Regulation of Na+/K+/Cl- cotransport and [3H]bumetanide binding site density by phorbol esters in HT29 cells.
The involvement of protein kinase C in the regulation of Na+/K+/Cl- cotransport was investigated in cultured HT29 human colonic adenocarcinoma cells. We have demonstrated previously the presence of a Na+/K+/Cl- cotransport pathway in HT29 cells (Kim, H.D., Tsai, Y-S., Franklin, C.C., and Turner, J.T. (1989) Biochim. Biophys. Acta 946, 397-404). Treatment of cells with the phorbol esters phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-dibutyrate (PDBu) caused an increase in membrane-associated protein kinase C activity that was accompanied by a concomitant decrease in cytosolic protein kinase C activity. PMA also produced a rapid transient increase in cotransport to 137% of control values by 5 min followed by a progressive decrease to 19% of control values by 2 h. To determine the underlying mechanism for the reduction in Na+/K+/Cl- cotransport, changes in cotransporter number and/or affinity were determined in radioligand binding studies using [3H]bumetanide. PMA and PDBu produced essentially identical time- and dose-dependent decreases in specific [3H]bumetanide binding that were similar to the observed decreases in cotransport. Analysis of saturation and competition binding data indicated that the decrease in binding was due to a lowered Bmax with no change in affinity. Both the decrease in binding and the changes in cotransport elicited by PMA were prevented by the protein kinase inhibitor H7. These findings suggest that phorbol esters cause a decrease in the number of cotransporters in HT29 cells, resulting in a reduction in Na+/K+/Cl- cotransport activity.
Franklin
C C
CC
Department of Pharmacology, School of Medicine, University of Missouri, Columbia 65212.
Turner
J T
JT
Kim
H D
HD
D001667:Binding, Competitive; D001692:Biological Transport; D002034:Bumetanide / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism*; D004232:Diuretics / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D010703:Phorbol Esters / Q000494:pharmacology*; D011188:Potassium / Q000378:metabolism*; D011493:Protein Kinase C / Q000502:physiology*; D012964:Sodium / Q000378:metabolism*; D013997:Time Factors; D014407:Tumor Cells, Cultured
D002712:Chlorides; D004232:Diuretics; D010703:Phorbol Esters; D002034:Bumetanide; D012964:Sodium; D011493:Protein Kinase C; D011188:Potassium