Nieto M, Kennedy E, Goldstein D, Brown J H
Department of Pharmacology, University of California, San Diego, La Jolla 92093.
Mol Pharmacol. 1994 Sep;46(3):406-13.
Activation of the M3 muscarinic receptor in 1321N1 human astrocytoma cells leads to increased phospholipase D (PLD)-catalyzed hydrolysis of phosphatidylcholine, which is maximal within 1 min of exposure to agonist. Studies examining the kinetics of phosphatidylethanol formation indicate that there is no further PLD activation beyond this time. Thrombin, a mitogen for quiescent 1321N1 cells, also activates PLD only transiently. The PLD response does not recover for up to 1 hr and cells that have been exposed to carbachol or thrombin do not respond to subsequent challenge with the heterologous agonist. In contrast to the desensitization observed with agonists, phorbol-12-myristate-13-acetate induces a sustained stimulation of PLD. In addition, cells pretreated with carbachol or thrombin show a normal response to phorbol-12-myristate-13-acetate, suggesting that the enzymatic activity of PLD is not compromised. Guanosine-5'-O-(3-thio)triphosphate activation of PLD in cell-free homogenates is also unaffected by prior treatment of the cells with agonist. Agonist-stimulated PLD activation in 1321N1 cells is mediated by protein kinase C (PKC). Thrombin and carbachol cause comparable changes in redistribution of both PKC-alpha and PKC-epsilon. The increase in membrane-associated PKC-alpha is transient and is reinitiated by addition of the heterologous agonist, whereas PKC-epsilon remains membrane associated for at least 60 min and is not further increased by addition of the heterologous agonist. We suggest that desensitization of PLD activation results from the down-regulation of an as yet undefined mediator required for agonist receptor coupling to PLD and that PKC-epsilon might participate in this down-regulation.
在1321N1人星形细胞瘤细胞中，M3毒蕈碱受体的激活会导致磷脂酶D（PLD）催化的磷脂酰胆碱水解增加，在暴露于激动剂1分钟内达到最大值。研究磷脂酰乙醇形成动力学表明，在此时间之后不会进一步激活PLD。凝血酶是静止1321N1细胞的促有丝分裂原，也仅短暂激活PLD。PLD反应在长达1小时内都不会恢复，并且已暴露于卡巴胆碱或凝血酶的细胞对随后的异源激动剂刺激无反应。与激动剂引起的脱敏作用相反，佛波醇-12-肉豆蔻酸酯-13-乙酸酯可诱导PLD的持续刺激。此外，用卡巴胆碱或凝血酶预处理的细胞对佛波醇-12-肉豆蔻酸酯-13-乙酸酯显示出正常反应，表明PLD的酶活性未受损害。在无细胞匀浆中，鸟苷-5'-O-（3-硫代）三磷酸对PLD的激活也不受细胞预先用激动剂处理的影响。1321N1细胞中激动剂刺激的PLD激活由蛋白激酶C（PKC）介导。凝血酶和卡巴胆碱会引起PKC-α和PKC-ε重新分布的类似变化。膜相关PKC-α的增加是短暂的，通过添加异源激动剂可重新引发，而PKC-ε至少在60分钟内仍与膜相关，并且添加异源激动剂不会使其进一步增加。我们认为，PLD激活的脱敏作用是由于激动剂受体与PLD偶联所需的一种尚未明确的介质下调所致，并且PKC-ε可能参与了这种下调过程。
