Muscarinic Agonist-Mediated Heterologous Desensitization in Isolated Ileum Requires Activation of Both Muscarinic M₂ and M₃ Receptors

Abstract

We investigated the subtypes of the muscarinic receptor mediating short-term heterologous desensitization in the isolated ileum. Treatment of the ileum from C57BL/6 mice with acetylcholine (30 M) for 20 min caused a subsequent decrease in contractile sensitivity to both prostaglandin F 2␣ (PGF 2␣ ) and the muscarinic agonist, oxotremorine-M. This subsensitivity was characterized by 7-and 3-fold increases in the EC 50 values of the agonists, respectively, with no significant effect on the maximal response. The subsensitivity to PGF 2␣ was prevented in both M 2 and M 3 muscarinic receptor knockout mice. Similarly, the subsensitivity to oxotremorine-M was prevented in M 2 knockout mice. Acetylcholine-mediated desensitization of histamine-induced contractions in the guinea pig ileum was inhibited by both M 2 -and M 3 -selective muscarinic antagonists with high potency, although careful analysis of the data suggested behavior more consistent with an M 2 antagonistic profile. Modeling studies showed that the competitive antagonism of response contingent upon activation of two receptor subtypes should exhibit a pharmacological profile similar to that of the least sensitive signaling pathway. Our results demonstrate that muscarinic agonist-mediated short-term heterologous desensitization of intestinal smooth muscle is contingent upon activation of both M 2 and M 3 muscarinic receptors and that activation of either receptor by itself is insufficient to cause desensitization.It has long been known that the sensitivity of gastrointestinal smooth muscle to the contractile effects of muscarinic agonists decreases after incubation of the tissue with moderate to high concentrations of agonist for relatively short periods (i.e., 5-30 min) (Cantoni and Eastman, 1946;Dale, 1958;Paton, 1961). The mechanism for this short-term desensitization seems to be at a locus downstream from the muscarinic receptor because muscarinic agonist-mediated desensitization is characterized by a decrease in sensitivity to other spasmogens as well. Such a generalized subsensitivity is not surprising considering the metabolic costs required to maintain tonic muscle contraction for several minutes. Thus, prolonged contraction caused by continuous exposure to a muscarinic agonist causes a decreased responsiveness of the contractile machinery to subsequent activation by muscarinic as well as other heterologous agents. It seems likely, therefore, that the muscarinic receptor subtypes mediating heterologous desensitization may be the same as those eliciting contraction.In isolated ileum, subtype-selective muscarinic antagonists inhibit the contractile response to agonists in a manner

Comparison of the Antimuscarinic Action of p-Fluorohexahydrosiladifenidol in Ileal and Tracheal Smooth Muscle

“…The pK B values estimated for 4-DAMP from the data just described as well as those in Fig. 3 are similar to the binding affinity of 4-DAMP estimated at human M 3 muscarinic receptors expressed in CHO cells (pK D ϭ 8.81), but not with that estimated at human M 2 muscarinic receptors (pK D ϭ 7.87) (Griffin et al, 2004). In these latter assays, binding was measured in a modified KRB buffer similar to that used in the present study.…”

“…To test this idea, we measured the competitive antagonism of oxotremorine-M-induced contractions by 4-DAMP in ileum and trachea. 4-DAMP exhibits 10-fold higher binding affinity for human M 3 muscarinic receptors (pK D ϭ 8.81) expressed in CHO cells compared with human M 2 receptors (pK D ϭ 7.87) (Griffin et al, 2004). At a concentration of 10 nM, 4-DAMP caused similar shifts (10.2-and 15.9-fold) in the oxotremorine-M concentration-response curve in ileum and trachea, respectively.…”

Muscarinic Agonists and Antagonists: Effects on Gastrointestinal Function

“…The contractions elicited by carbachol or oxotremorine-M in the ileum (Matsui et al 2002;Unno et al 2005;Griffin et al 2009) and gastric fundus and antrum (Stengel et al 2000(Stengel et al , 2002Kitazawa et al 2007) undergo a small loss of function in the M 2 KO mouse (two-fold increase in agonist EC 50 , with no decrease in E max ), a large loss of function in the M 3 KO mouse (50-70% reduction in E max ), and a complete loss in the M 2 /M 3 double KO mouse. The contractions measured in antrum, fundus, and ileum from the M 2 KO mouse are competitively inhibited by the antagonists, N,N-dimethyl-4-piperidinyl diphenylacetate (4-DAMP) (M 3 -over M 2 -selective) and AF-DX 116 (M 2 -over M 3 -selective) with pK B values (approximately 9.0 and 6.0, respectively) that agree with M 3 binding affinity [8.8 and 6.1, respectively (Esqueda et al 1996;Griffin et al 2004)]. Conversely, the pK B values of 4-DAMP and AF-DX 116 in the same tissues from the M 3 KO mouse (approximately 8.3 and 7.3, respectively) agree with M 2 -binding affinity [7.9 and 7.3, respectively (Esqueda et al 1996;Griffin et al 2004)].…”

M2 and M3 Muscarinic Receptor Activation of Urinary Bladder Contractile Signal Transduction. I. Normal Rat Bladder

“…We found that after five successive carbachol CRCs, the affinity of the M 3 -selective antagonist para-fluoro hexahydro siladifenadol was shifted to a lower value, consistent with M 2 receptors participating in the contraction (data not shown). Others have shown that the M 3 receptor subtype is subject to agonist-induced desensitization (Tobin et al, 1992;Willets et al, 2001Willets et al, , 2002Griffin et al, 2004). Therefore, M 3 receptor desensitization may occur during the repeated agonist CRCs, which could explain these differences.…”