β-adrenoceptor stimulation accelerates Ca²⁺ turnover through PKA-dependent phosphorylation in saponin-treated mouse myocardium

Abstract

Sarcoplasmic reticulum (SR) functions (Ca²⁺ uptake, content, release and leakage) are modulated by β-adrenoceptor stimulation (ARS) through phosphorylation of phospholamban and/or ryanodine receptor. In this study, we investigated the effect of β-ARS on SR functions using saponin-treated mouse myocardium. Thin trabeculae obtained from mice hearts were skinned with saponin (50 μg/ml) after treatment with isoproterenol (Iso) (1 μM, 30 min) or without Iso as control preparations. For the estimation of each SR function, Ca²⁺ remaining in SR after various maneuvers was released by caffeine (50 mM) and measured with fluo-3 (30 μM). Ca²⁺ uptake was estimated by measuring Ca²⁺ in SR after Ca²⁺ was loaded into SR by applying the solution containing ATP (4 mM) at various Ca²⁺ concentrations (pCa 8-5.6) for different periods (10-120 sec). Ca²⁺ leakage was estimated by measuring the remaining Ca²⁺ in SR after washing SR with the solution containing EGTA for various durations (15-300 sec) after Ca²⁺ loading (pCa 6.2, 120 sec). Iso-treatment increased Ca²⁺ uptake rate but did not alter maximal Ca²⁺ content. On the other hand, Ca²⁺ leakage was accelerated after Iso-treatment. These effects of β-ARS on SR were blocked by protein kinase A (PKA) inhibitor H-89 (2 μM). Thus, the PKA-dependent phosphorylation of phospholamban and ryanodine receptor in SR accelerates the turnover of Ca²⁺ in the myoplasm. [J Physiol Sci. 2008;58 Suppl:S179]