Increase in endotoxin-induced mucosal permeability is related to increased nitric oxide synthase activity using the Ussing chamber.
OBJECTIVE
To determine if nitric oxide production is associated with increased intestinal permeability after endotoxin challenge using the ex vivo Ussing chamber.


METHODS
Ileal mucosal membranes harvested from normal rats weighing 300 to 420 g.


METHODS
Endotoxin (lipopolysaccharide), 1, 10, 100 microg/ mL, or saline was placed on the serosal side of ileal mucosal membranes mounted in Ussing chambers after 10(9) Escherichia coli C-25 had been placed on the mucosal side of the ileal membranes (n = 6-7/group). In a second set of experiments, ileal membranes were exposed to 100 microg/mL lipopolysaccharide with or without the addition of the nitric oxide synthase inhibitor, N(G)-monomethyl-L-arginine at a concentration of 10 mM (n = 7-8/group).


METHODS
Bacterial translocation of E. coli C-25 from the mucosal to the serosal side of the ileal membrane was measured every hour during the 3-hr experimental period, as were serial measurements of the potential difference and resistance values of the ileal membranes. At the conclusion of the 3-hr period, the ileal membranes were harvested and levels of inducible nitric oxide synthase and constitutive nitric oxide synthase activity were measured.


RESULTS
The incidence of E. coli C-25 passage across the ileal membranes mounted in the Ussing chambers was significantly increased in the ileal membranes exposed to 10 or 100 microg/mL of lipopolysaccharide (71% and 86%, respectively) vs. the control membranes (0%) or the membranes exposed to 1 microg/mL of lipopolysaccharide (0%) (p < .05). This increase in E. coli C-25 passage in the ileal membranes exposed to 10 or 100 microg/mL of lipopolysaccharide was associated with a decrease in ileal membrane resistance and an increase in inducible nitric oxide synthase activity (p < .05). The addition of N(G)-monomethyl-L-arginine protected against lipopolysaccharide-induced bacterial translocation and prevented the lipopolysaccharide-induced increase in ileal membrane inducible nitric oxide synthase activity.


CONCLUSIONS
These results indicate that lipopolysaccharide induction of increased ileal inducible nitric oxide synthase activity is necessary for lipopolysaccharide-induced E. coli C-25 translocation to occur in normal ileal mucosal membranes tested in the Ussing chamber system.
Mishima
S
S
Department of Surgery, New Jersey Medical School, UMDNJ, Newark, USA.
Xu
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D
Deitch
E A
EA
D000818:Animals; D018988:Bacterial Translocation / Q000502:physiology*; D002463:Cell Membrane Permeability / Q000502:physiology*; D004195:Disease Models, Animal; D004926:Escherichia coli; D007082:Ileum; D066298:In Vitro Techniques; D007413:Intestinal Mucosa / Q000201:enzymology* / Q000382:microbiology*; D008070:Lipopolysaccharides / Q000009:adverse effects*; D008297:Male; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013997:Time Factors; D019323:omega-N-Methylarginine / Q000494:pharmacology
D008070:Lipopolysaccharides; D019323:omega-N-Methylarginine; D019001:Nitric Oxide Synthase