Protein kinase inhibitors reduce SR Ca transport in permeabilized cardiac myocytes.
Phosphorylation of the sarcoplasmic reticulum (SR) protein phospholamban by adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA) and Ca-calmodulin-dependent protein kinase (CaM-KII) stimulates Ca-adenosinetriphosphatase (ATPase) activity and SR Ca transport, but the role of CaM-KII-dependent phosphorylation is not well defined. We studied the PKA- and CaM-KII-dependent regulation of SR Ca transport in digitonin-permeabilized rabbit ventricular myocytes. SR Ca uptake and free Ca concentration were measured on line with indo 1 and Ca electrodes in the presence of 20 microM ruthenium red and 10 mM oxalate. neither N5,2'-w-dibutyryl-cAMP (up to 500 microM) nor the nonhydrolyzable cAMP agonist adenosine 3'5'-cyclic monophosphorothioate sodium salt (Sp-cAMP[S]; up to 275 microM) affected the maximum uptake rate (Vmax) or the dissociation constant (Kd) for Ca uptake. However, the PKA inhibitor H-89 significantly increased Kd (e.g., from 307 +/- 67 to 826 +/- 62 nM Ca at 40-65 microM H-89) without significantly affecting Vmax. Both CaM-KII inhibitors, KN-62 (60 microM) and a CaM-KII inhibitory peptide (10 microM), significantly decreased Vmax from 11.95 +/- 0.5 to 9.48 +/- 0.6 nmol.mg-1.min-1 and from 10.95 +/- 1.72 to 7.37 +/- 0.94 nmol.mg-1.min-1, respectively, without consistently changing Kd. The effects of H-89 on Kd and of KN-62 on Vmax were prevented by a monoclonal antibody to phospholamban 2D12 (consistent with the antibody removing the inhibitory effect of phospholamban on the SR Ca-ATPase).(ABSTRACT TRUNCATED AT 250 WORDS)
Mattiazzi
A
A
Department of Physiology, Loyola University Medical School, Maywood, Illinois 60153.
Hove-Madsen
L
L
Bers
D M
DM
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000031:analogs & derivatives*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000276:immunology; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D002463:Cell Membrane Permeability; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004789:Enzyme Activation; D007546:Isoquinolines / Q000037:antagonists & inhibitors; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D010879:Piperazines / Q000037:antagonists & inhibitors; D047428:Protein Kinase Inhibitors; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D013449:Sulfonamides; D000099211:Phospholamban
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000911:Antibodies, Monoclonal; D002118:Calcium; D002135:Calcium-Binding Proteins; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D007546:Isoquinolines; D010879:Piperazines; D047428:Protein Kinase Inhibitors; D013449:Sulfonamides; D000099211:Phospholamban; C063302:KN 62; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide