Lindeman R P, Chase H S
Department of Medicine, Columbia University, New York, New York 10032.
Am J Physiol. 1992 Jul;263(1 Pt 1):C140-6. doi: 10.1152/ajpcell.1992.263.1.C140.
We investigated the role of protein kinase C (PKC) in mediating carbachol's stimulation of transepithelial Cl- secretion in T84 cells. Direct PKC activation with phorbol 12-myristate 13-acetate (PMA) stimulated transepithelial Cl- transport (measured as the short-circuit current), demonstrating that PKC could interact with the secretory apparatus. Carbachol stimulated PKC activity, suggesting that the enzyme might participate in the hormone's action. Diacylglycerol metabolism inhibitors (DMIs), known to augment hormone-stimulated increases in diacylglycerol levels, potentiated the short-circuit current response to carbachol. The effect of DMIs was not due to amplification of carbachol-induced increases in PKC activity, however; PKC activity during carbachol stimulation was no higher in the presence of DMIs than in their absence. Augmentation of carbachol's action by DMIs appeared to be due to the direct activation of PKC which, like PMA, stimulated the Cl- conductance of the apical membrane (GCl). The effects of DMIs and carbachol on GCl were additive. Carbachol itself stimulated GCl but not by activating PKC; staurosporine did not blunt the effect of carbachol on GCl. Nor did staurosporine reduce the effect of carbachol on transepithelial Cl- secretion. These observations demonstrate that PKC does not participate in the secretory action of carbachol in T84 cells and suggest that direct PKC activation with DMIs and PMA stimulates an apical pool of PKC that is not accessible to carbachol applied to the basolateral membrane.
我们研究了蛋白激酶C（PKC）在介导卡巴胆碱刺激T84细胞跨上皮Cl⁻分泌中的作用。用佛波酯12 - 肉豆蔻酸酯13 - 乙酸酯（PMA）直接激活PKC可刺激跨上皮Cl⁻转运（以短路电流衡量），表明PKC可与分泌装置相互作用。卡巴胆碱刺激PKC活性，提示该酶可能参与激素的作用。已知可增强激素刺激的二酰甘油水平升高的二酰甘油代谢抑制剂（DMIs），增强了对卡巴胆碱的短路电流反应。然而，DMIs的作用并非由于卡巴胆碱诱导的PKC活性增加的放大；在存在DMIs的情况下，卡巴胆碱刺激期间的PKC活性并不比不存在时更高。DMIs增强卡巴胆碱的作用似乎是由于PKC的直接激活，与PMA一样，刺激顶端膜的Cl⁻电导（GCl）。DMIs和卡巴胆碱对GCl的作用是相加的。卡巴胆碱本身刺激GCl，但不是通过激活PKC；星形孢菌素不会减弱卡巴胆碱对GCl的作用。星形孢菌素也不会降低卡巴胆碱对跨上皮Cl⁻分泌的作用。这些观察结果表明，PKC不参与卡巴胆碱在T84细胞中的分泌作用，并提示用DMIs和PMA直接激活PKC会刺激一个顶端的PKC池，而应用于基底外侧膜的卡巴胆碱无法作用于该池。
