Liu Juan, Liu Huixia, Li Yanjing, Xu Xiaojian, Chen Zhengju, Liu Limei, Yu Xiaoxing, Gao Yuansheng, Dou Dou
*Department of Physiology and Pathophysiology, Peking University Health Science Center, Beijing, China; †Department of Physiology, Heze Medical College, Heze, China; and ‡Key Laboratory of Molecular Cardiovascular Science, Ministry of Education, Peking University, Beijing, China.
J Cardiovasc Pharmacol. 2014 Nov;64(5):452-9. doi: 10.1097/FJC.0000000000000137.
cGMP-dependent protein kinase (PKG) plays a crucial role in vasodilatation induced by cGMP-elevating agents. Akt has been demonstrated to be involved in modulating vasoreactivity. The present study was to determine the interaction between PKG and Akt and their influences on nitric oxide (NO)-induced vasodilatation. Isolated fourth-generation porcine pulmonary arteries were dissected from the lung and cut into rings in ice-cold modified Krebs-Ringer bicarbonate buffer. The relaxant responses of vessels were determined by organ chamber technique, cGMP was assayed by using enzyme-linked immunosorbent assay kit, the protein levels of phosphorylated Akt were examined by Western blotting, and the activity of phosphodiesterase type 5 (PDE5) was assayed by measuring the rate of cGMP degradation. Incubation with DETA NONOate (a stable NO donor) and 8-Br-cGMP (a cell membrane permeable analog of cGMP) attenuated Akt phosphorylation at Ser-473, which was prevented by Rp-8-Br-PET-cGMPS (a specific inhibitor of PKG) and calyculin A (an inhibitor of protein phosphatase 1 and 2A) but not by okadaic acid (a selective inhibitor of protein phosphatase 2A). Inhibition of Akt enhanced the relaxation and cGMP elevation of porcine pulmonary arteries induced by DETA NONOate or sodium nitroprusside, which was prevented by zaprinast, a specific inhibitor of PDE5. Incubation with LY294002 or Akt inhibitor reduced PDE5 activity in porcine pulmonary arteries. The present study indicates that PKG may attenuate Akt phosphorylation through protein phosphatase 1, which leads to an augmented cGMP elevation by inhibition of PDE5. The increased cGMP in turn activates PKG. Such a positive feedback may play an important role in NO-induced pulmonary vasodilatation.
环磷酸鸟苷（cGMP）依赖性蛋白激酶（PKG）在由升高cGMP的药物诱导的血管舒张中起关键作用。已证明Akt参与调节血管反应性。本研究旨在确定PKG与Akt之间的相互作用及其对一氧化氮（NO）诱导的血管舒张的影响。从猪肺中分离出第四代肺动脉，并在冰冷的改良Krebs-Ringer碳酸氢盐缓冲液中切成环。通过器官浴槽技术测定血管的舒张反应，使用酶联免疫吸附测定试剂盒测定cGMP，通过蛋白质印迹法检测磷酸化Akt的蛋白质水平，并通过测量cGMP降解速率来测定5型磷酸二酯酶（PDE5）的活性。用DETA NONOate（一种稳定的NO供体）和8-溴-cGMP（一种细胞膜可渗透的cGMP类似物）孵育可减弱Ser-473处的Akt磷酸化，这可被Rp-8-溴-PET-cGMPS（PKG的特异性抑制剂）和花萼海绵诱癌素A（一种蛋白磷酸酶1和2A的抑制剂）阻止，但不能被冈田酸（一种蛋白磷酸酶2A的选择性抑制剂）阻止。抑制Akt可增强DETA NONOate或硝普钠诱导的猪肺动脉舒张和cGMP升高，这可被PDE5的特异性抑制剂扎普司特阻止。用LY294002或Akt抑制剂孵育可降低猪肺动脉中的PDE5活性。本研究表明，PKG可能通过蛋白磷酸酶1减弱Akt磷酸化，这通过抑制PDE5导致cGMP升高增加。增加的cGMP反过来激活PKG。这种正反馈可能在NO诱导的肺血管舒张中起重要作用。
