AMPK mediates inhibition of electrolyte transport and NKCC1 activity by reactive oxygen species.

Reactive oxygen species such as H2O2 are believed to play a prominent role in the injury and loss of transport function that affect the intestinal epithelium in inflammatory conditions such as inflammatory bowel diseases. Defects in intestinal epithelial ion transport regulation contribute to dysbiosis and inflammatory phenotypes. We previously showed that H2O2 inhibits Ca2+-dependent Cl- secretion across intestinal epithelial cells (IECs) via a phosphatidylinositol 3-kinase (PI3K)- and extracellular signal-regulated kinase (ERK)-dependent mechanism that occurs, at least in part, through inhibition of the basolateral Na+-K+-2Cl- cotransporter NKCC1. NKCC1 governs Cl- entry into crypt IECs and thus plays a critical role in maintaining the driving force for Cl- secretion. Electrolyte transport consumes large amounts of cellular energy, and direct pharmacological activation of the cellular energy sensor AMP-activated protein kinase (AMPK) has been shown to inhibit a number of ion transport proteins. Here, we show that H2O2 activates AMPK in human IEC lines and ex vivo human colon. Moreover, we demonstrate that the inhibitory effect of H2O2 on Ca2+-dependent Cl- secretion and NKCC1 activity is AMPK-dependent. This inhibitory effect is associated with a physical interaction between AMPK and NKCC1, as well as increased phosphorylation (Thr212,217) of NKCC1, without causing NKCC1 internalization. These data identify a key role for AMPK-NKCC1 interaction as a point of convergence for suppression of colonic epithelial ion transport by inflammatory reactive oxygen species.NEW & NOTEWORTHY H2O2 inhibition of intestinal epithelial Ca2+-dependent Cl- secretion involves recruitment of AMP-activated protein kinase (AMPK) downstream of ERK and phosphatidylinositol 3-kinase signaling pathways, physical interaction of AMPK with the Na+-K+-2Cl- cotransporter NKCC1, and AMPK-dependent suppression of NKCC1-mediated electrolyte influx without causing NKCC1 internalization. It is intriguing that, in human intestinal epithelial cell lines and human colon, H2O2 activation of AMPK increased phosphorylation of NKCC1 residues required for promoting, not inhibiting, NKCC1 activity. These data identify an elevated complexity of AMPK regulation of NKCC1 in the setting of an inflammatory stimulus.

Hydrogen peroxide inhibits Ca2+-dependent chloride secretion across colonic epithelial cells via distinct kinase signaling pathways and ion transport proteins.

Reactive oxygen species (ROS) are key mediators in a number of inflammatory conditions, including inflammatory bowel disease (IBD). ROS, including hydrogen peroxide (H(2)O(2)), modulate intestinal epithelial ion transport and are believed to contribute to IBD-associated diarrhea. Intestinal crypt fluid secretion, driven by electrogenic Cl(-) secretion, hydrates and sterilizes the crypt, thus reducing bacterial adherence. Here, we show that pathophysiological concentrations of H(2)O(2) inhibit Ca(2+)-dependent Cl(-) secretion across T(84) colonic epithelial cells by elevating cytosolic Ca(2+), which contributes to activation of two distinct signaling pathways. One involves recruitment of the Ca(2+)-responsive kinases, Src and Pyk-2, as well as extracellular signal-regulated kinase (ERK). A separate pathway recruits p38 MAP kinase and phosphoinositide 3-kinase (PI3-K) signaling. The ion transport response to Ca(2+)-dependent stimuli is mediated in part by K(+) efflux through basolateral K(+) channels and Cl(-) uptake by the Na(+)-K(+)-2Cl(-) cotransporter, NKCC1. We demonstrate that H(2)O(2) inhibits Ca(2+)-dependent basolateral K(+) efflux and also inhibits NKCC1 activity independently of inhibitory effects on apical Cl(-) conductance. Thus, we have demonstrated that H(2)O(2) inhibits Ca(2+)-dependent Cl(-) secretion through multiple negative regulatory signaling pathways and inhibition of specific ion transporters. These findings increase our understanding of mechanisms by which inflammation disturbs intestinal epithelial function and contributes to intestinal pathophysiology.

Consequences of direct versus indirect activation of epidermal growth factor receptor in intestinal epithelial cells are dictated by protein-tyrosine phosphatase 1B.

The epidermal growth factor receptor (EGFR) is an integral regulator of many cellular functions. EGFR also acts as a central conduit for extracellular signals involving direct activation of the receptor by EGFR ligands or indirect activation by G protein-coupled receptor (GPCR)-stimulated transactivation of the EGFR. We have previously shown that EGFR negatively regulates epithelial chloride secretion as a result of transforming growth factor-alpha-mediated EGFR transactivation in response to muscarinic GPCR activation. Here we show that direct activation of the EGFR by EGFR ligands produces a different pattern of EGFR tyrosine phosphorylation and downstream phosphatidylinositol 3-kinase recruitment than GPCR-stimulated transactivation of the EGFR occurring via paracrine EGFR ligand release. Moreover, we demonstrate that this differential signaling and its consequences depend on protein-tyrosine phosphatase 1B activity. Thus protein-tyrosine phosphatase 1B governs differential recruitment of signaling pathways involved in EGFR regulation of epithelial ion transport. Our findings furthermore establish how divergent signaling outcomes can arise from the activation of a single receptor.