Termination of cAMP signals by Ca2+ and G(alpha)i via extracellular Ca2+ sensors: a link to intracellular Ca2+ oscillations.
Termination of cAMP signals by Ca2+ and G(alpha)i via extracellular Ca2+ sensors: a link to intracellular Ca2+ oscillations.
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DOI：
10.1083/jcb.200507054
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发表时间：
2005-10-24
影响因子：
7.8
通讯作者：
Hofer, Aldebaran M
中科院分区：
文献类型：
--
作者：
Gerbino, Andrea;Ruder, Warren C;Curci, Silvana;Pozzan, Tullio;Zaccolo, Manuela;Hofer, Aldebaran M
终止环磷酸腺苷（cAMP）信号通过细胞外Ca 2+敏感受体（CaR）可视化在单一的CaR表达的人胚肾（HEK）293细胞使用基于蛋白激酶A和Epac的比率荧光共振能量转移依赖性cAMP传感器。通过涉及百日咳毒素敏感性Gαi和CaR刺激的细胞内[Ca 2 +]增加的双重机制，CaR的刺激迅速逆转或阻止激动剂刺激的cAMP升高。在与Fura-2的平行测量中，CaR激活引起了强烈的Ca 2+振荡，在cAMP存在下频率增加，最终融合成持续的平台。考虑到这些细胞中cAMP积累的Ca 2+敏感性，在CaR刺激的初始阶段[cAMP]缺乏振荡令人困惑。额外的实验表明，低频，长时间的Ca 2+振荡在[cAMP]中产生动态阶梯模式，而高频尖峰没有影响。我们的数据表明，在HEK细胞中的cAMP机制作为一个低通滤波器忽略了相对快速的Ca 2+尖峰刺激的Ca 2+动员激动剂在生理条件下。
Termination of cyclic adenosine monophosphate (cAMP) signaling via the extracellular Ca2+-sensing receptor (CaR) was visualized in single CaR-expressing human embryonic kidney (HEK) 293 cells using ratiometric fluorescence resonance energy transfer–dependent cAMP sensors based on protein kinase A and Epac. Stimulation of CaR rapidly reversed or prevented agonist-stimulated elevation of cAMP through a dual mechanism involving pertussis toxin–sensitive Gαi and the CaR-stimulated increase in intracellular [Ca2+]. In parallel measurements with fura-2, CaR activation elicited robust Ca2+ oscillations that increased in frequency in the presence of cAMP, eventually fusing into a sustained plateau. Considering the Ca2+ sensitivity of cAMP accumulation in these cells, lack of oscillations in [cAMP] during the initial phases of CaR stimulation was puzzling. Additional experiments showed that low-frequency, long-duration Ca2+ oscillations generated a dynamic staircase pattern in [cAMP], whereas higher frequency spiking had no effect. Our data suggest that the cAMP machinery in HEK cells acts as a low-pass filter disregarding the relatively rapid Ca2+ spiking stimulated by Ca2+-mobilizing agonists under physiological conditions.