Natarajan V, Garcia J G
Department of Internal Medicine, Indiana University School of Medicine, Indianapolis.
J Lab Clin Med. 1993 Feb;121(2):337-47.
Regulation of phospholipase D (PLD) activity was investigated in cultured monolayers of bovine pulmonary artery endothelial cells (BPAECs). Agonists such as bradykinin, histamine, vasopressin, alpha-thrombin, and adenosine triphosphate (ATP) stimulated up to 15-fold accumulation of phosphatidylethanol (PEt) in the presence of ethanol through PLD-catalyzed phosphatidyltransferase activity. To examine mechanisms of PLD regulation, we investigated the role of protein kinase C (PKC) and Ca2+ fluxes in agonist-induced PLD activation. The PKC activator 12-O-tetradecanoylphorbol-13-acetate (TPA, 100 nmol/L) produced up to a 25-fold increase in PEt formation in a time- and dose-dependent manner. PEt production was also stimulated by other cell-permeant PKC activators such as 1,2 dioctanoylglycerol and 1-oleyl-2-acetylglycerol, whereas inactive phorbol derivatives 4-alpha-phorbol-12,13-didecanoate and 4-beta-phorbol showed no effect. The effect of TPA on PEt accumulation was inhibited by the PKC inhibitors staurosporine (5 mumol/L, 95% inhibition) and sphingosine (10 mumol/L, 50% inhibition). TPA-induced PEt accumulation was almost completely abolished (> 95% inhibition) by PKC down-regulation accomplished by long-term treatment with 100 nmol/L TPA. In contrast, bradykinin- or ATP-induced phosphorus 32-labeled PA and [32P]-labeled PEt formation was only partially blocked (70% inhibition) by either staurosporine (10 mumol/L) or PKC down-regulation, suggesting that part of agonist-stimulated PLD activity may occur in the absence of PKC activation. An increase in Cai2+ appears to be involved in agonist-induced PLD activation as bradykinin-, ATP-, or Ca2+ ionophore-induced [32P]. PEt production was attenuated by either depletion of extra-cellular Ca2+ with EGTA or chelation of intracellular Ca2+ by BAPTA. TPA-mediated PEt accumulation was not affected by EGTA treatment, whereas BAPTA reduced TPA-mediated PEt formation by 50%. These results suggest that direct PKC activation is a potent stimulus for PLD activity and that the major pathway for agonist-induced PLD activation involves PKC activation and is dependent on an increase in intracellular Ca2+. Further, these studies suggest that agonist-induced PLD activation may also involve a PKC-independent mechanism.
在牛肺动脉内皮细胞（BPAECs）的培养单层中研究了磷脂酶D（PLD）活性的调节。诸如缓激肽、组胺、血管加压素、α-凝血酶和三磷酸腺苷（ATP）等激动剂在乙醇存在下通过PLD催化的磷脂转移酶活性刺激磷脂酰乙醇（PEt）积累高达15倍。为了研究PLD调节的机制，我们研究了蛋白激酶C（PKC）和Ca2+通量在激动剂诱导的PLD激活中的作用。PKC激活剂12-O-十四烷酰佛波醇-13-乙酸酯（TPA，100 nmol/L）以时间和剂量依赖性方式使PEt形成增加高达25倍。其他细胞可渗透的PKC激活剂如1,2-二辛酰甘油和1-油酰-2-乙酰甘油也刺激了PEt的产生，而无活性的佛波醇衍生物4-α-佛波醇-12,13-二癸酸酯和4-β-佛波醇则无作用。TPA对PEt积累的作用被PKC抑制剂星形孢菌素（5 μmol/L，95%抑制）和鞘氨醇（10 μmol/L，50%抑制）抑制。通过用100 nmol/L TPA长期处理实现的PKC下调几乎完全消除了TPA诱导PEt积累（>95%抑制）。相反，缓激肽或ATP诱导的32P标记的磷脂酸（PA）和[³²P]标记的PEt形成仅被星形孢菌素（10 μmol/L）或PKC下调部分阻断（70%抑制），这表明部分激动剂刺激的PLD活性可能在没有PKC激活的情况下发生。细胞内Ca²⁺的增加似乎参与了激动剂诱导的PLD激活，因为缓激肽、ATP或Ca²⁺离子载体诱导的[³²P]。PEt产生被用EGTA耗尽细胞外Ca²⁺或用BAPTA螯合细胞内Ca²⁺所减弱。EGTA处理不影响TPA介导的PEt积累，而BAPTA使TPA介导的PEt形成减少50%。这些结果表明直接PKC激活是PLD活性的有效刺激，并且激动剂诱导的PLD激活的主要途径涉及PKC激活并且依赖于细胞内Ca²⁺的增加。此外，这些研究表明激动剂诱导的PLD激活也可能涉及一种不依赖PKC的机制。
