Phorbol 12-myristate 13-acetate-induced endocytosis of the Na-K-2Cl cotransporter in MDCK cells is associated with a clathrin-dependent pathway.
In secretory epithelial cells, the basolateral Na(+)-K(+)-2Cl(-) cotransporter (NKCC1) plays a major role in salt and fluid secretion. Our laboratory has identified NKCC1 surface expression as an important regulatory mechanism for Cl(-) secretion in the colonic crypt cell line T84, a process also present in native human colonic crypts. We previously showed that activation of protein kinase C (PKC) by carbachol and phorbol 12-myristate 13-acetate (PMA) decreases NKCC1 surface expression in T84 cells. However, the specific endocytic entry pathway has not been defined. We used a Madin-Darby canine kidney (MDCK) cell line stably transfected with enhanced green fluorescent protein (EGFP)-NKCC1 to map NKCC1 entry during PMA exposure. At given times, we fixed and stained the cells with specific markers (e.g., dynamin II, clathrin heavy chain, and caveolin-1). We also used chlorpromazine, methyl-beta-cyclodextrin, amiloride, and dynasore, blockers of the clathrin, caveolin, and macropinocytosis pathways and the vesicle "pinchase" dynamin, respectively. We found that PMA caused dose- and time-dependent NKCC1 endocytosis. After 2.5 min of PMA exposure, approximately 80% of EGFP-NKCC1 endocytic vesicles colocalized with clathrin and approximately 40% colocalized with dynamin II and with the transferrin receptor, the uptake of which is also mediated by clathrin-coated vesicles. We did not observe significant colocalization of EGFP-NKCC1 endocytic vesicles with caveolin-1, a marker of the caveolae-mediated endocytic pathway. We quantified the effect of each inhibitor on PMA-induced EGFP-NKCC1 endocytosis and found that only chlorpromazine and dynasore caused significant inhibition compared with the untreated control (61% and 25%, respectively, at 2.5 min). Together, these results strongly support the conclusion that PMA-stimulated NKCC1 endocytosis is associated with a clathrin pathway.
The Univ. of Chicago, 5841 S. Maryland Ave., MC 5029, Chicago, IL 60637, USA.
Andreas
A
Mykoniatis
Le
L
Shen
Mary
M
Fedor-Chaiken
Jun
J
Tang
Xu
X
Tang
Roger T
RT
Worrell
Eric
E
Delpire
Jerrold R
JR
Turner
Karl S
KS
Matlin
Patrice
P
Bouyer
Jeffrey B
JB
Matthews
D000818:Animals; D002460:Cell Line; D002966:Clathrin / Q000502:physiology*; D004285:Dogs; D004705:Endocytosis / Q000187:drug effects*; D005947:Glucose / Q000494:pharmacology; D007150:Immunohistochemistry; D007668:Kidney / Q000166:cytology / Q000187:drug effects / Q000502:physiology; D008856:Microscopy, Fluorescence; D028021:Sodium-Potassium-Chloride Symporters / Q000502:physiology*; D064506:Solute Carrier Family 12, Member 2; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D002966:Clathrin; C578877:SLC12A2 protein, human; D028021:Sodium-Potassium-Chloride Symporters; D064506:Solute Carrier Family 12, Member 2; D005947:Glucose; D013755:Tetradecanoylphorbol Acetate