Gene plasticity in colonic circular smooth muscle cells underlies motility dysfunction in a model of postinfective IBS

Abstract

The cellular mechanisms of motility dysfunction in postinfectious irritable bowel syndrome (PI-IBS) are not known. We used a rat model of neonatal inflammation to test the hypothesis that gene plasticity in colonic circular smooth muscle cells underlies motility dysfunction in PI-IBS. Mild/moderate or severe inflammation was induced in neonatal and adult rats. Experiments were performed in tissues obtained at 7 days (short term) and 6-8 wk (long term) after the induction of inflammation. Severe inflammation in neonatal rats induced persistent long-term smooth muscle hyperreactivity to acetylcholine (ACh), whereas that in adult rat caused smooth muscle hyporeactivity that showed partial recovery in the long term. Mild/moderate inflammation had no effect in neonatal rats, but it induced smooth muscle hyporeactivity to ACh in adult rats, which recovered fully in the long term. Smooth muscle hyperreactivity to ACh resulted in accelerated colonic transit and increase in defecation rate, whereas hyporeactivity had opposite effects. Smooth muscle hyperreactivity to ACh was associated with increase in transcription rate of key cell-signaling proteins of the excitation-contraction coupling alpha1C subunit of Cav1.2 (L-type) calcium channels, Galphaq, and 20-kDa myosin light chain (MLC20), whereas hyporeactivity was associated with their suppression. Inflammation in adult rats induced classical inflammatory response, which was absent in neonatal rats. Severe neonatal inflammation enhanced plasma norepinephrine and muscularis propria vasoactive intestinal polypeptide in the long term. We conclude that severe, but not mild/moderate, inflammation in a state of immature or impaired stress and immune response systems alters the transcription rate of key cell-signaling proteins of excitation-contraction coupling in colonic circular smooth muscle cells to enhance their contractility and accelerate colonic transit and defecation rate.

Fig. 1.

Outline of the protocols used in this study. Colonic inflammation in neonatal and adult rats was induced by two doses of 2,4,6-trinitrobenzenesulfonic acid (TNBS). Experiments were performed 7 days (short term) or 6–8 wk (long term) after the induction of inflammation.

Fig. 2.

Myeloperoxidase (MPO) levels in the muscularis propria in response to 50 mg/kg (T50) and 130 mg/kg (T130) doses of TNBS in neonatal and adult rats. *P < 0.05 compared with sham-treated age-matched controls (Ctr).

Fig. 3.

Short-term and long-term effects of low and high doses of TNBS on the contractile response of colonic circular smooth muscle strips to acetylcholine (ACh) in neonatal rats. N, neonatal rats; T, TNBS followed by dose in mg/kg; S, short term; L, long term; AUC, area under the contractions. *P < 0.05 compared with age-matched sham-treated controls.

Fig. 4.

Percent length shortening of single dispersed circular smooth muscle cells in response to ACh. The resting lengths of the single cells from the control and neonatal long-term rats were 114 ± 3.7 μm and 116.7 ± 4.1 μm, respectively. Their corresponding contracted lengths at 10^–5 M ACh were 78.3 ± 3.9 μm and 70.9 ± 3.1 μm, respectively. *P < 0.05 compared with cells from age-matched sham-treated controls.

Fig. 5.

Short-term and long-term effects of low and high doses of TNBS on the contractile response of colonic circular smooth muscle strips to ACh in adult rats (A). *P < 0.05 compared with age-matched sham-treated controls.

Fig. 6.

Short-term and long-term effects of two doses of TNBS on the expression of four proteins of the excitation-contraction coupling in neonatal and adult inflammation. Neo, neonatal rats. Short term and long term were 7 days and 6–8 wk after induction of inflammation, respectively. *P < 0.05 compared with age-matched sham-treated controls. MLC, myosin light chain; MLCK, MLC kinase.

Fig. 7.

Long-term effects of severe inflammatory insult in neonates on the mRNA expression of α_1C, Gα_q, MLC₂₀, and MLCK in the muscularis propria. *P < 0.05 compared with age-matched sham-treated controls.

Fig. 8.

Short-term and long-term effects of two doses of TNBS on the expression of inflammatory mediators in the muscularis propria of neonatal and adult rats. Short term and long term refer to 7 days and 6–8 wk after induction of inflammation, respectively. *P < 0.05 compared with age-matched sham-treated controls. MCP-1, monocyte chemoattractant protein-1.

Fig. 9.

A: long-term effects of severe inflammation on the plasma levels of norepinephrine (NE) in neonatal and adult rats. B: severe inflammation in neonatal rats enhanced the muscularis propria (MP) level of vasoactive intestinal polypeptide (VIP) in the long term. By contrast, severe inflammation in adult rats suppressed the muscularis propria level of VIP in the short term. *P < 0.05 compared with age-matched sham-treated controls.

Fig. 10.

A and B: long-term effect of neonatal severe inflammation on colonic transit measured by the geometric center method and defecation rate. The faster transit in rats with neonatal severe inflammation was reduced after 7-day continuous systemic infusion of VIP and pituitary adenylate cyclase-activating peptide receptors (VPAC)_1/2 receptor antagonist (VPAC_1/2R Ant.) by a surgically implanted osmotic pump (A). C and D: long-term effect of adult severe inflammation on colonic transit measured by the geometric center method and defecation rate. *P < 0.05 compared with age-matched sham-treated controls.