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MUCOSAL BIOLOGY

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

¹Department of Integrative Biology and Department of Pharmacology and Physiology, and ²Division of Gastroenterology, Hepatology, and Nutrition, Department of Internal Medicine, University of Texas Health Science Center Medical School, Houston, Texas

Submitted 28 July 2005 ; accepted in final form 16 March 2006

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 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 I_SCgeneration. 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--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 -, ₁-, -, and -isoform membrane partitioning and expression correlated with these findings. PMA-induced transcellular I_SC correlated with PKC- membrane association, whereas low doses of both agents inhibited transcellular and apical membrane I_SC-cAMP, increased PKC-₁, decreased PKC-₂membrane 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-₁ and a significant reduction in PKC- mass occurred. Thus activated PKC-₁ and/or PKC- prevented, whereas activated PKC- facilitated, apical membrane I_SC-cAMP. PKC--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 -isoform.

protein kinase C; short-circuit current; cystic fibrosis transmembrance conductance regulator; phorbol ester

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