A guinea pig heart Langendorif preparation was used in the present study to test the hypothesis that the coronary endothelium modulates coronary autoregulation through the production of nitric oxide (NO is an oversupply of oxygen, and diffusible metabolites are even more easily washed out of the heart. It is now clear that vascular endothelium modulates flow autoregulation. Mechanical denudation of the endothelium or endothelial injury by agents such as the combination of Evans blue plus irradiation by a heliumneon laser can enhance the myogenic response of isolated coronary8 and cerebral9 arteries. Endotheliumdependent relaxation factors, particularly nitric oxide (NO),10-12 participate in the setting of basal tone in many vascular beds, including the coronary circulation.'3-16 Competitively inhibiting the formation of NO from L-arginine, its natural precursor, 17,8 by means of analogues of L-arginine19 is an important tool for assessing the contribution of NO in vasodilator responses. Thus, Griffith and Edwards20 have shown that an isolated, buffer-perfused rabbit ear preparation that lacks the ability to autoregulate will develop the capacity to autoregulate under the combined influences of vasoconstriction by serotonin and the inhibition of NO production by NG-methyl-L-arginine.Our present article describes studies of isolated guinea pig hearts that test the hypothesis that NO modulates coronary autoregulation.Materials and Methods We used 28 Langendorff preparations from guinea pigs weighing 300-400 g. The perfusate consisted of (mM) NaCI 120, NaHCO3 27, KCI 3.7, MgSO4 0.64, CaCl2 1.3, sodium pyruvate 2.0, and glucose 5.0. The buffer was equilibrated with 95% 02-5% C02, warmed by guest on

The reaction of aliphatic aldehydes and ketones with 2-hydrazinoadenosine under relatively mild conditions (at room temperature or in refluxing methanol) formed 2-(N'-alkylidenehydrazino)-adenosines, 5-22, in good yields. Two kinds of adenosine receptors regulate cardiac and coronary physiology. In supraventricular tissues an A1AR coupled to muscarinic K channels mediates the negative chronotropic, dromotropic, and inotropic actions of adenosine, and an inhibitory A1AR coupled to adenylate cyclase mediates the "antiadrenergic" action of adenosine. One or more kinds of A2 receptors mediate coronary vasodilation. Bioassays employing a guinea pig heart Langendorff preparation showed that 5-22 weakly retard impulse conduction through the AV node (negative dromotropic effect), but several analogues were very active coronary vasodilators. The coronary vasoactivity of the (n-alkylidene- and of the (isoalkylidenehydrazino)adenosines paralleled the length of the alkyl chain, the EC50s of the of the most active n-pentylidene (8) and isopentylidene (18) congeners being 1 nM. The EC50s of the cyclohexylmethylene (9), cyclohexylethylidene (10), and cyclohex-3-enylmethylene (12), analogues were likewise < 1 nM, but the cyclohex-1-enylmethylene congener 12 was 10 times less active than 9. The unselective adenosine receptor antagonist 8-(p-sulfophenyl)theophylline (0.1 mM) raised the EC50s of the negative dromotropic effects of 8, 9, and 18 by 5-28-fold and the EC50s of coronary vasodilation of 22-90-fold. Catalytic reduction of 9 increased the hydrophobicity and changed the UV spectrum, suggesting reduction of the --CH = N-- bond. The product darkened on exposure to air and so was not characterized further. A new method for preparing 2',3',5'-tri-O-acetyl-2,6-dichloropurine riboside, a precursor in the synthesis of 2-hydrazinoadenosine, consists of the addition of tert-butyl nitrite to a mixture of 2',3',5'-tri-O-acetyl-6-chloroguanosine and CuCl in CHCl3 saturated with Cl2.

Experiments employing guinea pig heart Langendorff preparations compared the coronary vasoactivity of a functionalized congener of adenosine, 2-[(2-aminoethyl-aminocarbonylethyl)phenylethylamino]-5'-N-e thyl- carboxamidoadenosine, APEC, with the vasoactivity of the product of the reaction of APEC with 1,4-phenylene-diisothiocyanate, 4-isothiocyanatophenylaminothiocarbonyl-APEC (DITC-APEC). Previous experiments showed that whereas APEC binds reversibly to the A2A adenosine receptor of brain striatum, DITC-APEC binds irreversibly. APEC caused concentration-dependent coronary vasodilation that persisted unchanged when agonist administration continued for up to 165 min, but promptly faded when the agent was withdrawn. The unselective adenosine receptor antagonist 8-(4-sulfophenyl)theophyline (8-SPT) antagonized the vasoactivity of APEC. By contrast, DITC-APEC (0.125-1.0 nM) caused progressive, concentration-independent vasodilation that persisted unchanged for as long as 120 min after the agent was stopped and that was insensitive to antagonism by subsequently applied 8-SPT. However, perfusion of the heart with buffer containing 0.1 mM 8-SPT strongly antagonized the coronary vasodilatory action of DITC-APEC given subsequently. Such observations indicate that the covalent binding of DITC-APEC causes irreversible activation of the guinea pig coronary artery A2A adenosine receptor. Neither APEC nor DITC-APEC appeared to desensitize the coronary adenosine receptor during two or more hours of exposure to either agonist.

This study aimed at the development of 2-(N'-aralkylidenehydrazino)adenosines as coronary vasodilators. The reaction of aromatic aldehydes or ketones with 2-hydrazinoadenosine in refluxing methanol formed the target compounds 2-27 as crystalline products in good yields. Two kinds of receptors mediate the actions of adenosine on the heart. Retardation of impulse conduction through the atrioventricular node, the negative dromotropic action, is an example of adenosine's action at an A1 receptor (A1AR) and coronary vasodilation reflects adenosine's action at an A2 receptor (A2AR). Accordingly, bioassays employing guinea pig heart Langendorff preparations assessed the selectivity of 2-27 as coronary vasodilators. Analogues 2-27 were weak negative dromotropic agents; the EC50 of the most active analogue, 2-[N'-(1-naphthylmethylene)hydrazino]-adenosine, 23, was 0.8 microM, several orders of magnitude less than many A1AR agonists. Some of the analogues were quite active coronary vasodilators; 2-(N'-benzylidenehydrazino)adenosine, 2, and several of its para-substituted derivatives, namely, the fluoro (7), methyl (13), methoxy (16), and tert-butylcarbonylethyl, 31, had EC50s for coronary vasodilation in the range 1.7-3.2 nM. The selectivity ratios, EC50 (negative dromotropic)/EC50 (coronary vasodilatory), of these five analogues ranged between 5100 (analogue 31) and 43,000 (analogue 2). Phenyl ring substitutions of other kinds or at other positions, replacement of the phenyl ring by other aryl or heteroaryl groups, or the replacement of the benzylic H by a methyl group lowered coronary vasoactivity significantly. The unselective adenosine receptor antagonist 8-(p-sulfophenyl)theophylline raised the EC50 of the negative dromotropic activities of 2, 16, and 2-[N'-(2-naphthylmethylene)hydrazino]adenosine, 24, by 3-, 18-, and 7-fold, and raised the EC50s of coronary vasoactivity by 11-, 3-, and 30-fold, respectively evidence that vasoactivity was receptor-mediated.