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Interstitial levels and release of adenosine have been shown to be greater for aged adult hearts compared to young adult hearts. Furthermore, blockade of A1 adenosine receptors in the aged adult heart prevents the reduced contractile and metabolic response to isoproterenol. The aim of this study was to determine whether there is an enhanced antiadrenergic effect of adenosine in the aged adult heart. Ventricular membranes from young and aged adult hearts were incubated in the presence of isoproterenol (ISO) and phenylisopropyladenosine (PIA) either alone or in combination. Basal and ISO-enhanced adenylyl cyclase activity were significantly reduced in the membranes from aged rats. PIA alone, at 0.1 nM to 100 microM, had no direct effect on basal adenylyl cyclase activity in membranes from either group. In the presence of either 100 nM or 1 microM ISO, 100 microM PIA significantly attenuated ISO-enhanced adenylyl cyclase activity to a greater extent in the aged adult heart membranes (78 or 48% for the aged vs. 37 or 25% for the young). Moreover, in the presence of 100 nM ISO the IC50 for the PIA concentration response curve was shifted to the left for the aged ventricular membranes as compared to the membranes from young adults (1.62 x 10(-7) M vs 1.5 x 10(-6) M, aged vs young, respectively). The enhanced inhibition of adenylyl cyclase is associated with an increase in adenosine A1 receptor density (23.7 +/- 3.5 vs 14.7 +/- 1.7 fmol/mg, aged vs young) and Kd (6.1 +/- 1.7 vs 2.2 +/- 0.5 nM, aged vs young) in the aged adult heart membranes as determined by [3H]DPCPX binding. These results suggest that the reduced response to catecholamines in the aged adult heart may be due, at least in part, to an enhanced expression of the antiadrenergic effect of adenosine on beta-adrenergic receptor mediated activation of adenylyl cyclase.

High levels of norepinephrine in the heart are cardiotoxic resulting in contractile dysfunction and arrhythmic activity via beta-adrenoceptor mediated mechanisms. A low flow heart model perfused with physiological saline containing glucose and bubbled with an O2 gas mixture was used to determine whether adenosine, a nucleoside with antiadrenergic properties, could reduce the functional manifestations of catecholamine cardiotoxicity. Isolated rat hearts were treated with dipropylcyclopentylxanthine (DPCPX; 0.1 microM; A1 receptor antagonist) to block endogenous adenosine. In DPCPX-treated hearts stimulated with isoproterenol (ISO; 1 microM) during 45 min of low flow (0.5 ml/min) perfusion, the recovery of contractile function (ConF) at 15 min after the restoration of normal flow was 64% of control (before low flow) values as compared to 110% recovery of ConF in the absence of ISO. The incidence of arrhythmias observed upon restoration of control flow was increased by ISO when the action of endogenous adenosine was blocked with DPCPX. In the absence of DPCPX both the functional depression and arrhythmias induced by ISO were prevented in the presence of phenylisopropyladenosine (PIA; 1 microM; A1 receptor agonist). At 15 min after normal flow was restored. ConF in ISO-treated hearts with PIA was 53% greater than in the absence of PIA and presence of DPCPX. This enhancement of ConF by PIA was significantly reduced by DPCPX. By 30 min after flow restoration, these significant differences were absent. DPCPX reversed the PIA-induced reduction in arrhythmias observed upon restoration of normal flow. PIA and DPCPX alone in the absence of ISO, and ISO in the absence of PIA and DPCPX, did not result in altered ConF upon restoration of normal flow. These findings indicate that intense beta-adrenergic stimulation of the heart during low-flow perfusion in the absence of adenosine A1 receptor activity induces contractile depression and arrhythmicity subsequent to restoration of control perfusion. It is concluded that endogenous adenosine protects the heart against catecholamine toxicity via stimulation of adenosine A1 receptors.

Human diploid lung fibroblasts (IMR-90) were used to investigate the reported increase in beta-adrenergic-stimulated cyclic adenosine 3',5'-monophosphate (cAMP) levels in fibroblasts aged in culture. Under basal conditions cellular cAMP was 34.2 +/- 5.6 and 38.4 +/- 9.1 pmol/mg protein in early (PDL 22-24) and late (PDL 47-52) passage fibroblasts, respectively. Net release of cAMP from fibroblasts was 67.8 +/- 8.6 and 18.5 +/- 7.0 pmol/30 min/mg protein in early and late passage cultures, respectively. In confluent, early passage fibroblasts, cellular cAMP and net release of cAMP increased by 2.7-fold and 3.8-fold, respectively, after a 30 min incubation in 2 microM isoproterenol. In confluent late passage fibroblasts, isoproterenol incubation increased cellular cAMP and net release of cAMP by 7.8-fold and 26.1-fold, respectively. Adenosine failed to inhibit isoproterenol-induced stimulation of cAMP in early or late passage fibroblasts. There was no passage-related difference in basal, isoproterenol, or forskolin-stimulated adenylyl cyclase activity in crude fibroblast membrane preparations. The activity of cAMP-phosphodiesterase in sonicates of early and late passage IMR-90 was 9.61 +/- 1.15 and 5.81 +/- 1.11 pmol/min/mg protein respectively. Measurements of cAMP in subconfluent early passage fibroblasts indicated that mechanisms related to the reduced cell density in confluent late passage IMR-90 may, in part, account for the enhanced isoproterenol-induced cAMP levels observed in these cultures. The results suggest that the remainder of the enhanced cAMP response to isoproterenol of in vitro aged fibroblasts may be due to a lower cAMP phosphodiesterase activity in these cells.

The properties of adenosine inhibition of catecholamine-induced responses were investigated, using an isolated rat heart preparation. Perfusion of hearts with 0.1 microM isoproterenol increased myocardial cAMP content 2.8-fold, activation of cAMP-dependent protein kinase 4.4-fold, phosphorylase a formation 3.4-fold, left ventricular pressure 1.8-fold, rate of ventricular pressure development 2.1-fold, and rate of ventricular relaxation 2.2-fold within 1 minute. When perfused with the isoproterenol, 10 microM adenosine reduced the catecholamine-produced increase in cAMP, cAMP-dependent protein kinase, and phosphorylase by 30-40%, and the elevation in left ventricular pressure and rate of ventricular pressure development by 40-70% within 40 seconds. More than 2 minutes were required for the nucleoside to significantly reduce the isoproterenol-elicited increase in the rate of ventricular relaxation. Perfusion of adenosine alone at concentrations from 0.1 to 10 microM were without effect on the above parameters. Theophylline at 50 microM had no effect alone on the above parameters but blocked the inhibitory actions of adenosine on the isoproterenol-induced responses. In the presence of 15 mM Mg++ adenosine reduced by approximately 56% the 2-fold increase in myocardial membrane adenylate cyclase activity produced by 1 microM isoproterenol without affecting basal or fluoride-stimulated activity. Adenosine also reduced the isoproterenol-induced increase in enzyme activity assayed at 1-2 mM Mg++, a level that more closely approximates the intracellular activity of the ion. The results suggest that physiological concentrations of adenosine attenuate the catecholamine-induced increase in cAMP content, cAMP-dependent protein kinase activation, phosphorylase a formation, and contractile parameters in the working heart, via reducing the beta-adrenergic activation of adenylate cyclase.

Adenosine attenuates beta-adrenergic receptor mediated activation of adenylate cyclase in myocardial membranes via adenosine Ri receptors. The effects of adenosine analogs on the binding characteristics of beta-adrenergic receptors were examined in the present study utilizing rat ventricular membranes treated with adenosine deaminase. In 125I-cyanopindolol/isoproterenol competitive binding experiments phenylisopropyladenosine (PIA) significantly increased the IC50 for isoproterenol from 48 +/- 6 nM to 140 +/- 48 nM and steepened the slope of the competition curves from -0.56 +/- 0.03 to -0.90 +/- 0.21. Computer analysis of these curves indicated that binding of isoproterenol to the high affinity state of the beta-adrenergic receptor was eliminated in the presence of PIA. PIA had no effects in the presence of GPP(NH)P. 2-chloroadenosine, a less specific Ri agonist, caused smaller increases in IC50 and slope, without significantly affecting high affinity binding. 2',5'-dideoxyadenosine, a P-site agonist, had no significant effects on isoproterenol binding. During the time course of the competitive binding experiments the membranes displayed isoproterenol-sensitive adenylate cyclase activity in the absence of added GTP. These data suggest that adenosine attenuates catecholamine-induced activation of adenylate cyclase via Ri receptors by decreasing the ability of beta-adrenergic agonists to promote the formation of a high affinity complex composed of the agonist, receptor and stimulatory guanine nucleotide binding protein.

Adenosine inhibition of hormone-sensitive adenylate cyclase activity was investigated using isolated myocardial membranes prepared from rat hearts. When cyclase activity was determined in membranes, using [alpha-32P]ATP as substrate, 10(-5) M adenosine inhibited isoproterenol-stimulated adenylate cyclase activity by 25% but did not inhibit basal activity or fluoride (5 mM) activation of the enzyme. The adenosine reduction of isoproterenol-sensitive cyclase activity was dependent on GTP but was not prevented by 10(-3) M theophylline. Adenosine neither appeared to compete with ATP for the substrate converting site of the enzyme nor reduced 5'-guanylyl imidodiphosphate activation of the enzyme. Inasmuch as lower concentrations of adenosine had no influence on enzyme activity, endogenous adenosine may be present in the adenylate cyclase assay. To obviate the effects of endogenous adenosine, the adenylate cyclase assay was then modified to a 2'-deoxy system with [alpha-32P]dATP used as the substrate in the presence of adenosine deaminase. With this assay system, the 15% inhibition of isoproterenol-stimulated adenylate cyclase activity produced by the adenosine receptor agonists, 10(-8) M 2-chloroadenosine or phenylisopropyladenosine, was prevented by 10(-4) M 8-phenyltheophylline or isobutylmethylxanthine (IBMX), respectively. While under these assay conditions, 10(-7) M 2',5'-dideoxyadenosine, a P-site analogue, did not influence the hormone-sensitive cyclase activity. The 35% reduction of the hormone-sensitive enzyme produced by this analogue at 10(-5) M was not prevented by IBMX. These results suggest that nanomolar concentrations of adenosine analogues interact with a methylxanthine-sensitive adenosine receptor that mediates the attention of membrane hormone-sensitive adenylate cyclase activity.