Sena C M, Rosário L M, Parker P J, Patel V, Boarder M R
Department of Biochemistry, Faculty of Sciences and Technology, University of Coimbra, Portugal.
J Neurochem. 1996 Mar;66(3):1086-94. doi: 10.1046/j.1471-4159.1996.66031086.x.
In this report we investigate the isoforms of protein kinase C (PKC) present in cultured adrenal chromaffin cells with respect to their modulation by treatment with phorbol ester and their possible differential involvement in the regulation of responses to histamine and bradykinin. The presence of individual isoforms of PKC was investigated by using eight isoform specific antisera, as a result of which PKC-alpha, epsilon, and zeta were identified. To characterize down-regulation of these enzymes, cells were incubated for 6-48 h with 1 microM phorbol myristate acetate (PMA). PKC-epsilon down-regulated more rapidly than PKC-alpha. At 12 h, PMA pretreatment, for example, PKC-epsilon was maximally down-regulated (23 +/- 4% of controls), whereas PKC-alpha was unchanged. PKC-alpha showed partial down-regulation by 24 h of PMA pretreatment. PKC-zeta did not down-regulate at any of the times tested. Translocation from cytosol to membrane in response to PMA was also more rapid for PKC-epsilon than for PKC-alpha. The accumulation of total 3H-inositol (poly) phosphates in response to bradykinin or histamine was essentially abolished by prior treatment with 10-min PMA treatment (1 microM). However, with 12-h exposure to PMA, the bradykinin response was restored to the level seen with no prior PMA exposure. The histamine response showed no recovery by 12 h of PMA, but showed partial recovery by 24 h of PMA pretreatment. These observations showed that the restoration of the response to bradykinin corresponds to the loss of PKC-epsilon, whereas the restoration of the histamine response corresponds to the loss of PKC-alpha. This picture was confirmed with further studies on cytosolic Ca2+. The results show that chromaffin cells exhibit an unusual pattern of down-regulation of PKC isoforms on prolonged exposure to PMA, and that there is a differential effect of exposure to PMA on the histamine and bradykinin responses, suggesting that different PLC-linked receptors in chromafin cells are differentially regulated by PKC isoforms.
在本报告中，我们研究了培养的肾上腺嗜铬细胞中存在的蛋白激酶C（PKC）同工型，涉及它们受佛波酯处理的调节情况以及它们可能在调节对组胺和缓激肽反应中的不同作用。通过使用8种同工型特异性抗血清研究了PKC各同工型的存在情况，结果鉴定出了PKC-α、ε和ζ。为了表征这些酶的下调情况，将细胞与1 μM佛波醇肉豆蔻酸酯乙酸酯（PMA）孵育6 - 48小时。PKC-ε的下调比PKC-α更快。例如，在12小时时，PMA预处理后，PKC-ε最大程度地下调（为对照的23±4%），而PKC-α没有变化。PKC-α在PMA预处理24小时时显示出部分下调。PKC-ζ在任何测试时间都没有下调。PKC-ε对PMA的反应从胞质溶胶向膜的转位也比PKC-α更快。用1 μM PMA处理10分钟预处理基本消除了缓激肽或组胺刺激引起的总3H-肌醇（多）磷酸的积累。然而，在暴露于PMA 12小时后，缓激肽反应恢复到未预先暴露于PMA时的水平。组胺反应在PMA处理12小时时没有恢复，但在PMA预处理24小时时显示出部分恢复。这些观察结果表明，对缓激肽反应的恢复与PKC-ε的丧失相对应，而组胺反应的恢复与PKC-α的丧失相对应。通过对胞质Ca2+的进一步研究证实了这一情况。结果表明，嗜铬细胞在长时间暴露于PMA时表现出PKC同工型下调的异常模式，并且暴露于PMA对组胺和缓激肽反应有不同的影响，表明嗜铬细胞中不同的PLC连接受体受PKC同工型的调节存在差异。
