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

Objective: To determine ii 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 mu g/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 mu g/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). 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 mu g/mL of lipopolysaccharide (71% and 86%, respectively) vs. the control membranes (0%) or the membranes exposed to 1 mu g/mL of lipopolysaccharide (0%) (p < .05). This increase in E. coli C-25 passage in the ileal membranes exposed to 10 or 100 mu 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 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. 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.