Stimulation of cyclic AMP accumulation and phosphoinositide hydrolysis by M3 muscarinic receptors in the rat peripheral lung

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We investigated the contractile role of M 2 muscarinic receptors in mouse urinary bladder. When measured in the absence of other agents, contractions elicited to the muscarinic agonist oxotremorine-M exhibited properties consistent with that expected for an M 3 response in urinary bladder from wild-type and M 2 knockout (KO) mice. Evidence for a minor M 2 receptormediated contraction was revealed by a comparison of responses in M 3 knockout and M 2 /M 3 double knockout mice. Treatment of wild-type and M 2 knockout urinary bladder with N-2-chloroethyl-4-piperidinyl diphenylacetate (4-DAMP mustard) caused a large inhibition of the muscarinic contractile response. The residual contractions were much smaller in M 2 knockout bladder compared with wild type, suggesting that M 2 receptors rescue the muscarinic contractile response in wildtype bladder following inactivation of M 3 receptors with 4-DAMP mustard. When measured in the presence of prostaglandin F 2␣ and isoproterenol or forskolin, oxotremorine-M mediated a potent contractile response in urinary bladder from M 3 KO mice. This response exhibited an M 2 profile in competitive antagonism studies and was completely absent in M 2 /M 3 KO mice. Following 4-DAMP mustard treatment, oxotremorine-M elicited a contractile response in wild-type urinary bladder in the presence of KCl and isoproterenol or forskolin, and this response was diminished in M 2 KO mice. Our results show that the M 2 receptor mediates contractions indirectly in the urinary bladder by enhancing M 3 receptor-mediated contractions and inhibiting relaxation. We also show that it is difficult to detect M 2 receptor function in competitive antagonism studies under conditions where a simultaneous activation of M 2 and M 3 receptors occurs.Micturition is mediated through the actions of several neurotransmitters. Among those that directly influence the tone of urinary bladder smooth muscle, acetylcholine is important in contracting the reservoir and relaxing the outlet through activation of muscarinic receptors (de Groat and Yoshimura, 2001). Most evidence shows that it is the M 3 subtype of the muscarinic receptor that mediates the direct contractile response to acetylcholine in the urinary bladder. For example, the contractile response to muscarinic agonists exhibits an M 3 profile in competitive antagonism studies (Noronha-Blob et al., 1989;Longhurst et al., 1995;Choppin and Eglen, 2001), and these contractions are nearly absent in urinary bladder from M 3 muscarinic receptor knockout (M 3 KO) mice (Matsui et al., 2000). Male M 3 KO mice exhibit prominent urinary bladder distension in vivo, demonstrating the essential role of the M 3 receptor in micturition (Matsui et al., 2000). The small, direct contractile response that persists in urinary bladder from M 3 KO mice is completely lost in mice lacking both M 2 and M 3 muscarinic receptors (M 2 /M 3 KO mice), demonstrating that the M 2 receptor is capable of mediating very small contractions and that muscarinic recep-

Section: Resultsmentioning

confidence: 99%

The M₂ Muscarinic Receptor Mediates Contraction through Indirect Mechanisms in Mouse Urinary Bladder

Ehlert

Griffin

Abe

et al. 2004

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“…The Hill coefficients of the competition curves were estimated at 0.88 Ϯ 0.091 and 0.90 Ϯ 0.040, in CHO M 2 and CHO M 3 cells, respectively. We previously estimated the K i values of AF-DX 116 in competition experiments with [ 3 H]NMS using a modified KRB buffer (Esqueda et al, 1996). These K i values of AF-DX 116 for CHO M 2 and CHO M 3 cells are also listed in Table 3, together with those for 4-DAMP.…”

Section: Contractility Measurements In Muscarinic Receptormentioning

confidence: 99%

“…Such an approach is unaffected by the complex relationship between stimulus and response. Consequently, we used a mathematical modeling technique based Esqueda et al (1996). on receptor theory to generate agonist concentration-response curves that should simulate acetylcholine-mediated desensitization.…”

Section: Contractility Measurements In Muscarinic Receptormentioning

confidence: 99%

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

Griffin

Matsui

Shehnaz

et al. 2003

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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

“…The relatively small, 2.8-fold difference between the resultant pK B values of p-FHHSiD in ileum and trachea suggests a small difference in the potency of p-FHHSiD for antagonizing muscarinic receptors eliciting contraction in the two tissues. Such a difference might be attributed to a greater contribution of the M 2 muscarinic receptor to contraction in the trachea compared with the ileum, because p-FHHSiD exhibits approximately 16-fold higher binding affinity for M 3 receptors compared with M 2 (Esqueda et al, 1996). Consequently, we wondered whether another muscarinic antagonist with comparable selectivity for M 3 receptors over M 2 might exhibit a similar preference for the ileum relative to trachea.…”

Section: Resultsmentioning

confidence: 99%

“…Since both M 2 and M 3 muscarinic receptors have a contractile role in smooth muscle (for review, see Ehlert, 2003), and since p-FHHSiD exhibits approximately 16-fold higher affinity for M 3 muscarinic receptors over M 2 (Esqueda et al, 1996), it might seem that a greater contribution of the M 2 receptor to contraction in the trachea could explain the ileal selectivity of p-FHHSiD relative to trachea. However, we found that another muscarinic antagonist (4-DAMP) with 10-fold higher affinity for M 3 receptors over M 2 lacked ileal selectivity, which provides no support for the latter hypothesis.…”

Section: Discussionmentioning

confidence: 99%

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

Ehlert

Hsu²,

Leung³

et al. 2004

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We investigated the ability of the muscarinic antagonist pfluorohexahydrosiladifenidol to inhibit muscarinic agonist-induced contractions and phosphoinositide hydrolysis in the guinea pig ileum and trachea. This antagonist displayed higher potency at blocking oxotremorine-M-induced contractions of the ileum compared with those of the trachea. When estimated using a simple model for competitive antagonism, the observed dissociation constant of p-fluorohexahydrosiladifenidol exhibited approximately 12-fold higher potency in the ileum compared with the trachea. We also investigated the ability of p-fluorohexahydrosiladifenidol to affect the inhibition of contraction caused by the known competitive muscarinic antagonist atropine. Using resultant analysis to analyze this interaction, we found that the true dissociation constant of p-fluorohexahydrosiladifenidol for competitively antagonizing oxotremorine-M-induced contractions in the ileum exhibited significantly lower potency than when calculated assuming a simple competitive model. In contrast, resultant analysis showed little difference between the true and observed potencies of p-fluorohexahydrosiladifenidol for antagonizing oxotremorine-M-induced contractions in the trachea. Using a simple competitive model, we found little difference in the observed dissociation constant of p-fluorohexahydrosiladifenidol for antagonizing oxotremorine-M-induced phosphoinositide hydrolysis in guinea pig ileum and bovine trachea. We also noted that p-fluorohexahydrosiladifenidol (0.3-1.0 M) moderately inhibited histamine-induced contractions of ileum but not of trachea. Our results suggest that p-fluorohexahydrosiladifenidol does not discriminate markedly between M 3 muscarinic receptors in the ileum and trachea and that it may posses a more potent, nonmuscarinic inhibitory effect on contraction in the ileum.