The involvement of nitric oxide in the enhanced expression of mu-opioid receptors during intestinal inflammation in mice

Abstract

Intestinal inflammation enhances the potency of mu-opioid receptor (MOR) agonists inhibiting gastrointestinal transit and increases the expression of MOR in mice intestine. The precise mechanisms implicated in the increased expression of MOR during intestinal inflammation are not known. The aim of the study is to evaluate if nitric oxide released during intestinal inflammation could modulate MOR gene expression and affect gastrointestinal transit. Intestinal inflammation was induced by the intragastric administration of croton oil. In CD-1 mice, with and without inflammation, we evaluated the anti-transit effects of morphine in animals treated with NOS inhibitors (L-NAME and L-NIL) and the intestinal levels of iNOS enzyme mRNA. The anti-transit effects of morphine and the expression of MOR mRNA in the gut of wild-type (WT) and iNOS-/- mice were also assessed. Gastrointestinal transit was measured with charcoal meal and mRNA levels determined by real-time PCR. In CD-1 mice, inflammation induced a 10-fold increase (P<0.0001) in iNOS mRNA levels in the gut. The absence of iNOS gene and treatment of CD-1 mice with L-NAME or L-NIL abolished the increased antitransit effects of morphine observed during inflammation. Moreover, although the basal levels of MOR mRNA were similar in WT and iNOS animals (-/-), intestinal inflammation only increased the MOR expression in the gut of WT (P<0.01) but not in iNOS-/- mice. The results suggest that nitric oxide derived from the increased expression of iNOS is implicated in the enhanced effects of morphine and in the upregulation of MOR gene transcription observed during intestinal inflammation.

Figure 1

Percent of gastrointestinal transit in CD-1, WT and iNOS^−/−-deficient mice treated with saline (SS) or croton oil (CO). Each column represents the mean±s.e.m. of six to eight animals. ^*Compares CO with SS, P<0.02 and ^#compares iNOS-CO with CD-1-CO and WT-CO group, P<0.001 (Tukey's test).

Figure 2

Inhibition of gastrointestinal transit induced by the subcutaneous administration of morphine in controls (SS) and croton oil-(CO) treated animals, cotreated with L-NAME (a) or L-NIL (b). Each point represents the mean of six to eight animals; vertical lines show s.e.m. ^*P<0.01, indicates significant differences in the dose–response curve of morphine in croton oil-treated animals when compared to the other groups (Tukey's test).

Figure 3

Inhibition of gastrointestinal transit induced by the administration of a fixed dose of morphine (1 mg kg⁻¹) in WT and iNOS^−/− mice treated with saline (SS) or croton oil (CO). Each column represents the mean of six to eight animals; vertical lines show s.e.m., ^*compares CO with SS, P<0.001 and ^#compares iNOS-CO with WT-CO group, P<0.05 (Tukey's test).

Figure 4

(a) Is a graphical representation of the relative expression of iNOS mRNA in the jejunum samples of WT mice with (CO) and without intestinal inflammation (SS). Inflammation induced by croton oil enhances the iNOS mRNA levels. ^*P<0.0001 when croton oil-treated and saline-treated animals are compared (Student's t-test). (b) Represents the relative expression of MOR mRNA in the jejunum of WT and iNOS^−/− mice treated with vehicle (SS) or croton oil (CO). Inflammation enhances the intestinal MOR mRNA levels in WT but not in iNOS^−/− mice. For each genotype, ^*compares CO with SS, P<0.01 and ^#compares iNOS-CO with WT-CO group, P<0.05 (Tukey's test). Each column represents the mean±s.e.m. of four different samples from independent experiments.