Activation of phospholipase D in porcine tracheal smooth muscle: role of phosphatidylinositol 3-kinase and RhoA activation.
Activation of phospholipase D in porcine tracheal smooth muscle: role of phosphatidylinositol 3-kinase and RhoA activation.
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DOI：
10.1016/s0014-2999(01)01439-x
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发表时间：
2001-12
影响因子：
5
通讯作者：
A. Mamoon;Rodney C. Baker;Jerry M. Farley
中科院分区：
文献类型：
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作者：
A. Mamoon;Rodney C. Baker;Jerry M. Farley
毒蕈碱受体激动剂瞬时激活气管平滑肌中的磷脂酶D。该组织中磷脂酶D的毒蕈碱激活依赖于蛋白激酶C的激活和不依赖于蛋白激酶C的未鉴定途径。胆碱能药物也被证明通过与小G蛋白RhoA相关的途径激活磷脂酶D。本研究探讨了毒蕈碱激活磷脂酰肌醇3-激酶和激活RhoA之间的关系，并检查磷脂酶D激活是否依赖于气管平滑肌中的任一途径。Wortmannin或2-（4-morphonyl）-8-phenyl-4 H-1-benzopyran-4-one（LY-294002）是磷脂酰肌醇3-激酶的特异性抑制剂，可显著抑制乙酰胆碱诱导的磷脂酰乙醇的形成，并阻断乙酰胆碱诱导的RhoA向细胞膜的转位。在先前的实验中，蛋白激酶C抑制剂calphostin C部分抑制乙酰胆碱诱导的和佛波醇-12-肉豆蔻酸酯-13-乙酸酯（PMA）诱导的磷脂酰乙醇形成。在本研究中，calphostin C没有阻止乙酰胆碱诱导的RhoA易位到膜。然而，Rho激酶抑制剂N-（4-吡啶基）-4-（1-氨乙基）-环己烷甲酰胺（Y-27632），显着抑制乙酰胆碱诱导的磷脂酰乙醇的形成，但没有影响激活磷脂酶D的PMA。乙酰胆碱治疗也刺激磷脂酰肌醇3-激酶的110 kDa亚基的磷酸化。磷脂酰肌醇3-激酶110-kDa亚基的磷酸化可被wortmannin以浓度依赖性方式阻断，乙酰胆碱诱导的磷脂酰肌醇3-激酶活性被wortmannin显著抑制。LY-294002还抑制乙酰胆碱诱导的110-kDa亚基磷酸化和磷脂酰肌醇3-激酶的活化。这些结果表明，乙酰胆碱刺激易位RhoA的膜磷脂酰肌醇3-激酶依赖的机制和乙酰胆碱诱导的磷脂酶D刺激至少部分介导的磷脂酰肌醇3-激酶，然而，蛋白激酶C似乎激活磷脂酶D独立的磷脂酰肌醇3-激酶或RhoA激活猪气管平滑肌。
Muscarinic receptor agonists transiently activate phospholipase D in tracheal smooth muscle. Muscarinic activation of phospholipase D in this tissue is dependent on activation of protein kinase C and an unidentified pathway that is not protein kinase C dependent. Cholinergic agents have also been shown to activate phospholipase D by pathways linked to the small G protein, RhoA. This study explores the relationship between muscarinic activation of phophatidylinositol 3-kinase and activation of RhoA, and examines whether phospholipase D activation is dependent on either pathway in tracheal smooth muscle. Wortmannin or 2-(4-morphonyl)-8-phenyl-4H-1-benzopyran-4-one (LY-294002), putative specific inhibitors of phophatidylinositol 3-kinase, significantly inhibit acetylcholine-induced formation of phosphatidylethanol and also block acetylcholine-induced translocation of RhoA to the membrane. In previous experiments calphostin C, a protein kinase C inhibitor, partially inhibited both acetylcholine-induced and phorbol-12-myristate-13-acetate (PMA)-induced phosphatidylethanol formation. In the present study calphostin C did not block acetylcholine-induced RhoA translocation to the membrane. However, the Rho kinase inhibitor, N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide (Y-27632), significantly inhibited acetylcholine-induced phosphatidylethanol formation, but had no effect on activation of phospholipase D by PMA. Acetylcholine treatment also stimulated the phosphorylation of the 110-kDa subunit of phosphatidylinositol 3-kinase. Phosphorylation of phosphatidylinositol 3-kinase 110-kDa subunit could be blocked by wortmannin in a concentration-dependent manner, and acetylcholine-induced phosphatidylinositol 3-kinase activity was significantly inhibited by wortmannin. LY-294002 also inhibited acetylcholine-induced phosphorylation of 110-kDa subunit and activation of phosphatidylinositol 3-kinase. These results suggest that acetylcholine stimulation translocates RhoA to the membrane by a phosphatidylinositol 3-kinase-dependent mechanism and acetylcholine-induced phospholipase D stimulation is at least partly mediated via phosphatidylinositol 3-kinase, however, protein kinase C appears to activate phospholipase D independent of phosphatidylinositol 3-kinase or RhoA activation in porcine tracheal smooth muscle.