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

M. Artman, D. W. Robertson, L. Mahony, W. J. Thompson

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)302-311
Number of pages10
JournalMolecular Pharmacology
Volume36
Issue number2
StatePublished - 1989

Fingerprint

LY 186126
Cardiotonic Agents
indolidan
Phosphodiesterase Inhibitors
Sarcoplasmic Reticulum
Myocardium
Binding Sites
Rabbits
Type 4 Cyclic Nucleotide Phosphodiesterase
Milrinone
Sheep
Subcellular Fractions
Phosphoric Diester Hydrolases
Inhibitory Concentration 50
Rolipram
Magnesium
Anions
Chromatography

ASJC Scopus subject areas

  • Pharmacology

Cite this

Analysis of the binding sites for the cardiotonic phosphodiesterase inhibitor [3H]LY186126 in ventricular myocardium. / Artman, M.; Robertson, D. W.; Mahony, L.; Thompson, W. J.

In: Molecular Pharmacology, Vol. 36, No. 2, 1989, p. 302-311.

Research output: Contribution to journalArticle

@article{8398a39cdcbc442e8832cc2d34e4cc85,
title = "Analysis of the binding sites for the cardiotonic phosphodiesterase inhibitor [3H]LY186126 in ventricular myocardium",
abstract = "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.",
author = "M. Artman and Robertson, {D. W.} and L. Mahony and Thompson, {W. J.}",
year = "1989",
language = "English (US)",
volume = "36",
pages = "302--311",
journal = "Molecular Pharmacology",
issn = "0026-895X",
publisher = "American Society for Pharmacology and Experimental Therapeutics",
number = "2",

}

TY - JOUR

T1 - Analysis of the binding sites for the cardiotonic phosphodiesterase inhibitor [3H]LY186126 in ventricular myocardium

AU - Artman, M.

AU - Robertson, D. W.

AU - Mahony, L.

AU - Thompson, W. J.

PY - 1989

Y1 - 1989

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=0024417313&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0024417313&partnerID=8YFLogxK

M3 - Article

C2 - 2505059

AN - SCOPUS:0024417313

VL - 36

SP - 302

EP - 311

JO - Molecular Pharmacology

JF - Molecular Pharmacology

SN - 0026-895X

IS - 2

ER -