Co-ordinated changes in cAMP, phosphorylated phospholamban, Ca2+ and contraction following β-adrenergic stimulation of rat heart

Concentration-dependent changes in cyclic AMP (cAMP), site-specific phosphorylation of phospholamban, the intracellular calcium ([Ca2+](i)) transient and contraction were measured in isolated rat ventricular myocytes exposed to the beta-adrenoceptor agonist isoprenaline. Cyclic AMP was measured by [I-125]-cAMP scintillation proximity assay, phosphorylation of phospholamban at Ser(16) and Thr(17) was assessed using a pair of site-specific polyclonal antibodies, and [Ca2+](i) was monitored with the fluorescent dye fura 2. Cyclic AMP rose to twice basal levels in the presence of 10(-6) M isoprenaline. The maximum increase in phosphorylation at Ser(16) and Thr(17) of phospholamban was seen at 10(-7) M isoprenaline. At this stage Ser(16) phosphorylation was six times higher, and Thr(17) phosphorylation was three times higher than that recorded in the absence of isoprenaline. Phosphorylation at Ser(16) correlated more closely with changes in the [Ca2+](i) transient and contraction than did phosphorylation at Thr(17). This is the first study of its kind to measure simultaneous changes in cAMP, the phosphorylation of phospholamban, the [Ca2+](i) transient and contraction over a ranee of concentrations of beta-agonist. The results suggest that phosphorylation of phospholamban at Thr(17) is of lesser physiological relevance to the effects of beta-adrenergic stimulation on the heart than phosphorylation at Ser(16).