The human colonic carcinoma cell line HT-29cl.19A responds to the protein kinase C activator PDB (4-beta-phorbol 12,13-dibutyrate), as it does to forskolin (an activator of adenylyl cyclase), with a secretory response when the cells are grown on filters and studied at 36 degrees C. Previously, we showed that when cells were grown on Petri dishes and studied at about 25 degrees C with the cell-attached patch-clamp technique, forskolin, but not PDB, could activate 8-pS chloride channels (cystic fibrosis transmembrane conductance regulator, CFTR, channels). The present work was carried out to study this discrepancy. Experiments in Ussing chambers, at different temperatures, showed that the responses to PDB and forskolin differ in their temperature sensitivity. This was also found following conventional microelectrode and Ussing chamber studies with nystatin-permeabilized epithelial layers carried out at 25 degrees C and at 36 degrees C. Pre-incubation with the microtubular disruptive agents nocodazole or colcemid did not affect the response to PDB or forskolin, suggesting that chloride secretion induced by these agonists in these cells is independent of the microtubular structure. Pre-incubation with brefeldin A strongly inhibited the response to PDB, but the response to forskolin was hardly affected. The differing effect of temperature and brefeldin A on the responses to forskolin and PDB may be due to the activation of two distinct mechanisms by protein kinases A and C.
