Activated macrophages inhibit enterocyte gap junctions via the release of nitric oxide

Rahul J. Anand, Shipan Dai, Christopher Rippel, Cynthia Leaphart, Faisal Qureshi, Steven C. Gribar, Jeff W. Kohler, Jun Li, Donna Beer Stolz, Chhinder Sodhi, David J. Hackam

Research output: Contribution to journalArticle

22 Citations (Scopus)

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 ω-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.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Gastrointestinal and Liver Physiology
Volume294
Issue number1
DOIs
StatePublished - Dec 1 2007

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Enterocytes
Gap Junctions
Nitric Oxide
Macrophages
Connexin 43
Communication
Macrophage Activation
Nitric Oxide Synthase Type II
Lysine
S-Nitroso-N-Acetylpenicillamine
Necrotizing Enterocolitis
Nitric Oxide Donors
Wounds and Injuries
Microinjections
Coculture Techniques
Interferons
Lipopolysaccharides

Keywords

  • Enterocyte migration
  • Inflammation
  • Intestinal restitution
  • Necrotizing enterocolitis

ASJC Scopus subject areas

  • Gastroenterology
  • Physiology

Cite this

Activated macrophages inhibit enterocyte gap junctions via the release of nitric oxide. / Anand, Rahul J.; Dai, Shipan; Rippel, Christopher; Leaphart, Cynthia; Qureshi, Faisal; Gribar, Steven C.; Kohler, Jeff W.; Li, Jun; Stolz, Donna Beer; Sodhi, Chhinder; Hackam, David J.

In: American Journal of Physiology - Gastrointestinal and Liver Physiology, Vol. 294, No. 1, 01.12.2007.

Research output: Contribution to journalArticle

Anand, Rahul J. ; Dai, Shipan ; Rippel, Christopher ; Leaphart, Cynthia ; Qureshi, Faisal ; Gribar, Steven C. ; Kohler, Jeff W. ; Li, Jun ; Stolz, Donna Beer ; Sodhi, Chhinder ; Hackam, David J. / Activated macrophages inhibit enterocyte gap junctions via the release of nitric oxide. In: American Journal of Physiology - Gastrointestinal and Liver Physiology. 2007 ; Vol. 294, No. 1.
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AU - Dai, Shipan

AU - Rippel, Christopher

AU - Leaphart, Cynthia

AU - Qureshi, Faisal

AU - Gribar, Steven C.

AU - Kohler, Jeff W.

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AB - 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 ω-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.

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