Specific Binding of [H]LY186126, an Analogue of Indolidan (LY195115), to Cardiac Membranes Enriched in Sarcoplasmic Reticulum Vesicles

LY186126 was found to be a potent inhibitor of type IV cyclic AMP phosphodiesterase located in the sarcoplasmic reticulum of canine cardiac muscle. This compound, a close structural analogue of indolidan (LY195115), was prepared in high specific activity, tritiated form to study the positive inotropic receptor(s) for cardiotonic phosphodiesterase inhibitors such as indolidan and milrinone. A high-affinity binding site for [H]LY186126 was observed (Kd=4 nM) in purified preparations of canine cardiac sarcoplasmic reticulum vesicles. Binding was proportional to vesicle protein, was inactivated by subjecting membranes to proteolysis or boiling, and was dependent on added Mg. Scatchard analysis suggested the presence of a single class of binding sites in the membrane preparation. Indolidan, milrinone, and LY186126 (all at 1 /xM) produced essentially complete displacement of bound [H]LY186126, while nifedipine, propranolol, and prazosin had little or no effect at this concentration. This represents the first reported use of a radioactive analogue to label the inotropic receptor for cardiotonic phosphodiesterase inhibitors. The results suggest that [H]LY186126 is a useful radiotigand for examining the subcellular site(s) responsible for positive inotropic effects of these drugs. {Circulation Research 1989;64:1037-1040)