title: Characterization and pharmacological relevance of high affinity binding sites for [3H]LY186126, a cardiotonic phosphodiesterase inhibitor, in canine cardiac membranes.
abstract: [3H]LY186126, an analogue of the cardiotonic agent indolidan, was shown to bind reversibly and with high affinity (Kd = 4 nM) to a single class of binding sites within canine myocardial vesicles. Binding site density measured in various cardiac membrane fractions correlated well with Ca2+-ATPase activity (r = 0.94; p less than 0.01), but not with Na+,K+-ATPase or azide sensitive ATPase, indicating a localization of these sites within sarcoplasmic reticulum membranes. Divalent cations were required for binding and displayed the following order of activation: Zn2+ greater than Mn2+ greater than Mg2+ greater than Ca2+. Differential activation of [3H]LY186126 binding by various divalent cations was due to alterations in binding site density, rather than affinity. cGMP and selective inhibitors of type IV membrane-bound phosphodiesterase (SR-PDE), for example, indolidan, milrinone, imazodan, and enoximone, selectively displaced bound [3H]LY186126 caffeine, theophylline, and rolipram were relatively impotent as inhibitors of radiolabel binding. Kd values from displacement curves were highly correlated with IC50 values for inhibition of SR-PDE (r = 0.92; p less than 0.001). In addition, Kd values correlated well with published ED50 values for increases in cardiac contractility in pentobarbital-anesthetized dogs (r = 0.94; p less than 0.001). The results support the hypothesis that [3H]LY186126 labels the pharmacological receptor for the class of positive inotropic agents characterized as isozyme-selective phosphodiesterase inhibitors. Furthermore, the data suggest that the identity of the site labeled by [3H]LY186126 is SR-PDE, the type IV isozyme of cardiac phosphodiesterase located in the sarcoplasmic reticulum.
authors.affiliation: Department of Cardiovascular Pharmacology, Lilly Research Laboratories, Eli Lilly and Co, Lilly Corporate Center, Indianapolis, Indiana 46285.
authors.forename: R F
authors.initials: RF
authors.lastname: Kauffman
authors.forename: B G
authors.initials: BG
authors.lastname: Utterback
authors.forename: D W
authors.initials: DW
authors.lastname: Robertson
mesh_terms: D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002316:Cardiotonic Agents / Q000378:metabolism*; D006152:Cyclic GMP / Q000378:metabolism; D004285:Dogs; D004789:Enzyme Activation / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D007211:Indoles / Q000378:metabolism*; D007477:Ions; D008566:Membranes / Q000378:metabolism; D009206:Myocardium / Q000378:metabolism* / Q000648:ultrastructure; D000078183:Oxindoles; D010726:Phosphodiesterase Inhibitors / Q000378:metabolism*; D011724:Pyridazines / Q000378:metabolism*; D012519:Sarcoplasmic Reticulum / Q000378:metabolism
chemical_list: D002316:Cardiotonic Agents; D007211:Indoles; D007477:Ions; D000078183:Oxindoles; D010726:Phosphodiesterase Inhibitors; D011724:Pyridazines; C050325:indolidan; C059371:LY 186126; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D006152:Cyclic GMP