Activated Macrophages Inhibit Enterocyte Gap Junctions via the Release of Nitric Oxide

Entrocytes exist in close association with tissue macrophages, whose activation during inflammatory processes leads to the release of nitric oxide (NO). Repair from mucosal injury requires the migration of enterocytes into the mucosal defect, a process that requires connexin43 (Cx43)-mediated gap junction communication between adjacent enterocytes. Enterocyte migration is inhibited during inflammatory conditions including necrotizing enterocolitis, in part, through impaired gap junction communication.
In this study, we explored the idea that activated macrophages inhibit gap junctions of adjacent enterocytes and seek to determine whether NO release from macrophages was involved. Using a coculture system of enterocytes and macrophages, we now demonstrate that "activation" of macrophages with lipopolysaccharide and interferon reduces the phosphorylation of Cx43 in adjacent enterocytes, an event known to inhibit gap junction communication. The effects of macrophages on enterocyte gap junctions could be reversed by treatment of macrophages with the inducible nitric oxide synthase (iNOS) inhibitor l-Lysine omega-acetamidine hydrochloride (l-NIL) and by incubation with macrophages from iNOS(-/-) mice, implicating NO in the process.
Activated macrophages also caused a NO-dependent redistribution of connexin43 in adjacent enterocytes from the cell surface to an intracellular location, further suggesting NO release may inhibit gap junction function. Treatment of enterocytes with the NO donor S-nitroso-N-acetylpenicillamine (SNAP) markedly inhibited gap junction communication as determined using single cell microinjection of the gap junction tracer Lucifer yellow. Strikingly, activated macrophages inhibited enterocyte migration into a scraped wound, which was reversed by l-NIL pretreatment. These results implicate enterocyte gap junctions as a target of the NO-mediated effects of macrophages during intestinal inflammation, particularly where enterocyte migration is impaired.
As shown, NO inhibits gap junction communication between adjacent enterocytes. IEC-6 cells were plated on glass coverslips in the absence (A and B) or presence (C and D) of the NO donor SNAP (100 _M) and microinjected with the gap junction impermeant fluorescent dye Texas Red Dextran (Texas red) and the gap junction tracer Lucifer yellow (Lucifer yellow). Confocal micrographs were obtained 60 s later to reveal the localization of Texas Red Dextran within the injected cell only (A and C, asterisk indicates the location of injected cell) and transfer of Lucifer yellow to adjacent cells beyond the injected cell (B and D). Representative of at least 15 separate experiments with over 100 cells examined per experiment. E: quantification of gap junction communication as determined above in untreated cells (control), after treatment with the gap junction inhibitor oleamide (10 _M, OLM), or after exposure to SNAP (NO). Representative of at least 15 separate experiments. *P _ 0.05 vs. control. Size bar  10 _m.
Anand RJ, Dai S, Rippel C, Leaphart C, Qureshi F, Gribar SC, Kohler JW, Li J, Stolz DB, Sodhi C, Hackam DJ
Am J Physiol Gastrointest Liver Physiol
2008 Jan
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January 18, 2011
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Last Update
January 18, 2011