Lee H M, Fain J N
Department of Biochemistry, University of Tennessee, Memphis.
J Neurochem. 1991 May;56(5):1471-80. doi: 10.1111/j.1471-4159.1991.tb02040.x.
We examined the effect of phorbol esters on phospholipase C activation in rat brain cortical slices and membranes. There was little effect of concurrent addition of phorbol 12-myristate 13-acetate (PMA) with carbachol on phosphoinositide breakdown due to carbachol over a 1-h incubation of brain slices. However, if slices were preincubated for 3 h with 1 microM PMA or 200 microM sphingosine before addition of carbachol, there was a 35-50% inhibition of phosphoinositide breakdown. There was also a marked loss of protein kinase C (PKC) activity from both cytosol and membranes after a 3-h exposure to PMA. The loss in responsiveness to the muscarinic agonists in slices was not reflected in carbachol-stimulated phospholipase C activation using isolated membranes. However, the decrease in carbachol-induced phosphoinositide breakdown seen in slices after a 3-h exposure to PMA was abolished if the extracellular K+ concentration was elevated from 5.9 to 55mM. Because elevation of the K+ level induces depolarization and increases Ca2+ entry, we examined the effect of ionomycin, a Ca2+ ionophore. Ionomycin potentiated the effects of carbachol on phosphoinositide breakdown but was unable to reverse the effects of a 3-h incubation with PMA. Because apamin, an inhibitor of Ca2(+)-dependent K+ channels, mimicked the effects of exposure to PMA for 3 h, it is possible that these channels are involved in muscarinic cholinergic regulation of phosphoinositide breakdown in rat brain slices. These results support the hypothesis that prolonged PMA treatment in rat brain cortex has no direct effect on phospholipase C activation by muscarinic cholinergic stimulation.
我们研究了佛波酯对大鼠脑皮质切片和膜中磷脂酶C激活的影响。在脑切片1小时的孵育过程中，佛波醇12 -肉豆蔻酸酯13 -乙酸酯（PMA）与卡巴胆碱同时添加对卡巴胆碱引起的磷酸肌醇分解几乎没有影响。然而，如果在添加卡巴胆碱之前，将切片用1μM PMA或200μM鞘氨醇预孵育3小时，磷酸肌醇分解会受到35 - 50%的抑制。在暴露于PMA 3小时后，胞质溶胶和膜中的蛋白激酶C（PKC）活性也显著丧失。切片中对毒蕈碱激动剂反应性的丧失在使用分离膜的卡巴胆碱刺激的磷脂酶C激活中并未体现。然而，如果细胞外K⁺浓度从5.9mM升高到55mM，在暴露于PMA 3小时后切片中卡巴胆碱诱导的磷酸肌醇分解减少的现象就会被消除。由于K⁺水平升高会诱导去极化并增加Ca²⁺内流，我们研究了离子霉素（一种Ca²⁺离子载体）的作用。离子霉素增强了卡巴胆碱对磷酸肌醇分解的作用，但无法逆转与PMA孵育3小时的影响。因为钙依赖性钾通道抑制剂蜂毒明肽模拟了暴露于PMA 3小时的作用，所以这些通道可能参与了大鼠脑切片中磷酸肌醇分解的毒蕈碱胆碱能调节。这些结果支持了这样的假设，即大鼠脑皮质中长时间的PMA处理对毒蕈碱胆碱能刺激引起的磷脂酶C激活没有直接影响。
