Early inflammatory damage to intestinal neurons occurs via inducible nitric oxide synthase.

Intestinal inflammation affects the enteric nervous system (ENS) that lies adjacent to the smooth muscle layers. Previously, we showed that the loss of ENS neurons in animal models such as tri-nitrobenzene sulphonic acid (TNBS)-induced colitis was a limited and early event despite progressive worsening of inflammation. Here, we demonstrated that the rapid appearance of activated immune cells in the intestinal wall is selectively neurotoxic via iNOS-derived NO, using TNBS-induced colitis in both rats and mice, and a co-culture model of ENS neurons and smooth muscle. An influx of neutrophils and macrophages occurred within hours of initiation of rat colitis, correlating with iNOS expression, acutely elevated NO and neuronal death. In vitro, chemical donors of NO selectively caused axonal damage and neuronal death. These outcomes were similar to those seen with combined culture with either activated peritoneal immune cells or the immune cell lines RAW-264 and RBL-2H3. Immune cell-mediated neurotoxicity was blocked by the iNOS inhibitor L-NIL, and neuronal death was inhibited by the RIP-1 kinase inhibitor necrostatin. In a mouse model, the stereotypic loss of myenteric neurons by Day 4 post-TNBS was abrogated by the selective iNOS inhibitors L-NIL or 1400W without effect on other parameters of intestinal inflammation. Preservation of ENS neurons also ameliorated the hyperplasia of smooth muscle that is characteristic of intestinal inflammation, in line with prior work showing neural regulation of smooth muscle phenotype. This identifies a predominant pathway of immune cell damage to the ENS, where early, acute elevation of NO from iNOS can be cytotoxic to myenteric neurons.

Neuronal nitric oxide inhibits intestinal smooth muscle growth.

Hyperplasia of smooth muscle contributes to the thickening of the intestinal wall that is characteristic of inflammation, but the mechanisms of growth control are unknown. Nitric oxide (NO) from enteric neurons expressing neuronal NO synthase (nNOS) might normally inhibit intestinal smooth muscle cell (ISMC) growth, and this was tested in vitro. In ISMC from the circular smooth muscle of the adult rat colon, chemical NO donors inhibited [(3)H]thymidine uptake in response to FCS, reducing this to baseline without toxicity. This effect was inhibited by the guanylyl cyclase inhibitor ODQ and potentiated by the phosphodiesterase-5 inhibitor zaprinast. Inhibition was mimicked by 8-bromo (8-Br)-cGMP, and ELISA measurements showed increased levels of cGMP but not cAMP in response to sodium nitroprusside. However, 8-Br-cAMP and cilostamide also showed inhibitory actions, suggesting an additional role for cAMP. Via a coculture model of ISMC and myenteric neurons, immunocytochemistry and image analysis showed that innervation reduced bromodeoxyuridine uptake by ISMC. Specific blockers of nNOS (7-NI, NAAN) significantly increased [(3)H]thymidine uptake in response to a standard stimulus, showing that nNOS activity normally inhibits ISMC growth. In vivo, nNOS axon number was reduced threefold by day 1 of trinitrobenzene sulfonic acid-induced rat colitis, preceding the hyperplasia of ISMC described earlier in this model. We conclude that NO can inhibit ISMC growth primarily via a cGMP-dependent mechanism. Functional evidence that NO derived from nNOS causes inhibition of ISMC growth in vitro predicts that the loss of nNOS expression in colitis contributes to ISMC hyperplasia in vivo.