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CELLULAR AND MOLECULAR

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

Department of Physical Sciences, Chapman University, Orange, California (M.T.G.); Laboratory of Biomedical Genetics, Graduate School of Pharmaceutical Sciences, University of Tokyo, Bunkyo-ku, Tokyo, Japan (M.M., M.M.T.); Department of Pharmacology, College of Medicine, University of California, Irvine, Irvine, California (D.S., K.Z.A., F.J.E.); and Division of Neural Network, Department of Basic Medical Sciences, University of Tokyo, Tokyo, Japan (T.M.)

Received June 6, 2003; accepted August 22, 2003.

	Abstract

Top Abstract Materials and Methods Results Discussion APPENDIX References

 
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 µM) for 20 min caused a subsequent decrease in contractile sensitivity to both prostaglandin F₂ (PGF₂) and the muscarinic agonist, oxotremorine-M. This subsensitivity was characterized by 7- and 3-fold increases in the EC₅₀ values of the agonists, respectively, with no significant effect on the maximal response. The subsensitivity to PGF₂ was prevented in both M₂ and M₃ muscarinic receptor knockout mice. Similarly, the subsensitivity to oxotremorine-M was prevented in M₂ knockout mice. Acetylcholine-mediated desensitization of histamine-induced contractions in the guinea pig ileum was inhibited by both M₂- and M₃-selective muscarinic antagonists with high potency, although careful analysis of the data suggested behavior more consistent with an M₂ 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 M₂ and M₃ muscarinic receptors and that activation of either receptor by itself is insufficient to cause desensitization.

In isolated ileum, subtype-selective muscarinic antagonists inhibit the contractile response to agonists in a manner consistent with an M₃ mechanism (Lambrecht et al., 1989; Eglen et al., 1996; Ehlert et al., 1997b). Such behavior is consistent with the coupling of M₃ muscarinic receptors to phosphoinositide hydrolysis and the mobilization of Ca²⁺ in smooth muscle (Lazareno and Roberts, 1989; Candell et al., 1990). However, it is known that the most abundant muscarinic receptor subtype expressed in smooth muscle is the M₂, outnumbering the M₃ by a ratio of at least 4 to 1 (Choo et al., 1985; Giraldo et al., 1987; Michel and Whiting, 1987, 1988; Choo and Mitchelson, 1988). The M₂ subtype has been shown to mediate pertussis toxin-sensitive responses, including an inhibition of adenylyl cyclase (Peralta et al., 1988; Candell et al., 1990; Thomas and Ehlert, 1994) and Ca²⁺-activated potassium channels (Kotlikoff et al., 1992) and an activation of nonselective cation channels (Unno et al., 1995; Bolton and Zholos, 1997). It has been argued that the contribution of the M₂ receptor to contraction through these signaling mechanisms is conditional upon Ca²⁺ mobilization through another receptor, like the M₃ (Ehlert et al., 1997a,b, 1999; Ehlert, 2003). Accordingly, M₂ receptor activation in smooth muscle has been shown to inhibit the relaxant effects of forskolin and isoproterenol on contractions mediated by M₃ muscarinic and H₁ histamine receptors (Thomas et al., 1993; Thomas and Ehlert, 1994; Ostrom and Ehlert, 1998, 1999; Matsui et al., 2003) and to potentiate contractions mediated by M₃ receptors at high concentrations of muscarinic agonists (Sawyer and Ehlert, 1998, 1999). Thus, both M₂ and M₃ receptors seem to mediate contraction, albeit through different mechanisms, and this conclusion is consistent with recent studies on M₂ and M₃ receptor knockout (KO) mice (Matsui et al., 2000; Stengel et al., 2000). Modeling studies have shown that the competitive antagonism of a muscarinic response mediated through an interaction between M₂ and M₃ receptors should resemble the pharmacological profile of the directly acting receptor (i.e., the M₃) and not that of the receptor causing a conditional potentiation (i.e., M₂) (Ehlert et al., 1999; Sawyer and Ehlert, 1999; Ehlert, 2003). This theory can explain why subtype-selective muscarinic antagonists competitively inhibit the contractile response to muscarinic agonists in a manner consistent with an exclusive M₃ mechanism even though contraction may be mediated through an interaction between both M₂ and M₃ receptors.

The involvement of both M₂ and M₃ muscarinic receptors in contraction is consistent with a recent report suggesting that muscarinic receptor-mediated heterologous desensitization in guinea pig ileum is conditional upon activation of both M₂ and M₃ receptors (Ehlert et al., 2001; Shehnaz et al., 2001). Inactivation of M₂ receptor signaling by pertussis toxin treatment largely prevented acetylcholine-mediated desensitization of histamine-induced contractions. Also, selective inactivation of M₃ receptors with N-2-chloroethyl-4-piperidinyldiphenylacetate (4-DAMP mustard) was found to prevent heterologous desensitization by acetylcholine. In this report, we have found that acetylcholine-mediated desensitization of prostaglandin F₂ (PGF₂) induced contractions in ileum is prevented in both M₂ and M₃ muscarinic receptor KO mice. We also investigated the pharmacological antagonism of muscarinic agonist-mediated heterologous desensitization in guinea pig ileum and found evidence for a role of both M₂ and M₃ receptors. Our results are generally consistent with, but not identical to, a mathematical model predicting that the competitive antagonism of heterologous desensitization should resemble the pharmacological profile of the least sensitive signaling pathway (i.e., the M₂) (Ehlert et al., 1999).

	Materials and Methods

Top Abstract Materials and Methods Results Discussion APPENDIX References

 
Isolated Ileum. Segments (approximately 2.5 cm) of ileum from male guinea pigs (Hartely, 300-450 g) were set up in organ baths and bathed in 50 ml of Krebs-Ringer bicarbonate (KRB) buffer (124 mM NaCl, 5 mM KCl, 1.3 mM MgSO₄, 26 mM NaHCO₃, 1.2 mM KH₂PO₄, 1.8 mM CaCl₂, and 10 mM glucose) at 37°C and gassed with O₂/CO₂ (19:1) as described previously (Thomas et al., 1993). Indomethacin (1.0 µM) was present in the KRB buffer at all times. The tissues were connected to force-displacement transducers, and isometric tension was recorded using either a Polygraph (Grass Instruments, Quincy, MA) or PowerLab (ADInstruments, Grand Junction, CO) recording system. Resting tension was equivalent to that generated by a load of 0.5 g. The tissues were allowed to equilibrate for at least 60 min before the initiation of experiments.

For experiments on desensitization in guinea pig ileum, three test doses of histamine (40 nM) were applied sequentially. After the first two test doses, the tissues were washed with fresh KRB buffer and allowed to rest for approximately 5 to 10 min. After the last test dose, the tissues were allowed to rest for 20 min and then a cumulative concentration-response curve to histamine was measured, with the agonist concentrations being spaced 3-fold. Only the tonic phase of contraction was used in the calculation of the data for concentration-response curves. The tissues were washed extensively and allowed to equilibrate for 30 min. In some experiments, a muscarinic antagonist was included during this incubation period. After 30 min, acetylcholine was added to the bath without changing the buffer, and the ensuing contractile response was measured. After 20 min of exposure to acetylcholine, the tissues were washed three times, and resting tension was restored within 1 to 2 min. Five minutes after the removal of acetylcholine, a cumulative concentration-response curve to histamine was measured. In a given experiment, a total of four different concentrations of acetylcholine were used. Each concentration of acetylcholine was tested under two conditions in separate ilea. These conditions corresponded to the absence (control) and presence of a muscarinic antagonist. Thus, in a single experiment, a total of eight segments of ileum were used from a single guinea pig.

In some experiments on guinea pig ileum, the contractile response to acetylcholine was measured in the absence and presence of the muscarinic antagonists, [[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5,11-dihydro-6H-pyrido[2,3b][1,4]-benzodiazepine-6-one (AF-DX 116) and N,N-dimethyl-4-piperidinyldiphenylacetate (4-DAMP). For these experiments, three test doses of acetylcholine were first applied to the ileum as described above for histamine. The ileum was allowed to equilibrate for 20 min and then a cumulative concentration-response curve to acetylcholine was measured with the concentrations spaced 3-fold. The tissues were washed extensively and allowed to equilibrate for 30 min in the presence of muscarinic antagonist. A subsequent concentration-response curve to acetylcholine was measured.

M₂ muscarinic receptor knockout (M₂-/-) and M₃ muscarinic receptor knockout (M₃-/-) mice were generated in a mixed background between 129/SvJ and C57BL/6 as described in Matsui et al. (2002, 2000), respectively. These hybrid lines were backcrossed with C57BL/6 mice to yield an N3 generation of M₂-/- muscarinic receptor knockout mice and an N8 generation of M₃-/- muscarinic receptor knockout mice, which were used in the pharmacological studies described in this report. Only male knockout mice were used as well as male wild-type (M₂+/+, M₃ +/+) C57BL/6 mice. The mice were euthanized by CO₂ asphyxiation, and segments of the whole ileum (1.5-2 cm in length) were excised starting at a point approximately 5 cm rostral from the ileoceacal junction. The ilea were mounted in organ baths containing KRB buffer as described above. Resting tension was equivalent to that generated by a load of approximately 0.35 g. The tissues were allowed to equilibrate for at least 60 min and then three test doses of agonist were applied sequentially. The agonist used for the test doses was that same as that used in the experiment on desensitization (i.e., PGF₂ or oxotremorine-M). After the first two test doses, the tissues were washed with fresh KRB buffer and allowed to rest for approximately 5 to 10 min. After the third test dose, the tissues were allowed to rest for 20 min and then a noncumulative concentration-response curve to the agonist was measured, with the agonist concentrations being spaced 3-fold and applied in increasing order. Only the tonic phase of contraction was used in the calculation of the data for concentration-response curves. After each concentration of agonist, the tissues were washed with fresh KRB buffer and allowed to rest for approximately 10 min. After the last concentration of agonist, the tissues were washed extensively and allowed to equilibrate for 20 min. Acetylcholine (30 µM) was added to the bath, and the ensuing contractile response was measured. After 20 min of exposure to acetylcholine, the tissues were washed extensively. Five minutes after the removal of acetylcholine, the contractile response to a single concentration of agonist was measured, and the tension was ultimately expressed as a percentage of the maximal response measured before acetylcholine treatment. In a given experiment, a total of four ileal segments were used from the same mouse. Because the contractile response to only a single concentration of agonist was measured in each ileum after acetylcholine treatment, this strategy enabled us to measure four-point concentration-response curves after acetylcholine treatment.

Radioligand Binding. Chinese hamster ovary cells expressing the human M₂ (CHO M₂) and M₃ (CHO M₃) muscarinic receptors were obtained from Mark Brann (Acadia Pharmaceuticals Inc., San Diego, CA). These two cell lines were cultured and harvested in growth media (Dulbecco's modified Eagle's medium, high glucose plus L-glutamine, 7% fetal calf serum, and 100 units/ml penicillin and streptomycin) supplemented with 225 µg/ml neomycin (G 418 sulfate) as described previously (Ehlert et al., 1996). The specific binding of the muscarinic antagonist [³H]N-methylscopolamine ([[³H]NMS) to homogenates of these cell lines was measured as described previously (Griffin et al., 2003). Briefly, cellular homogenate was incubated at 37°C for 1 h with [³H]NMS (0.5 nM) and various concentrations of 4-DAMP in a final volume of 1 ml containing modified KRB buffer (124 mM NaCl, 5 mM KCl, 3 mM MgSO₄,26 mM NaHCO₃, 10 mM Na/HEPES, pH 7.4). Binding in the presence of atropine (10 µM) was defined as nonspecific. Bound [³H]NMS was trapped by filtration of the incubation mixture over glass fiber filters (GF-B; Whatman, Maidstone, UK) using a cell harvester (Brandel Inc., Gaithersburg, MD). All assays were run in triplicate.

Calculations. The EC₅₀ value (concentration of agonist eliciting half-maximal contraction) and the maximal response (E_max) to agonists were estimated from the concentration-response data by nonlinear regression analysis using an increasing logistic equation as described previously (Candell et al., 1990). For each experiment, the estimates of EC₅₀ were converted to negative logarithms (pEC₅₀), and the effect of acetylcholine on the EC₅₀ value was calculated as the difference in pEC₅₀ values (i.e., log EC₅₀ shift). To assess whether acetylcholine treatment had a significant effect on the EC₅₀ value, a t distribution was used to determine whether the log EC₅₀ shift values were significantly different from zero. A paired t test was used to determine whether acetylcholine treatment had a significant effect on the E_max. To determine whether these log EC₅₀ shift values or changes in E_max in muscarinic receptor knockout mice were significantly different from those measured in wild-type animals, an unpaired t test was used. The dissociation constants of muscarinic antagonists (K_B values), measured by competitive antagonism of functional responses, were estimated using the following equation (Arunlakshana and Schild, 1959):

(1)

in which B denotes the concentration of antagonist, and CR denotes the ratio of the EC₅₀ value of the agonist measured in the presence of the antagonist divided by that measured in its absence.

The IC₅₀ value (concentration of inhibitor causing 50% displacement of specific [³H]NMS binding) and Hill coefficient for the competitive inhibition of [³H]NMS binding by 4-DAMP were estimated by nonlinear regression analysis using a decreasing logistic equation as described previously (Candell et al., 1990). The IC₅₀ values were corrected for the competitive effect of [³H]NMS using the standard equation for competitive inhibition:

(2)

in which K_i denotes the dissociation constant of the competitive inhibitor, and [[³H]NMS] denotes the molar concentration of [³H]NMS used in the binding assay.

Mathematical Modeling. Mathematical simulations were carried out to model the competitive antagonism of a response mediated through an interaction between two different types of receptors. The nature of the interaction was defined such that the response under consideration is contingent upon activation of both receptor types and that activation of either type by itself does not cause the response. This type of interaction was evaluated because agonist-mediated heterologous desensitization can be considered to be a response, and the results described below indicate that this response is conditional upon activation of both M₂ and M₃ muscarinic receptors. In our analysis, we assumed that occupation of M₂ and M₃ receptors by a muscarinic agonist generates a stimulus as defined by Stephenson (1956) and Furchgott (1966). Competitive antagonists were assumed to interfere with the stimulus in the standard manner as described by Arunlakshana and Schild (1959). We used a logistic function to describe the relationship between the signaling effect and the stimulus because this type of function yields the typical sigmoid relationship between effect and log concentration of agonist (Furchgott, 1966; Kenakin and Beek, 1982; Black and Leff, 1983). To simulate the interaction between receptors, we multiplied the two independent signals from each receptor together to obtain the combined response. We used multiplication as the mathematical equivalent of the interaction because if either independent signaling effect had a value of zero, then the product of the two effects would be equal to zero, and hence the combined response would equal zero. In other words, the combined response is conditional upon activation both types of receptors. The details of our calculations are described in the Appendix.

Drugs and Chemicals. The reagents used in this study were obtained from the following sources. Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA); G 418 sulfate (Omega Scientific, Tarzana, CA); [³H]NMS (PerkinElmer Life Sciences, Boston, MA); AF-DX 116 (Boehringer Ingelheim USA, Ridgefield, CT); oxotremorine-M (Sigma. RBI, Natick, MA); and atropine, histamine, and PGF₂, (Sigma-Aldrich, St. Louis, MO). 4-DAMP was synthesized in our laboratory using a method similar to that described by Barlow et al. (1976).

	Results

Top Abstract Materials and Methods Results Discussion APPENDIX References

 
Contractility Measurements in Muscarinic Receptor Knockout Mice. In our previous studies on the guinea pig ileum, we investigated the effects of acetylcholine treatment on the contractile sensitivity of the ileum to the heterologous agonist histamine. However, histamine does not contract the mouse ileum; consequently, we used PGF₂ as the heterologous agonist in our studies in mice. PGF₂ elicited contractions in ilea from wild-type, M₂ KO, and M₃ KO mice with mean pEC₅₀ values ± S.E.M. of 6.89 ± 0.13, 7.38 ± 0.054, and 7.18 ± 0.11; and E_max values, expressed relative to the contraction elicited by KCl (50 mM) of 140 ± 18, 191 ± 26, and 249 ± 34%, respectively. Both the potency and maximal effect of PGF₂ were greater in M₂ and M₃ receptor KO mice compared with wild-type mice (Table 1). These differences reached statistical significance with regard to the pEC₅₀ in M₂ KO mice (P = 0.015) and the E_max in M₃ KO mice (P = 0.030), but not in with regard to the pEC₅₀ in M₃ KO mice (P = 0.15) and the E_max in M₂ KO mice (P = 0.16). After exposure of the isolated ileum from wild-type mice to acetylcholine (30 µM) for 20 min, the potency of PGF₂ decreased about 7-fold when the EC₅₀ value was estimated 5 min after washout of acetylcholine (Fig. 1a). Acetylcholine treatment was without effect on the maximal response. Similar results were obtained in the presence of tetrodotoxin (1 µM) (data not shown). In contrast, treatment with acetylcholine (30 µM) for 20 min had no effect on the contractile activity of PGF₂ in M₂ and M₃ receptor KO mice (Fig. 1, b and c, respectively). These results are summarized in Table 1.

View this table: [in this window] [in a new window]   TABLE 1 Effects of acetylcholine-treatment on the contractile response to PGF2 in the isolated mouse ileum The data were calculated from the experiments shown in Fig. 1. Mean values ± S.E.M. from four wild-type, four M2 KO, and four M3 KO mice are shown.

View larger version (16K): [in this window] [in a new window]   Fig. 1. Effects of acetylcholine treatment on the contractile response to PGF2 in the isolated ileum from wild-type (a), M2 KO (b), and M3 KO (c) mice. The contractile responses to PGF2 before () and after () treatment with acetylcholine are shown. The data represent mean values ± S.E.M. from four experiments on each strain of mouse.

We also investigated the ability of acetylcholine treatment to cause a desensitization of contractions elicited to the muscarinic agonist oxotremorine-M in wild type and M₂ receptor KO mice. Oxotremorine-M elicited a potent contractile response in the isolated ileum of wild-type mice with a mean pEC₅₀ value ± S.E.M. of 6.84 ± 0.084. The estimate of the E_max value expressed relative to the contractile response to KCl (50 mM) was 241 ± 30%, respectively. In M₂ receptor KO mice, the potency of oxotremorine-M was significantly less, corresponding to a 2.6-fold increase in the EC₅₀ value (P = 0.009). There was no significant difference in the E_max value of oxotremorine-M in M₂ KO mice relative to wild type (P = 0.34). After exposure of the isolated ileum from wild-type mice to acetylcholine (30 µM) for 20 min, the potency of oxotremorine-M decreased about 3-fold when the EC₅₀ value was estimated 5 min after washout of acetylcholine (Fig. 2a). Acetylcholine treatment was without effect on the maximal response. In contrast, treatment with acetylcholine (30 µM) for 20 min had no effect on the contractile activity of oxotremorine-M in M₂ receptor KO mice (Fig. 2b). These results are summarized in Table 2.

View larger version (19K): [in this window] [in a new window]   Fig. 2. Effects of acetylcholine treatment on the contractile response to oxotremorine-M in the isolated ileum from wild-type (a) and M2 KO (b) mice. The contractile responses to oxotremorine-M before () and after () treatment with acetylcholine are shown. The data represent mean values ± S.E.M. from five wild-type and four M2 KO mice.

Competitive Antagonism of Heterologous Desensitization. We have previously shown that acetylcholine-mediated desensitization of histamine-induced contractions in the guinea pig ileum is prevented by pertussis toxin treatment or by selective inactivation of M₃ receptors with the irreversible antagonist 4-DAMP mustard. Because pertussis toxin treatment has been shown to inactivate M₂ muscarinic receptor signaling mechanisms in the ileum, our previous results in the guinea pig are consistent with those described above in mice, indicating that heterologous desensitization is conditional upon activation of both M₂ and M₃ muscarinic receptors. Consequently, we investigated the nature of the competitive inhibition of this response with M₂- and M₃-selctive muscarinic antagonists. In these experiments, the ability of various concentrations of acetylcholine to cause desensitization of histamine-induced contractions was investigated. The contractile activity of histamine was measured before and after treatment with acetylcholine for 20 min. It can be seen in Fig. 3 that acetylcholine caused a concentration-dependent shift to the right in the histamine concentration-response curve without affecting the maximal response (Fig. 3, a-d). These experiments were repeated with the M₃-selective muscarinic antagonist 4-DAMP (10 nM), being present during the treatment phase with acetylcholine (Fig. 3, e-h). It can be seen that 4-DAMP partially blocked the desensitizing effects of acetylcholine treatment. To examine the potency of acetylcholine for causing desensitization, we plotted the log shift in the EC₅₀ value of the histamine concentration-response curve against the log concentration of acetylcholine as shown in Fig. 4a. It can be seen that acetylcholine caused a maximal 4.9-fold shift in the histamine concentration-response curve. The pEC₅₀ value of acetylcholine for causing this desensitization was 6.32 ± 0.26. The M₃-selective antagonist 4-DAMP shifted the acetylcholine concentration-response curve for desensitization to the right 8.4-fold. The calculated pK_B value of 4-DAMP for antagonizing desensitization was 8.83 ± 0.25 (eq. 1). The Hill coefficient of the acetylcholine concentration-response curve for desensitization was unaffected by 4-DAMP. The Hill coefficient in controls was 0.68 and that in the presence of 4-DAMP was 0.66.

View larger version (27K): [in this window] [in a new window]   Fig. 3. Effects of 4-DAMP on acetylcholine-mediated desensitization of the contractile response to histamine in the guinea pig ileum. The contractile responses to histamine before () and after () treatment with acetylcholine are shown. Isolated ilea were treated with acetylcholine at concentrations of 0.03 µM (a and e), 0.3 µM (b and f), 3.0 µM (c and g), and 30 µM (d and h). Acetylcholine treatment was carried out in the absence (a-d) and presence (e-h) of 4-DAMP (10 nM). The data represent mean values ± S.E.M. from six experiments.

View larger version (12K): [in this window] [in a new window]   Fig. 4. Competitive antagonism of acetylcholine-mediated desensitization of the contractile response to histamine by 4-DAMP (a) and AF-DX 116 (b) in the guinea pig ileum. The log shift in the EC50 value of histamine for eliciting contraction is plotted against the concentration of acetylcholine. The shift in the EC50 value is defined as the ratio of the EC50 value of histamine measured after acetylcholine treatment divided by that measured before acetylcholine treatment. Acetylcholine treatment was carried out in the absence () and presence of 4-DAMP () and AF-DX 116 (). The data represent mean values ± S.E.M. from six experiments with 4-DAMP and five experiments with AF-DX 116.

We also carried out similar experiments with AF-DX 116 to investigate the ability of this M₂-selective muscarinic antagonist to inhibit acetylcholine-mediated desensitization of histamine-induced contractions. The results of these experiments are summarized in Fig. 4b. In this set of experiments, acetylcholine caused a maximal 5.0-fold shift in the histamine concentration-response curve, with the pEC₅₀ value of acetylcholine for this effect being 7.07 ± 0.17. These effects are similar to those observed in Fig. 4a, though not identical, presumably because of experimental variation. The M₂-selective muscarinic antagonist AF-DX 116 (1 µM) caused a 54-fold shift in the acetylcholine concentration-response curve for desensitization without affecting the maximal response. The calculated pK_B value of AF-DX 116 for blocking desensitization was 7.73 ± 0.16. The estimates of the pK_B values of AF-DX 116 and 4-DAMP determined in these experiments on the competitive antagonism of desensitization are listed in Table 3. The Hill coefficient of the acetylcholine concentration-response curve for desensitization was reduced by AF-DX 116 from a control value of 0.99 to a value of 0.49 in the presence of AF-DX 116. Analysis of variance showed that this change was almost significant (F_1,48 = 3.77; P = 0.058). We have no explanation for this effect of AF-DX 116 but assume that it may be related to experimental error, given the difficulties in measuring accurate EC₅₀ ratios at the extremes of the curves. It is also possible that the decrease in the Hill coefficient caused by AF-DX 116 is the result of a disequilibrium between acetylcholine and AF-DX 116 as described in Discussion.

Competitive Antagonism of Contraction. We investigated the competitive inhibition of acetylcholine-mediated contractions of the isolated guinea pig ileum by 4-DAMP and AF-DX 116. Acetylcholine elicited a potent contractile response in the ileum characterized by a pEC₅₀ value of 7.44 ± 0.046 and a maximal response of 3.0 ± 0.18 g. The M₃-selective antagonist 4-DAMP (10 nM) caused a 20-fold increase in the EC₅₀ value of acetylcholine, yielding a calculated pK_B value of 9.28 ± 0.046 (Fig. 5a). The M₂-selective antagonist AF-DX 116 (1 µM) caused a 3.5-fold increase in the EC₅₀ value of acetylcholine, which yields a calculated pK_B value for AF-DX 116 of 6.41 ± 0.057 (Fig. 5b). Comparison of these estimates of antagonist affinity with those made in the desensitization experiments shows that AF-DX 116 exhibited 40-fold greater selectivity for antagonizing desensitization compared with contraction. 4-DAMP exhibited the converse selectivity although the difference in potency was not as great. Table 3 shows a comparison of the pK_B values of the antagonists together with their binding affinities at recombinant human M₂ and M₃ muscarinic receptors.

View larger version (15K): [in this window] [in a new window]   Fig. 5. Competitive antagonism of the contractile response to acetylcholine by 4-DAMP (a) and AF-DX 116 (b) in the guinea pig ileum. The contractile responses to acetylcholine in the absence () and presence of 4-DAMP () and AF-DX 116 () are shown. The data represent mean values ± S.E.M. from six experiments with 4-DAMP and five experiments with AF-DX 116.

Radioligand Binding. We measured the binding affinity of 4-DAMP for human M₂ and M₃ muscarinic receptors expressed in CHO cells using the specific muscarinic antagonist [³H]NMS. Binding assays were run in a modified KRB buffer similar to that used in our functional assays. This approach enabled us to compare the dissociation constants of 4-DAMP measured in functional assays (K_B values) with those measured in binding assays (K_D values) under essentially identical conditions. 4-DAMP caused a concentration-dependent inhibition of the binding of a fixed concentration of [³H]NMS (0.5 nM) in both CHO M₂ and CHO M₃ cells. The pIC₅₀ values of 4-DAMP in CHO M₂ and CHO M₃ cells were estimated at 7.60 ± 0.028 and 8.24 ± 0.064, respectively. These values were corrected for the competitive effect of [³H]NMS to yield the calculated K_i values shown in Table 3. The Hill coefficients of the competition curves were estimated at 0.88 ± 0.091 and 0.90 ± 0.040, in CHO M₂ and CHO M₃ cells, respectively. We previously estimated the K_i values of AF-DX 116 in competition experiments with [³H]NMS using a modified KRB buffer (Esqueda et al., 1996). These K_i values of AF-DX 116 for CHO M₂ and CHO M₃ cells are also listed in Table 3, together with those for 4-DAMP.

Mathematical Modeling. During heterologous desensitization, prolonged receptor activation generates a stimulus that ultimately causes a deficit in contractile function. This deficit can be assessed at some later time after the period of stimulation. As long as the agonist or both agonist and antagonist are at equilibrium during the stimulation phase, it should be appropriate to apply Schild's null method (Arunlakshana and Schild, 1959) for competitive antagonism to estimate the dissociation constant of the antagonist for blocking desensitization. Such an approach is unaffected by the complex relationship between stimulus and response. Consequently, we used a mathematical modeling technique based on receptor theory to generate agonist concentration-response curves that should simulate acetylcholine-mediated desensitization. As described in the Appendix, the response was considered to be conditional upon activation of both M₂ and M₃ receptors. In these simulations, the dissociation constants of 4-DAMP and AF-DX 116 were assigned values equivalent to those estimated in binding assays at recombinant human M₂ and M₃ receptors (Table 3). Acetylcholine was assumed to bind with equal affinity to both the M₂ and M₃ muscarinic receptors; however, the sensitivity or potency of the signaling pathways for M₂ and M₃ receptors were varied relative to each other.

The results of some of our simulations are shown in Fig. 6. In Fig. 6a, the competitive antagonism of the response to acetylcholine by 4-DAMP (10 nM) and AF-DX 116 (1 µM) is shown when the signaling pathway of the M₂ receptor is 30-fold more sensitive than that of the M₃ receptor (i.e., K_E-M3/K_E-M2 = 30; see Appendix, eq. 2. Under these conditions, 4-DAMP causes a 6.7-fold dextral shift in the acetylcholine concentration-response curve, whereas that caused by AF-DX 116 is only 3.2-fold. These shifts yield calculated pK_B values of 8.74 and 6.34 for 4-DAMP and AF-DX 116, respectively, very similar to those expected for an M₃ response (i.e., 8.77 and 6.10, respectively; Table 3). In Fig. 6c, the competitive antagonism is shown when the signaling pathway of the M₃ receptor is 30-fold more sensitive than that of the M₂ receptor (i.e., K_E-M3/K_E-M2 = 0.033). Under these conditions, 4-DAMP causes a 2.0-fold dextral shift in the acetylcholine concentration-response curve, whereas that caused by AF-DX 116 is 19-fold. These shifts yield calculated pK_B values of 8.02 and 7.24 for 4-DAMP and AF-DX 116, respectively, very similar to those expected for an M₂ response (i.e., 7.85 and 7.27, respectively; Table 2). Figure 6b, shows the competitive antagonism when the signaling pathways of the M₂ and M₃ receptors are the same (i.e., K_E-M3/K_E-M2 = 1.0). Under these conditions, 4-DAMP causes a 4.1-fold dextral shift in the acetylcholine concentration-response curve, whereas that caused by AF-DX 116 is 9.7-fold. These shifts yield calculated pK_B values of 8.49 and 6.94 for 4-DAMP and AF-DX 116, respectively. These values are mid-way between those expected for M₂ and M₃ receptors.

View larger version (18K): [in this window] [in a new window]   Fig. 6. Simulation of the competitive antagonism of a response that is conditional upon activation of two different receptors. The theoretical concentration-response curves were calculated as described in the Appendix for control conditions () and in the presence of 4-DAMP (10 nM) () and AF-DX 116 (1.0 µM) (). a, M2 signaling pathway is 30-fold more sensitive than that of the M3 receptor (KE-M3/KE-M2 = 30). b, signaling pathways of the M2 and M3 receptors are the same (KE-M3/KE-M2 = 1.0). c, M2 signaling pathway is only 1/30th as sensitive as that of the M3 receptors (KE-M3/KE-M2 = 0.033).

Figure 7 shows how the antagonist induced-shift in the acetylcholine concentration-response curve is affected by the relative sensitivities of the signaling pathways of M₂ and M₃ receptors. In Fig. 7a, the shift in the agonist concentration response curve caused by 4-DAMP (10 nM) is plotted against the sensitivity of the M₂ signaling pathway relative to the M₃. It can be seen that when the sensitivity of the M₂ signaling pathway is much less than that of the M₃, the shift caused by 4-DAMP is relatively small and is consistent with that expected for an M₂ receptor-mediated response. However, as the sensitivity of the M₂ pathway increases relative to that of the M₃, the shift caused by 4-DAMP increases and attains a value expected for an M₃ receptor-mediated response when the sensitivity of the M₂ pathway is much greater than that of the M₃. In contrast, the shifts caused by AF-DX 116 (1 µM) show the opposite dependence on the relative sensitivities of the M₂ and M₃ signaling pathways (Fig. 7b). When the sensitivity of the M₂ signaling pathway is much less than that of the M₃, the shift caused by AF-DX 116 is relatively large and is consistent with that expected for an M₂ receptor-mediated response. However, as the sensitivity of the M₂ pathway increases relative to that of the M₃, the shift caused by AF-DX 116 decreases and reaches a minimum value equivalent to that expected for an M₃ response when the sensitivity of the M₂ pathway is high relative to M₃. The results in Figs. 6 and 7 show that the competitive antagonism of a response conditional upon activation of two receptors has a tendency to resemble that expected for the less sensitive receptor-signaling pathway.

View larger version (11K): [in this window] [in a new window]   Fig. 7. Simulation of the competitive antagonism of a response that is conditional upon activation of two different receptors. The theoretical data were calculated as described in the Appendix. The shift in the agonist concentration response curve caused by the M3-selective antagonist 4-DAMP (a) and the M2-selective antagonist AF-DX 116 (b) are shown as a function of the sensitivity of the M2 signaling mechanism expressed relative to that of the M3 receptor. The shift value denotes the EC50 of the agonist measured in the presence of the antagonist divided by that measured in its absence. The dotted lines in a and b denote the shifts expected for a response mediated by only one of the two receptor subtypes. The M2 sensitivity is expressed relative to that of the M3 receptor and is defined as KE-M3/KE-M2.

In the simulations just described, the absolute sensitivity of the M₃ signaling pathway was kept constant at a high value corresponding to a K_E-M3 value of 0.003. To investigate how the absolute sensitivity of the M₃ signaling pathway might influence the pharmacological antagonism, we carried our additional simulations in which the sensitivity of the M₃ pathway was reduced by 1/10th and 1/100th (i.e., K_E-M3 = 0.03 and 0.3, respectively). In these simulations, the sensitivity of the M₂ pathway was set to values 30-fold greater, the same, and 1/30th that of the M₃ pathway. The results of this analysis showed that the antagonist-induced shifts in concentration-response curves were essentially identical to those shown in Figs. 6 and 7. Thus, the pharmacological antagonism of a response contingent upon activation of two receptor subtypes depends on the relative sensitivities of the two receptor signaling pathways, but not on their absolute sensitivity.

	Discussion

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We have investigated heterologous desensitization because it may provide indirect evidence for the receptors mediating contraction (see the introduction) and because the phenomenon is more amenable to experimental manipulation compared with homologous desensitization. In heterologous desensitization, the receptors that are activated to produce a desensitizing stimulus are different from those that are activated to monitor the degree of desensitization. Therefore, it is possible to modify the receptors activated in stimulation phase covalently, competitively or genetically without affecting the receptors whose response is desensitized.

Our studies on M₂ and M₃ muscarinic receptor knockout mice are consistent with our previous work on heterologous desensitization in the guinea pig ileum. We have shown that acetylcholine-mediated desensitization of histamine-induced contractions is inhibited by pertussis toxin treatment (Shehnaz et al., 2001). We have shown that pertussis toxin treatment inhibits a variety of M₂ responses in smooth muscle without inhibiting those of the M₃ receptor (Thomas and Ehlert, 1994; Ostrom and Ehlert, 1999; Sawyer et al., 2000; Ehlert et al., 2001). We have also shown that acetylcholine-mediated heterologous desensitization of histamine-induced contractions in the guinea pig ileum is prevented by selective inactivation of M₃ muscarinic receptors with 4-DAMP mustard (Shehnaz et al., 2001). Thus, collectively, our data from the guinea pig ileum show that acetylcholine-mediated desensitization of histamine-induced contractions is conditional upon activation of both M₂ and M₃ muscarinic receptors. Our present findings using muscarinic receptor knockout mice are consistent with our previous conclusions from work on wild-type guinea pigs.

Responses to postjunctional, muscarinic stimulation in the isolated ileum can be attributed to two muscarinic subtypes: M₂ and M₃. These receptors are the most abundant muscarinic subtypes expressed in the ileum, and second messenger responses to muscarinic stimulation of the smooth muscle can be attributed to M₂ and M₃ receptors, but not other muscarinic subtypes (Candell et al., 1990; Ehlert et al., 1997b). Finally, the contractile response to muscarinic agonists is completely eliminated in ileum from mice lacking both M₂ and M₃ muscarinic receptors (Matsui et al., 2002). Consequently, we have interpreted our competitive antagonism studies within the confines of a two-receptor system. When considered from this perspective, the potency of 4-DAMP for antagonizing desensitization (pK_B = 8.83) is in good agreement with its binding affinity at M₃ receptors (pK_D = 8.81), which suggests an M₃ mechanism (Fig. 8a). Nevertheless, the potency of the M₂-selective antagonist AF-DX 116 for antagonizing desensitization (pK_B = 7.73) is actually three-fold higher than its binding affinity at the M₂ receptor (pK_D = 7.27), for which it exhibits highest affinity among muscarinic subtypes (Fig. 8a). Thus, these data show that both M₂- and M₃-selective agents antagonized desensitization with high affinity. This behavior conflicts with the model for a response conditional upon activation of both M₂ and M₃ receptors. The model predicts high affinity for either M₂- or M₃-selective antagonists, but not both. Thus, our data may indicate that either the model is inaccurate or else that the conditions upon which the model is based were not met under the conditions of our assays.

View larger version (22K): [in this window] [in a new window]   Fig. 8. Comparison of the dissociation constants of AF-DX 116 and 4-DAMP determined in binding assays and in functional assays on desensitization and contraction in the guinea pig ileum. a, estimates of the negative logarithm of the dissociation constants are taken from Table 3. b, dissociation constants of AF-DX 116 in the various assays is expressed relative to those of 4-DAMP.

This latter interpretation seems reasonable considering the potential for a lack of equilibrium due to the inherent constraints of the desensitization assay. In our experiments, the smooth muscle was first incubated with antagonist for 30 min to allow the antagonist to reach equilibrium with receptors and then acetylcholine was added for 20 min to elicit desensitization. The problem is, once acetylcholine is added, it takes the system a while to reach a new equilibrium in the presence of both acetylcholine and 4-DAMP. If stimulation of M₂ and M₃ muscarinic receptors by acetylcholine during the entire 20-min incubation is important for desensitization, then acetylcholine would need to compete the antagonist off muscarinic receptors quickly if equilibrium is to be achieved during most of the 20-min stimulation phase. Because highly potent muscarinic antagonists, such as 4-DAMP, dissociate slowly from the muscarinic receptor, it seems unlikely that equilibrium was achieved quickly. Thus, one might expect the pK_B values to be overestimated in the desensitization assay and that the degree of overestimation should be proportional to antagonist potency. This latter relationship is expected because slower dissociation is correlated with higher potency. Because the errors in the estimates of the pK_B values of both antagonists are likely to be in the same direction (i.e., too high), one might expect that the error in the estimate of their ratio would be less. Consequently, we compared the ratio of binding affinities of the antagonists at M₂ and M₃ muscarinic receptors with their corresponding ratio of K_B values for antagonizing desensitization. We estimate a 3.8-fold higher binding affinity of 4-DAMP relative to AF-DX 116 at recombinant M₂ muscarinic receptors, whereas a 470-fold ratio was estimated at M₃ receptors (Fig. 8b). With regard to desensitization, we found that 4-DAMP exhibited 12.6-fold greater potency at blocking desensitization relative to AF-DX 116. Thus, this potency ratio is in much closer agreement with an M₂ profile than an M₃, particularly when one considers that there is a greater propensity to overestimate the antagonistic potency of the higher affinity 4-DAMP compared with that of the lower affinity AF-DX 116. This relationship also suggests that the data are consistent with a model based on the assumption that heterologous desensitization is conditional upon activation of both M₂ and M₃ receptors and that the sensitivity of the M₂ pathway is less than that of the M₃.

Because the incubation conditions of the contractile assay were the same as those of the desensitization assay, it might be argued that the pK_B values of 4-DAMP (9.28) and AF-DX 116 (6.41) for antagonizing contraction are also overestimated. However, this situation is unlikely because of the high potency of acetylcholine for eliciting contraction (EC₅₀ value = 36 nM). It only requires less than 1% receptor occupancy of M₃ receptors for acetylcholine to elicit a half-maximal contraction in the guinea pig ileum. Thus, at the concentration used, 4-DAMP should occupy about the same number of receptors at equilibrium in the absence of acetylcholine as it does in the presence of the low concentrations of acetylcholine used in the contractile assay. In contrast, the potency of acetylcholine for eliciting desensitization was much less, particularly in those experiments where we examined the antagonistic effects of 4-DAMP (control EC₅₀ value = 0.48 µM). This situation implies a greater level of receptor occupancy by acetylcholine, and hence, a greater potential for disequilibrium for the reasons described above.

The competitive antagonism of desensitization suggests a role for the M₂ receptor, and that the sensitivity of the M₂ component of desensitization is less than that of the M₃ receptor. Because several studies on heterologous desensitization suggest that a deficit in contractile function downstream from the receptor arises because of depletion in the requirements for contraction (Hishinuma and Uchida, 1988), it seems likely that the receptors that mediate contraction might also mediate desensitization. In this regard, we have previously shown that M₂ receptors mediate a conditional potentiation of the contractile response to M₃ receptor activation (Sawyer and Ehlert, 1999). We have also shown that the potency of muscarinic agonists for eliciting this conditional potentiation via the M₂ receptor is less than that of the direct M₃-receptor mediated contractile response. Thus, it is not surprising that both M₂ and M₃ receptors mediate heterologous desensitization and that the M₂ receptor mediates its desensitizing action through a less potent mechanism than that of the M₃ receptor.

Although we have not addressed the mechanism for desensitization, previous studies on smooth muscle have described mechanisms that could be involved in the interaction between M₂ and M₃ receptors in eliciting heterologous desensitization. It is well known that Ca²⁺-activated K⁺ channels are abundantly expressed in smooth muscle and are activated indirectly upon muscarinic stimulation due to the rise in intracellular Ca²⁺ (Cole et al., 1989; Kume et al., 1992; Wade and Sims, 1993; Carl et al., 1995). These channels represent an inhibitory feedback mechanism that limits contraction and Ca²⁺ influx, and this inhibitory action may be responsible for preventing heterologous desensitization caused activation of M₃ receptors only. Activation of M₂ receptors causes an inhibition of Ca²⁺-activated K⁺ channels (Kotlikoff et al., 1992), and thus, enhances the response to M₃ receptor activation. This synergistic interaction between M₂ and M₃ receptors may cause excessive cellular activation and Ca²⁺ mobilization that ultimately results in heterologous desensitization.

	APPENDIX

Top Abstract Materials and Methods Results Discussion APPENDIX References

 
The equations for simulating the data shown in Figs. 6 and 7 were derived using an approach similar to that described previously (Ehlert et al., 1999). It is assumed that the desensitizing response (R_Des) is equivalent to the product of the responses to M₂ and M₃ muscarinic receptor stimulation:

(3)

In this equation, R₂ and R₃ denote the responses to stimulation of M₂ and M₃ receptors, respectively. The response (R)to stimulation of the individual receptors was expressed as a logistic function of the stimulus (S) as previously shown by Furchgott (1966), Kenakin and Beek (1982), and Black and Leff (1983):

(4)

In this equation, K_E denotes a constant representing the sensitivity of the signaling pathway, and n denotes an exponent influencing the slope of the concentration-response curve. In the text and figure legends, we use subscripts to the K_E value to discriminate between the sensitivities of the M₂ (K_E-M2) and M₃ (K_E-M3) muscarinic receptor signaling pathways. The value of K_E-M3 was set equal to 0.003 for the simulations shown in Figs. 6 and 7. The value of K_EM2 is described in the text and figure legends. To simplify matters, n was assigned a value of 1 in all of our simulations. The S value was calculated as the product of receptor occupancy and intrinsic efficacy as described by Furchgott and Bursztyn (1967):

(5)

For the simulations, the value of for acetylcholine was assigned a value of 1 for both M₂ and M₃ receptors. Receptor occupancy by acetylcholine (A) in the presence of antagonist (I) was described by the following equation:

(6)

in which, K_D denotes the dissociation constant of acetylcholine, and K_I denotes the dissociation constant of the antagonist. The K_D value of acetylcholine was set to 10 µM for both M₂ and M₃ receptors. The K_I values of AF-DX 116 for M₂ and M₃ muscarinic receptors were set to 0.054 and 0.79 µM, respectively, whereas the corresponding values for 4-DAMP were 14.2 and 1.69 nM, respectively. These values were estimated in ligand binding assays on Chinese hamster ovary cells transfected with human M₂ and M₃ muscarinic receptors (Table 3). The simulated data shown in Figs. 6 and 7 were calculated using eq. 3 with the variables defined as in eqs. 4, 5, 6 described above. The maximum of the concentration-response curve was normalized to the maximal response of the curve in the absence of the antagonist.

	Acknowledgements

 
We thank T. Tamai, T. Ishikawa, and K. Takaku for technical advice; I. Ishii, A. Matsunaga, and A. Yokoi for blastocyst injections; and Y. Araki, S. Kobayashi, N. Matsubara, H. Karasawa, D. Motomura, and S. Takahashi for technical assistance.

	Footnotes

 
This work was supported by National Institutes of Health Grant NS30882, by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture (to M.M. and M.M.T.), by Industrial Technology Research Grant Program in 2000 and 2002 from the New Energy and Industrial Technology Development Organization of Japan (to M.M.), and by Grant from Organization for Pharmaceutical Safety and Research, Japan (to M.M.T.).

DOI: 10.1124/jpet.103.055327.

ABBREVIATIONS: KO, knockout; 4-DAMP, N,N-dimethyl-4-piperidinyldiphenylacetate; PGF_2á, prostaglandin F_2á; KRB, Krebs-Ringer bicarbonate; AF-DX 116, [[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5,11-dihydro-6H-pyrido[2,3b][1,4]-benzodiazepine-6-one; CHO, Chinese hamster ovary; NMS, N-methylscopolamine.

¹ Current address: Division of Neuronal Network, Department of Basic Medical Sciences, the Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639, Japan.

² Current address: Banting and Best Department of Medical Research, University of Toronto, Ontario, Canada M5G1L6.

³ Current address: Department of Pharmacology, Graduate School of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan.

Address correspondence to: Dr. Frederick J. Ehlert, Department of Pharmacology, University of California, Irvine, Irvine, California 92697-4625. E-mail: fjehlert{at}uci.edu

	References

Top Abstract Materials and Methods Results Discussion APPENDIX References

 

Arunlakshana O and Schild HO (1959) Some quantitative uses of drug antagonists. Br J Pharmacol 14: 48-58.[Medline]

Barlow RB, Berry KJ, Glenton PA, Nilolaou NM, and Soh KS (1976) A comparison of affinity constants for muscarine-sensitive acetylcholine. Br J Pharmacol 58: 613-620.[Medline]

Black JW and Leff P (1983) Operational models of pharmacological agonism. Proc R Soc Lond B Biol Soc 220: 141-162.[Medline]

Bolton TB and Zholos AV (1997) Activation of M₂ muscarinic receptors in guinea-pig ileum opens cationic channels modulated by M₃ muscarinic receptors. Life Sci 60: 1121-1128.[CrossRef][Medline]

Candell LM, Yun SH, Tran LL, and Ehlert FJ (1990) Differential coupling of subtypes of the muscarinic receptor to adenylate cyclase and phosphoinositide hydrolysis in the longitudinal muscle of the rat ileum. Mol Pharmacol 38: 689-697.[Abstract]

Cantoni GL and Eastman G (1946) On the response of the intestine to smooth muscle stimulants. J Pharmacol Exp Ther 87: 392-399.[Abstract/Free Full Text]

Carl A, Bayguinov O, Shuttleworth CW, Ward SM, and Sanders KM (1995) Role of Ca²⁺-activated K⁺ channels in electrical activity of longitudinal and circular muscle layers of canine colon. Am J Physiol 268: C619-C627.

Choo LK, Leung E, and Mitchelson F (1985) Failure of gallamine and pancuronium to inhibit selectively (-)-[3H]quinuclidinyl benzilate binding in guinea-pig atria. Can J Physiol Pharmacol 63: 200-208.[Medline]

Choo LK and Mitchelson F (1988) Binding studies on two functional cardioselective antimuscarinic compounds. J Pharm Pharmacol 40: 288-289.[Medline]

Cole WC, Carl A, and Sanders KM (1989) Muscarinic suppression of Ca²⁺-dependent K current in colonic smooth muscle. Am J Physiol 257: C481-C487.

Dale MM (1958) An inhibitory effect of acetylcholine on the response of the guineapig ileum to histamine. Br J Pharmacol 13: 17-19.[Medline]

Eglen RM, Hegde SS, and Watson N (1996) Muscarinic receptor subtypes and smooth muscle function. Pharmacol Rev 48: 531-565.[Medline]

Ehlert FJ (2003). Pharmacological analysis of the contractile role of M₂ and M₃ muscarinic receptor in smooth muscle. Receptors Channels 9: 261-277.[CrossRef][Medline]

Ehlert FJ, Ansari KZ, Shehnaz D, Sawyer GW, and Griffin MT (2001) Acetylcholine-induced desensitization of muscarinic contractile response in guinea pig ileum is inhibited by pertussis toxin treatment. J Pharmacol Exp Ther 299: 1126-1132.[Abstract/Free Full Text]

Ehlert FJ, Griffin MT, and Glidden PF (1996) The interaction of the enantiomers of aceclidine with subtypes of the muscarinic receptor. J Pharmacol Exp Ther 279: 1335-1344.[Abstract/Free Full Text]

Ehlert FJ, Ostrom RS, and Sawyer GW (1997a) Subtypes of the muscarinic receptor in smooth muscle. Life Sci 61: 1729-1740.[CrossRef][Medline]

Ehlert FJ, Sawyer GW, and Esqueda EE (1999) Contractile role of M₂ and M₃ muscarinic receptors in gastrointestinal smooth muscle [published erratum in Life Sci 1999;64:2535]. Life Sci 64: 387-394.[CrossRef][Medline]

Ehlert FJ, Thomas EA, Gerstin EH, and Griffin MT (1997b). Muscarinic receptors and gastrointestinal smooth muscle, in Muscarinic Receptor Subtypes in Smooth Muscle (Eglen RM ed) pp 92-147, CRC Press, Boca Raton, FL.

Esqueda EE, Gerstin EH Jr, Griffin MT, and Ehlert FJ (1996) Stimulation of cyclic AMP accumulation and phosphoinositide hydrolysis by M₃ muscarinic receptors in the rat peripheral lung. Biochem Pharmacol 52: 643-658.[CrossRef][Medline]

Furchgott RF (1966) The use of -haloalkylamines in the differentiation of receptors and in the determination of dissociation constants of receptor-agonist complexes. Adv Drug Res 3: 21-55.

Furchgott RF and Bursztyn P (1967) Comparison of dissociation constants and of relative efficacies of selected agonists acting on parasympathetic receptors. Ann NY Acad Sci 144: 882-899.[CrossRef]

Giraldo E, Monferini E, Ladinsky H, and Hammer R (1987) Muscarinic receptor heterogeneity in guinea pig intestinal smooth muscle: binding studies with AF-DX 116. Eur J Pharmacol 141: 475-477.[CrossRef][Medline]

Griffin MT, Hsu JC-H, Shehnaz D, and Ehlert FJ (2003) Comparison of pharmacological antagonism of M₂ and M₃ muscarinic receptors expressed in isolation and in combination. Biochem Pharmacol 65: 1227-1241.[CrossRef][Medline]

Hishinuma S and Uchida MK (1988) Short-term desensitization of guinea-pig taenia caecum induced by carbachol occurs at intracellular Ca stores and that by histamine at H₁-receptors. Br J Pharmacol 94: 882-889.[Medline]

Kenakin TP and Beek D (1982) In vitro studies on the cardiac activity of prenalterol with reference to use in congestive heart failure. J Pharmacol Exp Ther 220: 77-85.[Free Full Text]

Kotlikoff MI, Kume H, and Tomasic M (1992) Muscarinic regulation of membrane ion channels in airway smooth muscle cells. Biochem Pharmacol 43: 5-10.[CrossRef][Medline]

Kume H, Graziano MP, and Kotlikoff MI (1992) Stimulatory and inhibitory regulation of calcium-activated potassium channels by guanine nucleotide-binding proteins. Proc Natl Acad Sci USA 89: 11051-11055.[Abstract/Free Full Text]

Lambrecht G, Feifel R, Moser U, Wagner-Roder M, Choo LK, Camus J, Tastenoy M, Waelbroeck M, Strohmann C, Tacke R, et al. (1989) Pharmacology of hexahydrodifenidol, hexahydro-sila-difenidol and related selective muscarinic antagonists. Trends Pharmacol Sci Suppl: 60-64.

Lazareno S and Roberts FF (1989) Functional and binding studies with muscarinic M₂-subtype selective antagonists. Br J Pharmacol 98: 309-317.[Medline]

Matsui M, Griffin MT, Shehnaz D, Taketo MM, and Ehlert FJ (2003) Increased relaxant action of forskolin and isoproterenol against muscarinic agonist-induced contractions in smooth muscle from M₂ receptor knockout mice. J Pharmacol Exp Ther 305: 106-113.[Abstract/Free Full Text]

Matsui M, Motomura D, Fujikawa T, Jiang J, Takahashi S, Manabe T, and Taketo MM (2002) Mice lacking M₂ and M₃ muscarinic acetylcholine receptors are devoid of cholinergic smooth muscle contractions but still viable. J Neurosci 22: 10627-10632.[Abstract/Free Full Text]

Matsui M, Motomura D, Karasawa H, Fujikawa T, Jiang J, Komiya Y, Takahashi S, and Taketo MM (2000) Multiple functional defects in peripheral autonomic organs in mice lacking muscarinic acetylcholine receptor gene for the M₃ subtype. Proc Natl Acad Sci USA 97: 9579-9584.[Abstract/Free Full Text]

Michel AD and Whiting RL (1987) Direct binding studies on ileal and cardiac muscarinic receptors. Br J Pharmacol 92: 755-767.[Medline]

Michel AD and Whiting RL (1988) Methoctramine reveals heterogeneity of M₂ muscarinic receptors in longitudinal ileal smooth muscle membranes. Eur J Pharmacol 145: 305-311.[CrossRef]