Differential sensitivity to isoprenaline of troponin I and phospholamban phosphorylation in isolated rat hearts
- 15 February 1990
- journal article
- research article
- Published by Portland Press Ltd. in Biochemical Journal
- Vol. 266 (1), 115-122
- https://doi.org/10.1042/bj2660115
Abstract
Phosphorylation of phospholamban (PLB), a membrane-bound 15 kDa protein and troponin I (TNI) was studied in isolated perfused rat hearts by using the back-phosphorylation technique with [32P]ATP catalysed by an excess of exogenous catalytic subunit of cyclic AMP (cAMP)-dependent protein kinase, followed by protein separation. This standardized method allows the quantitative detection of protein phosphorylation specifically stimulated by cAMP. In control hearts the extent of specific phosphorylation was equivalent to 3.3 nmol of PLB and 11.0 mumol of TNI per g of cardiac tissue. In hearts freeze-clamped 30 s after exposure to isoprenaline (10 pM-10 microM), there was a dose-dependent decrease in phosphate incorporation in vitro, indicating a phosphorylation of the respective proteins in vivo. A differential sensitivity of TNI and PLB phosphorylation towards the beta-adrenergic agonist and the subsequent increase in tissue cAMP was found, favouring TNI phosphorylation. K0.5 values for isoprenaline were 2.94 +/- 0.04 nM and 4.46 +/- 0.24 nM for PLB and the 15 kDa protein, but 0.13 +/- 0.01 nM for TNI phosphorylation in the intact tissue. At an isoprenaline-induced increase in cAMP less than 3 pmol/mg of protein there was no or only a small increase in PLB phosphorylation, whereas TNI phosphorylation was nearly maximal. By plotting phosphorylation data against changes in contractile parameters a strong correlation was obtained for TNI (r = 0.95), assuming a linear relationship. For PLB a complex relationship is likely to exist. Our data (i) indicate a functional compartmentalization of the cAMP signal cascade and (ii) confirm that phosphorylation of TNI rather than of PLB is related to changes in mechanical myocardial responses.This publication has 43 references indexed in Scilit:
- Heterogeneous distribution of calmodulin‐and cAMP‐dependent regulation of Ca2+ uptake in cardiac sarcoplasmic reticulum subfracitonsEuropean Journal of Biochemistry, 1988
- The role of glutathione status in the protection against ischaemic and reperfusion damage: Effects of N-acetyl cysteineJournal of Molecular and Cellular Cardiology, 1988
- Dissociation between contraction and relaxation: The possible role of phospholamban phosphorylationBasic Research in Cardiology, 1987
- Complete complementary DNA-derived amino acid sequence of canine cardiac phospholamban.JCI Insight, 1987
- Dephosphorylation of myofibrillar proteins in actomyosin preparations and in isolated perfused rat hearts after β-agonist withdrawalJournal of Molecular and Cellular Cardiology, 1982
- Protein phosphorylation: Quantitative analysis in vivo and in intact cell systemsMolecular and Cellular Endocrinology, 1980
- Calcium sensitivity of the contractile system and phosphorylation of troponin in hyperpermeable cardiac cells.The Journal of general physiology, 1980
- Concerted regulation of cardiac sarcoplasmic reticulum calcium transport by cyclic adenosine monophosphate dependent and calcium-calmodulin-dependent phosphorylationsBiochemistry, 1979
- Phosphorylation of the inhibitory subunit of troponin and its effect on the calcium dependence of cardiac myofibril adenosine triphosphataseFEBS Letters, 1976
- Correlation between contraction and phosphorylation of the inhibitory subunit of troponin in perfused rat heartFEBS Letters, 1975