Dynamic regulation of Na(+)-K(+)-2Cl(-) cotransporter surface expression by PKC-{epsilon} in Cl(-)--secretory epithelia.
In secretory epithelia, activation of PKC by phorbol ester and carbachol negatively regulates Cl(-) secretion, the transport event of secretory diarrhea. Previous studies have implicated the basolateral Na(+)-K(+)-2Cl(-) cotransporter (NKCC1) as a target of PKC-dependent inhibition of Cl(-) secretion. In the present study, we examined the regulation of surface expression of NKCC1 in response to the activation of PKC. Treatment of confluent T84 intestinal epithelial cells with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (PMA) reduced the amount of NKCC1 accessible to basolateral surface biotinylation. Loss of cell surface NKCC1 was due to internalization as shown by 1) the resistance of biotinylated NKCC1 to surface biotin stripping after incubation with PMA and 2) indirect immunofluorescent labeling. PMA-induced internalization of NKCC1 is dependent on the epsilon-isoform of PKC as determined on the basis of sensitivity to a panel of PKC inhibitors. The effect of PMA on surface expression of NKCC1 was specific because PMA did not significantly alter the amount of Na(+)-K(+)-ATPase or E-cadherin available for surface biotinylation. After extended PMA exposure (>2 h), NKCC1 became degraded in a proteasome-dependent fashion. Like PMA, carbachol reduced the amount of NKCC1 accessible to basolateral surface biotinylation in a PKC-epsilon-dependent manner. However, long-term exposure to carbachol did not result in degradation of NKCC1; rather, NKCC1 that was internalized after exposure to carbachol was recycled back to the cell membrane. PKC-epsilon-dependent alteration of NKCC1 surface expression represents a novel mechanism for regulating Cl(-) secretion.
Dept. of Surgery, Univ. of Cincinnati Medical Center, 231 Albert B. Sabin Way, PO Box 670558, Cincinnati, OH 45267-0558, USA.
Isabel Calvo
IC
Del Castillo
Mary
M
Fedor-Chaiken
J Cecilia
JC
Song
Veronika
V
Starlinger
James
J
Yoo
Karl S
KS
Matlin
Jeffrey B
JB
Matthews
D002217:Carbachol / Q000494:pharmacology; D002478:Cells, Cultured; D002712:Chlorides / Q000378:metabolism*; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000502:physiology*; D006801:Humans; D007413:Intestinal Mucosa / Q000166:cytology; D011493:Protein Kinase C / Q000502:physiology*; D051744:Protein Kinase C-epsilon; D021381:Protein Transport; D028021:Sodium-Potassium-Chloride Symporters / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000031:analogs & derivatives / Q000494:pharmacology
D002712:Chlorides; D028021:Sodium-Potassium-Chloride Symporters; C032975:12-O-tetradeca-2,4,6,8-tetranoylphorbol-13-acetate; D002217:Carbachol; C497255:PRKCE protein, human; D011493:Protein Kinase C; D051744:Protein Kinase C-epsilon; D013755:Tetradecanoylphorbol Acetate