Objectives: To characterize the mechanisms leading to excessive production of nitric oxide within the gut as a consequence of endotoxemia. We sought to: a) determine the time course of inducible nitric oxide synthase (iNOS) messenger RNA (mRNA) expression in the intestine after challenging rats with lipopolysaccharide (LPS); and b) investigate whether there is differential expression of iNOS in enterocytes along the longitudinal or crypt-villus axes of the intestine in rats after LPS administration.
Design: Prospective, randomized, unblinded study.
Setting: Research laboratories at a large university-affiliated medical center.
Subjects: Male Sprague-Dawley rats.
Interventions: At T = 0 hr, rats were injected with O111:B4 Escherichia coli LPS (5 mg/kg) or a similar volume of the saline vehicle. At various time points thereafter, samples of duodenum, jejunum, ileum, colon, and liver were harvested for subsequent extraction of RNA. In some cases, populations of enterocytes enriched in either crypt or villus cells were harvested from the ileum. In some studies, rats were injected with cycloheximide (25 mg i.p.) 15 mins before being challenged with LPS or dexamethasone (2 mg i.p.) 30 mins before being injected with LPS.
Measurements and main results: iNOS mRNA was undetectable in ileal tissue from rats under basal conditions, but was evident by T = 1 hr and was maximal at T = 2 hrs after injection of LPS. Thereafter, ileal iNOS mRNA concentrations decreased and were undetectable again at T = 24 hrs. At T = 2 hrs after LPS injection, there was marked expression of iNOS mRNA in the ileum, whereas much lower concentrations of iNOS mRNA were detected in the jejunum and colon, and no iNOS mRNA was detected in the duodenum. At T = 3 hrs after LPS injection, expression of iNOS mRNA was up-regulated in both villus and crypt cells, although LPS-induced iNOS mRNA was more prominent in the former than the latter cell type. Pretreatment of rats with dexamethasone virtually abrogated the expression of iNOS mRNA in ileal samples obtained 3 hrs after the injection of LPS. Prior treatment of rats with the protein synthesis inhibitor, cycloheximide, also blunted LPS-induced iNOS mRNA expression.
Conclusions: LPS-induced iNOS expression is differentially regulated along both the longitudinal and crypt villus axes of the intestinal mucosa, being most prominent in the villus cells of the ileum. LPS-induced iNOS expression is blunted by pretreating rats with dexamethasone or cycloheximide. The latter finding suggests that LPS-induced expression of iNOS mRNA in the gut requires new protein synthesis. Differential regulation of nitric oxide production along the longitudinal and crypt-villus axes of the gut may be a determinant of the pattern of sepsis-induced intestinal damage.