Interplay of Ca2+ and cAMP signaling in the insulin-secreting MIN6 β-cell line
Interplay of Ca2+ and cAMP signaling in the insulin-secreting MIN6 β-cell line
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DOI：
10.1074/jbc.m505657200
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发表时间：
2005-09-02
影响因子：
4.8
通讯作者：
Roe, MW
中科院分区：
文献类型：
--
作者：
Landa, LR;Harbeck, M;Roe, MW
Ca ~（2+）和cAMP是调节多种细胞过程的重要第二信使。虽然以前的研究表明Ca 2+和cAMP信号通路之间的直接相互作用，潜在的机制仍然没有得到解决。特别是，在活细胞中Ca 2+调节cAMP产生的直接证据是不完整的。基于遗传编码的荧光共振能量转移的生物传感器已经使得单个活细胞中细胞内cAMP浓度的空间和时间梯度的实时成像成为可能。在这里，我们使用共聚焦显微镜，荧光共振能量转移，和胰岛素分泌MIN 6细胞表达Epac 1-营地，生物合成单分子cAMP指示剂，更好地了解细胞内Ca 2+在cAMP生产中的作用。我们报告，去极化与高外部K+，甲苯磺丁脲，或葡萄糖引起的cAMP的快速增加，依赖于细胞外Ca 2+和抑制尼群地平，Ca 2+通道阻滞剂，或2 '，5'-双脱氧腺苷，跨膜腺苷酸环化酶的P-位点拮抗剂。在四乙基氯化铵存在下，用葡萄糖刺激MIN 6细胞产生伴随的Ca 2+和cAMP振荡，在细胞外Ca 2+不存在的情况下，这些振荡被消除，并被磷酸二酯酶抑制剂2 '，5'-二脱氧腺苷或3-异丁基-1-甲基黄嘌呤阻断。同时测量钙和cAMP浓度与Fura-2和Epac 1-营地，分别揭示了一个密切的时间和因果关系的细胞质钙和cAMP水平的增加之间的相互关系膜去极化。这些研究结果表明，高度协调的相互作用之间的Ca 2+和cAMP信号在电兴奋性内分泌细胞和Ca 2+依赖的cAMP振荡来自腺苷酸环化酶活性的增加和周期性激活和cAMP水解磷酸二酯酶的失活。
Ca2+ and cAMP are important second messengers that regulate multiple cellular processes. Although previous studies have suggested direct interactions between Ca2+ and cAMP signaling pathways, the underlying mechanisms remain unresolved. In particular, direct evidence for Ca2+-regulated cAMP production in living cells is incomplete. Genetically encoded fluorescence resonance energy transfer-based biosensors have made possible real-time imaging of spatial and temporal gradients of intracellular cAMP concentration in single living cells. Here, we used confocal microscopy, fluorescence resonance energy transfer, and insulin-secreting MIN6 cells expressing Epac1-camps, a biosynthetic unimolecular cAMP indicator, to better understand the role of intracellular Ca2+ in cAMP production. We report that depolarization with high external K+, tolbutamide, or glucose caused a rapid increase in cAMP that was dependent on extracellular Ca2+ and inhibited by nitrendipine, a Ca2+ channel blocker, or 2', 5'-dideoxyadenosine, a P-site antagonist of transmembrane adenylate cyclases. Stimulation of MIN6 cells with glucose in the presence of tetraethylammonium chloride generated concomitant Ca2+ and cAMP oscillations that were abolished in the absence of extracellular Ca2+ and blocked by 2', 5'-dideoxyadenosine or 3- isobutyl-1-methylxanthine, an inhibitor of phosphodiesterase. Simultaneous measurements of Ca2+ and cAMP concentrations with Fura-2 and Epac1-camps, respectively, revealed a close temporal and causal interrelationship between the increases in cytoplasmic Ca2+ and cAMP levels following membrane depolarization. These findings indicate highly coordinated interplay between Ca2+ and cAMP signaling in electrically excitable endocrine cells and suggest that Ca2+-dependent cAMP oscillations are derived from an increase in adenylate cyclase activity and periodic activation and inactivation of cAMP-hydrolyzing phosphodiesterase.