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Intestine‐specific dual oxidase deficiency lacks ATP‐P2Y‐mediated luminal H 2 O 2 production in mice
Author(s) -
Maruta Koji,
Akiba Yasutada,
Kaji Izumi,
Kaunitz Jonathan D
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.856.20
Subject(s) - duodenum , secretion , medicine , ussing chamber , endocrinology , biology , oxidase test , extracellular , transepithelial potential difference , chemistry , ion transporter , biochemistry , enzyme , membrane
The intestinal epithelium affects luminal microbial composition via anion and mucus secretion, and the secretion of anti‐bacterial peptides or chemorepellents such as H 2 O 2 . Dual oxidase‐2 (Duox‐2) an enzyme predominantly expressed in the intestinal epithelium among NADPH oxidase family generates luminal H 2 O 2 via intracellular Ca 2+ increase. We found that luminal ATP‐P2Y signals increased luminal H 2 O 2 output in rat duodenum, which we hypothesized was due to the activity of Duox‐2. Lacking specific Duox inhibitors, we created mice selectively deficient for DuoxA1–A2 (accessory proteins essential for membrane trafficking of Duox‐1 and ‐ 2, respectively) by breeding villin‐Cre mice with mice bearing floxed DuoxA1–A2, needed since global Duox knockout (KO) causes hypothyroidism. We examined the effect of luminal ATP on H 2 O 2 production in wild type (WT) and Duox KO mice. We measured H 2 O 2 output using the fluorogenic substrate Amplex® and HCO 3 − secretion in an ATP‐perfused duodenal loop in vivo using flow‐through pH and CO 2 electrodes. We also measured luminal H 2 O 2 production in response to luminal ATP or ionomycin in Ussing chambered muscle‐stripped duodenum and cecum. Luminal perfusion of ATP (0.1 or 1 mM) augmented HCO 3 − secretion with increased luminal H 2 O 2 output in WT duodenum, whereas ATP‐induced HCO 3 − secretion was reduced and ATP‐induced H 2 O 2 output was abolished in KO duodenum. In Ussing chambered WT duodenum and cecum, basal H 2 O 2 concentrations were higher in the cecum than in the duodenum. Luminal H 2 O 2 production was similarly increased in the duodenum and cecum in response to luminal ATP (1 mM) and ionomycin (1 μM) in WT, whereas no additional H 2 O 2 production was observed in KO intestine. Furthermore, luminal pretreatment with the non‐specific P2Y receptor antagonist suramin (1 mM) abolished ATP‐induced H 2 O 2 production in WT. These results confirm that epithelial Duox‐2 generates luminal H 2 O 2 via luminal ATP‐P2Y signals. We propose that H 2 O 2 produced by Duox‐2 is a key factor governing the composition of the gut microbial flora. Support or Funding Information Supported by VA Merit Review, NIH R01 DK54221