Colonic Inflammation Increases the Contribution of Muscarinic M2 Receptors to Carbachol-Induced Contraction of the Rat Colon

Abstract

Objectives: Carbachol-induced contraction of the rat colon is impaired in rats with trinitrobenzene sulfonic acid (TNBS)-induced colitis. The main objective of this study was to examine the effect of colitis on the expression and function of muscarinic (M) receptor subtypes in the rat colon. Materials and Methods: Rats (n = 80) were treated with TNBS and used 5 days later for measurement of contractility, myeloperoxidase activity, histology and expression of muscarinic receptor isoforms using Western blot analysis. Results: Carbachol produced concentration-dependent contractions of colonic segments from control (n = 40) and TNBS-treated (n = 40) rats with no significant difference in potency. However, the maximum response to carbachol was significantly reduced in colon segments of TNBS-treated rats. The selective muscarinic receptor antagonists 4-diphenylacetoxy-N-methyl piperidine (4-DAMP, M₃), pirenzepine (M₁) and methoctramine (M₂) antagonized carbachol-induced contraction in control (9.1 ± 0.1, 6.7 ± 0.3 and 6.0 ± 0.1, respectively) and TNBS-treated rats (9.2 ± 0.2, 6.9 ± 0.2, 6.7 ± 0.2). The –logK_B values in control rats are consistent with an action of carbachol on muscarinic M₃ receptors. There was no significant difference in –logK_B values for 4-DAMP and pirenzepine in control and TNBS-treated rats, but methoctramine was fivefold more potent in TNBS-treated rats, possibly indicating an increased contribution of muscarinic M₂ receptors to carbachol-induced contraction in the inflamed colon. The expression of M₂ receptors was also significantly increased in colon segments from TNBS-treated rats, confirming the increased role of muscarinic M₂ receptors in the inflamed colon. Conclusions: The data show that while only M₃ receptors appeared to mediate carbachol-induced contraction in control segments, expression of both M₂ and M₃ receptors was increased in the inflamed rat colon.