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


King, Stephanie J; Bunz, Michael; Chappell, Alfred; Scharl, Michael; Docherty, Michael; Jung, Barbara; Lytle, Christian; McCole, Declan F (2019). AMPK mediates inhibition of electrolyte transport and NKCC1 activity by reactive oxygen species. American Journal Of Physiology. Gastrointestinal And Liver Physiology, 317(2):G171-G181.

Abstract

Reactive oxygen species such as H$_{2}$O$_{2}$ 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 H$_{2}$O$_{2}$ inhibits Ca$^{2+}$-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 H$_{2}$O$_{2}$ activates AMPK in human IEC lines and ex vivo human colon. Moreover, we demonstrate that the inhibitory effect of H$_{2}$O$_{2}$ on Ca$^{2+}$-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$^{212,217}$) 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 H$_{2}$O$_{2}$ inhibition of intestinal epithelial Ca$^{2+}$-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, H$_{2}$O$_{2}$ 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.

Abstract

Reactive oxygen species such as H$_{2}$O$_{2}$ 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 H$_{2}$O$_{2}$ inhibits Ca$^{2+}$-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 H$_{2}$O$_{2}$ activates AMPK in human IEC lines and ex vivo human colon. Moreover, we demonstrate that the inhibitory effect of H$_{2}$O$_{2}$ on Ca$^{2+}$-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$^{212,217}$) 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 H$_{2}$O$_{2}$ inhibition of intestinal epithelial Ca$^{2+}$-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, H$_{2}$O$_{2}$ 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.

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > University Hospital Zurich > Clinic for Gastroenterology and Hepatology
Dewey Decimal Classification:610 Medicine & health
Scopus Subject Areas:Life Sciences > Physiology
Health Sciences > Hepatology
Health Sciences > Gastroenterology
Health Sciences > Physiology (medical)
Language:English
Date:1 August 2019
Deposited On:13 Jan 2020 07:59
Last Modified:29 Jul 2020 12:24
Publisher:American Physiological Society
ISSN:0193-1857
OA Status:Closed
Free access at:Publisher DOI. An embargo period may apply.
Publisher DOI:https://doi.org/10.1152/ajpgi.00317.2018
PubMed ID:31070932

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