Regulation of cGMP-specific phosphodiesterase (PDE5) phosphorylation in smooth muscle cells
Regulation of cGMP-specific phosphodiesterase (PDE5) phosphorylation in smooth muscle cells
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DOI：
10.1074/jbc.m106562200
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发表时间：
2002-02-01
影响因子：
4.8
通讯作者：
Beavo, JA
中科院分区：
文献类型：
--
作者：
Rybalkin, SD;Rybalkina, IG;Beavo, JA
一氧化氮和内源性硝基血管舒张剂通过升高 cGMP 和激活环 GMP 依赖性蛋白激酶 (PKG) 来调节平滑肌张力。平滑肌中 cGMP 信号的幅度和持续时间在很大程度上由 cGMP 特异性环核苷酸磷酸二酯酶 (PDE5) 调节。先前的体外数据表明cAMP依赖性蛋白激酶和PKG都可以调节PDE5的活性。为了测试这种类型的调节在完整细胞中是否重要，我们产生了磷酸化 PDE5 特异性抗血清，并利用了来自 PKG I 基因破坏的小鼠的分离平滑肌细胞以及来自正常人平滑肌的细胞。数据显示，在人平滑肌细胞中，8-Br-cGMP 激活 PKG 导致 PDE5 磷酸化和激活。在相同的细胞中，8-Br-cAMP对PDE5磷酸化没有显着影响。用 8-Br-cGMP 处理野生型小鼠主动脉平滑肌细胞也诱导 PDE5 磷酸化，而在从 PKG I 基因已被破坏的小鼠中分离的平滑肌细胞中未观察到磷酸化。与人类细胞一样，在小鼠细胞中未观察到 8-Br-cAMP 引起的磷酸化。这些结果强烈表明，控制完整平滑肌中 PDE5 磷酸化和活性的主要调节途径是通过 PDE5 的 PKG 依赖性磷酸化。最后，花萼蛋白 A 和冈田酸的实验表明，肌球蛋白磷酸酶的催化亚基 PP1 磷酸酶可以调节 PDE5 去磷酸化。总之，数据表明 PKG I 对 PDE5 的磷酸化和激活以及随后肌球蛋白磷酸酶的去磷酸化可能是调节平滑肌松弛/收缩周期的关键步骤。
Nitric oxide and endogenous nitrovasodilators regulate smooth muscle tone by elevation of cGMP and activation of cyclic GMP-dependent protein kinase (PKG). The amplitude and duration of the cGMP signal in smooth muscle is regulated in large part by cGMP-specific cyclic nucleotide phosphodiesterase (PDE5). Previous in vitro data have suggested that both cAMP-dependent protein kinase and PKG can regulate the activity of PDE5. To test if this type of regulation is important in the intact cell, we have generated phospho-PDE5-specific antisera and have utilized isolated smooth muscle cells from mice having a disruption in the PKG I gene as well as cells from normal human smooth muscle. The data show that in human smooth muscle cells, activation of PKG by 8-Br-cGMP led to phosphorylation and activation of PDE5. In the same cells, 8-Br-cAMP had no significant effect on PDE5 phosphorylation. Treatment of wild-type mouse aortic smooth muscle cells with 8-Br-cGMP also induced the phosphorylation of PDE5, whereas no phosphorylation was seen in smooth muscle cells isolated from mice in which the gene for PKG I had been disrupted. As with the human cells, no phosphorylation was seen in the mouse cells in response to 8-Br-cAMP. These results strongly suggest that a major regulatory pathway for control of PDE5 phosphorylation and activity in intact smooth muscle is via PKG-dependent phosphorylation of PDE5. Finally, experiments with calyculin A and okadaic acid suggest that PP1 phosphatase, the catalytic subunit of myosin phosphatase, can regulate PDE5 dephosphorylation. Together, the data suggest that phosphorylation and activation of PDE5 by PKG I and its subsequent dephosphorylation by myosin phosphatase may be key steps in the regulation of relaxation/contraction cycles of smooth muscle.