Chung H C, Fleming N
Department of Oral Biology, University of Manitoba, Winnipeg, Canada.
Pflugers Arch. 1995 Dec;431(2):161-8. doi: 10.1007/BF00410187.
The characteristics of muscarinic cholinergic-induced phospholipase D (PLD) activation, and the involvement of the enzyme in the release of arachidonic acid were examined in rat submandibular acinar cells. Carbachol produced a dose-related activation of PLD to around fivefold control values at 100 microM agonist concentration. This was associated with the appearance of free choline, phosphatidic acid and arachidonic acid, indicating that the PLD substrate was phosphatidylcholine. The response to carbachol was inhibited by 60% by U73122, a blocker of a phospholipase C (PLC) specific to phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], suggesting that the cleavage of phosphatidylcholine by PLD was, at least in part, secondary to agonist-coupled hydrolysis of PtdIns(4,5)P2 by PLC. Consistent with this, PLD was also activated to levels comparable to those induced by carbachol, by the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), and the Ca2+ mobilizer, thapsigargin, two agents that respectively mimic the activation of protein kinase C (PKC) by diacylglycerol and the elevation of cytosolic Ca2+ by inositol 1,4,5-triphosphate [Ins(1,4,5)P3] in the phosphoinositide effect. The cell-permeant Ca2+ chelator 1,2-bis-(O-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, tetraacetoxymethyl ester (BAPTA/AM) abolished the thapsigargin-induced activation of PLD and inhibited the responses of PLD to carbachol and TPA by 60%. The PKC inhibitor, Ro-31-8220, also inhibited the activation of PLD by carbacol and TPA to a level of approximately double control values, but had no effect on the thapsigargin-induced elevation of PLD. A role for both the PKC-associated and Ca(2+)-mobilizing arms of the PtdIns(4,5)P2-PLC pathway in PLD regulation is thus suggested. Pretreatment of cells with the phosphatidate phosphohydrolase blocker, propranolol, significantly enhanced the carbachol-induced elevation of phosphatidic acid, but decreased agonist-stimulated production of diacylglycerol and arachidonic acid, indicating that phosphatidlycholine was the likely source of arachidonic acid. We therefore propose that, in submandibular mucous acinar cells, muscarinic activation of the PtdIns(4,5)P2-PLC pathway regulates phosphatidylcholine-specific PLD through both the PKC- and Ca(2+)-mobilizing arms of the phosphoinositide response, and that diacylglycerol, derived from phosphatidylcholine via phosphatidic acid, is a source of free arachidonic acid.
在大鼠下颌下腺腺泡细胞中，研究了毒蕈碱胆碱能诱导的磷脂酶D（PLD）激活的特征以及该酶在花生四烯酸释放中的作用。在100μM激动剂浓度下，卡巴胆碱使PLD产生剂量相关的激活，达到对照值的约五倍。这与游离胆碱、磷脂酸和花生四烯酸的出现相关，表明PLD的底物是磷脂酰胆碱。U73122（一种特异性作用于磷脂酰肌醇4,5-二磷酸[PtdIns(4,5)P2]的磷脂酶C（PLC）阻滞剂）可使对卡巴胆碱的反应抑制60%，这表明PLD对磷脂酰胆碱的裂解至少部分是由于激动剂偶联的PLC对PtdIns(4,5)P2的水解所致。与此一致的是，佛波酯12-O-十四酰佛波醇-13-乙酸酯（TPA）和Ca2+动员剂毒胡萝卜素也可将PLD激活至与卡巴胆碱诱导的水平相当，这两种试剂分别模拟了磷脂酰肌醇效应中由二酰基甘油激活蛋白激酶C（PKC）以及由肌醇1,4,5-三磷酸[Ins(1,4,5)P3]升高胞质Ca2+的过程。细胞可渗透的Ca2+螯合剂1,2-双-(O-氨基苯氧基)-乙烷-N,N,N',N'-四乙酸四乙酰氧甲酯（BAPTA/AM）消除了毒胡萝卜素诱导的PLD激活，并使PLD对卡巴胆碱和TPA的反应抑制60%。PKC抑制剂Ro-31-8220也将卡巴胆碱和TPA诱导的PLD激活抑制至约对照值的两倍，但对毒胡萝卜素诱导的PLD升高没有影响。因此提示PtdIns(4,5)P2-PLC途径中与PKC相关和Ca(2+)动员的分支在PLD调节中均起作用。用磷脂酸磷酸水解酶阻滞剂普萘洛尔预处理细胞可显著增强卡巴胆碱诱导的磷脂酸升高，但降低激动剂刺激的二酰基甘油和花生四烯酸的产生，表明磷脂酰胆碱可能是花生四烯酸的来源。因此我们提出，在下颌下黏液腺泡细胞中，PtdIns(4,5)P2-PLC途径的毒蕈碱激活通过磷脂酰肌醇反应中与PKC相关和Ca(2+)动员的分支调节磷脂酰胆碱特异性PLD，并且经由磷脂酸从磷脂酰胆碱衍生而来的二酰基甘油是游离花生四烯酸的一个来源。
