Co-ordinated changes in cAMP, phosphorylated phospholamban, Ca2+ and contraction following beta-adrenergic...

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 [125I]-cAMP scintillation proximity assay, phosphorylation of phospholamban at Ser16 and Thr17 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 Ser16 and Thr17 of phospholamban was seen at 10(-7) M isoprenaline. At this stage Ser16 phosphorylation was six times higher, and Thr17 phosphorylation was three times higher than that recorded in the absence of isoprenaline. Phosphorylation at Ser16 correlated more closely with changes in the [Ca2+]i transient and contraction than did phosphorylation at Thr17. 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 range of concentrations of beta-agonist. The results suggest that phosphorylation of phospholamban at Thr17 is of lesser physiological relevance to the effects of beta-adrenergic stimulation on the heart than phosphorylation at Ser16.