Phorbol 12-myristate 13-acetate-induced endocytosis of the Na-K-2Cl cotransporter in MDCK cells is associated with a clathrin-dependent pathway

Am J Physiol Cell Physiol. 2010 Jan;298(1):C85-97. doi: 10.1152/ajpcell.00118.2009. Epub 2009 Oct 28.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Line
  • Clathrin / physiology*
  • Dogs
  • Endocytosis / drug effects*
  • Glucose / pharmacology
  • Immunohistochemistry
  • Kidney / cytology
  • Kidney / drug effects
  • Kidney / physiology
  • Microscopy, Fluorescence
  • Sodium-Potassium-Chloride Symporters / physiology*
  • Solute Carrier Family 12, Member 2
  • Tetradecanoylphorbol Acetate / pharmacology*

Substances

  • Clathrin
  • SLC12A2 protein, human
  • Sodium-Potassium-Chloride Symporters
  • Solute Carrier Family 12, Member 2
  • Glucose
  • Tetradecanoylphorbol Acetate