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The guinea pig ileum lacks the direct, high-potency, M(2)-muscarinic, contractile mechanism characteristic of the mouse ileum.

Griffin MT, Matsui M, Ostrom RS, Ehlert FJ - Naunyn Schmiedebergs Arch. Pharmacol. (2009)

Bottom Line: Following 4-DAMP mustard treatment, the concentration-response curve of oxotremorine-M in wild-type ileum resembled that of the M(3) knockout mouse in terms of its pEC (50), E (max), and inhibition by selective muscarinic antagonists.Following 4-DAMP mustard treatment, the contractile response of the guinea pig ileum to oxotremorine-M exhibited low potency and a competitive-antagonism profile consistent with an M(3) response.The guinea pig ileum, therefore, lacks a direct, highly potent, M(2)-contractile component but may have a direct, lower potency M(2) component.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Chapman University, Orange, CA, USA.

ABSTRACT
We explored whether the M(2) muscarinic receptor in the guinea pig ileum elicits a highly potent, direct-contractile response, like that from the M(3) muscarinic receptor knockout mouse. First, we characterized the irreversible receptor-blocking activity of 4-DAMP mustard in ileum from muscarinic receptor knockout mice to verify its M(3) selectivity. Then, we used 4-DAMP mustard to inactivate M(3) responses in the guinea pig ileum to attempt to reveal direct, M(2) receptor-mediated contractions. The muscarinic agonist, oxotremorine-M, elicited potent contractions in ileum from wild-type, M(2) receptor knockout, and M(3) receptor knockout mice characterized by negative log EC(50) (pEC (50)) values +/- SEM of 6.75 +/- 0.03, 6.26 +/- 0.05, and 6.99 +/- 0.08, respectively. The corresponding E (max) values in wild-type and M(2) receptor knockout mice were approximately the same, but that in the M(3) receptor knockout mouse was only 36% of wild type. Following 4-DAMP mustard treatment, the concentration-response curve of oxotremorine-M in wild-type ileum resembled that of the M(3) knockout mouse in terms of its pEC (50), E (max), and inhibition by selective muscarinic antagonists. Thus, 4-DAMP mustard treatment appears to inactivate M(3) responses selectively and renders the muscarinic contractile behavior of the wild-type ileum similar to that of the M(3) knockout mouse. Following 4-DAMP mustard treatment, the contractile response of the guinea pig ileum to oxotremorine-M exhibited low potency and a competitive-antagonism profile consistent with an M(3) response. The guinea pig ileum, therefore, lacks a direct, highly potent, M(2)-contractile component but may have a direct, lower potency M(2) component.

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Competitive antagonism of the response to oxotremorine-M by AF-DX 116 (1 μM) and 4-DAMP (10 nM) in ilea from wild type (a), M2 KO (b), and M3 KO (c) mice. The data are normalized relative to the Emax of control and represent the mean values ± SEM from four to seven experiments
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Fig2: Competitive antagonism of the response to oxotremorine-M by AF-DX 116 (1 μM) and 4-DAMP (10 nM) in ilea from wild type (a), M2 KO (b), and M3 KO (c) mice. The data are normalized relative to the Emax of control and represent the mean values ± SEM from four to seven experiments

Mentions: Antagonism of the muscarinic response in mouse ileum We investigated two muscarinic antagonists (AF-DX 116 and 4-DAMP (N,N-dimethyl-4-piperidinyl diphenylacetate)) with known selectivity for receptor subtypes to determine if their inhibitory action in the mouse ileum is consistent with the picture of M2 and M3 receptor function described above in connection with our studies on KO mice. The binding affinities (pKD, negative log dissociation constant) of AF-DX 116 for the human M2 and M3 receptor subtypes are 7.27 ± 0.05 and 6.10 ± 0.06, and those of 4-DAMP are 7.87 ± 0.03 and 8.81 ± 0.05, respectively (Esqueda et al. 1996; Griffin et al. 2004). Thus, AF-DX 116 exhibits about 15-fold higher affinity for the M2 receptor relative to M3, whereas 4-DAMP exhibits an opposite tenfold selectivity. 4-DAMP actually exhibits high affinity for all subtypes of the muscarinic receptor except the M2. We tested each antagonist at a concentration approximately equal to the greater of its two KD values for M2 and M3 receptors. With this strategy, the M2 selective AF-DX 116 and the M3-selective 4-DAMP should only cause about twofold shifts in the concentration–response curve of an agonist for eliciting M3 and M2 responses, respectively, but much greater ten- to 15-fold shifts in responses mediated by the receptors for which they exhibit selectivity (i.e., M2 and M3, respectively).The results of antagonism studies using AF-DX 116 (1 μM) in the mouse ileum from wild-type, M2 KO, and M3 KO mice are show in Fig. 2a–c. In ileum from the M3 KO mouse (Fig. 2c), AF-DX 116 caused a 10.5-fold shift in the concentration response curve of oxotremorine-M, which yielded an estimated pKB value of (6.97 ± 0.03) similar to its binding affinity for the M2 receptor (pKD = 7.27 ± 0.05). In contrast, AF-DX 116 only caused 2.6- and 2.7-fold shifts in the concentration response curves of oxotremorine-M in wild-type and M2 KO mice, respectively. 4-DAMP exhibited the opposite selectivity (Fig. 2a–c). It caused a 6.8-fold shift in the concentration–response curve of oxotremorine-M in the M2 KO mouse (Fig. 2b), which yields an estimated pKB value (8.74 ± 0.19) similar to its binding affinity for the M3 receptor (8.81 ± 0.05, Griffin et al. 2004). A similar shift of 14-fold was observed in the wild-type mouse (Fig. 2a), whereas a much smaller shift (1.5-fold) was measured in ilea from the M3 KO mouse (Fig. 2c). These results are summarized in Table 3 and are consistent with the postulate mentioned above that the muscarinic contractile response in the mouse ileum includes major and minor, directly acting M3 and M2 components, respectively.Fig. 2


The guinea pig ileum lacks the direct, high-potency, M(2)-muscarinic, contractile mechanism characteristic of the mouse ileum.

Griffin MT, Matsui M, Ostrom RS, Ehlert FJ - Naunyn Schmiedebergs Arch. Pharmacol. (2009)

Competitive antagonism of the response to oxotremorine-M by AF-DX 116 (1 μM) and 4-DAMP (10 nM) in ilea from wild type (a), M2 KO (b), and M3 KO (c) mice. The data are normalized relative to the Emax of control and represent the mean values ± SEM from four to seven experiments
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Related In: Results  -  Collection

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Fig2: Competitive antagonism of the response to oxotremorine-M by AF-DX 116 (1 μM) and 4-DAMP (10 nM) in ilea from wild type (a), M2 KO (b), and M3 KO (c) mice. The data are normalized relative to the Emax of control and represent the mean values ± SEM from four to seven experiments
Mentions: Antagonism of the muscarinic response in mouse ileum We investigated two muscarinic antagonists (AF-DX 116 and 4-DAMP (N,N-dimethyl-4-piperidinyl diphenylacetate)) with known selectivity for receptor subtypes to determine if their inhibitory action in the mouse ileum is consistent with the picture of M2 and M3 receptor function described above in connection with our studies on KO mice. The binding affinities (pKD, negative log dissociation constant) of AF-DX 116 for the human M2 and M3 receptor subtypes are 7.27 ± 0.05 and 6.10 ± 0.06, and those of 4-DAMP are 7.87 ± 0.03 and 8.81 ± 0.05, respectively (Esqueda et al. 1996; Griffin et al. 2004). Thus, AF-DX 116 exhibits about 15-fold higher affinity for the M2 receptor relative to M3, whereas 4-DAMP exhibits an opposite tenfold selectivity. 4-DAMP actually exhibits high affinity for all subtypes of the muscarinic receptor except the M2. We tested each antagonist at a concentration approximately equal to the greater of its two KD values for M2 and M3 receptors. With this strategy, the M2 selective AF-DX 116 and the M3-selective 4-DAMP should only cause about twofold shifts in the concentration–response curve of an agonist for eliciting M3 and M2 responses, respectively, but much greater ten- to 15-fold shifts in responses mediated by the receptors for which they exhibit selectivity (i.e., M2 and M3, respectively).The results of antagonism studies using AF-DX 116 (1 μM) in the mouse ileum from wild-type, M2 KO, and M3 KO mice are show in Fig. 2a–c. In ileum from the M3 KO mouse (Fig. 2c), AF-DX 116 caused a 10.5-fold shift in the concentration response curve of oxotremorine-M, which yielded an estimated pKB value of (6.97 ± 0.03) similar to its binding affinity for the M2 receptor (pKD = 7.27 ± 0.05). In contrast, AF-DX 116 only caused 2.6- and 2.7-fold shifts in the concentration response curves of oxotremorine-M in wild-type and M2 KO mice, respectively. 4-DAMP exhibited the opposite selectivity (Fig. 2a–c). It caused a 6.8-fold shift in the concentration–response curve of oxotremorine-M in the M2 KO mouse (Fig. 2b), which yields an estimated pKB value (8.74 ± 0.19) similar to its binding affinity for the M3 receptor (8.81 ± 0.05, Griffin et al. 2004). A similar shift of 14-fold was observed in the wild-type mouse (Fig. 2a), whereas a much smaller shift (1.5-fold) was measured in ilea from the M3 KO mouse (Fig. 2c). These results are summarized in Table 3 and are consistent with the postulate mentioned above that the muscarinic contractile response in the mouse ileum includes major and minor, directly acting M3 and M2 components, respectively.Fig. 2

Bottom Line: Following 4-DAMP mustard treatment, the concentration-response curve of oxotremorine-M in wild-type ileum resembled that of the M(3) knockout mouse in terms of its pEC (50), E (max), and inhibition by selective muscarinic antagonists.Following 4-DAMP mustard treatment, the contractile response of the guinea pig ileum to oxotremorine-M exhibited low potency and a competitive-antagonism profile consistent with an M(3) response.The guinea pig ileum, therefore, lacks a direct, highly potent, M(2)-contractile component but may have a direct, lower potency M(2) component.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Chapman University, Orange, CA, USA.

ABSTRACT
We explored whether the M(2) muscarinic receptor in the guinea pig ileum elicits a highly potent, direct-contractile response, like that from the M(3) muscarinic receptor knockout mouse. First, we characterized the irreversible receptor-blocking activity of 4-DAMP mustard in ileum from muscarinic receptor knockout mice to verify its M(3) selectivity. Then, we used 4-DAMP mustard to inactivate M(3) responses in the guinea pig ileum to attempt to reveal direct, M(2) receptor-mediated contractions. The muscarinic agonist, oxotremorine-M, elicited potent contractions in ileum from wild-type, M(2) receptor knockout, and M(3) receptor knockout mice characterized by negative log EC(50) (pEC (50)) values +/- SEM of 6.75 +/- 0.03, 6.26 +/- 0.05, and 6.99 +/- 0.08, respectively. The corresponding E (max) values in wild-type and M(2) receptor knockout mice were approximately the same, but that in the M(3) receptor knockout mouse was only 36% of wild type. Following 4-DAMP mustard treatment, the concentration-response curve of oxotremorine-M in wild-type ileum resembled that of the M(3) knockout mouse in terms of its pEC (50), E (max), and inhibition by selective muscarinic antagonists. Thus, 4-DAMP mustard treatment appears to inactivate M(3) responses selectively and renders the muscarinic contractile behavior of the wild-type ileum similar to that of the M(3) knockout mouse. Following 4-DAMP mustard treatment, the contractile response of the guinea pig ileum to oxotremorine-M exhibited low potency and a competitive-antagonism profile consistent with an M(3) response. The guinea pig ileum, therefore, lacks a direct, highly potent, M(2)-contractile component but may have a direct, lower potency M(2) component.

Show MeSH
Related in: MedlinePlus