title: Activated macrophages inhibit enterocyte gap junctions via the release of nitric oxide.
abstract: Enterocytes 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. We now hypothesize 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.
authors.affiliation: Department of Surgery, Children's Hospital of Pittsburgh, Pittsburgh, PA 15213, USA.
authors.forename: Rahul J
authors.initials: RJ
authors.lastname: Anand
authors.forename: Shipan
authors.initials: S
authors.lastname: Dai
authors.forename: Christopher
authors.initials: C
authors.lastname: Rippel
authors.forename: Cynthia
authors.initials: C
authors.lastname: Leaphart
authors.forename: Faisal
authors.initials: F
authors.lastname: Qureshi
authors.forename: Steven C
authors.initials: SC
authors.lastname: Gribar
authors.forename: Jeff W
authors.initials: JW
authors.lastname: Kohler
authors.forename: Jun
authors.initials: J
authors.lastname: Li
authors.forename: Donna Beer
authors.initials: DB
authors.lastname: Stolz
authors.forename: Chhinder
authors.initials: C
authors.lastname: Sodhi
authors.forename: David J
authors.initials: DJ
authors.lastname: Hackam
mesh_terms: D000818:Animals; D002460:Cell Line; D002465:Cell Movement; D018920:Coculture Techniques; D018031:Connexin 43 / Q000378:metabolism; D020895:Enterocytes / Q000187:drug effects / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D017629:Gap Junctions / Q000187:drug effects / Q000378:metabolism*; D005759:Gastroenteritis / Q000201:enzymology / Q000378:metabolism*; D007372:Interferons / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D008239:Lysine / Q000031:analogs & derivatives / Q000494:pharmacology; D008262:Macrophage Activation / Q000187:drug effects; D008264:Macrophages / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D009569:Nitric Oxide / Q000378:metabolism*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D052247:Nitric Oxide Synthase Type II / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D019899:Paracrine Communication / Q000187:drug effects; D010766:Phosphorylation; D021381:Protein Transport; D051381:Rats; D026423:S-Nitroso-N-Acetylpenicillamine / Q000494:pharmacology; D013997:Time Factors
chemical_list: D018031:Connexin 43; D004791:Enzyme Inhibitors; D008070:Lipopolysaccharides; C090663:N(6)-(1-iminoethyl)lysine; D020030:Nitric Oxide Donors; D009569:Nitric Oxide; D026423:S-Nitroso-N-Acetylpenicillamine; D007372:Interferons; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; D008239:Lysine