Matthies H J, Palfrey H C, Hirning L D, Miller R J
J Neurosci. 1987 Apr;7(4):1198-206. doi: 10.1523/JNEUROSCI.07-04-01198.1987.
We investigated the effects of phorbol esters on protein kinase C (PKC) activity and on neurotransmitter release from cultured neuronal cells. Both differentiated and undifferentiated PC12 pheochromocytoma cells contained high levels of protein PKC. Under normal conditions all the enzyme activity was found in the cytoplasm. Addition of the phorbol esters phorbol 12-myristate-13-acetate (TPA) or phorbol 12,13-dibutyrate (PDBu) caused a rapid translocation of PKC from the cytoplasm to the particulate fraction. Continued culture of cells with these phorbol esters resulted in the decline of total PKC activity. After 10-20 hr of culture, both membrane and cytoplasmic PKC activity had declined to background levels. cAMP-dependent and Ca2+/calmodulin-dependent protein kinase activities were only slightly affected by chronic phorbol ester treatment. Addition of active phorbol esters to PC12 cells produced an enhancement of the depolarization-induced release of 3H-norepinephrine. Following chronic phorbol ester treatment, the ability of these substances to enhance evoked catecholamine release was lost. Furthermore, depolarizing stimuli released considerably less 3H-norepinephrine than in control untreated cells. Phorbol esters also enhanced depolarization-induced 3H-norepinephrine release from primary cultures of rat sympathetic neurons. Chronic treatment of these neurons with phorbol esters also resulted in the loss of their ability to enhance transmitter release and in a large reduction in the extent of depolarization-evoked transmitter release. Chronic phorbol ester treatment also resulted in the disappearance of PKC from sympathetic neurons, but had little effect on cAMP-dependent or Ca2+/calmodulin-dependent kinase activities. These results demonstrate that PKC-deficient neurons can be prepared. The data also demonstrate that depolarization-induced neurotransmitter release is mediated by both protein kinase C-dependent and independent pathways.
我们研究了佛波酯对蛋白激酶C（PKC）活性以及对培养的神经元细胞神经递质释放的影响。分化和未分化的PC12嗜铬细胞瘤细胞均含有高水平的蛋白PKC。在正常条件下，所有酶活性均存在于细胞质中。添加佛波酯佛波醇12-肉豆蔻酸酯-13-乙酸酯（TPA）或佛波醇12,13-二丁酸酯（PDBu）会导致PKC从细胞质快速易位至颗粒部分。用这些佛波酯持续培养细胞会导致总PKC活性下降。培养10 - 20小时后，膜和细胞质PKC活性均降至背景水平。慢性佛波酯处理对cAMP依赖性和Ca2+/钙调蛋白依赖性蛋白激酶活性仅有轻微影响。向PC12细胞中添加活性佛波酯会增强去极化诱导的3H-去甲肾上腺素释放。在慢性佛波酯处理后，这些物质增强诱发儿茶酚胺释放的能力丧失。此外，去极化刺激释放的3H-去甲肾上腺素比未处理的对照细胞少得多。佛波酯还增强了大鼠交感神经元原代培养物中去极化诱导的3H-去甲肾上腺素释放。用佛波酯对这些神经元进行慢性处理也导致它们增强递质释放的能力丧失，并且去极化诱发的递质释放程度大幅降低。慢性佛波酯处理还导致交感神经元中PKC消失，但对cAMP依赖性或Ca2+/钙调蛋白依赖性激酶活性影响很小。这些结果表明可以制备PKC缺陷的神经元。数据还表明，去极化诱导的神经递质释放由蛋白激酶C依赖性和非依赖性途径介导。
