Liles W C, Hunter D D, Meier K E, Nathanson N M
J Biol Chem. 1986 Apr 25;261(12):5307-13.
The tumor-promoting phorbol ester 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA), which activates protein kinase C, acted synergistically with A23187 to decrease muscarinic acetylcholine receptor (mAChR) number in neuroblastoma cells (clone N1E-115) as determined by a filter binding assay using [3H]quinuclidinyl benzilate in membrane homogenates. After a 6-h incubation, 10(-7) M PMA and 3 X 10(-7) M A23187 reduced mAChR number 30-40%, compared to the 40-50% reduction observed after treatment with 10(-3) M carbachol, a muscarinic agonist. Incubation with 3 X 10(-7) M A23187 and 10(-7) M 4 alpha-phorbol 12,13-didecanoate, an inactive phorbol ester, did not alter mAChR number. The addition of PMA and A23187 to cultures incubated with 10(-3) M carbachol caused only a modest 6% further reduction in mAChR number as compared to incubation with carbachol alone. The kinetics of the decrease in mAChR number produced by PMA/A23187 were similar to those seen after carbachol treatment. Recovery of mAChR number after treatment with either carbachol or PMA/A23187 was blocked by treatment with the protein synthesis inhibitor cycloheximide. Intact cell binding studies employing [3H]N-methylscopolamine showed that treatment with either PMA/A23187 or carbachol caused a rapid (within 15 min) loss of receptors from the cell surface prior to the decrease in total mAChR number. PMA (10(-7) M), but not 4 alpha-phorbol 12,13-didecanoate, promoted the translocation of protein kinase C activity from the cytosol to the membrane. Incubation with carbachol increased membrane-associated protein kinase C activity within 5 min with an EC50 of 3 X 10(-6) M. This increase persisted for at least 60 min in the continued presence of carbachol and was blocked by simultaneous incubation with atropine. These results suggest that activation of protein kinase C may be involved in the regulation of mAChR number in response to agonist.
可激活蛋白激酶C的促肿瘤佛波酯4β-佛波醇12β-肉豆蔻酸酯13α-乙酸酯（PMA）与A23187协同作用，可降低神经母细胞瘤细胞（克隆N1E-115）中的毒蕈碱型乙酰胆碱受体（mAChR）数量，这是通过在膜匀浆中使用[3H]喹核醇基苯甲酸酯进行滤膜结合试验测定的。孵育6小时后，10^(-7)M PMA和3×10^(-7)M A23187使mAChR数量减少30 - 40%，而用10^(-3)M毒蕈碱激动剂卡巴胆碱处理后减少40 - 50%。用3×10^(-7)M A23187和10^(-7)M 4α-佛波醇12,13 - 十二烷酸酯（一种无活性的佛波酯）孵育，不会改变mAChR数量。将PMA和A23187添加到用10^(-3)M卡巴胆碱孵育的培养物中，与单独用卡巴胆碱孵育相比，仅使mAChR数量进一步适度减少6%。PMA/A23187导致的mAChR数量减少的动力学与卡巴胆碱处理后相似。用蛋白合成抑制剂环己酰亚胺处理可阻断用卡巴胆碱或PMA/A23187处理后mAChR数量的恢复。采用[3H]N - 甲基东莨菪碱的完整细胞结合研究表明，用PMA/A23187或卡巴胆碱处理后，在总mAChR数量减少之前，细胞表面的受体迅速（15分钟内）丢失。10^(-7)M PMA可促进蛋白激酶C活性从胞质溶胶向膜的转位，但4α-佛波醇12,13 - 十二烷酸酯则不能。用卡巴胆碱孵育5分钟内可增加膜相关蛋白激酶C活性，EC50为3×10^(-6)M。在持续存在卡巴胆碱的情况下，这种增加至少持续60分钟，并且与阿托品同时孵育可阻断这种增加。这些结果表明，蛋白激酶C的激活可能参与了对激动剂应答时mAChR数量的调节。
