Phosphorylation of TRPC6 Channels at Thr69 Is Required for Anti-hypertrophic Effects of Phosphodiesterase 5 Inhibition
Phosphorylation of TRPC6 Channels at Thr69 Is Required for Anti-hypertrophic Effects of Phosphodiesterase 5 Inhibition
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DOI：
10.1074/jbc.m109.074104
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发表时间：
2010-04-23
影响因子：
4.8
通讯作者：
Kurose, Hitoshi
中科院分区：
文献类型：
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作者：
Nishida, Motohiro;Watanabe, Kenta;Kurose, Hitoshi
由受体刺激和机械应力诱导的Ca 2+信号激活在心肌肥厚的发展中起关键作用。典型的瞬时受体电位蛋白亚家族成员TRPC 6，其被二酰基甘油和机械拉伸激活，作为Ca 2+信号通路的上游调节剂起作用。尽管蛋白激酶G（PKG）的激活分别抑制TRPC 6通道活性和心肌肥大，但PKG是否通过抑制TRPC 6抑制心肌肥大尚不清楚。在这里，我们表明，cGMP选择性PDE 5（磷酸二酯酶5）的抑制抑制内皮素-1-，甘油二酯类似物，和机械牵张诱导的肥大，通过抑制钙离子流入大鼠新生心肌细胞。抑制PDE 5抑制激动剂或机械拉伸诱导的Ca 2+尖峰频率的增加。然而，PDE 5抑制并不抑制由高KCl或蛋白激酶C的活化诱导的肥大反应，表明PDE 5抑制抑制Ca 2+内流本身或Ca 2+内流上游的分子。PDE 5激活的PKG抑制TRPC 6蛋白在Thr（69）的磷酸化，并阻止TRPC 6介导的Ca 2+内流。用Ala取代TRPC 6中的Thr（69）消除了PDE 5抑制的抗肥大作用。此外，通过口服西地那非治疗的慢性PDE 5抑制实际上诱导了小鼠心脏中的TRPC 6磷酸化。RGS 2（G蛋白信号传导调节因子2）和RGS 4（两者均被PKG激活以减少G α（q）介导的信号传导）的敲低不影响PDE 5抑制对受体激活的Ca 2+内流的抑制。这些结果表明TRPC 6的磷酸化和功能抑制是通过PDE 5抑制预防病理性肥大的基础。
Activation of Ca2+ signaling induced by receptor stimulation and mechanical stress plays a critical role in the development of cardiac hypertrophy. A canonical transient receptor potential protein subfamily member, TRPC6, which is activated by diacylglycerol and mechanical stretch, works as an upstream regulator of the Ca2+ signaling pathway. Although activation of protein kinase G (PKG) inhibits TRPC6 channel activity and cardiac hypertrophy, respectively, it is unclear whether PKG suppresses cardiac hypertrophy through inhibition of TRPC6. Here, we show that inhibition of cGMP-selective PDE5 (phosphodiesterase 5) suppresses endothelin-1-,diacylglycerol analog-, and mechanical stretch-induced hypertrophy through inhibition of Ca2+ influx in rat neonatal cardiomyocytes. Inhibition of PDE5 suppressed the increase in frequency of Ca2+ spikes induced by agonists or mechanical stretch. However, PDE5 inhibition did not suppress the hypertrophic responses induced by high KCl or the activation of protein kinase C, suggesting that PDE5 inhibition suppresses Ca2+ influx itself or molecule(s) upstream of Ca2+ influx. PKG activated by PDE5 inhibition phosphorylated TRPC6 proteins at Thr(69) and prevented TRPC6-mediated Ca2+ influx. Substitution of Ala for Thr(69) in TRPC6 abolished the anti-hypertrophic effects of PDE5 inhibition. In addition, chronic PDE5 inhibition by oral sildenafil treatment actually induced TRPC6 phosphorylation in mouse hearts. Knockdown of RGS2 (regulator of G protein signaling 2) and RGS4, both of which are activated by PKG to reduce G alpha(q)-mediated signaling, did not affect the suppression of receptor-activated Ca2+ influx by PDE5 inhibition. These results suggest that phosphorylation and functional suppression of TRPC6 underlie prevention of pathological hypertrophy by PDE5 inhibition.