Co-ordinated changes in cAMP, phosphorylated phospholamban, Ca2+ and contraction following β-adrenergic stimulation of rat heart
Co-ordinated changes in cAMP, phosphorylated phospholamban, Ca2+ and contraction following β-adrenergic stimulation of rat heart
复制标题
DOI：
10.1007/s004240050728
复制
发表时间：
1998-11-01
影响因子：
4.5
通讯作者：
Colyer, J
中科院分区：
文献类型：
--
作者：
Calaghan, SC;White, E;Colyer, J
在暴露于 β-肾上腺素受体激动剂异丙肾上腺素的离体大鼠心室肌细胞中，测量了环 AMP (cAMP)、受磷蛋白位点特异性磷酸化、细胞内钙 ([Ca2+](i)) 瞬态和收缩的浓度依赖性变化。通过 [I-125]-cAMP 闪烁邻近测定法测量环 AMP，使用一对位点特异性多克隆抗体评估受磷蛋白 Ser(16) 和 Thr(17) 的磷酸化，并使用荧光染料 fura 2 监测 [Ca2+](i)。在 10(-6) M 异丙肾上腺素存在下，环 AMP 升至基础水平的两倍。在 10(-7) M 异丙肾上腺素时观察到受磷蛋白 Ser(16) 和 Thr(17) 磷酸化的最大增加。在此阶段，Ser(16) 磷酸化程度比不存在异丙肾上腺素时记录的高六倍，Thr(17) 磷酸化程度高三倍。 Ser(16) 的磷酸化与 [Ca2+](i) 瞬态和收缩的变化的相关性比 Thr(17) 的磷酸化更密切。这是同类研究中第一项同时测量 cAMP、受磷蛋白磷酸化、[Ca2+](i) 瞬变和β-激动剂浓度范围内收缩的研究。结果表明，与 Ser(16) 处的磷酸化相比，受磷蛋白 Thr(17) 处的磷酸化与 β-肾上腺素能刺激对心脏的影响的生理相关性较小。
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).