Singh A K, Taskén K, Walker W, Frizzell R A, Watkins S C, Bridges R J, Bradbury N A
Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, USA.
Am J Physiol. 1998 Aug;275(2):C562-70. doi: 10.1152/ajpcell.1998.275.2.C562.
Chloride exit across the apical membranes of secretory epithelial cells is acutely regulated by the cAMP-mediated second messenger cascade. To better understand the regulation of transepithelial chloride secretion, we have characterized the complement of cAMP-dependent protein kinase (PKA) isoforms present in the human colonic epithelial cell line T84. Our results show that both type I and type II PKA are present in T84 cells. Immunoprecipitation of 8-azido-[32P]cAMP-labeled cell lysates revealed that the major regulatory subunits of PKA were RIalpha and RIIalpha. In addition, immunogold electron microscopy showed that RIIalpha labeling was found on membranes of the trans Golgi network and on apical plasma membrane. In contrast, RIalpha was randomly distributed throughout the cytoplasm, with no discernible membrane association. Northern blot analysis of T84 RNA revealed that Calpha was the predominantly expressed catalytic subunit. Short-circuit current measurements were performed in the presence of combinations of site-selective cAMP analog pairs to preferentially activate either PKA type I or PKA type II in intact T84 cell monolayers. Maximal levels of chloride secretion (approximately 100 microA/cm2) were observed for both type I and type II PKA-selective analog pairs. Subsequent addition of forskolin was unable to further increase chloride secretion. Thus activation of either type I or type II PKA is able to maximally stimulate chloride secretion in T84 colonic epithelial cells.
氯离子通过分泌性上皮细胞的顶端膜排出受到cAMP介导的第二信使级联反应的急性调节。为了更好地理解跨上皮氯离子分泌的调节机制，我们对人结肠上皮细胞系T84中存在的cAMP依赖性蛋白激酶（PKA）同工型进行了表征。我们的结果表明，I型和II型PKA都存在于T84细胞中。对8-叠氮基-[32P]cAMP标记的细胞裂解物进行免疫沉淀显示，PKA的主要调节亚基是RIα和RIIα。此外，免疫金电子显微镜显示，在反式高尔基体网络的膜和顶端质膜上发现了RIIα标记。相比之下，RIα随机分布在整个细胞质中，没有明显的膜结合。对T84 RNA进行Northern印迹分析表明，Cα是主要表达的催化亚基。在完整的T84细胞单层中，使用位点选择性cAMP类似物对的组合进行短路电流测量，以优先激活I型或II型PKA。I型和II型PKA选择性类似物对都观察到了最大水平的氯离子分泌（约100 μA/cm2）。随后添加福斯可林无法进一步增加氯离子分泌。因此，激活I型或II型PKA都能够最大程度地刺激T84结肠上皮细胞中的氯离子分泌。
