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Real-time monitoring of phosphodiesterase inhibition in intact cells

机译：实时监测完整细胞中磷酸二酯酶的抑制作用

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Phosphodiesterases (PDEs) are hydrolytic enzymes, which convert cyclic AMP (cAMP) and cyclic GMP (cGMP) into their corresponding monophosphates. PDE-dependent hydrolysis shape gradients of these second messengers in cells, which may form the basis of their compartmentation and play a key role in a vast number of physiological and pathological processes. Here, we present a novel approach for real-time monitoring of local cAMP and cGMP levels associated with particular PDEs. We used HEK 293 cells expressing genetic constructs encoding a PDE of interest (PDE3A, PDE4A1 or PDE5A) fused to cAMP and cGMP sensors, which allow to directly visualize changes in cyclic nucleotide concentrations in the vicinity of PDE molecules by fluorescence resonance energy transfer (FRET). FRET was detected by imaging of single cells on 96-well plates and demonstrated specific effects of FIDE inhibitors on local cyclic nucleotide levels. In addition, this approach reported physiological regulation of PDE3A activity, its activation by PKA-dependent phosphorylation and inhibition by cGMP. In conclusion, our assay provides a unique and highly sensitive method to analyze FIDE activity in living cells. It allows to sense cAMP gradients around particular FIDE molecules and to study the pharmacological effects of selective inhibitors on localized cAMP signalling. (c) 2008 Elsevier Inc. All rights reserved.
机译：磷酸二酯酶（PDE）是水解酶，可将环状AMP（cAMP）和环状GMP（cGMP）转化为其相应的单磷酸盐。这些第二信使在细胞中的PDE依赖性水解形状梯度可能形成其分隔的基础，并在众多生理和病理过程中发挥关键作用。在这里，我们提出了一种实时监视与特定PDE相关的本地cAMP和cGMP水平的新颖方法。我们使用了HEK 293细胞来表达编码与PAMP和cGMP传感器融合的目标PDE（PDE3A，PDE4A1或PDE5A）的遗传构建体，从而可以通过荧光共振能量转移（FRET）直接可视化PDE分子附近的环状核苷酸浓度变化）。通过在96孔板上成像单个细胞来检测FRET，并证明FIDE抑制剂对局部环状核苷酸水平具有特定作用。此外，该方法报道了PDE3A活性的生理调节，PKA依赖的磷酸化激活和cGMP抑制。总之，我们的测定法提供了一种独特且高度灵敏的方法来分析活细胞中的FIDE活性。它可以检测特定FIDE分子周围的cAMP梯度，并研究选择性抑制剂对局部cAMP信号传导的药理作用。 （c）2008 Elsevier Inc.保留所有权利。

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