Hydrogen peroxide inhibits carbachol‐stimulated colonic epithelial ion transport by adenosine monophosphate‐activated protein kinase activation, without abolishing carbachol‐stimulated Ca2+ signal

Reactive oxygen species cause epithelial damage and dysfunction in inflammatory bowel disease (IBD). We have shown that hydrogen peroxide (H 2 O 2 ) inhibits intestinal Ca 2+ ‐dependent ion transport by PI3‐ kinase and MAP kinase activation. Here, we studied the role of the cellular energy sensor, adenosine monophosphate‐activated protein kinase (AMPK) in H 2 O 2 inhibition of carbachol (CCh)‐stimulated Ca 2+ ‐dependent Cl − secretion in colonic epithelial cells. We also investigated whether H 2 O 2 affects CCh‐stimulated cytosolic Ca 2+ concentration ([Ca 2+ ]cyt). Protein phosphorylation was measured by Western blot, and ion transport was measured in Ussing chambers. ([Ca 2+ ]cyt) was measured by digital Ca 2+ imaging. H 2 O 2 (500 μM) maximally increased AMPK‐α Tyr 172 phosphorylation in T 84 colonic epithelial monolayers at 5 min (p<0.01; n=3). AMPK activation occurs downstream of PI3‐K as the PI3‐K inhibitor, wortmannin (50 nM), reduced H 2 O 2 ‐stimulated AMPK phosphorylation. The AMPK inhibitor, Compound C (50 μM), reversed H 2 O 2 inhibition of CCh‐stimulated Cl − secretion across T 84 monolayers (p<0.05; n=4). Ca 2+ imaging showed that H 2 O 2 did not abolish CCh‐stimulated ([Ca 2+ ]cyt) increase in HT‐29 colonic epithelial cells. In summary, these data suggest that H 2 O 2 inhibits CCh‐stimulated Cl − secretion through inhibitory signaling pathways that act downstream of CCh‐induced Ca 2+ signals. Supported by the Crohn’s and Colitis Foundation of America and NIH.