Chronic PKC-β activation in HT-29 Cl.19a colonocytes prevents cAMP-mediated ion secretion by inhibiting apical membrane current generation

Chronic PKC-beta activation in HT-29 Cl.19a colonocytes prevents cAMP-mediated ion secretion by inhibiting apical membrane current generation. Am J Physiol Gastrointest Liver Physiol 291: G318-G330, 2006. First published March 30, 2006; doi:10.1152/ajpgi.00355.2005. - We investigated the effects of PKC-stimulating 12-deoxyphorbol 13-phenylacetate 20-acetate (DOPPA) and phorbol 12-myristate 13-acetate (PMA) phorbol esters on cAMP-dependent, forskolin (FSK)-stimulated, short-circuit Cl- current (I-SC-cAMP) generation by colonocyte mono-layers. These agonists elicited different actions depending on their dose and incubation time; PMA effects at the onset (< 5 min) were independent of cAMP agonist and were characterized by transient anion-dependent transcellular and apical membrane I-SC generation. DOPPA failed to elicit similar responses. Whereas chronic ( 24 h) exposure to both agents inhibited FSK-stimulated transcellular and apical membrane I-SC-cAMP, the effects of DOPPA were more complex: this conventional PKC-beta-specific agonist also stimulated Ba2+-sensitive basolateral membrane-dependent facilitation of transcellular I-SC-cAMP. PMA did not elicit a similar phenomenon. Prolonged exposure to high-dose PMA but not DOPPA led to apical membrane I-SC-cAMP recovery. Changes in PKC alpha-, beta(1)-, gamma-, and epsilon-isoform membrane partitioning and expression correlated with these findings. PMA-induced transcellular ISC correlated with PKC-alpha membrane association, whereas low doses of both agents inhibited transcellular and apical membrane I-SC-cAMP, increased PKC-beta(1), decreased PKC-beta(2) membrane association, and caused reciprocal changes in isoform mass. During the apical membrane I-SC-cAMP recovery after prolonged high-dose PMA exposure, an almost-complete depletion of cellular PKC-beta(1) and a significant reduction in PKC-beta(2) mass occurred. Thus activated PKC-beta(1) and/or PKC-beta(2) prevented, whereas activated PKC-alpha facilitated, apical membrane I-SC-cAMP. PKC-beta-dependent augmentation of transcellular I-SC-cAMP at the level of the basolateral membrane demonstrated that transport events with geographically distinct subcellular membranes can be independently regulated by the PKC beta-isoform.