AJP - Cell Physiology, Vol 265, Issue 4 C1109-C1117, Copyright © 1993 by American Physiological Society

ARTICLES

Phorbol ester sequentially downregulates cAMP-regulated basolateral and apical Cl- transport pathways in T84 cells

J. B. Matthews, C. S. Awtrey, G. Hecht, K. J. Tally, R. S. Thompson and J. L. Madara
Department of Surgery, Beth Israel Hospital, Boston, Massachusetts.

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 microM forskolin (half-maximal inhibition at a concentration of approximately 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), the cAMP-regulated Cl- efflux pathway was also eventually inhibited, and transepithelial electrical resistance progressively declined.(ABSTRACT TRUNCATED AT 250 WORDS)

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