Modulation of Na-K-2Cl cotransport by intracellular Cl(-) and protein kinase C-delta in Calu-3 cells.

In this study, we tested the hypothesis that intracellular Cl(-) (Cl) regulates the activity of protein kinase C (PKC)-delta and thus the activation of Na-K-Cl cotransport (NKCC1) in a Calu-3 cell line. The alpha(1)-adrenergic agonist methoxamine (MOX) and hypertonic sucrose increased Cl and increased or decreased intracellular volume, respectively, without changing Cl concentration ([Cl(-)](i)). Titration of [Cl(-)](i) from 20-140 mM in nystatin-permeabilized cell monolayers did not affect the baseline activity of PKC-delta, PKC-zeta, or rottlerin-sensitive NKCC1. At 200 mM Cl(-), rottlerin-sensitive NKCC1 was activated, and PKC isotypes were localized predominantly to a particulate fraction. MOX induced a biphasic increase in NKCC1 activity and PKC-delta in activity and particulate localization of PKC-delta and -zeta. Activity of NKCC1 and PKC-delta decreased with increasing Cl from 20 to 80 mM Cl then increased at 140-200 mM Cl apparently as an additive effect to high [Cl(-)](i) levels. Rottlerin inhibited the effects of MOX, which indicates that PKC-delta was required for activation of NKCC1. The results indicate that, in airway epithelial cells, a Cl electrochemical gradient alone is not sufficient to stimulate NKCC1 activity; rather, elevated activity of PKC-delta is necessary. Further, high Cl levels induce a subcellular redistribution of PKC-delta, which results in increased enzyme activity.

Activation of NKCC1 by hyperosmotic stress in human tracheal epithelial cells involves PKC-delta and ERK.

Hyperosmotic stress activates Na+-K+-2Cl- cotransport (NKCC1) in secretory epithelia of the airways. NKCC1 activation was studied as uptake of 36Cl or 86Rb in human tracheal epithelial cells (HTEC). Application of hypertonic sucrose or NaCl increased bumetanide-sensitive ion uptake but did not affect Na+/H+ and Cl-/OH-(HCO3-) exchange carriers. Hyperosmolarity decreased intracellular volume (Vi) after 10 min from 7.8 to 5.4 microl/mg protein and increased intracellular Cl- (Cl-i) from 353 to 532 nmol/mg protein. Treatment with an alpha-adrenergic agent rapidly increased Cl-i and Vi in a bumetanide-sensitive manner, indicating uptake of ions by NKCC1 followed by osmotically obligated water. These results indicate that HTEC act as osmometers but lose intracellular water slowly. Hyperosmotic stress also increased the activity of PKC-delta and of the extracellular signal-regulated kinase ERK subgroup of the MAPK family. Activity of stress-activated protein kinase JNK was not affected by hyperosmolarity. PD-98059, an inhibitor of the ERK cascade, reduced ERK activity and bumetanide-sensitive 36Cl uptake. PKC inhibitors blocked activation of ERK indicating that PKC may be a downstream activator of ERK. The results indicate that hyperosmotic stress activates NKCC1 and this activation is regulated by PKC-delta and ERK.