Phosphorylation of phospholamban in intact myocardium. Role of Ca2+-calmodulin-dependent mechanisms.
Phospholamban, a putative regulator of cardiac sarcoplasmic reticulum Ca2+ transport, has been shown to be phosphorylated in vitro by cAMP-dependent protein kinase and an intrinsic Ca2+-calmodulin-dependent protein kinase activity. This study was conducted to determine if Ca2+-calmodulin-dependent phosphorylation of phospholamban occurs in response to physiologic increases in intracellular Ca2+ in intact myocardium. Isolated guinea pig and rat ventricles were perfused with 32Pi after which membrane vesicles were isolated from individual hearts by differential centrifugation. Administration of isoproterenol (10 nM) to perfused hearts stimulated 32P incorporation into phospholamban, Ca2+-ATPase activity, and Ca2+ uptake of sarcoplasmic reticulum isolated from these hearts. These biochemical changes were associated with increases in contractility and shortening of the t 1/2 of relaxation. Elevated extracellular Ca2+ produced comparable increases in contractility but failed to stimulate phospholamban phosphorylation or Ca2+ transport and did not alter the t 1/2 of relaxation. Inhibition of trans-sarcolemmal Ca2+ influx by perfusing the ventricles with reduced extracellular Ca2+ (50 microM) attenuated the increases in 32P incorporation produced by 10 nM isoproterenol. Trifluoperazine (10 microM) also attenuated isoproterenol-induced increases in 32P incorporation into phospholamban. In both cases, Ca2+ transport was reduced to a degree comparable to the reduction in phospholamban phosphorylation. These results suggest that direct physiologic increases in intracellular Ca2+ concentration do not stimulate phospholamban phosphorylation in intact functioning myocardium. Ca2+-calmodulin-dependent phosphorylation of phospholamban may occur in response to agents which stimulate cAMP-dependent mechanisms in intact myocardium.
Lindemann
J P
JP
Watanabe
A M
AM
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism*; D005260:Female; D006168:Guinea Pigs; D007545:Isoproterenol / Q000494:pharmacology; D008297:Male; D008970:Molecular Weight; D009200:Myocardial Contraction; D009206:Myocardium / Q000378:metabolism*; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000378:metabolism*; D014268:Trifluoperazine / Q000494:pharmacology; D000099211:Phospholamban
D002118:Calcium; D002135:Calcium-Binding Proteins; D000252:Calcium-Transporting ATPases; D002147:Calmodulin; D007545:Isoproterenol; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases; D014268:Trifluoperazine; D000099211:Phospholamban