Korean J Physiol Pharmacol.  2012 Dec;16(6):437-446. 10.4196/kjpp.2012.16.6.437.

Mechanisms of Motility Change on Trinitrobenzenesulfonic Acid-Induced Colonic Inflammation in Mice

• Cheon GJ
• Cui Y
• Yeon DS
• Kwon SC
• Park BG

• KMID: 2285453
• DOI: http://doi.org/10.4196/kjpp.2012.16.6.437

Abstract

Ulcerative colitis is an inflammatory bowel disease (IBD) characterized by recurrent episodes of colonic inflammation and tissue degeneration in human or animal models. The contractile force generated by the smooth muscle is significantly attenuated, resulting in altered motility leading to diarrhea or constipation in IBD. The aim of this study is to clarify the altered contractility of circular and longitudinal smooth muscle layers in proximal colon of trinitrobenzen sulfonic acid (TNBS)-induced colitis mouse. Colitis was induced by direct injection of TNBS (120 mg/kg, 50% ethanol) in proximal colon of ICR mouse using a 30 G needle anesthetized with ketamin (50 mg/kg), whereas animals in the control group were injected of 50% ethanol alone. In TNBS-induced colitis, the wall of the proximal colon is diffusely thickened with loss of haustration, and showed mucosal and mucular edema with inflammatory infiltration. The colonic inflammation is significantly induced the reduction of colonic contractile activity including spontaneous contractile activity, depolarization-induced contractility, and muscarinic acetylcholine receptor-mediated contractile response in circular muscle layer compared to the longitudinal muscle layer. The inward rectification of currents, especially, important to Ca2+ and Na+ influx-induced depolarization and contraction, was markedly reduced in the TNBS-induced colitis compared to the control. The muscarinic acetylcholine-mediated contractile responses were significantly attenuated in the circular and longitudinal smooth muscle strips induced by the reduction of membrane expression of canonical transient receptor potential (TRPC) channel isoforms from the proximal colon of the TNBS-induced colitis mouse than the control.

Keyword

Cation channel; Colitis; Inflammation; Smooth muscle; TRPC

MeSH Terms

Acetylcholine
Animals
Colitis
Colitis, Ulcerative
Colon
Constipation
Contracts
Diarrhea
Edema
Ethanol
Humans
Inflammation
Inflammatory Bowel Diseases
Membranes
Mice
Mice, Inbred ICR
Models, Animal
Muscle, Smooth
Muscles
Needles
Protein Isoforms
Acetylcholine
Ethanol
Protein Isoforms

Figure

• 

  Fig. 1 Photomicrographs of sections from colon of a control (A) and TNBS-induced colitis mouse (B) stained with hematoxylin-eosin. Note the loss of haustration and folding, submucosal and mucosal edema, distortion of mucosal architecture, thickening of the muscularis mucosae, and appearance of infiltration by inflammatory cells in the muscle layer. Each frame indicates a typical trace out of a five independent experiments.

• 

  Fig. 2 Effects of TNBS-induced colonic inflammation on the SCA of the circular and longitudinal muscle layer from the proximal colon of the control and TNBS-induced colitis mouse. Representative traces for SCA of the control (A) and TNBS-induced colitis (B). SCA was recorded with a preload of 1 g along their circular axis in a bath filled with Krebs solution maintained at 37℃ in an atmosphere of 95% O2-5% CO2. The square box (right panel) present magnified rhythmic SCA traces (A, B). Summary for effects of TNBS-induced colonic inflammation on the average cycle rate and amplitude of SCA of the circular (C, D) and longitudinal (E, F) muscle layer from proximal colon. The data are represented to the mean±SD. Numbers in parenthesis are the number in a series of experiments. **p<0.01, ***p<0.005 vs control.

• 

  Fig. 3 Effects of TNBS-induced colonic inflammation on depolarization-induced contractility. Representative traces for high K+ (50 mM)-induced contractile response of the circular (A) and longitudinal (B) smooth muscle layer from the proximal colon of the control (left panel) and TNBS-induced colitis mouse (right panel). Contractile response was recorded with a preload of 1 g along their circular or longitudinal axis in a bath filled with Krebs solution maintained at 37℃ in an atmosphere of 95% O2-5% CO2. Summary of the contractile response induced by high K+ (50 mM) in circular (C) and longitudinal (D) smooth muscle layer from the proximal colon of the control and TNBS-induced colitis mouse. The data are represented to the mean±SD. Numbers in parenthesis are the number in a series of experiments. *p<0.05, ***p<0.005 vs control.

• 

  Fig. 4 Effects of TNBS-induced colonic inflammation on the carbachol-induced contractions in the circular (A, B) and longitudinal (C, D) smooth muscle layer from the proximal colon. Representative traces for contractile response induced by log scale concentration of carbachol (3×10-7~10-5 M) in proximal colonic circular (A, B) and longitudinal (C, D) smooth muscle layer from the control (A, C) and TNBS-induced colitis mouse (B, D).

• 

  Fig. 5 Summary of the carbachol- and oxotremorine M-induced contractile responses in proximal colon. The response evoked by application of carbachol (A, B) in log scale concentration (3×10-7~3×10-5 M) and oxotremorine M (C, D) in log scale concentration (3×10-8~3×10-6 M) in the circular (A, C) and longitudinal (B, D) smooth muscle layer from the proximal colon of the control (○) and TNBS-induced colitis mouse (□). The data are represented to the mean±SD from seven experiments.

• 

  Fig. 6 Effects of TNBS-induced colonic inflammation on the NSCC currents in the circular smooth muscle cells. Whole cell currents were recorded under the condition of 145 mM Cs+ in internal solution. Slow ramp depolarizations from +100 mV to -100 mV were applied at a holding potential of -60 mV. Representative traces of the current-voltage relationship were obtained from the circular smooth muscle cells in control and inflamed proximal colon (A). Bar graphs show mean current amplitudes of carbachol-induced NSCC currents at -60 mV (B). The data are represented to the mean±SD. Numbers in parenthesis are the number in a series of experiments. ***p<0.005 vs control.

• 

  Fig. 7 Qualitative RT-PCR analysis of mRNA encoding TRPC channel isoforms expressed in whole brain (upper), control (middle), and TNBS-induced inflamed colon (lower panel). Each total RNA isolated from whole brain and dissociated circular smooth muscle cells of the control and TNBS-induced colitis mouse proximal colon were reverse transcribed, and amplified by PCR with TRPC channel isoform-specific primers (Table 1). The resultant PCR product were separated and visualized on an agarose gel containing ethidium bromide (EtBR). GapDH RNA as an internal control and whole brain RNA as a positive control were used.

• 

  Fig. 8 Fold changes in gene expression of TRPC channel isoforms assessed by real-time quantitative RT-PCR. To analyze the relative changes in gene expression from the real-time PCR experiments, the comparative 2-△△CT method was used. The △CT value for each isoform gene was calculated using the equation: △CT (control or TNBS-induced colitis)=CT (TRPC isoform)-CT (reference, i.e., GapDH). The averaged CT value was obtained from triplicate of each PCR reaction. The fold change in gene expression of a TRPC isoform was finally obtained from the formula 2-△△CT, where the △△CT (TRPC isoform) value was the difference between △CT (colitis) and △CT (control) values. The point on the Y-axis (dotted line) indicates expression amounts in the whole brain. n=5. *p<0.05, **p<0.01, ***p<0.005 vs control.

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