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.
Differential sensitivity to isoprenaline of troponin I and phospholamban phosphorylation in isolated rat hearts
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P Karczewski, S Bartel, E G Krause; Differential sensitivity to isoprenaline of troponin I and phospholamban phosphorylation in isolated rat hearts. Biochem J 15 February 1990; 266 (1): 115–122. doi: https://doi.org/10.1042/bj2660115
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