PKC AND THE SECRETION AND BIOSYNTHESIS OF NEUROPEPTIDES IN ATT-20 CELLS

蛋白磷酸化增强是细胞内事件之一，它 总是伴随着膜受体的激活。最近确定的 蛋白激酶C(PKC)是一种独特的酶，它能磷酸化特定的 蛋白质，其功能尚待确定。PKC需要 磷脂和二酰甘油(DAG)具有最大的活性。DAG是 膜多聚磷脂酰肌醇的降解产物，发生 在膜受体被占据之后，这是 信号转导。PI周转期间DAG的释放大幅增加 刺激PKC活性高于在静止细胞中观察到的活性。已知的 共致癌物或促癌剂TPA特异性地增加细胞膜PKC 活动。与这种膜PKC活性增加相关的是一种 胞浆PKC活性降低，提示细胞易位 PKC从胞浆到胞膜是一个重要的细胞内事件。 使用对各种膜和细胞内有反应的ATT-20细胞 通过释放POMC衍生的多肽，我们探索了 PKC通过监测β释放参与激素分泌 内啡肽(BE)，以及膜和胞质PKC活性。双酚A 治疗导致BE分泌与剂量相关的增加和 PKC从胞浆向膜部分的移位。一个 最大刺激浓度TPA增强膜PKC活性 1分钟内2倍，3分钟内5倍。AtT-20细胞的预培养 使用乙醇(0.1-0.6%)24小时导致与剂量相关的2-5倍 胞质PKC活性降低，膜PKC活性降低50% 乙醇浓度为0.4%时的活性。仅限24小时的乙醇预处理 略微降低了最大刺激浓度的能力 TPA诱导PKC易位。

Enhanced protein phosphorylation is one of the intracellular events which invariably follows membrane receptor activation. Recently identified Protein Kinase C (PKC) is a unique enzyme which phosphorylates specific proteins, whose functions remain to be determined. PKC requires a phospholipid and a diacylglycerol (DAG) for maximal activity. DAG is the hydrolytic product of membrane polyphosphoinositide breakdown, which occurs following membrane receptor occupancy, and is one of the initial events in signal transduction. The release of DAG during PI turnover substantially stimulates PKC activity over that observed in quiescent cells. The known co-carcinogen or tumor promoter, TPA, specifically increases membrane PKC activity. Associated with this increased membrane PKC activity is a reduction of cytosolic PKC activity, which suggests that translocation of PKC from the cytosol to the membrane is an important intracellular event. Using AtT-20 cells that respond to a variety of membrane and intracellular activators by releasing POMC-derived peptides, we have explored the involvement of PKC in hormone secretion by monitoring the release of beta endorphin (BE), as well as membrane and cytosolic PKC activity. TPA treatment resulted in a dose-related increase in BE secretion and translocation of PKC from the cytosolic to the membrane fraction. A maximally-stimulating concentration of TPA enhanced membrane PKC activity 2-fold within 1 min and 5-fold within 3 min. Preincubation of AtT-20 cells with ethanol (0.1-0.6%) for 24 hours resulted in a dose-related 2-5 fold reduction in cytosolic PKC activity and a 50% reduction in membrane PKC activity at 0.4% ethanol. Ethanol pretreatment for 24 hours only marginally reduced the ability of a maximal stimulatory concentration of TPA to induce translocation of PKC.