Liu G L, Shaw L, Heagerty A M, Ohanian V, Ohanian J
Department of Medicine, Manchester Royal Infirmary, Manchester, UK.
J Vasc Res. 1999 Jan-Feb;36(1):35-46. doi: 10.1159/000025624.
A characteristic of endothelin-1 (ET-1)-induced contraction is the prolonged duration of the response. Phosphatidylcholine (PC) hydrolysis has been implicated in sustained agonist-induced effects through the formation of the second messengers phosphatidic acid (PA) and diacylglycerol (DAG). Therefore, we have investigated the activation of PC-phospholipase D (PLD) and PC-phospholipase C (PLC) in intact rat mesenteric small arteries stimulated with ET-1 and determined whether PC-derived DAG is necessary for ET-1-induced contraction. PLD activity, measured as phosphatidylethanol (PEt) production in vessels labelled with [3H] or [14C]myristate, was increased in a concentration- and time-dependent manner following ET-1 stimulation, as were [3H]PA levels, peaking at 10 min (ET-1 100 nM) and remaining above control levels for up to 20 min. Inclusion of 0.5% ethanol during ET-1 stimulation reduced [3H]PA levels, but did not alter the time course of formation. In addition, [14C]choline release was increased, confirming PLD-mediated PC hydrolysis. In contrast, DAG levels, measured by [3H]myristate labelling and mass assay, increased transiently at 5 min of ET-1 stimulation only. Analysis of the subclasses of DAG demonstrated an increase in all types of DAG without any enrichment with arachidonate-containing species, indicating PC, not inositol lipid hydrolysis was the source of DAG. The PC-PLC inhibitor D609, 2.5 microg/ml, completely abolished the ET-1-induced increase in [14C]DAG without affecting the increase in [14C]PA, PLD activity or the contractile response. In [14C]choline-labelled vessels, [14C]phosphocholine levels were increased by ET-1 with a similar time course to DAG production. Removal of extracellular Ca2+ and addition of 0.1 or 2 mM EGTA completely inhibited ET-1-stimulated PLD activity. The tyrosine kinase inhibitor tyrphostin A23 (100 microM) abolished ET-1-induced [3H]PA, [3H]PEt and [14C]DAG increases, whilst the negative analogue A1 was without effect. These data suggest that ET-1 couples via a calcium-dependent tyrosine kinase mechanism to PLD and PC-PLC in vascular smooth muscle. PC-derived DAG did not appear to be necessary for the contractile response, whereas sustained formation of PA, generated by PLD activity, implies that this lipid second messenger could be involved in the prolonged contractile response to this peptide.
内皮素 -1（ET -1）诱导的收缩的一个特点是反应持续时间延长。磷脂酰胆碱（PC）水解通过第二信使磷脂酸（PA）和二酰甘油（DAG）的形成参与激动剂诱导的持续效应。因此，我们研究了用ET -1刺激完整大鼠肠系膜小动脉时PC -磷脂酶D（PLD）和PC -磷脂酶C（PLC）的激活情况，并确定PC衍生的DAG是否是ET -1诱导收缩所必需的。用[³H]或[¹⁴C]肉豆蔻酸标记血管，以磷脂酰乙醇（PEt）生成量来衡量PLD活性，ET -1刺激后，PLD活性呈浓度和时间依赖性增加，[³H]PA水平也是如此，在10分钟时达到峰值（ET -1 100 nM），并在长达20分钟内保持高于对照水平。在ET -1刺激期间加入0.5%乙醇可降低[³H]PA水平，但不改变其形成的时间进程。此外，[¹⁴C]胆碱释放增加，证实了PLD介导的PC水解。相比之下，通过[³H]肉豆蔻酸标记和质量测定法测得的DAG水平仅在ET -1刺激5分钟时短暂增加。对DAG亚类的分析表明，所有类型的DAG均增加，且不含花生四烯酸的物种没有任何富集，这表明DAG的来源是PC而非肌醇脂质水解。PC - PLC抑制剂D609（2.5 μg/ml）完全消除了ET -1诱导的[¹⁴C]DAG增加，而不影响[¹⁴C]PA增加、PLD活性或收缩反应。在[¹⁴C]胆碱标记的血管中，ET -1使[¹⁴C]磷酸胆碱水平升高，其时间进程与DAG产生相似。去除细胞外Ca²⁺并加入0.1或2 mM EGTA可完全抑制ET -1刺激的PLD活性。酪氨酸激酶抑制剂 tyrphostin A23（100 μM）消除了ET -1诱导的[³H]PA、[³H]PEt和[¹⁴C]DAG增加，而阴性类似物A1则无此作用。这些数据表明，ET -1通过钙依赖性酪氨酸激酶机制与血管平滑肌中的PLD和PC - PLC偶联。PC衍生的DAG似乎不是收缩反应所必需的，而PLD活性产生的PA的持续形成意味着这种脂质第二信使可能参与了对该肽的延长收缩反应。
