title: Protein kinase C activation downregulates the expression and function of the basolateral Na+/K+/2Cl(-) cotransporter.
abstract: The basolateral Na+/K+/2Cl(-) cotransporter (NKCC1) has been shown to be an independent regulatory site for electrogenic Cl(-) secretion. The proinflammatory phorbol ester, phorbol 12-myristate 13-acetate (PMA), which activates protein kinase C (PKC), inhibits basal and cyclic adenosine monophosphate (cAMP)-stimulated NKCC1 activity in T84 intestinal epithelial cells and decreases the steady state levels of NKCC1 mRNA in a time- and dose-dependent manner. The levels of NKCC1 protein also fall in accordance with the NKCC1 mRNA transcript and these levels are unaffected by 4alpha-phorbol, which does not activate PKC. Inhibition of maximal (cAMP-stimulated) NKCC1 functional activity by PMA was first detected by 1 h, whereas decreases in the steady state levels of NKCC1 mRNA were not detectable until 4 h. NKCC1 mRNA expression recovers toward control levels with extended treatment of cells with PMA suggesting that the PMA effects on NKCC1 expression are mediated through activation of PKC. Although NKCC1 mRNA and protein levels return to control values after extended PMA exposure, NKCC1 functional activity does not recover. Immunofluorescence imaging suggest that the absence of functional recovery is due to failure of newly synthesized NKKC1 protein to reach the cell surface. We conclude that NKCC1 has the capacity to be regulated at the level of de novo expression by PKC, although decreased NKCC1 expression alone cannot account for either early or late loss of NKCC1 function.
authors.affiliation: Division of General and Gastrointestinal Surgery, Department of Surgery, Beth Israel Deaconess Medical Center, Boston, Massachusetts 02215, USA.
authors.forename: O C
authors.initials: OC
authors.lastname: Farokhzad
authors.forename: G D
authors.initials: GD
authors.lastname: Sagar
authors.forename: E C
authors.initials: EC
authors.lastname: Mun
authors.forename: J K
authors.initials: JK
authors.lastname: Sicklick
authors.forename: M
authors.initials: M
authors.lastname: Lotz
authors.forename: J A
authors.initials: JA
authors.lastname: Smith
authors.forename: J C
authors.initials: JC
authors.lastname: Song
authors.forename: T C
authors.initials: TC
authors.lastname: O'Brien
authors.forename: C P
authors.initials: CP
authors.lastname: Sharma
authors.forename: T B
authors.initials: TB
authors.lastname: Kinane
authors.forename: R A
authors.initials: RA
authors.lastname: Hodin
authors.forename: J B
authors.initials: JB
authors.lastname: Matthews
mesh_terms: D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism*; D002455:Cell Division; D002460:Cell Line; D015536:Down-Regulation / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D011493:Protein Kinase C / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D028021:Sodium-Potassium-Chloride Symporters; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D002352:Carrier Proteins; D012333:RNA, Messenger; D028021:Sodium-Potassium-Chloride Symporters; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate