Analysis of the binding sites for the cardiotonic phosphodiesterase inhibitor [³H]LY186126 in ventricular myocardium

The positive inotropic action of the newer cardiotonic phosphodiesterase inhibitors such as indolidan, milrinone, and imazodan has been previously attributed to selective inhibition of cGMP-inhibitable Type IV (high affinity) cAMP phosphodiesterase activity. However, the subcellular binding site(s) for this class of compounds has not been defined. We have characterized the binding of [³H]LY186126, an analogue of indolidan, in subcellular fractions prepared from rabbit and sheep ventricular myocardium. Binding required magnesium ion and exhibited rapid association and dissociation kinetics. Specific binding (defined by ligand displacement with 5 μM indolidan) to enriched rabbit sarcoplasmic reticulum (SR) membrane vesicles was saturable (B(max) = 714 ± 77 fmol/mg of protein) and of high affinity (K(d) = 6.2 ± 1.4 nM). Linear and nonlinear analyses of the binding isotherms fit a single-site model. Mixed SR preparations from sheep myocardium exhibited binding characteristics (B(max) = 944 ± 115 fmol/mg; K(d) = 8.5 ± 2.3 nM) comparable to those of rabbit cardiac SR. Further subfractionataion of sheep SR indicated that the binding sites were equally distributed between free (B(max) = 630 fmol/mg; K(d) = 4.4 nM) and junctional SR (B(max) = 569 fmol/mg; K(d) = 10.9 nM). Specific binding of [³H]LY186126 was also demonstrated in the cytosolic subfraction of rabbit myocardium that contained Type IV phosphodiesterase activity (Peak III from anion exchange chromatography). Competition for [³H]LY186126 binding studied in rabbit SR showed that, of the compounds tested, lixazinone (RS 82856) competed most effectively (IC₅₀ = 0.030 ± 0.008 nM), followed by indolidan (0.14 ± 0.05 nM), cGMP (17.8 ± 2.6 nM), milrinone (39.3 ± 13.2 nM), and imazodan (192 ± 73 nM). In contrast, rolipram, which does not inhibit SR-associated Type IV phosphodiesterase activity, was not effective at competing for [³H]LY186126 binding (IC₅₀ > 30 μM). These results indicate that [³H]LY186126 has specific binding sites in myocardial subcellular fractions that contain cGMP-inhibitable Type IV (high affinity) cAMP phosphodiesterase activity.