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

Reactive oxygen species such as HO 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 HO inhibits Ca-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 HO activates AMPK in human IEC lines and ex vivo human colon. Moreover, we demonstrate that the inhibitory effect of HO on Ca-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 (Thr) 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. HO inhibition of intestinal epithelial Ca-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, HO 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.