Paradoxical regulation of ChAT and nNOS expression in animal models of Crohn's colitis and ulcerative colitis

Abstract

Morphological and functional changes in the enteric nervous system (ENS) have been reported in inflammatory bowel disease. We examined the effects of inflammation on the expression of choline acetyltransferase (ChAT) and nNOS in the muscularis externae of two models of colonic inflammation, trinitrobenzene sulfonic acid (TNBS)-induced colitis, which models Crohn's disease-like inflammation, and DSS-induced colitis, which models ulcerative Colitis-like inflammation. In TNBS colitis, we observed significant decline in ChAT, nNOS, and protein gene product (PGP) 9.5 protein and mRNA levels. In DSS colitis, ChAT and PGP9.5 were significantly upregulated while nNOS levels did not change. The nNOS dimer-to-monomer ratio decreased significantly in DSS- but not in TNBS-induced colitis. No differences were observed in the percentage of either ChAT (31 vs. 33%)- or nNOS (37 vs. 41%)-immunopositive neurons per ganglia or the mean number of neurons per ganglia (55 ± 5 vs. 59 ± 5, P > 0.05). Incubation of the distal colon muscularis externae in vitro with different types of inflammatory mediators showed that cytokines decreased ChAT and nNOS expression, whereas H₂O₂, a component of oxidative stress, increased their expression. NF-κB inhibitor MG-132 did not prevent the IL-1β-induced decline in either ChAT or nNOS expression. These findings showed that TNBS- and DSS-induced inflammation differentially regulates the expression of two critical proteins expressed in the colonic myenteric neurons. These differences are likely due to the exposure of the myenteric plexus neurons to different combinations of Th1-type inflammatory mediators and H₂O₂ in each model.

Keywords: choline acetyltransferase; enteric neurons; motility; neuronal nitric oxide synthase; smooth muscle.

Fig. 1.

Differential regulation of choline acetyltransferase (ChAT) and nNOS in dextran sodium sulfate (DSS)-induced colitis. A: Western blots showing that expression of ChAT and the neuronal marker protein gene product (PGP) 9.5 were significantly increased in the distal colon muscularis externae of rats that received 5% DSS in their water for 7 days; normalized to β-actin (*P < 0.05) and compared with naïve rats (n = 4); nNOS protein was not significantly different from control (P > 0.05). Ctr, control. B: in real-time RT-PCR experiments, we observed an increase in ChAT mRNA levels in DSS-treated rats (P < 0.05). The increase in PGP9.5 mRNA did not reach statistical significance. *P < 0.05 vs. control. C: there was a significant decline in the nNOS dimer-to-monomer ratio in DSS-treated rats (*P < 0.05).

Fig. 2.

ChAT (blue), nNOS (red), and the neuronal marker Hu C/D (green) immunofluorescence on whole mount longitudinal muscle myenteric plexus preparations from control and DSS distal colon. No significant differences were observed in the mean number of ChAT- or nNOS-immunoreactive neurons per ganglia.

Fig. 3.

Downregulation of ChAT, nNOS, and PGP9.5 in trinitrobenzene sulfonic acid (TNBS)-induced colitis. A: Western blots showing that TNBS treatment at 50 (T50) or 80 (T80) mg/kg suppressed the expression of ChAT, nNOS, and the neuronal marker PGP9.5 in the distal colon muscularis externae of rats (*P < 0.05); normalized to β-actin (*P < 0.05) and compared with naïve rats (n = 4). B: bar graph displaying the results of quantitative RT-PCR assays measuring relative mRNA levels of ChAT and PGP9.5, normalized to 18S RNA, in muscularis externa of rats treated with either 50 or 80 mg/kg TNBS and controls.

Fig. 4.

Effects of oxidative stress on ChAT and nNOS expression in vitro. A: muscularis externae strips from distal colons of naïve rats were incubated in vitro for 6 h with vehicle or various concentrations of H₂O₂. Top, Western blot results. Bottom, compiled results in bar graph. We observed significant increase in ChAT, nNOS, and PGP9.5 expression compared with control at 50 μM H₂O₂ (*P > 0.05, n = 6). B: bar graphs showing results of quantitative RT-PCR measurements of ChAT and nNOS mRNA in muscularis externae strips from distal colons of naïve rats that were incubated in vitro for 1, 3, or 6 h with 50 μM H₂O₂. Both ChAT and nNOS mRNA levels were significantly increased, n = 4, *P < 0.05.

Fig. 5.

Effects of inflammatory cytokines on ChAT and nNOS expression in vitro. A: Western blots showing ChAT, nNOS, and PGP9.5 protein expression in distal colonic muscularis externae from naïve rats that were incubated for 24 h with various concentrations of IL-1β (0.1, 0.3, 1, 5, or 20 ng/ml). Results compiled in bar graph show that ChAT and nNOS protein expressions were significantly decreased, n = 4, *P < 0.05. B: incubation of distal colon muscularis externae strips with a combination of 20 ng/ml IL-1β (I) and 50 μM H₂O₂ (H) produced a significant decrease in both ChAT and nNOS expression. *P < 0.05 vs. control.

Fig. 6.

Effects of NF-κB inhibitor on inflammatory IL-1β- and H₂O₂-induced alterations in ChAT and nNOS expression. Distal colon muscle strips from naïve rats were incubated for 24 h with either IL-1β (20 ng/ml) or H₂O₂ (50 μM) in the presence of the NF-κB inhibitor MG-132. MG-132 failed to prevent changes in ChAT or nNOS expression in response to incubation with IL-1β or H₂O₂ (*P < 0.05 vs. control).

Fig. 7.

Summary of alterations in enteric neural and smooth muscle regulation of motility function induced by TNBS and DSS inflammation. Mucosal stimulation by digesta stimulates intrinsic sensory neurons that transmit the signal to excitatory and inhibitory motor neurons. The release of ACh and NO induces excitation-contraction and excitation-inhibition couplings, respectively, in smooth muscle cells that compete to determine the amplitude, duration, and type of contraction generated in smooth muscle cells. A: 1) TNBS inflammation increases excitability and synaptic neurotransmission of intrinsic sensory neurons (22, 26). 2) It suppresses the expression of ChAT and nNOS as well as the release of ACh and NO by electrical field stimulation that would reduce the excitatory and inhibitory inputs to smooth muscle cells. 3) At the next level, TNBS inflammation impairs excitation-contraction coupling in smooth muscle cells that reduces smooth muscle reactivity to Ach, resulting in suppression of rhythmic phasic contractions (RPCs) and tone (45). 4) This type of inflammation increases SP and neurokinin 1 receptors and upregulates SP-immunoreactive fibers (14, 28, 48). GMCs, giant migrating contractions. B: 1) The effects of ulcerative colitis-like inflammation induced by DSS treatment on the intrinsic sensory neuron excitability or neurotransmission remain unknown. 2) DSS inflammation upregulates the expression of ChAT that has the potential to increase RPCs. 3) At the smooth muscle level, DSS inflammation suppresses the smooth muscle reactivity to ACh; the overall effect of the two opposing alterations is to suppress RPCs in this type of inflammation (13, 45). 4) DSS inflammation increases the proportion of SP immunoreactive in the myenteric plexus of ulcerative colitis patients (31). In addition, H₂O₂ generated by oxidative stress has been shown to increase the frequency of GMCs (13). ISN, intrinsic sensory neuron.