Calaghan S C, White E, Colyer J
School of Biomedical Sciences, Worsley Building, University of Leeds, Leeds LS2 9NQ, UK.
Pflugers Arch. 1998 Nov;436(6):948-56. doi: 10.1007/s004240050728.
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.
在分离的大鼠心室肌细胞中，测量了暴露于β - 肾上腺素能受体激动剂异丙肾上腺素后，环磷酸腺苷（cAMP）浓度依赖性变化、受磷蛋白的位点特异性磷酸化、细胞内钙（[Ca2+]i）瞬变和收缩情况。通过[125I]-cAMP闪烁邻近分析法测量cAMP，使用一对位点特异性多克隆抗体评估受磷蛋白在丝氨酸16和苏氨酸17处的磷酸化，并用荧光染料fura 2监测[Ca2+]i。在10(-6) M异丙肾上腺素存在下，cAMP升高至基础水平的两倍。在10(-7) M异丙肾上腺素时，观察到受磷蛋白丝氨酸16和苏氨酸17处磷酸化的最大增加。此时，丝氨酸16磷酸化比无异丙肾上腺素时记录的值高6倍，苏氨酸17磷酸化高3倍。与苏氨酸17处的磷酸化相比，丝氨酸16处的磷酸化与[Ca2+]i瞬变和收缩的变化相关性更密切。这是同类研究中首次在一系列β - 激动剂浓度下同时测量cAMP、受磷蛋白磷酸化、[Ca2+]i瞬变和收缩的变化。结果表明，与丝氨酸16处的磷酸化相比，苏氨酸17处受磷蛋白的磷酸化对β - 肾上腺素能刺激心脏效应的生理相关性较小。
