Phorbol ester sequentially downregulates cAMP-regulated basolateral and apical Cl- transport pathways in T84 cells
Matthews, J.B.; Awtrey, C.S.; Hecht, G.; Tally, K.J.; Thompson, R.S.; Madara, J.L.
American Journal of Physiology 265(4 Pt 1): C1109-C1117
1993
ISSN/ISBN: 0002-9513 PMID: 8238301 Document Number: 405817
The effect of phorbol esters on adenosine 3',5'-cyclic monophosphate (cAMP)-regulated epithelial Cl- transport was studied in T84 cells, a human colonic cell line that serves as a model for electrogenic Cl- secretion. Preincubation of T84 cell monolayers with phorbol 12-myristate 13-acetate (PMA) caused a time- and dose-dependent inhibition of the net transepithelial secretory response to 10 mu-M forskolin (half-maximal inhibition at a concentration of apprx 10 nM PMA and a time of 45 min). Similar inhibition was observed with phorbol 12,13-dibutyrate but not the inactive phorbol ester phorbol 12,13-diacetate. Na+K+-2Cl- cotransporter activity, assessed by bumetanide-sensitive 86Rb+ uptake, and K+ conductance, assessed by 86Rb+ efflux, were both found to be markedly reduced by PMA with a time course that paralleled the loss of the cAMP-regulated Cl-secretory response. One- and four-hour treatment of T84 cells with 100 nM PMA caused a sustained increase in the membrane-bound fraction of protein kinase C (PKC) but a decrease in total cellular PKC. Although, at these time points, the Na+K+-2Cl- cotransporter and K+ efflux pathways were markedly inhibited (associated with inhibition of the forskolin-stimulated transepithelial Cl- secretory response), the activity of the cAMP-regulated Cl- efflux pathway, assessed by 125I-labeled efflux, remained unaffected. With prolonged exposure to PMA (up to 10 h), the cAMP-regulated Cl- efflux pathway was also eventually inhibited, and transepithelial electrical resistance progressively declined. We conclude that treatment of T84 cell monolayers with PMA results in a progressive inhibition of cAMP-stimulated electrogenic Cl- secretion, due to effects on two basolateral transport elements (Na+-K+-2Cl- cotransporter and K+ conductance). Such data emphasize the potential for regulation of basolateral ion transport processes separately from regulation of apical Cl- efflux pathways, as well as the potential for electrogenic Cl- secretion to be regulated at basolateral sites.