Characterization of PKA isoforms and kinase-dependent activation of chloride secretion in T84 cells.

摘要

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.