Chakraborti Sajal, Sarkar Jaganmay, Chowdhury Animesh, Chakraborti Tapati
Department of Biochemistry and Biophysics, University of Kalyani, Kalyani 741235, West Bengal, India.
Arch Biochem Biophys. 2017 Nov 1;633:1-14. doi: 10.1016/j.abb.2017.08.012. Epub 2017 Aug 16.
Treatment of human pulmonary artery smooth muscle cells (HPASMCs) with the thromboxane A2 receptor antagonist, SQ29548 inhibited U46619 stimulation of phospholipase D (PLD) and NADPH oxidase activities in the cell membrane. Pretreatment with apocynin inhibited U46619 induced increase in NADPH oxidase activity. The cell membrane contains predominantly PLD2 along with PLD1 isoforms of PLD. Pretreatment with pharmacological and genetic inhibitors of PLD2, but not PLD1, attenuated U46619 stimulation of NADPH oxidase activity. U46619 stimulation of PLD and NADPH oxidase activities were insensitive to BFA and Clostridium botulinum C3 toxin; however, pretreatment with secinH3 inhibited U46619 induced increase in PLD and NADPH oxidase activities suggesting a major role of cytohesin in U46619-induced increase in PLD and NADPH oxidase activities. Arf-1, Arf-6, cytohesin-1 and cytohesin-2 were observed in the cytosolic fraction, but only Arf-6 and cytohesin-1 were translocated to the cell membrane upon treatment with U46619. Coimmunoprecipitation study showed association of Arf-6 with cytohesin-1 in the cell membrane fraction. In vitro binding of GTPγS with Arf-6 required the presence of cytohesin-1 and that occurs in BFA insensitive manner. Overall, BFA insensitive Arf6-cytohesin1 signaling axis plays a pivotal role in U46619-mediated activation of PLD leading to stimulation of NADPH oxidase activity in HPASMCs.
用血栓素A2受体拮抗剂SQ29548处理人肺动脉平滑肌细胞（HPASMCs），可抑制U46619对细胞膜中磷脂酶D（PLD）和NADPH氧化酶活性的刺激。阿朴吗啡预处理可抑制U46619诱导的NADPH氧化酶活性增加。细胞膜中主要含有PLD2以及PLD的PLD1亚型。用PLD2而非PLD1的药理学和基因抑制剂预处理可减弱U46619对NADPH氧化酶活性的刺激。U46619对PLD和NADPH氧化酶活性的刺激对BFA和肉毒杆菌C3毒素不敏感；然而，用secinH3预处理可抑制U46619诱导的PLD和NADPH氧化酶活性增加，提示胞膜结合蛋白在U46619诱导的PLD和NADPH氧化酶活性增加中起主要作用。在细胞溶质部分观察到了Arf-1、Arf-6、胞膜结合蛋白-1和胞膜结合蛋白-2，但在用U46619处理后，只有Arf-6和胞膜结合蛋白-1转位到细胞膜。免疫共沉淀研究显示在细胞膜部分Arf-6与胞膜结合蛋白-1存在关联。GTPγS与Arf-6的体外结合需要胞膜结合蛋白-1的存在，且以对BFA不敏感的方式发生。总体而言，对BFA不敏感的Arf6-胞膜结合蛋白1信号轴在U46619介导的PLD激活中起关键作用，从而导致HPASMCs中NADPH氧化酶活性受到刺激。
