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

Abstract

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 (ISC-cAMP) generation by colonocyte monolayers. 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 ISC generation. DOPPA failed to elicit similar responses. Whereas chronic (24 h) exposure to both agents inhibited FSK-stimulated transcellular and apical membrane ISC-cAMP, the effects of DOPPA were more complex: this conventional PKC-beta-specific agonist also stimulated Ba2+-sensitive basolateral membrane-dependent facilitation of transcellular ISC-cAMP. PMA did not elicit a similar phenomenon. Prolonged exposure to high-dose PMA but not DOPPA led to apical membrane ISC-cAMP recovery. Changes in PKC alpha-, beta1-, 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 ISC-cAMP, increased PKC-beta1, decreased PKC-beta2 membrane association, and caused reciprocal changes in isoform mass. During the apical membrane ISC-cAMP recovery after prolonged high-dose PMA exposure, an almost-complete depletion of cellular PKC-beta1 and a significant reduction in PKC-epsilon mass occurred. Thus activated PKC-beta1 and/or PKC-epsilon prevented, whereas activated PKC-alpha facilitated, apical membrane ISC-cAMP. PKC-beta-dependent augmentation of transcellular ISC-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.