Phosphorylation of Phospholamban at Threonine-17 in the Absence and Presence of β -Adrenergic Stimulation in Neonatal Rat Cardiomyocytes

The site-specific phospholamban phosphorylation was studied with respect to the interplay of cAMP- and Ca²⁺signaling in neonatal rat cardiomyocytes. To elucidate the signal pathway(s) for the activation of Ca²⁺/calmodulin-dependent protein kinase (CaMKII) we studied Thr17 phosphorylation of phospholamban in dependence of Ca²⁺channel activation by S(-)-Bay K8644 and in dependence of the depletion of the sarcoplasmic reticulum Ca²⁺stores by ryanodine or thapsigargin in the absence or presence of β -adrenergic stimulation. The isoproterenol (0.1 μ)-induced Thr17 phosphorylation was potentiated 2.5-fold in presence of 1 μ S(-)-Bay K8644. Interestingly, S(-)-Bay K8644 alone was also able to induce Thr17 phosphorylation in a dose- and time-dependent fashion. Ryanodine (1.0 μ) reduced both the isoproterenol (0.1μ ) and S(-)-Bay K8644-(1 μ) mediated Thr17 phosphorylation by about 90%. Thapsigargin (1 μ) diminished the S(-)-Bay K8644 and isoproterenol-associated Thr17 phosphorylation by 53.5±6.3% and 92.5±11.1%, respectively. Ser16 phosphorylation was not affected under these conditions. KN-93 reduced the Thr17 phosphorylation by S(-)-Bay K8644 and isoproterenol to levels of 1.1±0.3% and 8.6±2.1%, respectively. However, the effect of KN-93 was attenuated (47.8±3.6%) in isoproterenol prestimulated cells. Protein phosphatase inhibition by okadaic acid increased exclusively the Ser16 phosphorylation. In summary, our results reflect a cross-talk between β -adrenoceptor stimulation and intracellular Ca²⁺at the level of CaMKII-mediated phospholamban phosphorylation in neonatal rat cardiomyocytes. We report conditions which exclusively produce Thr17 or Ser16 phosphorylation. We postulate that Ca²⁺transport systems of the sarcoplasmic reticulum are critical determinants for the activation of CaMKII that catalyzes phosphorylation of phospholamban.