Invited Lectures

2006 Purinergic Signalling Purinergic Signalling  
Following a brief account of the early history of the discovery of purinergic signalling, a personal view of some of the exciting cutting-edge directions being taken by research in the field will be considered. In particular, emphasis will be placed on the pathophysiology of purinergic signalling and its therapeutic potential. Recent advances in understanding the role of adenosine and its receptors in physiology and pathophysiology as well as new developments in medicinal chemistry of these
more » ... ptors have made it possible to begin to realize the therapeutic potential of adenosine and its receptors. Currently adenosine itself is targeted at the heart; a bolus of adenosine intravenously is commonly used to treat supraventricular tachycardia whereas an infusion of adenosine is used as a coronary vasodilator during pharmacologic stress testing. Non-selective adenosine receptor antagonists are used to maintain wakefulness (caffeine), as an analgesic (caffeine) and, less commonly at present, to treat bronchospasm (theophylline, aminophylline, enprofylline). Currently a number of new selective adenosine receptor agonists and antagonists are in testing for a variety of new indications and one adenosinetargeted indication. The older indication is pharmacologic stress testing and new selective A 2A receptor agonists, ATL146e (Apadenoson, Adenosine Therapeutics) and Regadenoson (CV Therapeutics), are currently under study (Phase II-III) for this indication. In addition, a selective A 1 receptor agonist is in trials for supraventricular tachycardia (tecadenoson, CV Therapeutics), Currently a number of companies are testing selective adenosine receptor agonists for newer indications including Rheumatoid Arthritis, cancer and wound healing, Adenosine A 3 receptor agonists are in trials for the treatment of Rheumatoid Arthritis and Cancer (CF101, CF102, Can-Fite Biopharmaceuticals) and preliminary promising results in patients with RA have been reported. Topical application of an adenosine A 2A receptor agonist to promote healing of diabetic foot ulcers is currently in Phase II trials after the successful completion of a Phase I safety/early efficacy trial (MRE94, King Pharmaceuticals). An allosteric enhancer at the adenosine A 1 receptor is in trials for neuropathic pain (T-62, King Pharmaceuticals). Because adenosine receptor stimulation may also be involved in disease pathogenesis selective adenosine receptor antagonists are being studied for select indications as well. Istradefylline (KW-6002, Kyowa Pharmaceuticals), a selective adenosine A 2A receptor antagonist, is now in late stage clinical trials for the treatment of Parkinson"s Disease. Another approach to targeting adenosine receptors is to increase extracellular adenosine concentrations, mechanisms shown to mediate the anti-inflammatory effects of two drugs commonly used in the treatment of Rheumatoid Arthritis, methotrexate and sulfasalazine. Dipyridamole inhibits adenosine uptake and this clearly underlies its effect as a coronary vasodilator during pharmacologic cardiac stressing. New methotrexate analogues are in therapeutic trials in Rheumatoid Arthritis and recent pre-clinical studies of an adenosine uptake inhibitor for the treatment of inflammatory arthritis have been reported (KF24345, Kyowa Hakka Kogyo). In the future other therapeutic indications for adenosine receptor targeting may include fibrosis and scarring in the skin or liver, inflammation and inflammatory diseases, chronic pain syndromes and other yet to be described conditions. Agonists of adenosine A 2A receptors (A 2A R) such as ATL146e can substantially reduce necrosis of liver, heart and other tissues when added at the time of reperfusion following ischemia. Protection is attenuated by A 2A R antagonists and is absent in A 2A R KO mice. We prepared bone marrow chimera mice by transplanting bone marrow from A 2A R KO mice to previously irradiated WT syngenic recipients (KO/WT chimera) or vice versa. Experiments with these mouse chimera revealed that A 2A R-mediated protection from liver or heart reperfusion injury is entirely dependent on A 2A Rs on bone marrow-derived cells. A mouse line with a floxed adora2a gene was constructed to enable specific deletion of receptors in tissues expressing Cre recombinase. The lysM promoter was used to express Cre selectively in macrophages and neutrophils. LysMCre-A 2A R f/f mice effectively delete the A 2A R gene, adora2a, in macrophages and neutrophils but not T cells based both on functional assays and PCR based assays to quantify the floxed and recombined genes in purified cell populations. ATL146e was able to inhibit reperfusion injury in LysMCre-A 2A R f/f mice, indicating that bone marrow derived cells other than neutrophils and macrophages contribute to protection. Macrophages participate in liver reperfusion injury because injury is reduced by depletion of macrophages with liposomal clodronate. Lymphocytes also appear to participate in reperfusion injury because Rag1 KO mice that lack lymphocytes and B cells are protected. Reperfusion injury is restored in Rag1 KO mice by adoptive transfer of CD4 + , but not CD8 + T cells, and injury is not restored by CD4 + cells derived from INF+ KO mice. Assays of CD4 + T cells in vitro indicates that activation of the TCR causes rapid induction of A 2A R mRNA, and that ATL146e binding to the A 2A R can reduce TCRstimulated INF+ production by 98%. ATL146e reduces reperfusion injury in Rag1 KO mice following adoptive transfer of WT, but not A 2A R KO CD4 + cells purified from donor spleens. As confirmation that CD4 + cells are important in reperfusion injury we found that depletion of these cells with anti-CD4 + antibodies also reduced liver or hear reperfusion injury but anti-CD8 + antibodies that effectively deplete CD8 + cells from blood are ineffective. Immunohistochemistry was used to measure the time course of accumulation of T cells into the heart and liver after initiating reperfusion following ischemia. In both tissues there is a small but significant accumulation of T cells within minutes that is largely attenuated by ATL146e. These data suggest that an important means by which A 2A R activation reduces reperfusion injury is by activating receptors on CD4 + T lymphocytes. This reduces macrophage-dependent T cell activation and INF+ release required for further rapid recruitment of additional lymphocytes and/or subsequent recruitment of neutrophils into the tissue. In ongoing work we are attempting to determine what factors are responsible for activating CD4 + T cells during reperfusion injury and if a particular subpopulation of CD4 + T cells are selectively activated. A 2A agonists may be clinically useful for the treatment of ischemia-reperfusion injury, e.g. during coronary reperfusion following myocardial infarction, or tissue transplantation. Cultured dorsal root ganglion (DRG) neurons of rats are known to be endowed at their cell bodies as well as at their peripheral and central processes with P2X 3 receptors mediating rapidly desensitizing inward current responses to ATP or its structure analogue !,"-methylene ATP (!,"-meATP). In addition, we described the presence of P2Y 1 receptors at these neurons, negatively coupled to N-type voltage-sensitive Ca 2+ channels. Whereas P2X 3 receptor-activation is supposed to induce propagated action potentials and thereby pain sensation, P2Y receptor-activation may cause an opposite effect, by decreasing the Ca 2+ dependent release of sensory transmitters from the central terminals of DRG neurons. According to the known transduction mechanisms of P2X 3 and P2Y 1 receptors an early and rapidly declining pronociceptive action of ATP is expected to be followed by a more protracted antinociceptive action of the same compound. In support of this assumption, we found a negative interaction between P2Y 1 and P2X 3 receptors at rat DRG neurons, which was due to G protein activation, but did not utilize any of the hitherto known second-messenger mechanisms including phospholipase C, phosphatidylinositol 3 kinase, protein kinase C, inositoltriphosphate or Ca 2+ /calmodulin kinase II. In accordance with our in vitro data the intrathecal application of P2Y receptor agonists exhibited analgesic activity Invited Lectures 3 in an acute pain model (tail-flick test). In conclusion, the endogenous P2X/P2Y receptor agonist ATP either by itself or via its degradation product ADP may on the one hand induce pain and on the other hand may limit an overt pain reaction. These two opposing mechanisms may operate with a considerable time-lag between each other. P2X receptors belong to a superfamily of membrane proteins that are characterised by two membrane-spanning domains and a cysteine-rich ectodomain. Other members of this superfamily include (a) the nucleoside triphosphate diphosphohydrolase enzymes (NTPDase 1-8) which catalyse the extracellular degradation of ATP and ADP, and (b) the family of ion channels that includes the acid-sensing ion channels (ASIC 1-4), epithelial sodium channels (ENaC !-%) and degenerins of C. elegans (e.g. MEC-4, MEC-10, DEG-1, DEL-1, UNC-8, UNC-105). The three families of proteins are unrelated in primary structure. In terms of quaternary structure, P2X receptors are considered to be trimers, whereas the body of evidence suggest that ENaC/ASIC channels are tetramers. This lecture will summarise and review experiments in which site-directed mutagenesis and functional expression have been used to deduce those parts of the P2X receptor involved in (a) ATP binding, (b) ion permeation, and (c) interactions with other proteins. The principal focus will be on P2X 1 , P2X 2 , P2X 2/3 and P2X 4 subunits, with comparisons from studies on other family members where appropriate. Extracellular nucleotides (e.g. ATP, ADP, UTP) activate type-2 purinergic/pyrimidinergic (P2Y and P2X) receptors on platelets, endothelium and leukocytes. Ecto-nucleotidases hydrolyze these mediators, ultimately to the respective nucleosides, to regulate P2-signaling. Ecto-nucleotidases of the CD39/E-NTPDase family are expressed at high levels in the vasculature and immune systems. In addition to catalytic functions of the ectodomain of CD39, the palmitoylated intracytoplasmic N-terminus has been shown to structurally and functionally associate with a Ran binding protein, termed RanBPM. This multi-adaptor, scaffolding membrane protein regulates small GTPases and influences integrin signaling. We have proposed that spatial and temporal expression of CD39/NTPDase1 within the vasculature, by immune cells and/or derived microparticles (viz. membrane vesicles) could regulate inflammatory processes, immune reactions and also impact development of cancers. Expression of vascular CD39 appears crucial in regulating innate immunity, platelet thrombotic reactions, acute ischemic insults, altered vascular permeability, angiogenesis and tumor growth. For example, as visualized by in vivo video-microscopy, laser-induced arteriolar thrombus is characterized by rapid accumulation of platelets and microparticles. This process is then stabilized by platelet disaggregation with decreases in thrombus mass. The accumulation of NTPDase1 within thrombi blocks further ADP-mediated platelet activation. Mutant mice null for Cd39 and transgenic over-expressors of CD39 show the predicted abnormalities with marked differences in clot formation in vivo. Dendritic cell expression of CD39 influences antigen presentation and T cellular responses that are crucial in the evolution of adaptive immunity e.g. alloimmune reactions. CD39 is both an important surface marker of T Abstract not received The G q/11 -coupled P2Y 2 nucleotide receptor (P2Y 2 R) for ATP and UTP activates phospholipase C leading to an increase in IP 3 -dependent calcium mobilization and diacylglycerol-dependent activation of protein kinase C, responses that regulate the activity of phospholipases A 2 in primary murine astrocytes. Recent studies indicate that P2Y 2 Rs possess novel molecular determinants that enable them to activate other signaling pathways independent of G q proteins. For example, data indicate that proline rich Src-homology-3 (SH3) binding domains in the intracellular C-terminus of the P2Y 2 R can mediate the transactivation of growth factor receptors (e.g., vascular endothelial growth factor receptor-2) to promote the up-regulation of cell adhesion molecules (e.g., vascular cell adhesion molecule-1) in endothelium that promote the binding and vascular infiltration of monocytes Invited Lectures 7 The P2Y 1 receptor (P2Y 1 -R) is expressed broadly in mammalian tissues, for example on the surface of platelets where it is essential for ADP-promoted aggregation. Our laboratories have focused on the development of P2Y 1 -R-selective antagonists, agonists, and radioligands as reagents for delineating the physiological and molecular properties of this important cell signaling protein. Synthesis of a series of selective, high affinity, non-nucleotide competitive antagonists for the P2Y 1 -R led to development of [ 3 H]MRS2279 as a radioligand for reliable 8 Invited Lectures 1. Muller T, Bayer H, Myrtek D et al. Am J Respir Cell Mol Biol 2005. 2. Moore DJ, Murdock PR, Watson JM et al. Brain Res Mol Brain Res 2003; 118(1-2): 10-23. 3. Kreda S, Okada S, van Heusden C et al. Abstract to the 8th International Symposium on Adenosine and Adenine Nucleotides (Ferrara, 2006). As the epithelium is the most external layer of the cornea, it is often damaged by several factors causing corneal wounds. These wounds are repaired in a three-step process called corneal wound healing. Diadenosine polyphosphates, ApnA, are present in rabbit and human tears. This fact invites to think that these dinucleotides may participate in ocular surface processes such as corneal wound healing. Therefore, we have investigated the possible role of diadenosine polyphosphates on corneal wound healing by studying the changes in the rate of corneal epithelial cell migration after dinucleotide applications. Primary corneal epithelial cell cultures were obtained from New Zealand white rabbits. Immunocytochemical experiments were carried out by fixing the cells with 4% PFA and incubated with citokeratine 3 primary antibody which was subsequently incubated with a secondary Ig-G mouse labelled with FITC an cells were observed under confocal microscopy. Migration studies were carried out by taking confluent monolayers which were wounded with a pipette tip and challenged with different di-and mononucleotides (Ap 3 A, Ap 4 A, Ap 5 A, Up 4 U, ATP, UTP, ADP and UDP). For concentration-response analysis compounds were tested in doses ranging from 10 j8 to 10 j3 M. When the P2 antagonists, PPADS, suramin and reactive blue 2, were used, they were assayed at 100 2M, and from 10 j7 to 10 j3 M in concentration-response studies. In order to study the intracellular pathways involved in cell migration, several MAPK and citoskeleton inhibitors (U0126 100 2M, Y27632 100 2M, AG1478 100 2M, PAO (phenylarsine oxide) 5 2M, (j)-Blebblistatin 10 2M and ML7 25 2M) were assayed in the presence or absence of Ap 4 A and Ap 3 A both 100 2M. Stability of the dinucleotides was assayed by HPLC using an isocratic method. Cells under study were identified as corneal epithelial cells due to this positive labelling to cytokeratine-3 in the immunocytochemical analysis. Cell migration experiments showed that Ap 4 A, UTP and ATP accelerate the rate of healing, while Ap 3 A, Ap 5 A and UDP delay it. ADP and Up 4 U did not modify the rate of migration. For further experiments we took Ap 4 A (which accelerates the rate of wound) and Ap 3 A which delays it. P2Y antagonists presented small differences between Ap 4 A and Ap 3 A. The assays with MAPK and citoskeleton inhibitors, revealed that both, MAPK pathway and cdc-42/RAC/RhoA/ROCK pathways are involved in the epithelial migration. In this sense, Ap 4 A activating cdc-42 cascade increase the rate of corneal epithelial cell migration, while the delay caused by Ap 3 A is mainly due to the MAPK pathway Finally, concerning the possible degradation of the dinucleotides it was almost impossible to detect any product as a consequence of their cleavage. Degradation of 1-3% of the dinucleotides in 2 min perfusion indicates that the active molecules are the diadenosine polyphosphates and not the generated mononucleotides. In summary, analysing the pharmacological profile of all the compounds tested we can conclude that diadenosine polyphosphates activate two main P2Y receptors: a P2Y 2 receptor accelerating the rate of healing and a P2Y 6 receptor which delays this process. ADP serves as an endogenous agonist for both P2Y 1 and P2Y 12 on platelets, and antagonism of its action at either receptor has been shown to inhibit platelet aggregation. Inhibition of platelet aggregation using irreversible P2Y 12 antagonists has proven to be a useful therapy to treat Acute Coronary Syndrome (ACS). Previously we had Our project focussed on the synthesis of base-modified uracil nucleotides and metabolically stable nucleotide mimetics as ligands for the uracil nucleotide-sensitive P2Y receptor subtypes. A series of nucleotides and analogs was synthesized, investigated at recombinant human P2Y 2 , P2Y 4 , and P2Y 6 receptors, and structure-activity relationships were analyzed. Alkyl substituents in the 6-position of UTP and UDP were not tolerated by the three receptor subtypes. Large, aromatic substituents at N3 of UDP were well tolerated by the P2Y 6 receptor leading to potent, P2Y 6 -selective agonists. Modification of the triphosphate chain by introduction of a ",+-dichloromethylene bridge in UTP derivatives, resulting in compounds with enhanced metabolic stability, was well tolerated by all three receptor subtypes. Radioligands are valuable tools for studying GPCRs, however, selective radioligands have only been available for the P2Y 1 receptor subtype so far. We have recently developed the first selective radioligand for the P2Y 12 receptor subtype [2], an ADP receptor predominantly expressed on platelets, and in lower density in the brain. The P2Y 12 antagonist radioligand [ 3 H]PSB-0413 (2-propylthioadenosine-5 0 -adenylic acid (1,1-dichloro-1-phosphonomethyl-1-phosphonyl) anhydride, AR-C67085MX) has been successfully used for characterizing P2Y 12 receptors on the protein level in various tissues and cells. The radioligand allowed to set up a fast and efficient screening assay for identifying novel lead structures for the development of P2Y 12 antagonists, which are potent antithrombotic agents. Extracellular nucleotides are quickly hydrolyzed by ectonucleotidases to limit their action. Inhibitors of such enzymes would act as indirect P2Y receptor agonists by prolonging the effects of endogenously released nucleotides. We have developed a fast and convenient nanoscale capillary electrophoresis assay to screen and characterize inhibitors of ectonucleotidases [3]. The enzymatic reaction is performed within the capillary followed by electrophoretic separation and quantification (by UV) of the reaction products. A novel class of ectonucleotidase inhibitors has been discovered by this approach. Brunschweiger A, Mü ller CE. The nucleoside adenosine has been suggested to play a major role in the regulation of coronary blood flow during metabolic stress (i.e., hypoxia and ischemia) by the preferential dilation of small coronary arterioles primarily through the activation of adenosine A 2A receptors. However, the signaling pathways leading to this dilation are incompletely understood. Since tissue acidosis and alteration in local hemodynamics are associated with metabolic stress, it is important to examine whether the adenosine-induced vasodilatory response can be influenced by these factors. Using an isolated vessel approach and videomicroscopic techniques, these issues were addressed recently in our laboratory. It was found that disruption of endothelium, blockade of endothelial ATP-sensitive potassium (K ATP ) channels by glibenclamide, inhibition of nitric oxide (NO) synthase by L-NAME and of soluble guanylyl cyclase by ODQ produced identical attenuation of vasodilation to adenosine. Combined administration of these inhibitors did not further attenuate the vasodilatory response. Pertussis toxin (PTX), but not cholera toxin, significantly inhibited vasodilation to adenosine, and this inhibitory effect was only evident in vessels with an intact endothelium. Both glibenclamide and a high concentration of extraluminal KCl abolished vasodilation of denuded vessels to adenosine; however, inhibition of calcium-activated potassium channels had no effect on this dilation. Rp-8-Br-cAMPS, a cAMP antagonist, inhibited vasodilation to cAMP analog 8-Br-cAMP but failed to block adenosine-induced dilation. These results suggest that adenosine activates both endothelial and smooth muscle pathways to exert its vasomotor activity. On one hand, adenosine opens endothelial K ATP channels through activation of PTX-sensitive G-proteins. This signaling leads to the influx of Ca 2+ for NO synthesis, which subsequently increases smooth muscle cGMP for vasodilation. On the other hand, adenosine activates smooth muscle K ATP channels and leads to vasodilation through hyperpolarization. The latter pathway is independent of G-proteins and cAMP/cGMP signaling. In the presence of a threshold concentration of adenosine (0.1 nM), the vasodilation in response to increased lumenal flow was significantly enhanced. Inhibiting endothelial K ATP channels (by lumenal glibenclamide) or depolarizing endothelial cells (by lumenal KCl, 40 mM) abolished the effect of adenosine. Reducing intraluminal pressure from 80 to 40 cmH 2 O significantly enhanced vasodilations to adenosine and pinacidil (K ATP channel activator). These enhanced responses were not affected by endothelial removal but were abolished by a subthreshold concentration of glibenclamide. In contrast to adenosine, sodium nitroprusside (SNP)-induced dilation was not affected by pressure changes. In the acidic solution (pH = 7.3), the dilations to both adenosine and pinacidil, but not to SNP and KCl (10 to 20 mM), were enhanced in a glibenclamide-sensitive manner. In summary, these findings suggest that adenosine elicits coronary arteriolar dilation by activating K ATP channels for membrane hyperpolarization in the endothelial (i.e., NO release) and smooth muscle cells. Adenosine, by activating endothelial K ATP channels, can potentiate coronary arteriolar dilation in response to increased flow. In addition, activation of smooth muscle K ATP channels, by a mild acidosis or lowering intraluminal pressure, increases coronary arteriolar dilation to adenosine. It appears that adenosine can interact with local hemodynamics and chemical factors for integrative vasomotor regulation by selectively modulating resting K ATP channel activity in either endothelial or smooth muscle cells. It is well-established that metabolic factors have a critical role in the regulation of angiogenesis [1]. An increase in metabolic activity leads to a decrease in tissue oxygenation causing tissues to become hypoxic. The hypoxia 1. Adair TH, Gay WJ. Montani JP. Growth regulation of the vascular system: evidence for a metabolic hypothesis. Am J Physiol 1990; 259: R393-404. 2. Adair TH. Growth regulation of the vascular system: an emerging role for adenosine. Am J Physiol 2005; 289: R283-296. Adenosine is produced inside the cell by action of 5 0 -nucleotidase(s) and S-adenosylhomocysteine hydrolase. In addition, there is compelling evidence for extracellular adenosine production by ecto-5 0 -nucleotidase. A previous analysis of complex data sets of cardiac adenosine production has provided strong evidence that the prevailing path of adenosine production in the heart is intracellular (Deussen et al., Circulation 1999; 99: 2041-47). However, it was also found that intracellular removal of adenosine via adenosine kinase and adenosine deaminase was very rapid, and for physiological baseline conditions a transmembraneous adenosine concentration gradient was predicted, which was directed from extra-to intracellular. This scenario opens the possibility that extracellular adenosine production by ecto-5 0 -nucleotidase (CD73) is an important source of adenosine, which may significantly modulate the extracellular adenosine concentration. This hypothesis was tested in isolated endothelial cells and isolated perfused mouse hearts with respect to an activation of CD73, pharmacological block of the enzyme and a genetic knock out approach. Baseline activity of CD73 was assessed by measurement of the conversion of 1,N 6 -etheno-AMP to 1,N 6 -etheno-adenosine in intact cells or hearts (HPLC assay with fluorescence detection). While conversion of the etheno-labeled AMP provided a read out of CD73 activity of intact cells or tissues, additional measurement of native adenosine release (HPLC-1. Rongen GA, Smits P, Ver Donck K, Willemsen JJ, de Abreu RA, Van Belle H, Thien T. Hemodynamic and neurohumoral effects of various grades of selective adenosine transport inhibition in humans. Implications for its future role in cardioprotection. J Clin Invest 1995; 95: 658-68. 2. Rongen GA, Brooks SC, Ando S, Abramson BL, Floras JS. Angiotensin AT1 receptor blockade abolishes the reflex sympatho-excitatory response to adenosine. J Clin Invest 1998; 101: 769-76. Invited Lectures 19 3. Smits P, Williams SB, Lipson DE, Banitt P, Rongen GA, Creager MA. Endothelial release of nitric oxide contributes to the vasodilator effect of adenosine in humans. Circulation 1995; 92: 2135-41. 4. Bijlstra P, van Ginneken EEM, Huls M, van Dijk R, Smits P, Rongen GA. Glyburide inhibits dipyridamole-induced forearm vasodilation but not adenosine-induced forearm vasodilation. Clin Pharmacol Ther 2004; 75: 147-56. 5. Riksen NP, Rongen GA, Boers GH, Blom HJ, van den Broek PH, Smits P. Enhanced cellular adenosine uptake limits adenosine receptor stimulation in patients with hyperhomocysteinemia. Arterioscler. Thromb Vasc Biol 2005; 25(1): 109-14. 6. Riksen NP, Barrera P, van den Broek PH, van RP, Smits P, Ron-gen G. Methotrexate modulates the kinetics of adenosine in humans in vivo. Ann Rheum Dis 2006, in press. 7. Rongen GA, Lenders JWM, Lambrou G, Willemsen JJ, Van Belle H, Thien T, Smits P. Presynaptic inhibition of norepinephrine release from sympathetic nerve endings by endogenous adenosine. Hypertension 1996; 27: 933-8. 8. Riksen NP, Oyen WJ, Ramakers BP, van den Broek PH, Engbersen R, Boerman OC, Smits P, Rongen GA. Oral therapy with dipyridamole limits ischemia-reperfusion injury in humans. Clin Pharmacol Ther 2005; 78 (1): 52-9. 9. Rongen GA, Oyen WJG, Ramakers BP, Riksen NP, Boerman OC, Steinmetz N, Smits P. Annexin A5 scintigraphy of forearm as a novel in-vivo model of skeletal muscle preconditioning in man. Adenosine, a purine nucleoside acts through its cell surface receptors (A 1 , A 2A , A 2B , and A 3 ) via its coupling to G-proteins. Adenosine causes an increase in coronary flow mostly through A 2A adenosine receptors. However, the involvement of other adenosine receptors in the modulation of coronary flow regulation is not well understood. Using adenosine receptor knockout mice, we have investigated the roles of each adenosine receptor subtype in the regulation of coronary flow. Recently, using the isolated mouse heart preparation from A 3 adenosine receptor knockout mice, we reported an increased A 2A receptor-mediated coronary flow [1]. Similarly, we found an increase in coronary flow in A 1 adenosine receptor knockout mice [2]. These data support inhibitory roles for A 3 and A 1 adenosine receptors in the regulation of coronary flow. Additionally, in A 2A adenosine receptor knockout mice, the response to A 2A selective agonist (CGS-21680) was totally abolished [3]. On the other hand, adenosine A 2A receptor knockout mice showed a decrease in blood flow to NECA (non-selective agonist). Currently, we are investigating the coronary flow responses to both NECA and CGS-21680 in A 2B adenosine receptor knockout mice. Further, real time-PCR for all four adenosine receptor knockout mice showed For years the effects of adenosine in ventricular myocardium (intact hearts and myocytes) were attributed solely to A 1 receptor subtypes. However evidence generated by numerous laboratories over the past several years In our presentation we review our experience in the use of adenosine-compounds for diagnostic purposes in cardiac arrhythmias. Adenosine-compounds are the treatment of choice for common supraventricular tachyarrhythmias involving the atrio-ventricular node. Moreover, adenosine-compounds can also be used for diagnostic purposes. Injection of adenosine compounds during sinus rhythm leads to short lasting bradycardia (sinus arrest or block of conduction along the AV-node). This effect is short lasting and invariably resolves within seconds. The induced bradyarrhythmia is always followed by sinus tachycardia. This unique effects of adenosine compounds may be used for the following diagnostic purposes: 1) to reveal the presence of Wolff-Parkinson-White in patients with minor preexcitation due to a left sided location of the accessory pathway and fast AV nodal conduction, 2) to reveal the presence of dual AV node physiology in patients with AV-node reentry tachycardia, 3) to reveal the presence of concealed accessory pathways in patients with AV reentry tachycardia. The last two effects can be useful when evaluating patients who have a history of palpitations but no documented arrhythmias. Finally, since 1. Schö fl C, Cuthbertson KS, Walsh CA, Mayne C, Cobbold P, von zur Mü hlen A, Hesch RD, Gallagher JA. Evidence for P 2purinoceptors on human osteoblast-like cells. J Bone Miner Res 1992; 7: 485-91. Since ATP is released from cells in response to mechanical stimulation, it has been proposed that P2 nucleotide receptors mediate mechanotransduction in bone. 1 Mice with targeted disruption of the P2X7 receptor gene exhibit diminished periosteal bone formation together with excessive trabecular bone resorption 2 and impaired response to mechanical loading. 3 Our goal was to understand the roles of P2X7 receptors in osteoblasts (bone forming cells) and osteoclasts (bone resorbing cells). Whereas the expression of functional P2X7 receptors has been established in osteoclasts, 4 their existence in osteoblasts has been controversial. Osteoblast-enriched cultures were obtained from calvariae of newborn rats, and wild-type (WT) and P2X7 receptor knockout (KO) mice. Osteoblast-enriched cultures were supplemented with ascorbic acid and "-glycerophosphate to induce formation of mineralized nodules. Cells expressing functional P2X7 receptors were associated with bone nodules. Benzoylbenzoyl-ATP (BzATP, P2X7 agonist), but not UTP, promoted mineralization in rat calvarial cell cultures. Moreover, mineralization in KO cultures was markedly reduced compared to WT, consistent with a role for P2X7 receptors. Expression of osteoblast marker genes (encoding Runx2, Osterix, osteocalcin and bone sialoprotein) was assessed by real-time PCR. Transcript levels were significantly enhanced by BzATP in rat calvarial cell cultures and suppressed in cultures from KO mice compared to WT (except Runx2). Cyclooxygenase inhibitors abolished the stimulatory effect of BzATP on bone formation without affecting mineralization in untreated cultures. Thus, P2X7 receptors stimulate osteoblast differentiation through a cell-autonomous, cyclooxygenase-dependent mechanism. The lifespan of osteoclasts is an important regulator of bone resorption. Osteoclasts were isolated from long bones of newborn WT and KO mice and cell survival was assessed. In the absence of exogenous nucleotides, higher numbers of WT than KO osteoclasts were apoptotic at 6 h. Consistent with this observation, fewer WT osteoclasts survived at 12 h. Interestingly, BzATP had no additional effect on either apoptosis or survival. Control experiments revealed that WT and KO osteoclasts were equally susceptible to apoptosis induced by the proapoptotic agent staurosporine. Brilliant blue G (P2X7 antagonist) decreased apoptosis and increased survival of WT osteoclasts, providing additional evidence for involvement of the P2X7 receptor. Treatment of WT osteoclasts with alkaline phosphatase (to hydrolyze endogenous nucleotides) increased their survival to levels comparable to KO osteoclasts, consistent with activation of P2X7 receptors by constitutively released nucleotides. In summary, P2X7 receptors stimulate osteoblast differentiation and induce osteoclast apoptosis. Since the P2X7 receptor both enhances bone formation and suppresses bone resorption, it represents a potential target for the development of drugs with combined anabolic and antiresorptive effects. Invited Lectures 25 diabetes with vehicle and diabetes with ATL146e, a selective A 2A agonist (10 ng/kg/min) via osmotic pump over 6 weeks. Results Blood glucose increased in the diabetes and treatment groups 48 hours after STZ-induction of diabetes. Renal medullary blood flow and urinary NOX excretion were reduced significantly in diabetes group (67%; p < 0.001 and 60%; p < 0.01 from control) and restored to normal levels by ATL146e (86% and 100% from control), respectively. There was a significant correlation between urinary albumin excretion rate and urinary NOX (r = 0.84; p < 0.0001). We next sought to determine renal NO synthase (NOS) isoforms altered in DN. By using realtime PCR, diabetes led to a decrease in renal eNOS mRNA expression (67% from control) that was reversed by ATL146e (137% from control; p < 0.05 from diabetes). iNOS mRNA expression was not detectable in control but was induced in diabetes, an effect that was reversed by ATL146e. Immunohistochemistry with iNOS antibody of the kidney tissue confirmed the result of mRNA expression and was corelated with increased macrophages infiltration, a major source of iNOS, in diabetic kidneys. In contrast, there was no significant change in nNOS gene expression between groups. Summary These results demonstrate that DN is associated with decreased renal production of NO mainly through decreased expression of eNOS an effect reversed by A 2A agonists. Furthermore an increase iNOS due in part to macrophage infiltration in DN is reversed with ATL146e. Conclusion The effects of A 2A agonists on ameliorating the previously described functional and morphological changes associated with DN may in part be due to effects on NO. Adenosine promotes the healing of wounds and matrix deposition in the skin, actions known to be mediated through the A 2A receptor. The release of adenosine also accounts for the anti-inflammatory activity of methotrexate, one of the most widely used anti-rheumatic agents. We hypothesized that ligation of the A 2A receptor is responsible for the development of hepatic fibrosis, one of the most serious complications of chronic methotrexate therapy, as well as cirrhosis arising from other causes such as ethanol consumption. Epidemiological studies have demonstrated a protective effect of caffeine in hepatic cirrhosis and we speculate that this protective effect is also related to the antagonistic activity of caffeine at the adenosine receptor. The capacity of adenosine for modulating fibrous tissue development in the skin is also relevant to the pathogenesis of another rheumatic disease, scleroderma, where skin fibrosis is a prominent feature. Using in vitro and in vivo models, we have demonstrated a role for the A 2A receptor in the pathogenesis of these disorders. These findings provide a strong rationale for the exploitation of adenosine receptor antagonist therapies in future treatment of fibrotic diseases.
doi:10.1007/s11302-006-9006-2 pmid:18404494 pmcid:PMC2096525 fatcat:ul2ea34gmndrfiaaqjq2rdkdhi