Ward D T, Ohanian J, Heagerty A M, Ohanian V
Department of Medicine, University Hospital of South Manchester, U.K.
Biochem J. 1995 Apr 15;307 ( Pt 2)(Pt 2):451-6. doi: 10.1042/bj3070451.
To investigate membrane lipid metabolism during smooth-muscle activation, the role of phospholipase D (PLD) in the production of phosphatidate (PA) was studied in rat small arteries stimulated with noradrenaline. Incubation with [3H]myristate preferentially labelled phosphatidylcholine (PtdCho), and in the presence of 0.5% ethanol [3H]phosphatidylethanol ([3H]PEt) was formed, demonstrating PLD activity. Noradrenaline (NA) stimulation resulted in an increase in PtdCho derived [3H]PA and [3H]PEt formation, indicating PLD activation. Stimulation of [14C]choline release confirmed PLD-mediated hydrolysis of PtdCho. Propranolol, an inhibitor of PA phosphohydrolase, increased [3H]PA levels in non-stimulated tissue and decreased the rate of degradation of both [3H]PA and [3H]PEt, implying that this is an active route for PA metabolism in small arteries. However, [3H]diacylglycerol levels were not increased during NA stimulation. Fluoroaluminate increased [3H]PEt formation and [14C]choline release, whereas high K+ in the presence of alpha 1-adrenoceptor blockade did not. Pervanadate increased phosphotyrosine levels in small arteries, and markedly stimulated [3H]PEt formation and [14C]choline release. The combination of pervanadate and NA stimulation resulted in a dramatic increase in [3H]PEt formation, which was greater than the sum of the individual responses to the two agonists. Pervanadate and fluoroaluminate in combination appeared to give an additive response, whereas high K+ did not alter the pervanadate-induced formation of [3H]PEt. Phosphotyrosine levels were increased by NA in the presence of tyrosine phosphatase inhibitors. This effect was blocked by genistein, a tyrosine kinase inhibitor. These data demonstrate that in NA-stimulated small arteries PLD-induced PtdCho hydrolysis contributes to accumulation of PA, but not of diacylglycerol. Furthermore, regulation of PLD activity appears to require G-protein and tyrosine-phosphorylation-linked pathways.
为研究平滑肌激活过程中的膜脂代谢，我们在去甲肾上腺素刺激的大鼠小动脉中研究了磷脂酶D（PLD）在磷脂酸（PA）生成中的作用。用[3H]肉豆蔻酸盐孵育可优先标记磷脂酰胆碱（PtdCho），在0.5%乙醇存在的情况下会形成[3H]磷脂酰乙醇（[3H]PEt），这证明了PLD活性。去甲肾上腺素（NA）刺激导致源自PtdCho的[3H]PA和[3H]PEt生成增加，表明PLD被激活。对[14C]胆碱释放的刺激证实了PLD介导的PtdCho水解。PA磷酸水解酶抑制剂普萘洛尔增加了未刺激组织中的[3H]PA水平，并降低了[3H]PA和[3H]PEt的降解速率，这意味着这是小动脉中PA代谢的一条活跃途径。然而，在NA刺激期间[3H]二酰基甘油水平并未增加。氟铝酸盐增加了[3H]PEt生成和[14C]胆碱释放，而在α1 -肾上腺素能受体阻断存在的情况下高钾则没有。过氧钒酸盐增加了小动脉中的磷酸酪氨酸水平，并显著刺激了[3H]PEt生成和[14C]胆碱释放。过氧钒酸盐和NA刺激相结合导致[3H]PEt生成急剧增加，这大于对两种激动剂各自反应的总和。过氧钒酸盐和氟铝酸盐联合使用似乎产生了相加反应，而高钾并未改变过氧钒酸盐诱导的[3H]PEt生成。在酪氨酸磷酸酶抑制剂存在的情况下，NA增加了磷酸酪氨酸水平。这种效应被酪氨酸激酶抑制剂染料木黄酮阻断。这些数据表明，在NA刺激的小动脉中，PLD诱导的PtdCho水解有助于PA的积累，但无助于二酰基甘油的积累。此外，PLD活性的调节似乎需要G蛋白和酪氨酸磷酸化相关途径。
