ACTIVATION OF ION-TRANSPORT BY COMBINED EFFECTS OF IONOMYCIN, FORSKOLIN AND PHORBOL ESTER ON CULTURED HT-29CL.19A HUMAN COLONOCYTES

The differentiated clone 19A of the HT-29 human colon carcinoma cell l ine was used as a model to study the intracellular electrophysiologica l effects of interaction of the cAMP, the protein kinase C (PKC) and t he Ca2+ pathways. (a) A synergistic effect between ionomycin and forsk olin was observed. From intracellular responses it was concluded that the synergistic effect is caused by activation of an apical Cl- conduc tance by protein kinase A and a basolateral K+ conductance by Ca2+. (b ) A transient synergistic effect of ionomycin and the phorbol ester ph orbol dibutyrate (PDB) was found. The decrease of the response appeare d to be due to PKC-dependent inactivation of the basolateral K+ conduc tance. The synergism is caused by PKC-dependent increase of the apical Cl- conductance and Ca2+-dependent increase of the basolateral K+ con ductance. (c) The effects of carbachol and PDB were not fully additive presumably because of their convergence on PKC activation. (d) Forsko lin and PDB, when added in this order, had a less than additive effect . Results of cell-attached patch-clamp studies, presented in the accom panying paper, showed a synergistic effect of forskolin and PDB on non -rectifying small-conductance Cl- channels. Assuming that these channe ls are involved in the transepithelial responses it is suggested that forskolin and PDB induce a modulatory, synergistic increase of the api cal Cl- conductance when both pathways are activated simultaneously. ( e) The HT-29cl.19A cells differ from T-84 cells in that the latter did not respond with an increase of the short-circuit current to addition of phorbol ester. This may be due to a very low expression of PKC alp ha.