Nitric oxide increases the permeability of caco-2 cell monolayer in association with dephosphorylation of occludin


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Aim:Interferon-gamma (IFN) is well known to chronically down-regulate intestinal epithelial ion transport and barrier function, largely through specific decreases in protein expression such as the alpha subunit of the Na pump, NalK/2CI co-transporter, occ1udin, and ZO proteins.Although the cellular events mediating these effects were not known, we observed that IFN causes an acute inhibition of Na,K-ATPase (Na pump) activity.Therefore, we tested the hypothesis that this event, which leads to increases intracellular Na (Na.) and cell volume, mediates the chronic IFN effects on epithelial function.Methods: I) Na pump activity, Na., and cell volume were measured in T84 colon cells incubated with lOOOIU IFN for 6 or 24 hours.2) Expression of transport-and barrier-function associated proteins were determined by Western blot after 6 or 24 hours in control cells and cells subjected to low extracellular Na (20mM), hypotonic stress, ouabain, and monensin.Results: Treatment of cells with IFN, ouabain, and monensin caused significant increases in Nai and cell volume and nearly identical downregulatory effects by 24 h on barrier-and transport-related proteins.These effects were blocked by incubating cells in 20mM Na-containing media, which prevented IFN-induced effects on Na, and cell volume.These results strongly implicated the downregulation of Na pump activity as an important initiating signal.To determine the role of increased cell volume in mediating the IFN and Ouabain effects, cell were subjected to chronic hypotonic stress.Hypotonic-induced cell swelling had similar downregulatory effects as these agents, but notably did not affect the expression of the alpha! subunit of the Na pump.Thus, increased Na, resulting from chronic IFN or Ouabain treatment may be a primary determinant of the downregulated alpha] subunit of the Na pump.Conclusion:The selective downregulation of intestinal epithelial transport-and barrier-function related proteins by IFN is secondary to an initial inhibition of Na,K-ATPase activity.This event results in increased Na, and cell volume which are distinct signaling events that ultimately downregulate specific intestinal epithelial functions.These effects may be important for phenotypic transformation or for reducing metabolic requirements during periods of sustained inflammation-associated stress.
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nitric oxide
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