Evidence for Isoproterenol-Induced Phosphorylation of Phosphatase Inhibitor-1 in the Intact Heart

• Published in: Circulation research 1991-12, Vol.69 (6), p.1450-1457
• Main Authors: Neumann, Joachim, Gupta, Ramesh C, Schmitz, Wilhelm, Scholz, Hasso, Nairn, Angus C, Watanabe, August M
• Format: Article
• Language: English
• Subjects: Animals; Biological and medical sciences; Blotting, Western; Carrier Proteins; Cyclic AMP - physiology; Enzyme Inhibitors - chemistry; Enzyme Inhibitors - immunology; Enzyme Inhibitors - metabolism; Female; Fundamental and applied biological sciences. Psychology; Guinea Pigs; Heart; Intracellular Signaling Peptides and Proteins; Isoproterenol - pharmacology; Male; Molecular Weight; Myocardium - metabolism; Phosphoprotein Phosphatases - antagonists & inhibitors; Phosphorylation; Protein Kinases - metabolism; Proteins - chemistry; Proteins - immunology; Proteins - metabolism; Vertebrates: cardiovascular system
• ISSN: 0009-7330; 1524-4571
• DOI: 10.1161/01.RES.69.6.1450

The positive inotropic effect of the β-adrenoceptor agonist isoproterenol is accompanied by inhibition of phosphatase type 1 activity in myocardium. Indirect assays suggest that this effect is due to activation of protein phosphatase inhibitor-1, which inhibits phosphatase activity only when phosphorylated. To test this hypothesis directly, electrically stimulated (3 Hz) guinea pig ventricular preparations were perfused according to the Langendorif method with physiological buffers with or without 5 mCi P/heart, and then various concentrations of isoproterenol were applied. Contractility was recorded. Hearts were freeze-clamped and cAMP and inhibitor-1 activities were measured. In P-labeled hearts a protein at about 26 kd on autoradiograms of 12% sodium dodecyl sulfate gels was detected. Isoproterenol (1 μM) increased rate of tension development to 238% of the predrug value, cAMP concentrations 1.5-fold, and inhibitor-1 activity threefold. Concomitantly, there was an increase in a P-labeled band at about 26 kd from 380 to 540 pmol P/mg protein. This protein at about 26 kd, after transfer to nitrocellulose, was recognized by an antiserum prepared against rabbit skeletal muscle inhibitor-1. More radioactive protein of about 26 kd could be immunoprecipitated by the antiserum from isoproterenol-treated than from untreated hearts. It is concluded that a protein, probably identical to phosphatase inhibitor-1, is phosphorylated in vivo in the heart in the presence of isoproterenol. Phosphorylation of inhibitor-1 with consequent modification of type 1 phosphatase activity may contribute to the effects of isoproterenol in the heart.