Spatiotemporal dynamics of β-adrenergic cAMP signals and L-type Ca2+ channel regulation in adult rat ventricular myocytes -: Role of Phosphodiesterases
Spatiotemporal dynamics of β-adrenergic cAMP signals and L-type Ca2+ channel regulation in adult rat ventricular myocytes -: Role of Phosphodiesterases
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DOI：
10.1161/circresaha.107.167817
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发表时间：
2008-05-09
影响因子：
20.1
通讯作者：
Vandecasteele, Gregoire
中科院分区：
文献类型：
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作者：
Leroy, Jerome;Abi-Gerges, Aniella;Vandecasteele, Gregoire
心脏β-肾上腺素能受体的稳态激活导致环核苷酸磷酸二酯酶(PDE)的局部活性导致cAMP在细胞内的分区。为了评价不同脑室cAMP变化的时程，对成年大鼠心肌细胞施加短暂(15秒)的异丙肾上腺素(100nmol/L)脉冲，同时使用工程环核苷酸门控通道和基于荧光共振能量转移的传感器Epac2-cAMP监测细胞膜下和胞浆内cAMP的变化。比较两个脑区cAMP动力学与L钙通道电流(I-Ca，I-L)幅值的时程变化。CAMP瞬变在质膜上的开始和恢复分别比在胞浆中快30%和50%，这与第二信使在质膜上的快速产生和降解以及cAMP在胞浆中的有限扩散相一致。ICa、L在膜上的幅度增加比cAMP慢两倍，cAMP恢复到基础水平后，电流持续升高约5min，表明cAMP的变化在通道磷酸化/去磷酸化过程中不是限速的。阻断PDE4(10 mU/L Ro20-1724)使cAMP信号的幅度增加，cAMP信号的起始和恢复明显减慢，而阻断PDE3(1 mU/L西洛胺)仅对肌膜下cAMP无明显影响。但当PDE_3和PDE_4均被抑制时，或用3-异丁基-1-甲基黄嘌呤(300 mU/L)阻断所有PDE时，cAMP信号和ICa均呈下降趋势，且下降的时间常数为>；蛋白激酶抑制剂对cAMP依赖的蛋白激酶的抑制作用与PDE4对胞浆cAMP瞬变的部分抑制作用相似。在短暂(15秒)暴露于异丙肾上腺素的ARVM上，cAMP-PDE分析显示总PDE活性显著(高达约50%)呈剂量依赖性增加，这主要归因于PDE4的激活。这些结果揭示了ARVM中不同的β-肾上腺素能受体cAMP区段，并为PDE3和PDE4的复杂作用提供了新的线索。
Steady-state activation of cardiac beta-adrenergic receptors leads to an intracellular compartmentation of cAMP resulting from localized cyclic nucleotide phosphodiesterase (PDE) activity. To evaluate the time course of the cAMP changes in the different compartments, brief (15 seconds) pulses of isoprenaline (100 nmol/L) were applied to adult rat ventricular myocytes (ARVMs) while monitoring cAMP changes beneath the membrane using engineered cyclic nucleotide-gated channels and within the cytosol with the fluorescence resonance energy transfer-based sensor, Epac2-camps. cAMP kinetics in the two compartments were compared to the time course of the L-type Ca2+ channel current (I-Ca,I-L) amplitude. The onset and recovery of cAMP transients were, respectively, 30% and 50% faster at the plasma membrane than in the cytosol, in agreement with a rapid production and degradation of the second messenger at the plasma membrane and a restricted diffusion of cAMP to the cytosol. ICa, L amplitude increased twice slower than cAMP at the membrane, and the current remained elevated for approximate to 5 minutes after cAMP had already returned to basal level, indicating that cAMP changes are not rate-limiting in channel phosphorylation/dephosphorylation. Inhibition of PDE4 (with 10 mu mol/L Ro 20-1724) increased the amplitude and dramatically slowed down the onset and recovery of cAMP signals, whereas PDE3 blockade (with 1 mu mol/L cilostamide) had a minor effect only on subsarcolemmal cAMP. However, when both PDE3 and PDE4 were inhibited, or when all PDEs were blocked using 3-isobutyl-1-methylxanthine (300 mu mol/L), cAMP signals and ICa, L declined with a time constant >10 minutes. cAMP-dependent protein kinase inhibition with protein kinase inhibitor produced a similar effect as a partial inhibition of PDE4 on the cytosolic cAMP transient. Consistently, cAMP-PDE assay on ARVMs briefly (15 seconds) exposed to isoprenaline showed a pronounced (up to approximate to 50%) dose-dependent increase in total PDE activity, which was mainly attributable to activation of PDE4. These results reveal temporally distinct beta-adrenergic receptor cAMP compartments in ARVMs and shed new light on the intricate roles of PDE3 and PDE4.