adenosine or thymidine resulting from a tsx mutation and confers sensitivity to high concentrations of adenosine or thymidine. The ImpX protein, when tagged with a 3xFLAG epitope, is functional and associates with the inner membrane; impX mutants are defective in the export of 3H-radiolabeled thymidine. Taken together, these and other results suggest that the S. Typhi Tsx porin and ImpX inner membrane protein facilitate competing mechanisms of thymidine influx and efflux, respectively, to maintain the steady-state levels of internal nucleoside pools.

Adenosine A1 receptor antagonist improves intradialytic hypotension

Intradialytic hypotension is a most frequent complication of hemodialysis and may contribute to cardiovascular events and high mortality. There is a hypothesis that an increase in adenosine generation during hemodialysis may cause vasodilation and a decrease in cardiac output, which results in systemic hypotension. We studied whether this can be blocked by an adenosine A1 receptor antagonist. We investigated the effects of an A1 antagonist, FK352, injection in 30 chronic hemodialysis patients with frequent intradialytic hypotension by a prospective, multicenter, double-blind placebo-controlled study for 4 weeks after 4 weeks of the observation period. Intradialytic hypotension was defined as systolic blood pressure (SBP) less than 110 mmHg, with SBP drop of more than 30 mmHg from the predialysis level. The efficacy of FK352 was primarily assessed by the reduction rate of dialysis hypotension between the FK352 and placebo groups. Incidence of emergency treatments caused by hypotension was evaluated. FK352 (50 mg, intravenous) or an equivalent placebo was injected into the dialysis circuit 1 h after starting dialysis. Blood pressure and heart rate were monitored every 30 min during dialysis. FK352 significantly improved intradialytic hypotension (P=0.046), in that the reduction rates of intradialytic hypotension in the FK352 and placebo groups were -12.8% (Q1 (first quantile), Q3 (third quantile): -27.5, -1.7), and +8.3% (Q1, Q3: -16.6, +16.7), respectively. The frequency of discontinuation of dialysis was significantly reduced by FK352. No apparent side effects were observed from treatment with FK352. In conclusion, the A1 antagonist FK352 may offer a novel therapeutic option for chronic dialysis patients associated with intradialytic hypotension.

Presynaptic Control of Striatal Glutamatergic Neurotransmission by Adenosine A1–A2AReceptor Heteromers

The functional role of heteromers of G-protein-coupled receptors is a matter of debate. In the present study, we demonstrate that heteromerization of adenosine A1 receptors (A1Rs) and A2A receptors (A2ARs) allows adenosine to exert a fine-tuning modulation of glutamatergic neurotransmission. By means of coimmunoprecipitation, bioluminescence and time-resolved fluorescence resonance energy transfer techniques, we showed the existence of A1R-A2AR heteromers in the cell surface of cotransfected cells. Immunogold detection and coimmunoprecipitation experiments indicated that A1R and A2AR are colocalized in the same striatal glutamatergic nerve terminals. Radioligand-binding experiments in cotransfected cells and rat striatum showed that a main biochemical characteristic of the A1R-A2AR heteromer is the ability of A2AR activation to reduce the affinity of the A1R for agonists. This provides a switch mechanism by which low and high concentrations of adenosine inhibit and stimulate, respectively, glutamate release. Furthermore, it is also shown that A1R-A2AR heteromers constitute a unique target for caffeine and that chronic caffeine treatment leads to modifications in the function of the A1R-A2AR heteromer that could underlie the strong tolerance to the psychomotor effects of caffeine.

Mechanisms of adenosine‐induced cytotoxicity and their clinical and physiological implications

Extracellular ATP (ATPo) and adenosine are cytotoxic to several cancer cell lines, suggesting their potential use for anticancer therapy. Adenosine causes cytotoxicity, either when added exogenously or when generated from ATPo hydrolysis, via mechanisms which are not mutually exclusive and which involve, adenosine receptor activation, pyrimidine starvation and/or increases in intracellular S-adenosylhomocysteine: S-adenosylmethionine ratio. Given that adenosine also appears to protect against cytotoxicity via mechanisms including immunity against damage by oxygen free radicals, an understanding of the contribution of adenosine to ATPo-induced cytotoxicity is thus crucial, when considering any potential therapeutic use for these compounds. However, such an understanding has been largely hindered by the fact that many studies have not focused enough on the possibility that both ATPo and adenosine may mediate cytotoxicity in the same system. Such studies can benefit from use a range of ATPo concentrations when assessing the contribution of adenosine to ATPo-induced cytotoxicity. Whilst future molecular and pharmacological studies are needed to establish the nature of the cytotoxic adenosine receptor, it is possible that more than just one adenosine receptor type is involved and that the cytotoxic receptor(s) type is more likely to have a low affinity for adenosine. Activation of the adenosine receptor(s) would thus lead to cytotoxicity only at relatively high adenosine concentrations, while lower adenosine concentrations mediate non-cytotoxic physiological effects.

Kinetic study of adenosine concentrations and the expression of adenosine deaminase in mononuclear cells during hemodialysis

We previously showed that high intralymphocytic adenosine (Ado) concentrations are found in hemodialyzed patients due to the reduced activity of mononuclear cell adenosine deaminase (MCADA). These abnormalities contribute to the immune defect observed in HD patients. The kinetics of these abnormalities and the causes of the low MCADA activity, however, have not been investigated. Here, we addressed this question. Since interferon gamma (IFNgamma) partially modulates MCADA, we also evaluated the effect of IFNgamma on MCADA activity in vitro. The study included 12 patients (eight men and four women) who were observed from the first to the 36th hemodialysis (HD) session, and eight healthy subjects (controls). MCADA activity and Ado concentrations were normal before the first HD session. Ado concentrations progressively increased from the first (10.5 +/- 3.1 pmol/10(7) cells) to the fourth session (26.7 +/- 3 pmol/10(7) cells), before stabilizing at a high level. MCADA activity increased transiently until the second session (2.2 +/- 0.5 IU/10(7) cells before HD vs. 2.8 +/- 0.6 IU/10(7)cells), and then decreased and stabilized at a low level (1.0 +/- 0.5 IU/10(7)cells). The amount of MCADA mRNA transiently increased until the third session (mRNA MCADA/mRNA beta-actin: 0.6 +/- 0.2 vs. 0.8 +/- 0.2), and then decreased to 0.3 +/- 0.1 at the 36th session. MCADA activity underwent a dose-dependent increase after IFNgamma stimulation. HD affects the transcription of the gene encoding MCADA after just three HD sessions, leading to decreased MCADA activity and increased plasma concentration of Ado.