Evidence for isoproterenol-induced phosphorylation of phosphatase inhibitor-1 in the intact heart.
The positive inotropic effect of the beta-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 Langendorff method with physiological buffers with or without 5 mCi 32P/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 32P-labeled hearts a protein at about 26 kd on autoradiograms of 12% sodium dodecyl sulfate gels was detected. Isoproterenol (1 microM) 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 32P-labeled band at about 26 kd from 380 to 540 pmol 32P/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.
Neumann
J
J
Department of Medicine, Krannert Institute of Cardiology, Indiana University School of Medicine, Indianapolis.
Gupta
R C
RC
Schmitz
W
W
Scholz
H
H
Nairn
A C
AC
Watanabe
A M
AM
D000818:Animals; D015153:Blotting, Western; D002352:Carrier Proteins; D000242:Cyclic AMP / Q000502:physiology; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D005260:Female; D006168:Guinea Pigs; D047908:Intracellular Signaling Peptides and Proteins; D007545:Isoproterenol / Q000494:pharmacology*; D008297:Male; D008970:Molecular Weight; D009206:Myocardium / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D011506:Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*
D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D011506:Proteins; C021042:protein phosphatase inhibitor-1; D000242:Cyclic AMP; D011494:Protein Kinases; D010749:Phosphoprotein Phosphatases; D007545:Isoproterenol