Secretagogue-induced protein phosphorylation and chloride transport in Caco-2 cells

Abstract

The effects of vasoactive intestinal polypeptide (VIP), 16,16-dimethyl prostaglandin E2 (DMPGE2) and dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP) on protein phosphorylation were studied in relation to stimulation of chloride transport in cell suspensions of the human colon epithelial cell line Caco-2. In 36Cl-loaded cells, VIP and DMPGE2 within 1 min decreased cellular chloride content 35-40%, with half-maximal effects being elicited at 1.0 and 85 nM concentration, respectively. A similar effect on chloride content occurred after 10 min of treatment with 0.5 mM DBcAMP. For all three secretagogues, decreases in cellular chloride content were associated with increases in membrane permeability to chloride. DMPGE2 and VIP within 1 min, and DBcAMP within 10 min, increased the phosphorylation of an unidentified soluble protein of Mr = 42,000 and pI = 6.1, and of a protein of Mr = 20,200 and pI = 4.9 identified as myosin regulatory light chain. Between 10 and 30 min of stimulation, however, phosphorylation of the Mr = 42,000 protein and chloride transport activity remained elevated in DMPGE2- and DBcAMP-treated cells, whereas light chain phosphorylation returned to control level. No effect of secretagogues on phosphorylation was detected in the total particulate fraction or an integral membrane protein fraction. It is concluded that increased membrane permeability to chloride induced by cAMP-mediated secretagogues in Caco-2 is temporally associated with the increased phosphorylation of a Mr = 42,000 soluble protein.