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首页> 外文期刊>American Journal of Physiology >Cellular mechanisms underlying prostaglandin-induced transient cAMP signals near the plasma membrane of HEK-293 cells.
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Cellular mechanisms underlying prostaglandin-induced transient cAMP signals near the plasma membrane of HEK-293 cells.

机译：HEK-293细胞质膜附近的前列腺素诱导的瞬时cAMP信号的细胞机制。

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We have previously used cyclic nucleotide-gated (CNG) channels as sensors to measure cAMP signals in human embryonic kidney (HEK)-293 cells. We found that prostaglandin E(1) (PGE(1)) triggered transient increases in cAMP concentration near the plasma membrane, whereas total cAMP levels rose to a steady plateau over the same time course. In addition, we presented evidence that the decline in the near-membrane cAMP levels was due primarily to a PGE(1)-induced stimulation of phosphodiesterase (PDE) activity, and that the differences between near-membrane and total cAMP levels were largely due to diffusional barriers and differential PDE activity. Here, we examine the mechanisms regulating transient, near-membrane cAMP signals. We observed that 5-min stimulation of HEK-293 cells with prostaglandins triggered a two- to threefold increase in PDE4 activity. Extracellular application of H89 (a PKA inhibitor) inhibited stimulation of PDE4 activity. Similarly, when we used CNG channels to monitor cAMP signals wefound that both extracellular and intracellular (via the whole-cell patch pipette) application of H89, or the highly selective PKA inhibitor, PKI, prevented the decline in prostaglandin-induced responses. Following pretreatment with rolipram (a PDE4 inhibitor), H89 had little or no effect on near-membrane or total cAMP levels. Furthermore, disrupting the subcellular localization of PKA with the A-kinase anchoring protein (AKAP) disruptor Ht31 prevented the decline in the transient response. Based on these data we developed a plausible kinetic model that describes prostaglandin-induced cAMP signals. This model has allowed us to quantitatively demonstrate the importance of PKA-mediated stimulation of PDE4 activity in shaping near-membrane cAMP signals.
机译：我们以前使用环状核苷酸门控（CNG）通道作为传感器来测量人胚胎肾脏（HEK）-293细胞中的cAMP信号。我们发现前列腺素E（1）（PGE（1））触发了质膜附近cAMP浓度的瞬时增加，而总cAMP水平在同一时间过程中上升到稳定的水平。此外，我们提供的证据表明，近膜cAMP水平的下降主要归因于PGE（1）诱导的磷酸二酯酶（PDE）活性的刺激，并且近膜和总cAMP水平之间的差异在很大程度上是由于扩散障碍和不同的PDE活性。在这里，我们研究了调节瞬态，近膜cAMP信号的机制。我们观察到用前列腺素刺激HEK-293细胞5分钟会触发PDE4活性增加2至3倍。 H89（一种PKA抑制剂）的细胞外应用抑制了PDE4活性的刺激。同样，当我们使用CNG通道监测cAMP信号时，我们发现H89或高选择性PKA抑制剂PKI在细胞外和细胞内（通过全细胞膜移液器）的应用均能防止前列腺素诱导的反应下降。用咯利普兰（PDE4抑制剂）预处理后，H89对近膜或总cAMP水平影响很小或没有影响。此外，用A激酶锚定蛋白（AKAP）破坏者Ht31破坏PKA的亚细胞定位可防止瞬时反应的下降。基于这些数据，我们开发了描述前列腺素诱导的cAMP信号的合理动力学模型。该模型使我们能够定量证明PKA介导的PDE4活性刺激在塑造近膜cAMP信号中的重要性。

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