Differential sensitivity to isoprenaline of troponin I and phospholamban phosphorylation in isolated rat hearts

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.

The cardiac sarcoplasmic reticulum phospholamban kinase is a distinct δ‐CaM kinase isozyme

“…For measurement of [32pIP i incorporation into native PLB, SR proteins were separated on Urea/SDS gels [24]. Autoradiograms were quantitated with a PDI (NY, USA) densitometer or bands of interest were cut out and counted by liquid scintillation.…”

Phosphorylation of the L‐type calcium channel β subunit is involved in β‐adrenergic signal transduction in canine myocardium

“…Fractions of total cardiac membranes were prepared from frozen tissue samples as described in [30]. Cardiac membranes (3 mg of protein) were phosphorylated, if not stated otherwise, in a final volume of 1.5 ml with 0.63 PM catalytic subunit of PKA and 10 PM [r-"P]ATP (2-3 nCi/pmol) for 6 min at 30°C in a medium containing 40 mM HEPESiTris-buffer, pH 7.4, 15 mM MgCl,, 0.1% digitonin, 1 mM EGTA, 5 mM EDTA, 12.5 mM NaF and 25 mM Pi.…”

“…Tissue levels of CAMP were analysed in neutralized trichloroacetic acid extracts as described in [30]. The activity of CAMP-PK was measured as in [33] and is expressed as the ratio between activities assayed without and with CAMP (-cAMP/+cAMP).…”

“…This is based on the in vitro toppingup of phosphorylation of cardiac membrane proteins in the presence of an excess of the catalytic subunit of PKA and [Y-~'P]ATP. The induced in vitro 32Pi incorporation is inversely related to the in vivo phosphorylation of proteins [15,29,30,38,39]. In this report we have combined the in vitro back-phosphorylation with the immunoprecipitation standardized as described above.…”

Myofilament proteins: From cardiac disorders to proteomic changes

“…Therefore, determination of phosphorylation states solely by relative positions on 1-D-IEF gels may not be completely accurate, and additional tests, for example, parallel analysis of dephosphorylated samples, are needed to verify the presence of phosphorylation. On the contrary, in vitro labeling of proteins with [ 32 P] followed by autoradiography is a definitive and very sensitive method to detect protein phosphorylation [90], and basal phosphorylation levels can be compared by using the so-called "back-phosphorylation" [91] method in which amounts of [ 32 P] incorporated into unoccupied phosphorylation sites are compared. Using this method, we determined that TnI and LC2 are hyperphosphorylated in 12-month PKCe TG mice [92].…”