Protein kinase C activation downregulates the expression and function of the basolateral Na+/K+/2Cl(-) cotransporter

The basolateral Na+/K+/2Cl(-) cotransporter (NKCC1) has been shown to be an independent regulatory site for electrogenic Cl- secretion. The proinflamm atory 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 dec reases 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 4 alpha-phorbol, which d oes not activate PKC. Inhibition of maximal (cAMP-stimulated) NKCC1 functio nal activity by PMA was first detected by 1 h, whereas decreases in the ste ady stale levels of NKCC1 mRNA were not detectable until 4 h. NKCC1 mRNA ex pression recovers toward control levels with extended treatment of cells wi th PMA suggesting that the PMA effects on NKCC1 expression are mediated thr ough activation of PKC. Although NKCC1 mRNA and protein levels return to co ntrol values after extended PMA exposure, NKCC1 functional activity does no t recover. Immunofluorescence imaging suggest that the absence of functiona l recovery is due to failure of newly synthesized NKKC1 protein to reach th e 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. (C) 1999 Wiley-Liss, Inc.