Phorbol ester sequentially downregulates cAMP-regulated basolateral and apical Cl- transport pathways in T84 cells.
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)
Matthews
J B
JB
Department of Surgery, Beth Israel Hospital, Boston, Massachusetts.
Awtrey
C S
CS
Hecht
G
G
Tally
K J
KJ
Thompson
R S
RS
Madara
J L
JL
D001692:Biological Transport; D002352:Carrier Proteins / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002712:Chlorides / Q000378:metabolism*; D005576:Colforsin / Q000494:pharmacology; D003106:Colon / Q000166:cytology / Q000378:metabolism / Q000502:physiology; D000242:Cyclic AMP / Q000502:physiology*; D004594:Electrophysiology; D006801:Humans; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology*; D011188:Potassium / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D028021:Sodium-Potassium-Chloride Symporters; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D013997:Time Factors
D002352:Carrier Proteins; D002712:Chlorides; D028021:Sodium-Potassium-Chloride Symporters; D005576:Colforsin; D015240:Phorbol 12,13-Dibutyrate; D000242:Cyclic AMP; D011493:Protein Kinase C; D000254:Sodium-Potassium-Exchanging ATPase; D013755:Tetradecanoylphorbol Acetate; D011188:Potassium