Increase in endotoxin-induced mucosal permeability is related to increased nitric oxide synthase activity using the Ussing chamber

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Abstract

Objective

To determine if nitric oxide production is associated with increased intestinal permeability after endotoxin challenge using the ex vivo Ussing chamber.

Subjects

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

Interventions

Endotoxin (lipopolysaccharide), 1, 10, 100 [micro sign]g/mL, or saline was placed on the serosal side of ileal mucosal membranes mounted in Ussing chambers after 109 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 [micro sign]g/mL lipopolysaccharide with or without the addition of the nitric oxide synthase inhibitor, NG-monomethyl-L-arginine at a concentration of 10 mM (n = 7-8/group).

Main Outcome Measure

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 [micro sign]g/mL of lipopolysaccharide (71% and 86%, respectively) vs. the control membranes (0%) or the membranes exposed to 1 [micro sign]g/mL of lipopolysaccharide (0%) (p < .05). This increase in E. coli C-25 passage in the ileal membranes exposed to 10 or 100 [micro sign]g/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 NG-monomethyl-L-arginine protected against lipopolysaccharide-induced bacterial translocation and prevented the lipopolysaccharide-induced increase in ileal membrane inducible nitric oxide synthase activity.

Conclusion

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. (Crit Care Med 1999; 27:880-886)

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