REGULATION OF PROTEIN-PHOSPHORYLATION BY INOTROPIC AGENTS IN ISOLATED RAT MYOCARDIAL-CELLS

Isolated myocardial cells from rat heart were prepared and these cells were used to study the possibility that protein phosphorylation may play a role in cAMP-mediated effects on cardiac contractility. Cell suspensions were preincubated with 32Pi and various drugs were tested for their effects on 32P incorporation into proteins. The .beta.-adrenergic agonist (-)-isoproterenol caused increased 32P incorporation into 5 proteins. 32P incorporation was maximal within 45 s, and half-maximal incorporation was observed with 5 nM (-)-isoproterenol. Epinephrine, norepinephrine, dibutyryl cAMP, isobutylmethylxanthine and glucagon all had similar effects on 32P incorporation into phosphoproteins. The effects of isoproterenol could be completely inhibited by simultaneous addition of (.+-.)-propranolol. Ouabain, an inotropic agent whose effects are not mediated by cAMP, failed to alter the phosphorylation pattern. The 5 phosphoproteins, which were detected by autoradiography of sodium dodecyl sulfate-polyacrylamide gels, had apparent MW of 12,000, 28,000, 33,000 94,000 and 150,000. When propranolol was added after maximal stimulation with isoproterenol, only the MW = 12,000 and 94,000 proteins showed rapid reversal of the increased 32P incorporation. The possible role of these phosphoproteins as mediators of the inotropic and relaxant effects of .beta.-adrenergic agonists and related drugs are discussed. In addition to its effects on protein phosphorylation, (-)-isoproterenol (1 .mu.M, 1 min) caused a 2-fold increase in cAMP content, indicating that the cells possess functional .beta.-adrenergic receptors. The phosphodiesterase inhibitor isobutylmethylxanthine (0.1 mM) also caused a 2-fold increase in cAMP content, and addition of the 2 drugs together caused an almost 10-fold increase. The isolated cells were rod-shaped, possessed typical muscle cell morphology and were tolerant to millimolar concentrations of Ca. Following a 60 min incubation in medium containing 2 mM CaCl2, at least 75% of the cells remained viable and had intact sarcolemmal membranes as judged by a variety of techniques.