2-chloro-n(6)-(3-iodobenzyl)adenosine-5--n-methyluronamide and Myocardial-Ischemia

Adenosine released during myocardial ischemia mediates cardioprotective preconditioning. Multivalent drugs covalently bound to nanocarriers may differ greatly in chemical and biological properties from the corresponding monomeric agents. Here, we conjugated chemically functionalized nucleosides to poly(amidoamine) (PAMAM) dendrimeric polymers and investigated their effects in rat primary cardiac cell cultures and in the isolated heart. Three conjugates of A₃ adenosine receptor (AR) agonists, chain-functionalized at the C2 or N⁶ position, were cardioprotective, with greater potency than monomeric agonist Cl-IB-MECA. Multivalent amide-linked MRS5216 was selective for A₁ and A₃ARs, and triazole-linked MRS5246 and MRS5539 (optionally containing fluorescent label) were A₃AR-selective. The conjugates protected ischemic rat cardiomyocytes, an effect blocked by an A₃AR antagonist MRS1523, and isolated hearts with significantly improved infarct size, rate of pressure product, and rate of contraction and relaxation. Thus, strategically derivatized nucleosides tethered to biocompatible polymeric carriers display enhanced cardioprotective potency via activation of A₃AR on the cardiomyocyte surface.

Topics: Adenosine; Adenosine A3 Receptor Agonists; Animals; Cardiotonic Agents; Cells, Cultured; Dendrimers; Heart; Male; Myocardial Ischemia; Myocytes, Cardiac; Rats; Rats, Sprague-Dawley; Receptor, Adenosine A3

The aim of the present study was to investigate the protective role of pharmacological preconditioning on antioxidant enzymes using A(1) and A(3) adenosine receptor agonists in the recovery of the isolated myocardium after cardioplegic ischemia. Two different modes of preconditioning were studied: isolated rat hearts were perfused with A(1) receptor agonist 2-chloro-N(6)-cyclopentyladenosine (CCPA) or A(3) 2-chloro-N(6)-(3-iodobenzyl) adenosine-5'-N-methyluronamide (Cl-IB-MECA) (1 nM), followed by cardioplegic ischemia and reperfusion (30 min each) (perfusion mode), or CCPA or Cl-IB-MECA (100 micro g/kg) were injected intravenously 24 h before the experiment (injection mode). Hearts treated with CCPA improved in terms of mechanical function, infarct size, ATP levels, superoxide dismutase, and catalase (p < 0.005) in both modes of administration. Cl-IB-MECA was beneficial mainly in the injected group. Reduced damage to the mitochondria in the CCPA-treated hearts was observed using electron microscopy evaluation. In the Cl-IB-MECA-injected hearts, mitochondrial damage was moderate. CCPA in both modes of treatment and Cl-IB-MECA in the injected mode were beneficial in protecting the perfused isolated rat heart, subjected to normothermic cardioplegic ischemia. This protection was partially related to the higher myocardial activity of superoxide dismutase and catalase.

Topics: Adenosine; Adenosine A1 Receptor Agonists; Adenosine A3 Receptor Agonists; Adenosine Triphosphate; Animals; Antioxidants; Catalase; Glutathione Peroxidase; Heart; Hemodynamics; In Vitro Techniques; Ischemic Preconditioning, Myocardial; Male; Myocardial Ischemia; Myocardial Reperfusion; Myocardium; Rats; Rats, Wistar; Receptor, Adenosine A1; Receptor, Adenosine A3; Superoxide Dismutase

It is well established that adenosine receptors are involved in cardioprotection and that protein kinase B (PKB) is associated with cell survival. Therefore, in this study we have investigated whether adenosine receptors (A(1), A(2A) and A(3)) activate PKB by Western blotting and determined the involvement of phosphatidylinositol 3-kinase (PI-3K)/PKB in adenosine-induced preconditioning in cultured newborn rat cardiomyocytes. Adenosine (non-selective agonist), CPA (A(1) selective agonist) and Cl-IB-MECA (A(3) selective agonist) all increased PKB phosphorylation in a time- and concentration-dependent manner. The combined maximal response to CPA and Cl-IB-MECA was similar to the increase in PKB phosphorylation induced by adenosine alone. CGS 21680 (A(2A) selective agonist) did not stimulate an increase in PKB phosphorylation. Adenosine, CPA and Cl-IB-MECA-mediated PKB phosphorylation were inhibited by pertussis toxin (PTX blocks G(i)/G(o)-protein), genistein (tyrosine kinase inhibitor), PP2 (Src tyrosine kinase inhibitor) and by the epidermal growth factor (EGF) receptor tyrosine kinase inhibitor AG 1478. The PI-3K inhibitors wortmannin and LY 294002 blocked A(1) and A(3) receptor-mediated PKB phosphorylation. The role of PI-3K/PKB in adenosine-induced preconditioning was assessed by monitoring Caspase 3 activity and lactate dehydrogenase (LDH) release induced by exposure of cardiomyocytes to 4 h hypoxia (0.5% O(2)) followed by 18 h reoxygenation (HX4/R). Pre-treatment with wortmannin had no significant effect on the ability of adenosine-induced preconditioning to reduce the release of LDH or Caspase 3 activation following HX4/R. In conclusion, we have shown for the first time that adenosine A(1) and A(3) receptors trigger increases in PKB phosphorylation in rat cardiomyocytes via a G(i)/G(o)-protein and tyrosine kinase-dependent pathway. However, the PI-3K/PKB pathway does not appear to be involved in adenosine-induced cardioprotection by preconditioning.

Topics: Adenosine; Adenosine A1 Receptor Agonists; Adenosine A3 Receptor Agonists; Animals; Animals, Newborn; Cardiotonic Agents; Cell Hypoxia; Cells, Cultured; GTP-Binding Protein alpha Subunits, Gi-Go; Mitogen-Activated Protein Kinase 3; Myocardial Ischemia; Myocardial Reperfusion Injury; Myocytes, Cardiac; Pertussis Toxin; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Rats; Receptor, Adenosine A1; Receptor, Adenosine A3

Adenosine (ADO) is a well-known regulator of a variety of physiological functions in the heart. In stress conditions, like hypoxia or ischemia, the concentration of adenosine in the extracellular fluid rises dramatically, mainly through the breakdown of ATP. The degradation of adenosine in the ischemic myocytes induced damage in these cells, but it may simultaneously exert protective effects in the heart by activation of the adenosine receptors. The contribution of ADO to stimulation of protective effects was reported in human and animal hearts, but not in rat hearts. The aim of this study was to evaluate the role of adenosine A1 and A3 receptors (A1R and A3R), in protection of isolated cardiac myocytes of newborn rats from ischemic injury. The hypoxic conditions were simulated by exposure of cultured rat cardiomyocytes (4-5 days in vitro), to an atmosphere of a N2 (95%) and CO2 (5%) mixture, in glucose-free medium for 90 min. The cardiotoxic and cardioprotective effects of ADO ligands were measured by the release of lactate dehydrogenase (LDH) into the medium. Morphological investigation includes immunohistochemistry, image analysis of living and fixed cells and electron microscopy were executed. Pretreatment with the adenosine deaminase considerably increased the hypoxic damage in the cardiomyocytes indicating the importance of extracellular adenosine. Blocking adenosine receptors with selective A1 and A3 receptor antagonists abolished the protective effects of adenosine. A1R and A3R activation during the hypoxic insult delays onset of irreversible cell injury and collapse of mitochondrial membrane potential as assessed using DASPMI fluorochrom. Cardioprotection induced by the A1R agonist, CCPA, was abolished by an A1R antagonist, DPCPX, and was not affected by an A3R antagonist, MRS 1523. Cardioprotection caused by the A3R agonist, Cl-IB-MECA, was antagonized completely by MRS 1523 and only partially by DPCPX. Activation of both A1R and A3R together was more efficient in protection against hypoxia than by each one alone. Our study indicates that activation of either A1 or A3 adenosine receptors in the rat can attenuate myocyte injury during hypoxia. Highly selective A1R and A3R agonists may have potential as cardioprotective agents against ischemia or heart surgery.

Topics: Adenosine; Animals; Animals, Newborn; Cell Hypoxia; Cells, Cultured; L-Lactate Dehydrogenase; Microscopy, Electron; Myocardial Ischemia; Myocardium; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Pyridines; Rats; Receptor, Adenosine A3; Receptors, Purinergic P1; Xanthines

We tested the hypothesis that selective adenosine A(3)-receptor stimulation reduces postischemic contractile dysfunction through activation of ATP-sensitive potassium (K(ATP)) channels. Isolated, buffer-perfused rat hearts (n = 8/group) were not drug pretreated (control) or were pretreated with adenosine (20 microM), 2-chloro-N(6)-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (Cl-IB-MECA; A(3) agonist, 100 nM), Cl-IB-MECA + 8-(3-noradamantyl)-1,3-dipropylxanthine (KW-3902; A(1) antagonist, 5 microM), Cl-IB-MECA + glibenclamide (Glib; K(ATP)-channel blocker, 0. 3 microM), or Glib alone for 12 min before 30 min of global normothermic ischemia followed by 2 h of reperfusion. After 2 h of reperfusion, left ventricular developed pressure (LVDP, %baseline) in control hearts was depressed to 34 +/- 2%. In hearts pretreated with Cl-IB-MECA, there was a statistically significant increase in LVDP (50 +/- 6%), which was reversed with coadministration of Glib (37 +/- 1%). Control hearts also showed similar decreases in left ventricular peak positive rate of change in pressure (dP/dt). Therefore, the A(3) agonist significantly attenuated postischemic cardiodynamic injury compared with the control, which was reversed by Glib. Cumulative creatine kinase (CK in U/min) activity was most pronounced in the control group (10.4 +/- 0.6) and was significantly decreased by Cl-IB-MECA (7.5 +/- 0.4), which was reversed by coadministration of Glib (9.4 +/- 0.2). Coronary flow was increased during adenosine infusion (160% of baseline) but not during Cl-IB-MECA infusion. Effects of Cl-IB-MECA were not reversed by the specific A(1) antagonist KW-3902. We conclude that cardioprotection afforded by A(3)-receptor stimulation may be mediated in part by K(ATP) channels. Cl-IB-MECA may be an effective pretreatment agent that attenuates postischemic cardiodynamic dysfunction and CK release without the vasodilator liability of other adenosine agonists.

Topics: Adenosine; Adenosine Triphosphate; Animals; Coronary Circulation; Creatine Kinase; Diastole; Glyburide; In Vitro Techniques; Myocardial Ischemia; Potassium Channels; Rats; Rats, Sprague-Dawley; Receptor, Adenosine A3; Receptors, Purinergic P1; Ventricular Function, Left

The possible cardioprotective roles of adenosine A1 and A3 receptors were investigated in a cardiac myocyte model of injury. The adenosine A3 receptor is a novel cardiac receptor capable of mediating potentially important cardioprotective functions. Prolonged hypoxia with glucose deprivation was used to simulate ischemia and to induce injury in cardiac ventricular myocytes cultured from chick embryos 14 days in ovo. When present during the prolonged hypoxia, the adenosine A3 agonists N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide (IB-MECA) and 2-chloro-N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide (CI-IB-MECA) caused a dose-dependent reduction in the extent of hypoxia-induced injury as manifested by a decrease in the amount of creatine kinase released and the percentage of myocytes killed. The adenosine A1 agonists 2-chloro-N6-cyclopentyladenosine (CCPA), N6-cyclohexyladenosine, and adenosine amine congener were also able to cause a decrease in the extent of myocyte injury. The A1 receptor-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine blocked the cardioprotective effect of the A1 but not of the A3 agonists. Conversely, the selective A3 antagonists MRS-1191 and MRS-1097 blocked the protection induced by CI-IB-MECA but had minimal effect on that caused by CCPA. Thus the cardioprotective effects of A1 and A3 agonists were mediated by their respective receptors. This study defines a novel cardioprotective function of the cardiac A3 receptor and provides conclusive evidence that activation of both A1 and A3 receptors during hypoxia can attenuate myocyte injury.

Topics: Adenosine; Animals; Cardiotonic Agents; Cell Hypoxia; Cells, Cultured; Chick Embryo; Dihydropyridines; Heart; Heart Ventricles; Myocardial Ischemia; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Receptor, Adenosine A3; Receptors, Purinergic P1; Xanthines