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Aims The purpose of this study was to investigate the functional role of G-protein-coupled inward rectifier potassium (GIRK) channels in the cardiac ventricle. Methods and results Immunofluorescence experiments demonstrated that GIRK4 was localized in outer sarcolemmas and t-tubules in GIRK1 knockout (KO) mice, whereas GIRK4 labelling was not detected in GIRK4 KO mice. GIRK4 was localized in intercalated discs in rat ventricle, whereas it was expressed in intercalated discs and outer sarcolemmas in rat atrium. GIRK4 was localized in t-tubules and intercalated discs in human ventricular endocardium and epicardium, but absent in mid-myocardium. Electrophysiological recordings in rat ventricular tissue ex vivo showed that the adenosine A1 receptor agonist N 6-cyclopentyladenosine (CPA) and acetylcholine (ACh) shortened action potential duration (APD), and that the APD shortening was reversed by either the GIRK channel blocker tertiapin-Q, the adenosine A1 receptor antagonist DPCPX or by the muscarinic M2 receptor antagonist AF-DX 116. Tertiapin-Q prolonged APD in the absence of the exogenous receptor activation. Furthermore, CPA and ACh decreased the effective refractory period and the effect was reversed by either tertiapin-Q, DPCPX or AF-DX 116. Receptor activation also hyperpolarized the resting membrane potential, an effect that was reversed by tertiapin-Q. In contrast, tertiapin-Q depolarized the resting membrane potential in the absence of the exogenous receptor activation. Conclusion Confocal microscopy shows that among species GIRK4 is differentially localized in the cardiac ventricle, and that it is heterogeneously expressed across human ventricular wall. Electrophysiological recordings reveal that GIRK current may contribute significantly to ventricular repolarization and thereby to cardiac electrical stability.

This editorial refers to an article by S. Philipp et al. [1][1] published in Cardiovascular Research in 2006 (see [Box 1][2]). It is accompanied by an editorial by K. Przyklenk, pp. 195–197, this issue, as part of this Spotlight on Landmark Papers in Cardiovascular Research . Our interest in adenosine A2B receptors is based on studies reported in a paper we published in this journal in 2006.1 Below we describe the largely unplanned and serendipitous events that led us to conclude that the A2B receptor was an important player. There are four known adenosine receptor subtypes: A1, A2A, A2B, and A3. Interestingly, all four have been proposed to be involved in protecting the heart against ischaemic injury. All subtypes are G protein-coupled receptors of the serpentine 7 family. The A1 and A3 receptors are clearly Gi-coupled, whereas the A2A receptor is Gs-coupled. The A2B receptor is also Gs-coupled, but a Gi (or Go) coupling can also be demonstrated.2 The A2B receptor also has a much lower affinity for adenosine than the other three subtypes. The cardiomyocyte expresses all four subtypes, but their physiological effects are diverse. A1 receptor occupation reduces the heart rate by modulating sinoatrial node activity and depresses conduction through the AV node. A2A agonists are powerful vasodilators in most organs because of their Gs coupling. On the other hand, these agonists have a negligible effect on contractility in the heart, probably a reflection of their fairly low density relative to β-adrenergic receptors. A2B agonists have little haemodynamic effect. In 1986 Murry et al .3 discovered ischaemic preconditioning. This is a phenomenon whereby a brief period of myocardial ischaemia … [1]: #ref-1 [2]: #F1

Aims ATP-sensitive potassium (KATP) channels in the heart are critical regulators of cellular excitability and action potentials during ischaemia. However, little is known about subcellular localization of these channels and their regulation. The present study was designed to explore the potential role of caveolae in the regulation of KATP channels in cardiac ventricular myocytes. Methods and results Both adult and neonatal rat cardiomyocytes were used. Subcellular fractionation by density gradient centrifugation, western blotting, co-immunoprecipitation, and immunofluorescence confocal microscopy were employed in combination with whole-cell voltage clamp recordings and siRNA gene silencing. We detected that the majority of KATP channels on the plasma membrane of cardiac myocytes were localized in caveolin-3-enriched microdomains by cell fractionation and ultracentrifugation followed by western blotting. Immunofluorescence confocal microscopy revealed extensive colocalization of KATP channel pore-forming subunit Kir6.2 and caveolin-3 along the plasma membrane. Co-immunoprecipitation of cardiac myocytes showed significant association of Kir6.2, adenosine A1 receptors, and caveolin-3. Furthermore, whole-cell voltage clamp studies suggested that adenosine A1 receptor-mediated activation of KATP channels was largely eliminated by disrupting caveolae with methyl-β-cyclodextrin or by small interfering RNA, whereas pinacidil-induced KATP activation was not altered. Conclusion We demonstrate that KATP channels are localized to caveolin-enriched microdomains. This microdomain association is essential for adenosine receptor-mediated regulation of KATP channels in cardiac myocytes.

This editorial refers to an article by S. Philipp et al . [16][1] published in Cardiovascular Research in 2006. It is accompanied by a retrospective editorial by two authors of that original article, J.M. Downey and M.V. Cohen (doi:10.1093/cvr/CVS214), as part of this Spotlight on Landmark Papers in Cardiovascular Research . Adenosine has a long and storied history in the field of cardioprotection. A wealth of evidence accrued over three decades has demonstrated the ability of adenosine-based pharmacological strategies (and, in some instances, adenosine per se ) to attenuate multiple facets of myocardial ischaemia-reperfusion injury.1–15 It is equally well established that the favourable effects of adenosine and adenosine agonists are receptor-mediated and initiated by the stimulation of one or more of the family of four adenosine receptor subtypes (A1, A3, A2A, A2B; reviewed in1–3). Initial attention focused on the A1 subtype, with a host of studies reporting that pre-treatment with A1 receptor agonists significantly attenuated post-ischaemic contractile dysfunction and reduced the myocardial infarct size. Subsequent studies utilized both pharmacological and genetic approaches to interrogate the A1, A2A, and A3 receptors, and, in particular, the potential role of these receptor subtypes in the endogenous cardioprotection afforded by ischaemic preconditioning, postconditioning, and remote conditioning.1– … [1]: #ref-16

Background: Nitric oxide (NO), synthesised from the inducible isoform of nitric oxide synthase (iNOS), is implicated in mediating second window of protection (SWOP)/delayed ischemic preconditioning. However the role of NO and iNOS in delayed pharmacological protection remains unclear and is the subject of this investigation. Methods: To test the hypothesis that iNOS is necessary for delayed pharmacological preconditioning, the adenosine A1 receptor agonist, 2-chloro N6 cyclopentyl adenosine (CCPA) (25 μg/kg i.v.) or saline was administered to wild type (WT) or iNOS gene knockout mice (KO). Twenty-four hours later, the hearts were isolated, Langendorff perfused and subjected to 35 min ischemia/30 min reperfusion prior to infarct size determination. Results: WT and KO control hearts had identical infarct sizes of 37±3% and 37±2%, respectively. CCPA significantly reduced infarct size in WT hearts to 22±2% and also, unexpectedly, in KO hearts (27±2%). This protection was abrogated with the non-specific NOS inhibitor, Nω nitro l-arginine methyl ester (l-NAME, 100 μM), and could be mimicked in naive hearts with the NO donor, donor S -nitroso N -acetyl dl penicillamine (SNAP, 1 μM). Delayed protection appeared to be mediated by NO synthesis in both WT and KO hearts. Additional studies using Western blot analysis demonstrated endothelial NOS (eNOS) upregulation and increased NO x release in both WT and KO hearts. Conclusions: This is the first study to demonstrate a role for eNOS in delayed A1 receptor triggered (pharmacological) preconditioning, potentially representing a new pharmacological target for protecting the ischemic heart.

Glycolysis-from-glycose may be more beneficial than glycogenolysis in protecting hearts against ischemia. We tested the hypothesis that ischemic preconditioning is mediated by increased exogenous glucose use during low-flow ischemia, an effect triggered by adenosine A1 receptor activation.Langendorff rat hearts were subjected to 25 min low-flow ischemia (0.6 ml/min) and 30 min reperfusion. Prior to underperfusion, hearts (n = 6 per group) were subjected to two cycles of either preconditioning ischemia (PC), infusion of the adenosine A1 agonist 2-chloro-N6-cyclopentyladenosine (CCPA; 0.25 mumol/l), or PC in the presence of the adenosine antagonist 8-(p-sulfophenyl)theophylline (SPT; 50 mumol/l). Glycolysis-from-glucose during underperfusion was measured using D-[2-3H]glucose.At the end of reperfusion, recovery of rate-force product was enhanced in the PC and CCPA groups (62 and 67% of preischemic value) compared to the ischemic control hearts (IC, 32%; P0.05), whereas protection was abolished in the SPT hearts (20%; P0.05 vs. PC). PC improved total glycolysis-from-glucose during underperfusion by 31% (P0.05 vs. IC); SPT abolished this increase. CCPA reduced total lactate release and glucose uptake during ischemia by 47% and 61%, respectively (P0.05 vs. IC). Abolishment of the preconditioning-associated increase in glucose uptake during underperfusion, by switching to a low glucose buffer, resulted in a loss of functional protection.This study strongly suggests that increased exogenous glucose utilization during low-flow ischemia mediates ischemic preconditioning without increasing total anaerobic glycolytic flux. Although adenosine A1 receptor activation reduces ischemic injury, it does not facilitate the increased glucose uptake observed with ischemic preconditioning, suggesting a different mechanism of protection.

Objective: The aim of this study was to characterize the adenosine A3 receptor agonist, N 6-(3-chlorobenzyl)-5′- N -methylcarboxamidoadenosine (CB-MECA), evaluate its ability to reduce myocardial ischemia/reperfusion injury and determine the role of KATP-channel activation in A3 receptor-mediated cardioprotection. Methods: Binding affinities and adenylate cyclase inhibition were examined in CHO cells expressing rabbit recombinant adenosine A1 or A3 receptors. Infarct size (normalized for area-at-risk;% IA/AAR) was measured in buffer-perfused rabbit hearts exposed to 30-min regional ischemia and 120 min of reperfusion. Results: CB-MECA was 100-fold selective for A3 vs. A1 receptors (A3 K i: 1 nM; A1 K i: 105 nM). Five-min perfusion with CB-MECA before ischemia/reperfusion elicited a concentration-dependent reduction in infarct size (EC50: 0.3 nM). The CB-MECA-dependent cardioprotection (control: 58±2; CB-MECA: 21±3% IA/AAR) was unchanged by an A1-selective concentration of the antagonist, BWA1433, but was completely prevented ( P

Objective: Adenosine receptor activation has been implicated in the mechanism of ischaemic preconditioning protection. Evidence suggests adenosine A1 receptor involvement, and possibly A3 receptor involvement in the rabbit. This study investigated the roles of these receptors in human preconditioning. Human A1- and A3-selective compounds were chosen based on K i values for inhibition of N 6-(4-amino-3-[125I]iodobenzyl)adenosine (125I-ABA) binding to stably expressed recombinant human A1 and A3 receptors. Cyclopentyladenosine (CPA), a 194-fold selective A1 agonist, and iodobenzylmethylcarboxamidoadenosine (IBMECA), a 10-fold selective A3 agonist were used alone and in combination with dipropylcyclopentylxanthine (DPCPX) a 62-fold selective A1 antagonist. Methods: Human atrial trabeculae were superfused with oxygenated Tyrode's solution. After stabilisation, muscles underwent one of 8 protocols ( n = 6 per group), followed by 90 min of simulated ischaemia and 120 min of reoxygenation. The experimental endpoint was recovery of contractile function, presented as percentage baseline function. Results: 5 nM CPA (52.2±3.1%), 30 nM IBMECA (49.7±3.8%) and preconditioning (55.3±2.5%) produced similar functional recoveries at 120 min of reoxygenation; significantly different to controls (27.7±1.0%; P

Objective: This study was undertaken to investigate the functional significance of adenosine A2 receptor stimulation in a mammalian ventricular myocyte preparation. Methods: Isolated contracting rat ventricular myocytes were employed to assess the contractile, adenylyl cyclase and cyclic AMP responses to adenosine receptor stimulation. Results: In single myocytes the presence of A1 receptors was confirmed, as indicated by the A1 receptor agonist, phenylisopropyladenosine (PIA), reducing by 60 and 74% the inotropic response and activation of adenylyl cyclase, respectively, elicited by the β-adrenergic agonist, isoproterenol. An A1 receptor antagonist, dipropylcyclopentylxanthine (DPCPX), prevented the antiadrenergic action of PIA. The A2 receptor agonist, carboxyethylphenethyl-aminoethylcarboxamido-adenosine (CGS-21680; 0.01–10 μM) increased myocyte inotropy in a concentration-dependent manner, reaching a maximum of 41–45%. Ethylcarboxamidoadenosine (NECA), naphthyl-substituted aralkoxy-adenosine (SHA-082) and adenosine in the presence of DPCPX also increased myocyte inotropy, as evidenced by increases in myocyte shortening, duration of shortening, time-to-peak shortening, time-to-75% relaxation and rate of maximal shortening. The agonists, however, did not effect the maximal rate of relaxation. The A2 receptor antagonists, chlorofuranyldihydrotri-azoloquinazolinimine (CGS-15943) and chlorostyrylcaffeine (CSC), the latter selective for the A2a receptor, prevented the contractile responses elicited by the A2 agonists. Compared to the concentrations of A2 receptor agonists necessary to increase myocyte contractile variables, 3–12 times greater concentrations of the agonist were required to increase myocyte adenylyl cyclase activity and cAMP levels. Conclusion: The results suggest the presence of adenosine A2a receptors in the rat ventricular myocyte that appear to be responsible for an increase in inotropy via cAMP-dependent and -independent mechanisms. © 1997 Elsevier Science B.V.

Objective: This study tested the hypothesis that endogenous adenosine depresses anaerobic glycolysis in preischaemic and moderately ischaemic myocardium. Methods: Isolated, working guinea-pig hearts, perfused with glucose-fortified Krebs-Henseleit buffer, were subjected to 15 min mild hypoperfusion (coronary flow 60% of baseline) followed by 10 min ischaemia (coronary flow 20% of baseline). Adenosine A1 receptors were blocked with 8- p -sulfophenyl theophylline (8-SPT; 20 μM). Glucose oxidation and lactate production from exogenous glucose were assessed from 14CO2 and [14C]lactate formation, respectively, from [U-14C]glucose. Energy metabolites, glycolytic intermediates and glycogen were measured in extracts of stop-frozen preischaemic, mildly hypoperfused and ischaemic myocardium. Results: Adenosine receptor blockade did not affect left ventricular function assessed from heart rate × pressure product and pressure × volume work although coronary flow was slightly reduced. Adenosine receptor blockade increased glucose uptake ( P < 0.05) by 100% during preischaemia and by 74% during mild hypoperfusion, and increased lactate production from exogenous glucose ( P < 0.05) by 89% during preischaemia and fourfold during mild hypoperfusion, but did not stimulate glucose oxidation under any condition. Glycogen degradation was not increased by adenosine receptor blockade during ischaemia. Crossover plots of glycolytic intermediates revealed that phosphofructokinase was activated by adenosine receptor blockade at all three levels of perfusion. Conclusion: Endogenous adenosine attenuates anaerobic glycolysis in normally perfused, hypoperfused and ischaemic myocardium by blunting phosphofructokinase activity; this effect is mediated by adenosine A1 receptors.

Objectives: An interaction between adenosine A1 receptors and ATP-sensitive potassium channels (KATP) has been hypothesized to mediate preconditioning in several species. Unlike other species tested, KATP blockers and A1 antagonists do not abolish preconditioning in rat hearts. The purpose of this study was to determine if KATP and A1 receptors are pharmacologically linked in rat hearts as they are in other species. Methods: Isolated rat hearts were given 0.03–1.00 μM R-PIA (adenosine A1 receptor agonist) with or without concomitant 0.3 μM glyburide starting 10 min pre-ischemia. After 25 min global ischemia, the hearts were reperfused for 30 min. Rat hearts were also treated with 1–30 μM cromakalim in the presence of 10 μM DPCPX (adenosine A1 antagonist). Results: R-PIA produced a concentration-dependent bradycardia before ischemia which was blocked by DPCPX. R-PIA increased the time to onset of contracture in a concentration-dependent manner (EC25 = 0.13 μM) and this was unaffected by 0.3 μM glyburide (EC25 = 0.20 μM). This concentration of glyburide completely abolished the protective effects of 10 μM cromakalim. R-PIA also significantly enhanced post-ischemic recovery of function and reduced LDH release, and glyburide did not alter these responses. Cromakalim significantly increased the time to onset of contracture (EC25 = 4.5 μM) and 10 μM DPCPX had no effect on this (EC25 = 5.6 μM). Cromakalim also significantly enhanced post-ischemic recovery of function and reduced LDH release. DPCPX did not alter these cardioprotective effects while glyburide completely abolished the cardioprotective effects of cromakalim. Conclusions: While both cromakalim and R-PIA are cardioprotective in isolated rat hearts, they are not pharmacologically linked, possibly explaining why preconditioning may be different in this species.

See article by Fabritz et al. [1] (pages 500-508) in this issue. In this issue of Cardiovascular Research, a paper by Fabritz et al. [1] investigates the effects of controlled overexpression of the A3 adenosine receptor (A3AR) in the mouse heart. Their findings will feed further the current debate on the use of adenosine receptors (AR) as targets for gene therapy aimed at protecting the myocardium against ischemic damage. Furthermore, Fabritz et al. report interesting observations on the functional role of adenosine receptors in the regulation of heart rate. In the heart, adenosine regulates pacemaker activity as well as different cellular functions through the A1, A2, and A3AR. ARs are expressed on different cell types, including myocytes, vascular and endothelial cells, fibroblasts, and neutrophils (for review, see Ref. [2]). Furthermore, adenosine activates the cellular mechanisms of protection from ischemic damage. Endogenous release of adenosine during myocardial ischemia induces a potent protective effect in both a paracine and autocrine way [3]. Particularly, adenosine reduces the infarct size and delays the onset of ischemic contracture by reducing the rate of ATP catabolism. It also improves the postischemic contractile function and attenuates myocardial stunning. At the cellular level, adenosine induces cardioprotection by directly acting on cardiomyocytes, by inhibiting platelet aggregation and adhesion of inflammatory cells to the endothelium, and by reducing the production of superoxide by neutrophils [3,4]. Administration of adenosine is effective in both ischemic and pharmacological preconditioning [5] and throughout the early [6] and delayed [7] windows of preconditioning. The observation that exogenous administration of adenosine has antiischemic properties during coronary angioplasty has suggested the possibility of developing clinical therapies that … *Corresponding author. Tel.: +33-4-99-61-99-39; fax: +33-4-99-61-99-01. Email address: matteo.mangoni{at}igh.cnrs.fr

Objective: To study the actions of adenosine on the electrophysiology of spontaneously active, rod-shaped cells enzymatically isolated from rabbit atrioventricular (AV) node. Methods: Calcium-tolerant myocytes were isolated from the region of the AV node by enzymatic and mechanical dispersion. They were rod- or spindle-shaped, with spontaneous activity at 35–37 °C, and had higher membrane resistances (776 ± 283 MΩ, n = 13), compared to atrial cells (41 ± 18.2 MΩ, n = 7; P < 0.001). Membrane potential, spontaneous action potentials and transmembrane ionic currents were studied using the whole-cell patch-clamp technique, in current-clamp and voltage-clamp mode. Results: Adenosine (0.1–50 μM) slowed or abolished the spontaneous activity, with hyperpolarisation of the membrane potential. Voltage-clamp experiments showed that adenosine induced an inwardly rectifying time-independent current. The adenosine-induced current was shown to be carried by potassium ions by the effect of increasing external potassium, which altered the reversal potential in accordance with the calculated potassium equilibrium potential. The A1 adenosine receptor antagonist, CPDPX (8-cyclopentyl-1,3-dypropylxanthine), reversed the effects of adenosine and an A1 receptor agonist, R-PIA [ R (−) N 6-(2-phenylisopropyl)adenosine] had effects similar to adenosine. Adenosine also caused a small decrease in inward calcium current ( I Ca) in some AV nodal cells. Conclusions: These results indicate that adenosine acts at A1 adenosine receptors to suppress spontaneous activity, hyperpolarise membrane potential and induce a time-independent potassium current in AV nodal cells. These actions, combined with reduction in inward calcium current in some cells, may underlie the negative chronotropic and dromotropic actions of adenosine on rabbit AV nodal cells.

Objective: The aim was to establish whether polymorphonuclear neutrophils can, by themselves, elicit depression of postischaemic heart function immediately after short periods of ischaemia, and to examine the involvement of endogenous adenosine and platelet activating factor (PAF) in the observed phenomena. Methods: Isolated buffer perfused guinea pig hearts performing pres sure-volume work under standardised conditions were subjected to 15 min of global ischaemia. Constant flow reperfusion (5 ml · min−1) was carried out in the presence or absence of homologous neutrophils (approximately 2000 cells · μl−1 perfusate). After 15 min of reflow, work was resumed and functional recovery assessed another 20 min later. Results: In hearts perfused only with Krebs-Henseleit buffer, postischaemic heart function recovered to 67(SEM 3)% (n = 13) of the preischaemic value. As early as the first minute of reperfusion, the application of neutrophils already led to a significant decrease in recovery to 39(3)% (n = 12; P < 0.05). Without ischaemia, neutrophils did not have any deleterious effect, recovery of external heart work amounting to 91(4)% (n = 6). Adenosine concentrations measured in the coronary effluent after ischaemia were substantially increased during reperfusion from preischaemic values of

Objective: The primary aim was to determine the action of pathophysiologically relevant adenosine concentrations (0.1-1 μM) on adhesion of neutrophils to coronary endothelium. Further aims were to evaluate the nature and localisation of the adenosine receptor involved, and to assess the effect of endogenous adenosine. Methods: Adhesion was studied in isolated perfused guinea pig hearts by determining the number of cells emerging in the coronary effluent after intracoronary bolus injections of 600 000 neutrophils prepared from guinea pig or human blood. The system was characterised by the use of the proadhesive stimulus thrombin. Results: A 5 min infusion of adenosine (0.1-0.3 μM) or the A1 receptor agonist N6−cyclopentyladenosine (CPA, 0.01 μM) significantly increased adhesion from about 20% (control) to 30%. This effect was prevented by the A1 receptor antagonist dipropyl-8-cyclopentylxanthine (DPCPX, 0.1 μM). It was not diminished by cessation of adenosine infusion 90 s prior to neutrophil injection. At a higher concentration of adenosine (1 μM), adhesion did not seem to be enhanced. However, coinfusion of the A1 receptor antagonist 3,7-dimethyl-l-propargylxanthine (DMPX. 0.1 μM) with 1 μM adenosine unmasked the A1 action, adhesion rising to 39%. Adenosine had a quantitatively identical effect on adhesion of human neutrophils. Total ischaemia of 15 min duration raised adhesion of subsequently applied neutrophils to 35%. This effect was completely blocked by DPCPX, as well as by ischaemic preconditioning (3 × 3 min). Preconditioning raised initial postischaemic coronary effluent adenosine from about 0.8 μM to 1.5 μM. Conclusions: The findings suggest a bimodal participation of adenosine in the development of postischaemic dysfunction by an endothelium dependent modulation of neutrophil adhesion. Stimulation occurs via endothelial A1 receptors at submicromolar adenosine levels, whereas cardio-protection by adenosine may in part relate to the use of pharmacologically high concentrations of adenosine or enhanced endogenous production after preconditioning. Cardiovascular Research 1994; 28 :1366-1372

Objective: Agonists selective for the A1 adenosine receptor mimic the protective effect of ischaemic preconditioning against infarction in the rabbit heart. Unselective adenosine antagonists block this protection but, paradoxically, the A1 adenosine receptor selective antagonist 8-cyclopentyl-l,3-dipropylxanthine (DPCPX) does not. The aim of this study was to test the hypothesis that the newly described A3 adenosine receptor, which has an agonist profile similar to the A1 receptor but is insensitive to DPCPX, might mediate preconditioning. Methods: Isolated rabbit hearts perfused with Krebs buffer experienced 30 min of regional ischaemia followed by 120 min of reperfusion. Infarct size was measured by tetrazolium staining. Results: In control hearts infarction was 32.2(SEM 1.5)% of the risk zone. Preconditioning by 5 min ischaemia and 10 min reperfusion reduced infarct size to 8.8(2.3)%. Replacing the regional ischaemia with 5 min perfusion with 10 μM adenosine or 65 nM N6-[2-(4-aminophenyl)ethyl]adenosine (APNEA), an adenosine A3 receptor agonist, was equally protective. The unselective antagonist 8-p-sulphophenyl theophylline at 100 μM abolished protection by preconditioning, adenosine, and APNEA, but 200 nM DPCPX did not block protection by any of the interventions. Likewise the potent but unselective A3 receptor antagonist 8-(4-carboxyethenylphenyl)-1,3-dipropylxanthine (BW A1433) completely blocked protection from ischaemic preconditioning. Conclusions: Because protection against infarction afforded by ischaemic preconditioning, adenosine, or the A3 receptor agonist APNEA could not be blocked by DPCPX and because the potent A3 receptor antagonist BW A1433 blocked protection from ischaemic preconditioning, these data indicate that the protection of preconditioning is not exclusively mediated by the adenosine A1 receptor in rabbit heart and could involve the A3 receptor. Cardiovascular Research 1994; 28: 1057-1061

Objective: A preconditioning mimetic agent could be useful therapy for cardiac ischaemic events; stimulation of adenosine receptors has been proposed as a preconditioning mediator. The ability of adenosine-receptor activation to mimic ischaemic preconditioning was tested in an in vivo rabbit model. Methods: Adenosine (15 mg. a maximally tolerated dose, n = 10) was infused over six minutes via a coronary artery and compared with saline (n = 12) in anaesthetised rabbits. Five minutes after infusion, a coronary artery was occluded for 40 minutes followed by three hours of reperfusion. In a second study, preischaemic intravenous treatment with adenosine (25 mg·kg−1 n = 9). or an Aj-adenosine agonist. R(-)-N-(2-phenylisopropyl)-adenosine (PIA, 900 μg·kg−1. n= 12). were compared with saline (n = 12), when given before 40 minutes of coronary artery ligation and three hours of reperfusion in anaesthetised rabbits. Results: Intracoronary adenosine reduced mean arterial pressure during infusion (48(3) v 80(4) mm Hg. control, p

Objective: The aim was to determine if isolated rat cardiomycytes could be protected from ischaemic cell death by preincubation with adenosine or adenosine agonists. Methods: Cardiomyocytes isolated from rat hearts were preincubated in the presence of adenosine, CCPA (2-chloro-N6-cyclopentyladenosine), or carbachol prior to concentration into an ischaemic slurry. Effects of glycolysis and of isoprenaline were determined by addition of iodoacetic acid or isoprenaline to the ischaemic incubates and by exclusion of glucose from all media. Rates of ischaemic contracture were determined and survival of the myocytes versus paired control preparations was determined after various times of ischaemia, following resuspension of the cells in isotonic or hypotonic media. Results: Adenosine and CCPA produced only a small reduction of the rates of contracture and death of isolated myocytes. Carbachol gave no significant protection. Neither the degree of injury of control cells nor the amount of protection by CCPA was altered in the presence of added isoprenaline. Protection was abolished by the A1 receptor blocker sulphophenyl theophylline, iodoacetic acid, and exclusion of glucose. Conclusions: Adenosine and adenosine agonists afford a minimal degree of protection to ischaemic isolated myocytes by a glucose dependent mechanism. This protection does not appear to account for the larger degree of protection seen in intact hearts, following similar preconditioning protocols. The failure of adenosine to protect may be related to the quiescent state of isolated cardiomyocytes, or be species specific in that adenosine may not be the trigger for preconditioning in rats. Cardiovascular Research 1993; 27 :1670-1676

Objective: The highly selective adenosine A1 receptor agonist, 2-chloro-N6-cyclopentyl-adenosine (CCPA), has been shown to be as cardioprotective as ischaemic preconditioning when evaluated with an early staining method using tetrazolium. However, tetrazolium-positive tissue measured 3 h after reperfusion may still overestimate the long term salvage. To test for this possible artefact, a 72 h reperfusion rabbit model of myocardial infarction was used, and infarct size was assessed by histology. Methods: Myocardial infarction was induced by a 30 min coronary occlusion. Rabbits were assigned to a control group receiving no treatment, pretreatment with 0.125 mg·kg−1 CCPA, or 0.25 mg·kg−1 pretreatment with CCPA (0.25 mg·kg−1) followed by an A1 selective antagonist, 8-cyclopentyl-l,3-dipropylxanthine (DPCPX) 30 min after reperfusion to reverse the haemodynamic side effects. Results: In the 0.125 mg·kg−1 CCPA group, 30.8(SEM 4.2)% of the ischaemic zone was infarcted, which was significantly less than that seen in the control group [46.5(3.0)%; p

Adenosine is recognised as an important regulator of myocardial function and coronary vascular tone in the ischaemic myocardium. It is produced by the enzymatic dephosphorylation of 5'-AMP by 5'-nucleotidase and the hydrolysis of SAH by SAH-hydrolase. 5'-Nucleotidase is thought to contribute to adenosine production aside from the accumulation of 5'-AMP in the ischaemic myocardium, while the hydrolysis of SAH plays a major role in adenosine production in the normoxic myocardium. 5'-Nucleotidase activity is reported to increase adenosine production through accumulation of ATP, ADP, H+, Mg2+ and inorganic phosphate during ischaemia. In addition, we have found that alpha 1 adrenergic receptors, activated in ischaemic hearts, increase both 5'-nucleotidase activity and adenosine production. Inactivation of adenosine deaminase and adenosine kinase may also contribute to adenosine production. On the other hand, the major role of endogenous adenosine is to increase coronary blood flow. This adenosine induced coronary vasodilatation is amplified by alpha 2 adrenoceptor stimulation. Adenosine induced vasodilatation is also enhanced by increasing H+ and opening ATP sensitive K+ channels, which occurs in the ischaemic myocardium. However, coronary vasodilatation is not the only effect of adenosine in the ischaemic myocardium. Stimulation of adenosine A2 receptors coupled to Gs proteins attenuates both free radical generation by activated leucocytes and aggregation of platelets. Adenosine A1 receptor activation coupled to G(i) proteins attenuates beta adrenoceptor mediated increases in myocardial contractility, Ca2+ influx into myocytes, and noradrenaline release from the presynaptic nerves. Any or all of these effects may attenuate ischaemic and reperfusion injury.(ABSTRACT TRUNCATED AT 250 WORDS)

Objective: The aim was to examine the effect of a selective A1 adenosine receptor antagonist (N-0861) on postdefibrillation cardiovascular depression to determine whether antagonist mediated enhanced postdefibrillation haemodynamic recovery is due to reversal of the antiadrenergic action of adenosine, an A1 receptor mediated effect. Methods: Halothane-nitrous oxide anaesthetised Duroc pigs of either sex, weight 22-25 kg, were subjected to sequential episodes of induced ventricular fibrillation lasting either 15 s (terminated by a suprathreshold shock at 60-70 A) or 35 s (subjected to a subthreshold shock at 20 A followed by a suprathreshold shock at 60-70 A) during intravenous placebo infusion (n=10), N-0861 infusion (0.1 mg·kg−1·min−1, n=10), and N-0861 infusion plus propranolol (2 mg·kg−1, n=6). Results: N-0861 significantly enhanced immediate postdefibrillation electrophysiological and haemodynamic recovery compared to placebo for ventricular fibrillation episodes lasting 35 s. Over the first 15 s postdefibrillation, N-0861 significantly (p

Objective: The aim was to characterise the process and the mechanisms of sensitisation of the atrial myocyte to adenosine receptor agonist. Methods: The ability of adenosine A1 receptor to mediate inhibition of adenylyl cyclase activity and myocyte contractility was determined in atrial myocytes cultured from 14 d chick embryos. Under conditions in which the myocytes were sensitised to the effects of A1 agonist, changes in the levels of adenosine A1 receptor and pertussis toxin sensitive G proteins were determined and correlated with alterations in the adenylyl cyclase activity and contractile responses of the myocyte to the A1 agonist. Results: Removal of adenosine from the culture medium with adenosine deaminase resulted in an enhanced ability of the adenosine A1 receptor agonist R-N6-(2-phenylisopropyl)-adenosine to exert a direct, negative inotropic effect and to inhibit isoprenaline stimulated adenylyl cyclase activity. The increase in the extent of maximum inhibition of adenylyl cyclase activity and of myocyte contractility was 215(30), n=5, and 90(10)%, n=14, respectively. Binding of the antagonist radioligand [3H]-8-cyclopentyl-1,3-dipropylxanthine in membranes from myocytes pre-exposed to adenosine deaminase showed a 70% increase in the adenosine A1 receptor density and a 54% increase in the proportion of the high affinity adenosine A1 receptor: control 33(5)%, n=5, versus sensitised 49(3)%, n=5, p

Objective: Adenosine deaminase (ADA) may be multifunctional, regulating adenosine levels and adenosine receptor (AR) agonism, and potentially modifying AR functionality. Herein we assess effects of ADA (and A1AR) deficiency on AR-mediated responses and ischaemic tolerance. Methods: Normoxic function and responses to 20 or 25 min ischaemia and 45 min reperfusion were studied in isolated hearts from wild-type mice and from mice deficient in ADA and/or A1ARs. Results: Neither ADA or A1AR deficiency significantly modified basal contractility, although ADA deficiency reduced resting heart rate (an effect abrogated by A1AR deficiency). Bradycardia and vasodilation in response to AR agonism (2-chloroadenosine) were unaltered by ADA deficiency, while A1AR deficiency eliminated the heart rate response. Adenosine efflux increased 10- to 20-fold with ADA deficiency (at the expense of inosine). Deletion of ADA improved outcome from 25 min ischaemia, reducing ventricular diastolic pressure (by 45%; 21 ± 4 vs. 38 ± 3 mm Hg) and lactate dehydrogenase (LDH) efflux (by 40%; 0.12 ± 0.01 vs. 0.21 ± 0.02 U/g/min ischaemia), and enhancing pressure development (by 35%; 89 ± 6 vs. 66 ± 5 mm Hg). Similar protection was evident after 20 min ischaemia, and was mimicked by the ADA inhibitor EHNA (5 μM). Deletion of ADA also enhanced tolerance in A1AR deficient hearts, though effects on diastolic pressure were eliminated. Conclusions: Deficiency of ADA does not alter sensitivities of cardiovascular A1 or A2ARs (despite markedly elevated [adenosine]), but significantly improves ischaemic tolerance. Conversely, A1AR deficiency impairs ischaemic tolerance. Effects of ADA deficiency on diastolic pressure appear solely A1AR-dependent while other ARs or processes additionally contribute to improved contractile recovery and reduced cell death.

Objectives: To assess the impact of ischemia-reperfusion (I/R) on coronary function, and the role of endogenous adenosine in modifying post-ischemic vascular function in asanguinous hearts. Methods : Vascular function was studied in Langendorff perfused mouse hearts subjected to 20–25-min ischemia and 30-min reperfusion. Results: Ischemia altered the dependence of flow on work-rate observed in normoxic hearts, and inhibited reflow by mechanisms additional to diastolic compression. Coronary responses were selectively reduced: 2-chloroadenosine and ADP dilated with pEC50s of 8.4±0.1 and 7.4±0.1 in non-ischemic hearts versus 7.7±0.1 and 7.1±0.1 after 20-min ischemia ( P

Purpose: Adenosine fine-tuning regulates cardiovascular function. The negative chronotropic effect of adenosine depends on the activation of the most abundant adenosine receptor in the heart, the A1AR, leading to GIRK/Kir3.1/3.4 channels opening in the SA node. The A1AR-mediated responses in the atrial muscle also depend on KCa2/SK channel function (unpublished observations). Since these channels are involved in atrial repolarization and in human rhythm disturbances, we aimed at studying the interplay between A1AR and SK channels both in situ and single-cell preparations of rat atria. Methods: Isometric contraction experiments were performed on isolated spontaneously beating atria from Wistar rats. For whole-cell voltage-clamp recordings, atrial myocytes were enzymatically isolated by retrograde perfusion. Immunolocalization studies (A1AR, GIRK1, KCa2.2 and KCa2.3) were evaluated by confocal microscopy. The SA node was identified as a connexin-43 low / neurofilament-160 high immunoreactive region. Results: The selective A1AR agonist R-PIA (0.001-1μM) decreased atrial chronotropism and inotropism in concentration-dependent manner. However, the negative chronotropic effect of R-PIA (IC50~0.03 μM) was evidenced at much lower (~30 times) concentrations than the negative inotropic action (IC50~1 μM). Selective blockade of A1AR and of GIRK/Kir3.1/3.4 channels, respectively with DPCPX (100 nM) and tertiapin Q (300 nM), significantly attenuated the effects R-PIA. Blockade of KCa2/SK channels with apamin (30 nM) potentiated the negative inotropic response of R-PIA, without affecting atrial rate. Apamin-induced atrial sensitization to R-PIA was not observed with other cardiodepressant agents, namely oxotremorine (0.01-3 μM). Voltage-clamp experiments demonstrated that adenosine, via A1AR, plays a dual role in atrial cardiomyocytes by activating inwardly rectifying GIRK/Kir3.1/3.4 and inactivating outward Ca2+-activated KCa2/SK currents upon cell depolarization. Immunolabelling of A1AR, KIR3.1, KCa2.2 and KCa2.3 was observed throughout the right atria. Conclusion: Data demonstrate for the first time that A1AR has opposing effects on distinct K+ currents operating atrial repolarization. While A1AR activation favors the opening of inwardly rectifying GIRK/KIR3.1/3.4 channels modulating atrial automatism, it may also inactivate outward KCa2/SK currents. Taking into consideration that KCa2/SK channel dysfunction has pro-arrhythmic effects drugs targeting the A1AR for supraventricular tachycardia must be used with caution as they might precipitate atrial arrhythmias.

Adenosine (Ado) accumulates in tissues under metabolic stress. On myocardial cells, the nucleoside interacts with various receptor subtypes (A(1), A(3), and probably A(2A) and A(2B)) that are coupled, via G proteins, to multiple effectors, including enzymes, channels, transporters and cytoskeletal components. Studies using Ado receptor agonists and antagonists, as well as animals overexpressing the A(1) receptor indicate that Ado exerts anti-ischemic action. Ado released during preconditioning (PC) by short periods of ischemia followed by reperfusion induces cardioprotection to a subsequent sustained ischemia. This protective action is mediated by A(1) and A(3) receptor subtypes and involves the activation and translocation of PKC to sarcolemmal and to mitochondrial membranes. PKC activation leads to an increased opening of ATP-sensitive K(+) (K(ATP)) channels. Recent studies implicate mitochondrial rather than sarcolemmal K(ATP) channels in the protective action of PC. Other effectors possibly contributing to cardioprotection by Ado or PC, and which seem particularly involved in the delayed (second window of) protection, include MAP kinases, heat shock proteins and iNOS. Because of its anti-ischemic effects, Ado has been tested as a protective agent in clinical interventions such as PTCA, CABG and tissue preservation, and was found in most cases to enhance the post-ischemic recovery of function. The mechanisms underlying the role of Ado and of mitochondrial function in PC are not completely clear, and uncertainties remain concerning the role played by newly identified potential effectors such as free radicals, the sarcoplasmic reticulum, etc. In addition, more studies are needed to clarify the signalling mechanisms by which A(3) receptor activation or overexpression may promote apoptosis and cellular injury, as reported by a few recent studies.

Objective: In congestive heart failure (CHF), a desensitisation of stimulatory β-receptors and of adenylyl cyclase in the heart is associated with an increase in inhibitory Gi proteins. To investigate whether the regulation of the Gi-mediated inhibitory side of the adenylyl cyclase system may be of functional importance in the failing myocardium, the contractile response of isolated adult cardiomyocytes to stimulation of inhibitory muscarinic M2 and A1 adenosine receptors was analysed. Methods: CHF was induced in rats by banding of the ascending aorta and was verified by doubling of lung wet weight. After four weeks, contraction amplitude (Δ L ) and the velocity (d L /d t max) of isolated ventricular cardiomyocytes during electrical field stimulation in the presence of 1 mM Ca2+ were measured using video micrometry. Results: Contractile responses of failing cardiomyocytes to 5 mM Ca2+ were unchanged. The response to increasing concentrations of the β-adrenergic agonist, isoproterenol (0.1–30 nM), and to forskolin (0.1 nM–1 μM) were significantly blunted. When A1 receptors were activated with N 6-( R -phenyl-isopropyl)-adenosine (PIA; 0.01–1 μM) in the presence of 3 nM isoproterenol, contractility was unchanged in cells compared with those from sham-operated rats, but Δ L was reduced by up to 23% and d L /d t max by 35% in failing cardiomyocytes ( P

A recent paper in Cardiovascular Research from Hove-Madsen and colleagues [1] has investigated the role of the adenosine A2A receptor in human atrial myocytes. Elegant confocal images show that this receptor is localized at the level of the Z line in the myocyte. Their paper then proceeds to address an important physiological question; the role of this receptor. No effect was found on the amplitude or voltage-dependence of the L-type Ca current. However agonists of the adenosine A2A receptor increased the frequency of both Ca sparks and Ca waves, phenomena that result from Ca induced Ca release (CICR) from the sarcoplasmic reticulum (SR). There was no change in either the amount of Ca extruded from the cell during each Ca wave or the SR Ca content (as assessed by the integral of the caffeine evoked Na–Ca exchange, NCX, current). Based on these …

Objective: The aim was to investigate the effects of adenosine on nitric oxide (NO) synthesis in vascular smooth muscle cells. Methods: NO and cAMP synthesis was measured in confluent rat vascular smooth muscle cells in culture at passage 5–10, using Griess reagent and an enzyme immunoassay kit, respectively. The expression of inducible NO synthase mRNA was assayed by Northern blotting. Results: Incubation of cultures with interleukin-1β (10 ng/ml) for 24 h caused a significant increase in nitrite production. The interleukin-1β-induced nitrite production by vascular smooth muscle cells was significantly increased by adenosine or its stable analogue, 2-chloroadenosine, in a dose-dependent manner. The adenosine A2a receptor antagonist, KF17837, but not the A1 receptor antagonist, DPCPX, significantly inhibited 2-chloroadenosine-mediated nitrite production. The 2-chloroadenosine-induced nitrite production by interleukin-1β-stimulated cells was accompanied by increased inducible NO synthase mRNA accumulation. In the presence of dibutyryl-cAMP (1 mM), interleukin-1β-induced nitrite accumulation was further increased, but the effect of 2-chloroadenosine was not additive or synergistic. Addition of 2-chloroadenosine dose-dependently increased intracellular cAMP levels of vascular smooth muscle cells. Conclusions: These results indicate that adenosine acts on A2 receptors and augments NO synthesis in interleukin-1β-stimulated vascular smooth muscle cells, at least partially through a cAMP-dependent pathway.

Objective: The aim of this study was to determine whether selective activation of the adenosine A3 receptor reduces infarct size in a Langendorff model of myocardial ischemia-reperfusion injury. Methods: Buffer-perfused rabbit hearts were exposed to 30 min regional ischemia and 120 min of reperfusion. Infarct size was measured by tetrazolium staining and normalized for area-at-risk (IA/AAR). Results: Preconditioning by 5 min global ischemia and 10 min reperfusion reduced infarct size (IA/AAR) to 19±4% (controls: 67±5%). Replacing global ischemia with 5 min perfusion of the rabbit A3-selective agonist, IB-MECA (A3 Ki : 2 nM; A1 Ki : 30 nM) elicited a concentration-dependent reduction in infarct size; 50 nM IB-MECA reduced IA/AAR to 24±4%. The A1-selective agonist, R-PIA (25 nM) reduced IA/AAR to a similar extent (21±6%). However, while the cardioprotective effect of R-PIA was significantly inhibited (54±7% IA/AAR) by the rabbit A1-selective antagonist, BWA1433 (50 nM), the IB-MECA-dependent cardioprotection was unaffected (28±6% IA/AAR). A non-selective (A1 vs. A3) concentration of BWA1433 (5 μ M) significantly attenuated the IB-MECA-dependent cardioprotection (61±7% IA/AAR). Conclusions: These data clearly demonstrate that selective A3 receptor activation provides cardioprotection from ischemia-reperfusion injury in the rabbit heart. Furthermore, the degree of A3-dependent cardioprotection is similar to that provided by A1 receptor stimulation or ischemic preconditioning.

Objective: The aim was to determine a role of ATP sensitive potassium (KATP) channels in adenosine A2 receptor mediated coronary vasodilatation in anaesthetised dogs in vivo. Methods: Coronary blood flow in the left circumflex coronary artery, aortic pressure, and left ventricular pressure were measured during intracoronary infusions of the drugs into the left circumflex artery. Results: A non-selective A2 receptor agonist NECA (5'-N-ethylcarboxamidoadenosine) at 10−10-10−8 mol·min−1 before and after an A, receptor antagonist DPCPX (8-cyclopentyl-l,3-dipropylxanthine) increased coronary blood flow in a dose dependent manner, without affecting other haemodynamic variables. Glibenclamide at 10 μg·kg−1·min−1, which did not alter baseline haemodynamic variables, markedly inhibited the increases in coronary blood flow caused by NECA alone and after DPCPX (p

Objective: To study antagonism of A1 adenosine receptors in an anaesthetised open chest swine model, the selective A1 receptor antagonist, N6-endonorbornan-2-yl-9-methyladenine (N-0861), was examined to see if it attenuates bradycardia, augments reflex tachycardia associated with adenosine infusion, or both. Its effects were compared with those of the non-selective antagonist of adenosine A1 and A2 receptors, 8-(p-sulphophenyl)- theophylline (8-pST). Methods: Twenty nine pigs were studied. The prolongation of P-R interval mediated by A1 receptors, the increase in left anterior descending coronary artery blood flow mediated by A2 receptors, and decreases in systemic and left ventricular pressures and first derivative of left ventricular pressure (dP/dt) were monitored in each animal assigned to one of three protocols. (1) Adenosine, 40 to 180 (μg·kg−1·min−1, was infused for more than 6 min before and immediately after rapid infusion of 8-pST, 5 mg·kg−1 intravenously, or a solvent that did or did not contain N-0861, 0.2 mg·kg−1 intravenously (n = 14). (2) In the same animal, we compared N-0861 and 8-pST in reversing responses mediated by A1 and A2 receptors during two 10 min infusions of adenosine separated by a 1 h washout period (n = 7). (3) N-0861 with adenosine (mean dose, 0.4 mg·kg−1) was infused with or without complete autonomic blockade with atropine (2.5 mg·kg−1) and propranolol (2 mg·kg−1) (n = 8). Results: Adenosine prolonged P-R interval (and cycle length in non-paced hearts), increased coronary flow, and decreased calculated coronary resistance. N-0861 alone did not affect any variable, but N-0861 with adenosine prevented or reversed A1 receptor mediated prolongation of P-R interval in paced hearts and cycle length in non-paced hearts and enhanced A2 receptor mediated coronary vasodilatation. Left ventricular dP/dt and rate-pressure product were maintained with N-0861 and adenosine, and N-0861 unmasked a postadenosine reflex tachycardia. Prolonged PR interval, decreased heart rate, and increased coronary flow were prevented or reversed by the non-selective antagonist 8-pST. Conclusions: Selectivity of N-0861 for the adenosine A1 receptor may, without reducing coronary blood flow, ameliorate bradyarrhythmia and maintain the positive inotropic response when exogenous adenosine is given or when interstitial myocardial adenosine is increased. Cardiovascular Research 1994; 28 :621-628

Objective: The aim was to test the hypothesis that A1 receptor mediated cardioprotection by endogenous adenosine is exerted during ischaemia rather than reperfusion. Methods: Anaesthetised open chest rabbits were subjected to 30 min regional ischaemia and 120 min reperfusion, and randomised to one of six groups: group I — saline vehicle (VEH) (n=12) to allow A1 and A2 adenosine receptor interactions during ischaemia and reperfusion: group II — both A1 and A2 receptors were antagonised during ischaemia and reperfusion with 8-p-sulphophenyltheophylline (SPT) (10 mg·kg−1) (SPTIR, n = 14); groups III and IV — the selective A1 adenosine receptor antagonist 8-(3-noradamantyl)-l,3-dipropylxanthine (KW-3902) was given during ischaemia-reperfusion in low dose (1 mg·kg−1. LA1-IR, n = 11) and higher dose (2 mg·kg−1, HA1-IR, n = 6); group V - KW-3902 (1 mg·kg−1) was given only during reperfusion (A1-R, n = 12); group VI — SPT was given only at reperfusion (SPTR, n = 11). Results: In in vitro studies, (1) KW-3902 completely inhibited negative inotropic effects of the A1 agonist R(-)N6-(2 phenylisopropyl) adenosine (R-PIA) in catecholamine stimulated papillary muscles, and (2) had no effect on concentration dependent vasorelaxation to adenosine or R-PIA. In in vivo studies, transmural myocardial blood flow in the area at risk (determined using 15 μm radiolabelled microspheres) was reduced by 98% in all groups from 139(SEM 15.8) to 2.7(1.1) ml·min−1·100 g−1 (p

Objective: The aims were (1) to determine whether ischaemic and hypoxic preconditioning enhance recovery of left ventricular function after global ischaemia in the rat, and (2) to evaluate the effects of selective adenosine A1 receptor antagonists on ischaemic and hypoxic preconditioning. Methods: Isolated rat hearts, perfused at constant pressure, were subjected to 30 min ischaemia and 30 min reperfusion. Control hearts were compared to hearts preconditioned with 5 min ischaemia in the presence or absence of the adenosine A1 antagonist AI433U (10 μM), and hearts preconditioned with 5 min hypoxia in the presence or absence of the adenosine A1 antagonist 8-cyclopentyl-l,3-dipropylxanthine (DPCPX, 5 μM). Recovery of left ventricular function was assessed by percent recovery of preischaemic rate-pressure product. Results: Control hearts recovered 64(SEM 4)% of preischaemic rate-pressure product after 30 min reperfusion, whereas ischaemic and hypoxic preconditioned hearts recovered 86(3)% and 94(5)%, respectively (p

Objective: The aim was to test the hypothesis that adenosine A1 receptor activation triggers the cardioprotective effects of ischaemic preconditioning, by determining whether pretreatment with acadesine (5-amino-4-imidazolecarboxamide riboside), an agent which increases cardiac adenosine level during ischaemia, could alter the threshold for preconditioning. Methods: A branch of the left coronary artery of rabbit hearts was occluded for 30 min and reperfused for 3 h. Infarct size and risk zone size were determined with tetrazolium and fluorescent particles, respectively. Four groups were studied: untreated controls, a group which was pretreated with acadesine (2.5 mg·kg−1·min−1 for 5 min followed by 0.5 mg·kg−1·min−1 for 30 min ending 10 min prior to ischaemia), a group which was preconditioned with 2 min coronary branch occlusion + 10 min reper-fusion, and a group which received pretreatment with acadesine prior to 2 min ischaemic preconditioning. Results: Percent infarction, normalised as a percentage of the ischaemic zone, in the 2 min preconditioning group was 43.2(SEM 5.1)% which was not different from control [40.2(3.5)%]. Two minutes of preconditioning was not long enough to confer the cardioprotective effect of preconditioning. Acadesine alone had no protective effect on infarct size [38.5(4.5)%], but acadesine + 2 min preconditioning significantly limited infarction [18.1(2.7)%; p

Four subtypes of adenosine receptors have recently been cloned from thyroid, brain and testis. In this review we have summarised properties of these purinergic receptors. The cloned A1 and A2 subtypes are probably similar or identical to receptors that exist on cardiac and vascular tissues, respectively. A comparison of the amino acid sequences of A1, A2a, and A2b receptors reveals several stretches of conserved amino acids that are unique to adenosine receptors, primarily in the membrane spanning regions. Species differences in A1 receptors indicate that minor changes in receptor structure can produce marked changes in ligand binding properties and may facilitate the identification of amino acids involved in ligand recognition. A confusing A1 receptor subclassification system of putative A1a, A1b, and A3 subtypes has emerged based on subtle rank order potency differences for various ligands among tissues. cDNAs corresponding to these A1 subtypes have not yet been isolated. Atrial A1 receptors activate K+ channels and inhibit adenylyl cyclase. These two pathways appear to be independently up and down regulated, suggesting the existence either of atrial A1 receptor subtypes or of differential regulation of the coupling of a single receptor to distinct GTP binding proteins. An adenosine receptor distinct from A1 and A2 receptors has been cloned from testis and designated TGPCR, or A3, although it differs from the pharmacologically defined A3 receptor. We suggest that the current A1/A3 receptor subtype nomenclature be abandoned and superseded by a nomenclature based solely on receptor cDNAs. In addition to the cloned adenosine receptors, a novel A4 subtype has been proposed based on pharmacological and electrophysiological criteria.(ABSTRACT TRUNCATED AT 250 WORDS)

Attempts to identify mechanisms by which calcium antagonists might influence intracellular metabolism have not yet yielded conclusive findings. In this study bepridil, verapamil, nifedipine, and nisoldipine were found to have no influence on the rate of rat heart myosin adenosine triphosphatase or the calcium dependence of myofibrillar adenosine triphosphatase. None of these calcium antagonists alters the rate of reaction of any of the adenine nucleotide catabolic or adenosine salvage enzymes, adenylate kinase, creatine kinase, adenosine kinase, adenosine deaminase, or 5' nucleotidase, in extracts of rat heart. All four compounds, however, reduced, apparently in a non-specific manner, the rate of uptake of adenosine by myocytes isolated from rat heart. It is concluded that calcium antagonists may, through intercalation with the sarcolemmal membrane, inhibit efflux of adenosine formed by catabolism of adenine nucleotides in ischaemic myocytes. This might offer therapeutic advantage since the intracellular concentration of adenosine would thereby be increased, allowing an increased rate of incorporation of adenosine into the adenosine triphosphate pool in reoxygenated myocardium.

The influence of aminophylline, a competitive antagonist of adenosine, on the chronotropic and dromotropic effects of adenosine and adenosine triphosphate was studied in pentobarbital anaesthetised dogs under various modifications of the autonomic nervous tone. Adenosine and adenosine triphosphate (3 mumol.kg-1 each) were rapidly (greater than or equal to 1 s) injected into the right atrium during both sinus rhythm and right atrial pacing (cycle length 300 ms) before and after infusion of aminophylline (5 mg.kg-1) (n = 21) as well as after increasing doses of aminophylline (n = 10). Some dogs underwent either muscarinic blockade with atropine (0.2 mg.kg-1) (n = 10), or beta adrenergic blockade with propranolol (1 mg.kg-1) (n = 10), or complete autonomic blockade with atropine and propranolol (n = 10). Aminophylline (5 mg.kg-1) antagonised the negative chronotropic and dromotropic effects of adenosine triphosphate and adenosine in dogs pretreated with atropine or atropine plus propranolol but did not affect them in autonomically intact dogs. In addition, the electrophysiological effects of adenosine were antagonised by only the highest doses of aminophylline in autonomically intact dogs and by aminophylline (5 mg X kg-1) in dogs pretreated with propranolol. It was concluded that (a) alteration of the electrophysiological effects of adenosine triphosphate and adenosine by aminophylline is appreciably influenced by the autonomic nervous tone and (b) autonomic blockade is required for the manifestation of the antagonism by aminophylline of the electrophysiological action of adenosine and adenosine triphosphate.

Quantitative determination of myocardial adenosine formation and breakdown is necessary to gain insight into the mechanism and regulation of its physiological actions. Deamination of adenosine was studied in isolated perfused rat hearts by infusion of adenosine (1 to 20 mumol X litre-1). All catabolites in the perfusates (inosine, hypoxanthine, xanthine and uric acid) were measured, as well as unchanged adenosine. Apparent uptake of adenosine was determined; it increased linearly with the concentration of adenosine infused. Adenosine was predominantly deaminated, even at low (1 mumol X litre-1) concentration. The inhibitory capacity of the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) was determined, while 5 mumol X litre-1 adenosine was infused. EHNA inhibited the apparent adenosine deaminase activity for 62 and 92% at 5 and 50 mumol X litre-1, respectively. When 50 mumol X litre-1 EHNA was infused into normoxic hearts, release of adenosine was significantly elevated, as was coronary flow. Induction of ischaemia increased total purine release four-to fivefold. Infusion of EHNA into ischaemic hearts did not alter total purine release, but adenosine release increased from 15 to 60% of total purines. However, when EHNA was present, a large part of total purine release still existed of inosine, hypoxanthine, xanthiner and uric acid. This was 83% during normoxia and 40% during ischaemia. These results suggest significant contribution of IMP and GMP breakdown to purine release from isolated perfused rat hearts.

A study was carried out to investigate whether or not an impairment of the adenosine mediated negative inotropic effect in the presence of beta adrenoceptor stimulation plays a role in the pathogenesis of the hereditary cardiomyopathy of the Syrian hamster. In electrically driven papillary muscles isolated from the hearts of cardiomyopathic (strain BIO 8262) and age matched healthy control Syrian hamsters the effects of isoprenaline, adenosine, and adenosine in the presence of isoprenaline were studied within the first 30 days of life (the prenecrotic stage of the disorder). In both cardiomyopathic and control hamsters adenosine antagonised the positive inotropic effect of isoprenaline, whereas adenosine alone had no or, only a weak, inhibitory effect on the force of contraction. The effects in both groups were similar. The effect of isoprenaline on the force of contraction also did not differ in the two groups. The data show that in both cardiomyopathic and control hamsters adenosine reduces the force of contraction during beta adrenergic stimulation. The potency or efficacy of adenosine did not differ in the two groups. An impaired adenosine mediated feedback control of the heart does not therefore seem to play a role in the pathogenesis of the hereditary dystrophic cardiomyopathy of the Syrian hamster.