Boyano-Adánez M C, Lundqvist C, Larsson C, Gustavsson L
Department of Medical Neurochemistry, Institute of Laboratory Medicine, Lund University Hospital, Sweden.
Neuropharmacology. 1997 Mar;36(3):295-304. doi: 10.1016/s0028-3908(96)00178-5.
The cholinergic regulation of phospholipase D activity was studied in SH-SY5Y human neuroblastoma cells with phosphatidylethanol formation as a specific marker for the enzyme activity. The muscarinic antagonists, hexahydrosiladifenidol and pirenzepine, inhibited carbachol-induced phosphatidylethanol formation in a concentration-dependent manner and the inhibitory constants indicated that muscarinic M1 receptors are responsible for the major part of the phospholipase D activation. The mechanism of receptor-mediated phospholipase D activation varies between different cell types and receptors. In SH-SY5Y cells, the carbachol-induced phospholipase D activity was inhibited by protein kinase C inhibitors. Since both phospholipases D and C are activated by muscarinic stimulation in SH-SY5Y cells, most of the phospholipase D activation is probably secondary to the protein kinase C activation that follows phospholipase C-mediated increase in diacylglycerols. Other kinases may be involved in the regulation since also a tyrosine kinase inhibitor decreased the phosphatidylethanol formation. Stimulation of G-protein(s) and increase in the intracellular Ca2+ concentration activated phospholipase D and may be additional mechanisms for the muscarinic regulation of phospholipase D in SH-SY5Y cells. Propranolol, an inhibitor of phosphatidic acid phosphohydrolase, increased the carbachol-induced formation of phosphatidic acid at the expense of 1,2-diacylglycerol. This indicates that phospholipase D contributes to the formation of 1,2-diacylglycerol after carbachol stimulation in SH-SY5Y cells.
以磷脂酰乙醇的形成作为磷脂酶D活性的特异性标志物，在SH-SY5Y人神经母细胞瘤细胞中研究了磷脂酶D活性的胆碱能调节。毒蕈碱拮抗剂六氢硅二苯酯和哌仑西平以浓度依赖性方式抑制卡巴胆碱诱导的磷脂酰乙醇形成，抑制常数表明毒蕈碱M1受体是磷脂酶D激活的主要原因。受体介导的磷脂酶D激活机制在不同细胞类型和受体之间有所不同。在SH-SY5Y细胞中，卡巴胆碱诱导的磷脂酶D活性被蛋白激酶C抑制剂抑制。由于磷脂酶D和C在SH-SY5Y细胞中均由毒蕈碱刺激激活，因此大部分磷脂酶D激活可能继发于磷脂酶C介导的二酰基甘油增加后蛋白激酶C的激活。其他激酶可能也参与调节，因为酪氨酸激酶抑制剂也降低了磷脂酰乙醇的形成。G蛋白的刺激和细胞内Ca2+浓度的增加激活了磷脂酶D，这可能是SH-SY5Y细胞中毒蕈碱调节磷脂酶D的额外机制。磷脂酸磷酸水解酶抑制剂普萘洛尔以1,2-二酰基甘油为代价增加了卡巴胆碱诱导的磷脂酸形成。这表明在SH-SY5Y细胞中卡巴胆碱刺激后磷脂酶D有助于1,2-二酰基甘油的形成。
