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.

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

“…Collectively, these results show that exogenous NKCC1 is normally distributed in MDCK cells. PMA caused a dose-and time-dependent NKCC1 endocytosis in MDCK cells, as previously observed in T84 cells (11,46), demonstrating that our cell system is valid to investigate the internalization pathway of NKCC1 during PMA exposure.…”

“…In T84 cells, we have extensively characterized the functional inhibition of NKCC1 induced by the diacylglycerol mimetic PMA. Early inhibition of NKCC1 activity by PMA correlates with a reduction in the number of binding sites for radiolabeled bumetanide and endocytosis of NKCC1 (8,11,35,37).…”

“…Over the past few years, PKC has emerged as a hub in controlling endocytosis of channels, transporters, and receptors (1,11,18,22,25,40,42). Distinct constitutive and PKCmediated endocytic signals have been identified, for example, in the dopamine transporter (20), but in the case of NKCC1, no PKC phosphorylation sites are present.…”

“…In our laboratory, we previously demonstrated that, in the Cl Ϫ secretory human colonic cancer cell line T84, activation of protein kinase C (PKC) by phorbol ester or the acetylcholine analog carbachol induces rapid endocytosis of NKCC1 and leads to a dramatic reduction in transepithelial Cl Ϫ secretion (8,11,46). These findings were subsequently confirmed in native human colonic crypts (43).…”

Activated PKCδ and PKCϵ Inhibit Epithelial Chloride Secretion Response to cAMP via Inducing Internalization of the Na+-K+-2Cl− Cotransporter NKCC1

“…Previous studies from our laboratory showed that activation of PKC by phorbol 12-myristate 13-acetate (PMA) and the non-phorbol PKC activator bryostatin-1 and carbachol (CCh, a M3 muscarinic receptor agonist) inhibits epithelial chloride secretion in the human colonic crypt T84 epithelial cell line (12)(13)(14). PKC-dependent inhibition of chloride secretion correlated closely with a marked reduction of NKCC1 function, which in turn was paralleled by a loss of NKCC1 units from the basolateral membrane, and, subsequently, a reduction in gene expression (12,15).…”