Compartmentalized cAMP responses to prostaglandin EP2 receptor activation in human airway smooth muscle cells
Compartmentalized cAMP responses to prostaglandin EP2 receptor activation in human airway smooth muscle cells
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DOI：
10.1111/bph.13904
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发表时间：
2017-08-01
影响因子：
7.3
通讯作者：
Harvey, Robert D.
中科院分区：
文献类型：
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作者：
Agarwal, Shailesh R.;Miyashiro, Kathryn;Harvey, Robert D.
背景和结论以前的研究表明，前列腺素EP 2受体选择性地偶联到气道平滑肌（ASM）细胞的非脂筏结构域中的AC 2，在那里它们调节特异性cAMP依赖性反应。本研究的目的是确定EP 2受体刺激cAMP production.Experimental方法的cAMP生物传感器的细胞微域针对不同的亚细胞位置的原代人ASM细胞。Epac 2-camps生物传感器在整个细胞中表达，用于测量大量细胞质响应。Epac 2-MyrPalm和Epac 2-CAAX分别用于测量与质膜的脂筏和非筏区域相关的响应。用毛喉素激活AC或用异丙肾上腺素激活β（2）-肾上腺素能受体可增加所有亚细胞位置的cAMP。用布他前列素激活EP 2受体产生的cAMP反应最容易被非筏传感器和核传感器检测到，但仅被胞质传感器微弱检测到，并且根本不能被脂筏传感器检测到。暴露于rolipram，一种PDE 4抑制剂，揭示了EP 2受体增加与脂筏结构域相关的cAMP水平的能力。AC 2的过表达选择性地增加EP 2受体刺激的cAMP在non-raft membrane domains.CONCLUSIONS AND IMPLICATIONSEP 2受体激活AC 2导致cAMP在non-raft和人ASMs核隔室的生产，而β（2）肾上腺素受体信号被广泛检测到跨microdomains。PDE 4的活性似乎在维持这些细胞中区室化EP 2受体反应的完整性方面发挥作用。
BACKGROUND AND PURPOSEPrevious studies indicate that prostaglandin EP2 receptors selectively couple to AC2 in non-lipid raft domains of airway smooth muscle (ASM) cells, where they regulate specific cAMP-dependent responses. The goal of the present study was to identify the cellular microdomains where EP2 receptors stimulate cAMP production.EXPERIMENTAL APPROACHFRET-based cAMP biosensors were targeted to different subcellular locations of primary human ASM cells. The Epac2-camps biosensor, which expresses throughout the cell, was used to measure bulk cytoplasmic responses. Epac2-MyrPalm and Epac2-CAAX were used to measure responses associated with lipid raft and non-raft regions of the plasma membrane respectively. Epac2-NLS was used to monitor responses at the nucleus.KEY RESULTSActivation of AC with forskolin or beta(2)-adrenoceptors with isoprenaline increased cAMP in all subcellular locations. Activation of EP2 receptors with butaprost produced cAMP responses that were most readily detected by the non-raft and nuclear sensors, but only weakly detected by the cytosolic sensor and not detected at all by the lipid raft sensor. Exposure to rolipram, a PDE4 inhibitor, unmasked the ability of EP2 receptors to increase cAMP levels associated with lipid raft domains. Overexpression of AC2 selectively increased EP2 receptor-stimulated production of cAMP in non-raft membrane domains.CONCLUSIONS AND IMPLICATIONSEP2 receptor activation of AC2 leads to cAMP production in non-raft and nuclear compartments of human ASMs, while beta(2) adrenoceptor signalling is broadly detected across microdomains. The activity of PDE4 appears to play a role in maintaining the integrity of compartmentalized EP2 receptor responses in these cells.