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

Abstract

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.

CLIC1 Inhibition Protects Against Cellular Senescence and Endothelial Dysfunction Via the Nrf2/HO-1 Pathway

“…All histograms were presented as mean ± SD, n = 3, * p ≤ 0.05, ** p ≤ 0.01 versus control. # p ≤ 0.05, ## p ≤ 0.01 versus H 2 O 2 treatment regulation of chloride channels, such as CFTR Chloride Channel [35], and Na(+)-K(+)-Cl(−) co-transporter-1 (NKCC1) [36]. CLIC1 expression is regulated by cAMP through the protein kinase A (PKA) pathway [37].…”

Cochlear Inflammaging in Relation to Ion Channels and Mitochondrial Functions

“…Similarly, there are reports confirming the involvement of NKCC1 in chronic inflammation in various body parts [128][129][130]. NKCC1 regulation has been experimentally correlated with inflammation through nuclear factor kappa B (NF-κB) signaling pathways.…”

The Mitochondrial Permeability Transition: Nexus of Aging, Disease and Longevity

“…AMPK phosphorylates ULK1, thereby initiating autophagy [ 141 ]. Other activators of AMPK are a reduction in glucose concentration and, most importantly in the context of aging and disease, elevated levels of ROS [ 142 , 143 , 144 ]. AMPK phosphorylates many other key enzymes within the autophagy pathways, including key enzymes of mitophagy [ 145 , 146 , 147 ].…”