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， 总是伴随着膜受体的激活。 最近发现 蛋白激酶C（Protein Kinase C，PKC）是一种独特的磷酸化特异性 蛋白质，其功能仍有待确定。 PKC需要一个 磷脂和二酰基甘油（DAG）的混合物以获得最大活性。 DAG是 膜聚磷酸肌醇分解水解产物， 在膜受体占据之后，并且是细胞内的初始事件之一。 信号转导 在PI周转期间释放DAG， 刺激PKC活性超过在静止细胞中观察到的。 已知 辅助致癌物或肿瘤促进剂TPA特异性增加膜PKC 活动 与这种增加的膜PKC活性相关的是 胞浆PKC活性的降低，这表明 PKC从胞浆到细胞膜是一个重要的细胞内事件。 使用AtT-20细胞，该细胞对多种膜和细胞内 通过释放POMC衍生肽的激活剂，我们已经探索了 PKC通过监测β-受体的释放参与激素分泌 内啡肽（BE），以及膜和胞质PKC活性。 TPA 治疗导致BE分泌的剂量相关性增加， PKC从胞质到膜组分的易位。 一 TPA最大刺激浓度增强膜PKC活性 2-AtT-20细胞的预孵育 用乙醇（0.1-0.6%）处理24小时，导致剂量相关的2-5倍 胞浆PKC活性降低，膜PKC活性降低50% 在0.4%乙醇中的活性。 仅乙醇预处理24小时 轻微降低了最大刺激浓度的能力， TPA诱导PKC转位。

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.