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CELLULAR AND MOLECULAR

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

Department of Pharmacology, College of Medicine, University of California, Irvine, Irvine, California (F.J.E., D.M.A., T.H.V.); Department of Physical Sciences, Chapman University, Orange, California (M.T.G.); Department of Pharmacology, Graduate School of Medicine, Kyoto University, Sakyo-ku, Kyoto, Japan (M.M.T.); Division of Neuronal Network, Department of Basic Medical Sciences, the Institute of Medical Science, the University of Tokyo, Minato-ku, Tokyo, Japan (T.M.); and Laboratory of Biomedical Genetics, Graduate School of Pharmaceutical Sciences, the University of Tokyo, Bunkyo-ku, Tokyo, Japan (M.M.)

Received for publication September 14, 2004
Accepted November 11, 2004.

	Abstract

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We investigated the contractile role of M₂ 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 M₃ response in urinary bladder from wild-type and M₂ knockout (KO) mice. Evidence for a minor M₂ receptor-mediated contraction was revealed by a comparison of responses in M₃ knockout and M₂/M₃ double knockout mice. Treatment of wild-type and M₂ 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 M₂ knockout bladder compared with wild type, suggesting that M₂ receptors rescue the muscarinic contractile response in wild-type bladder following inactivation of M₃ receptors with 4-DAMP mustard. When measured in the presence of prostaglandin F₂ and isoproterenol or forskolin, oxotremorine-M mediated a potent contractile response in urinary bladder from M₃ KO mice. This response exhibited an M₂ profile in competitive antagonism studies and was completely absent in M₂/M₃ 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 M₂ KO mice. Our results show that the M₂ receptor mediates contractions indirectly in the urinary bladder by enhancing M₃ receptor-mediated contractions and inhibiting relaxation. We also show that it is difficult to detect M₂ receptor function in competitive antagonism studies under conditions where a simultaneous activation of M₂ and M₃ receptors occurs.

The signaling mechanisms of M₂ and M₃ muscarinic receptors in smooth muscle are consistent with their respective roles in contraction. The M₃ receptor interacts with G_q/11 to mediate phosphoinositide hydrolysis (Noronha-Blob et al., 1989; Candell et al., 1990; Roffel et al., 1990; Zhang and Buxton, 1991) and Ca²⁺ mobilization, which is essential for contraction, whereas the M₂ receptor interacts with G_i/o to mediate responses that are ultimately contingent upon activation of other Ca²⁺-mobilizing receptors, such as the M₃. For example, M₂ receptors mediate an inhibition of adenylyl cyclase (Noronha-Blob et al., 1989; Candell et al., 1990; Yang et al., 1991; Zhang and Buxton, 1991). In smooth muscle, cAMP causes relaxation (Conti and Adelstein, 1980; Kerrick and Hoar, 1981; Ruegg et al., 1981); thus, activation of the M₂ receptor has the potential to mediate an inhibition of the relaxant effects of forskolin or -adrenoceptors on contractions elicited through activation of a G_q-linked receptor. We refer to this type of contractile mechanism as indirect, because it represents an inhibition of relaxation and not a direct mediation of contraction. The M₂ receptor has also been shown to mediate an inhibition of Ca²⁺-activated K⁺ channels (Cole et al., 1989; Kume et al., 1992; Wade and Sims, 1993). Through this mechanism, the M₂ receptor would be expected to diminish the inhibitory effects of Ca²⁺-activated K⁺ channels on the contraction mediated by other Ca²⁺-mobilizing receptors. This potential muscarinic mechanism is also indirect, because M₂ receptor activation is expected to have little effect by itself but nonetheless enhances the effect of other contractile receptors. It has been demonstrated through mathematical modeling that a response mediated through an interaction between directly and indirectly acting receptors has a tendency to display the pharmacological profile of the directly acting receptor (i.e., M₃) in competitive antagonism studies and not that of the indirectly acting receptor (i.e., M₂) (Sawyer and Ehlert, 1999a; Ehlert, 2003b). This model explains why it is difficult, if not impossible, to detect a role for the M₂ receptor using competitive antagonists in experiments where a simultaneous activation of both M₂ and M₃ receptors occurs. This rationale also explains the large loss of the muscarinic contractile function in smooth muscle from M₃ KO mice. If the action of the M₂ receptor is contingent upon M₃ receptor activation, then the M₂ response will also be lost in the M₃ KO mouse.

Because of these limitations, a method was developed to isolate the indirect contractile response of the M₂ receptor from the direct response of the M₃ in competitive antagonism experiments on isolated smooth muscle (Thomas et al., 1993). The method involves first inactivating M₃ receptors with a selective nitrogen mustard derivative (4-DAMP mustard) and then measuring the contractile response to a muscarinic agonist in the presence of heterologous contractile (e.g., histamine) and relaxant (forskolin) agents. Using this approach, it has been demonstrated that M₂ receptors mediate an inhibition of the relaxant effect of forskolin, and in some instances isoproterenol, on histamine-induced contractions of the colon (Sawyer and Ehlert, 1998, 1999b), ileum (Thomas et al., 1993; Thomas and Ehlert, 1994), esophagus (Eglen et al., 1996), and trachea (Thomas and Ehlert, 1996; Ostrom and Ehlert, 1998, 1999) and also on KCl-induced contractions of rat urinary bladder (Hegde et al., 1997). However, no indirect mechanism for the M₂ receptor was detected in mouse urinary bladder using the same approach (Choppin and Eglen, 2001).

In the present report, we have investigated an indirect role for the M₂ receptor in mouse urinary bladder using tissue from wild-type, M₂ KO, M₃ KO, and M₂/M₃ KO mice. Our results are consistent with the postulate that the M₂ receptor mediates an inhibition of the relaxant effects of isoproterenol and forskolin on contractions mediated by prostaglandin FP and muscarinic M₃ receptors. We also obtained evidence that M₂ receptors enhance the contractile response to M₃ receptor activation in the absence of relaxant agents. Our results are consistent with a recent report demonstrating that the relaxant effects of isoproterenol and forskolin against muscarinic agonist-induced contractions are enhanced in urinary bladder from M₂ KO mice (Matsui et al., 2003).

	Materials and Methods

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Isolated Urinary Bladder. M₂ muscarinic receptor knockout (M₂-/-;M₂ KO), M₃ muscarinic receptor knockout (M₃-/-;M₃ KO), and M₂/M₃ muscarinic receptor double knockout (M₂-/-, M₃-/-; M₂/M₃ KO) mice were generated in a mixed background between 129/SvJ and C57BL/6 as described in Matsui et al. (2002) and Matsui et al. (2000), respectively. These hybrid lines were backcrossed with C57BL/6 mice to yield an N4 generation of M₂ KO mice, an N8 generation of M₃ KO mice, and an N2 generation of M₂/M₃ KO mice, which were used in the pharmacological studies described herein. Only male knockout and wild-type (M₂+/+, M₃+/+) C57BL/6 mice were used. The mice were euthanized by CO₂ asphyxiation, and the whole urinary bladder was excised and used in contractile studies.

In most experiments, the whole urinary bladder was mounted longitudinally in tissue baths (50-ml capacity) with silk thread attached at the apex and at the outlet of the urinary bladder. In some experiments, the bladder was cut in half, and each half was mounted longitudinally in a similar fashion. The tissues were bathed at 37°C in 50 ml of Krebs-Ringer bicarbonate (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; KRB) buffer containing 1 µM indomethacin and connected to force-displacement transducers. Isometric tension was recorded using a PowerLab (ADInstruments, Grand Junction, CO) recording system. Resting tension was adjusted to that generated by a mass of 1 g for the whole urinary bladder and to 0.5 g for the half urinary bladder strip. The tissues were allowed to equilibrate for at least 60 min prior to measurement of contractile responses. Three test doses of 50 mM KCl were applied to the tissues first, and subsequent contractile measurements were normalized relative to the third KCl test dose. Concentration-response curves to the muscarinic agonist oxotremorine-M were measured using a cumulative technique, essentially the same as that described previously (Matsui et al., 2003). The bladder was allowed to rest for at least 30 min between consecutive measurements of the concentration-response curve to oxotremorine-M. When present, competitive antagonists were incubated with the bladder for 30 min prior to the measurement of contractile responses. In experiments where the irreversible muscarinic antagonist 4-DAMP mustard was used, the compound was first incubated at 37°C for 30 min in 10 mM sodium-potassium phosphate buffer, pH 7.4, to allow formation of the reactive aziridinium ion, essentially as described previously (Thomas et al., 1992). Solutions of cyclized 4-DAMP mustard were kept on ice and used as soon as possible. Following treatment of the bladder with a combination of 4-DAMP mustard and AF-DX 116, the tissue was washed four times over a period of 30 min to remove AF-DX 116 and the transformation products of 4-DAMP mustard.

Calculations. An increasing logistic equation was fitted to the oxotremorine-M concentration-response curve by nonlinear regression analysis to estimate the maximal response (E_max), the concentration of oxotremorine-M eliciting a half-maximal response (EC₅₀), and the Hill slope, as described previously (Candell et al., 1990). The dissociation constant (K_B) of competitive antagonists were estimated in contractile studies using K_B = [B]/CR-1 (Arunlakshana and Schild, 1959), in which B denotes the concentration of the antagonist and CR denotes the EC₅₀ value of oxotremorine-M measured in the presence of the antagonist divided by that measured in its absence. The K_B and EC₅₀ values were estimated in molar units and then converted to negative logarithms before statistical analysis. In most instances, a two-tailed Student's t test was used to determine the statistical significance of differences between parameter estimates.

Drugs and Chemicals. The reagents used in this study were obtained from the following sources: AF-DX 116, Boehringer Ingleheim USA (Ridgefield, CT); oxotremorine-M, Sigma RBI (Natick, MA); atropine, isoproterenol, and PGF₂, Sigma-Aldrich (St. Louis, MO); and forskolin, Calbiochem (San Diego, CA). 4-DAMP was synthesized in our laboratory using a method similar to that described by Barlow et al. (1976), and 4-DAMP mustard was synthesized as described previously (Thomas et al., 1992).

	Results

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Contractile Activity of Oxotremorine-M and PGF_2a. Oxotremorine-M elicited contractions with high potency (pEC₅₀ = 6.54) in urinary bladder from wild-type mice (Fig. 1a). When expressed relative to the contraction elicited to 50 mM KCl, the E_max value was calculated to be 326%. Oxotremorine-M exhibited similar contractile activity in urinary bladder from M₂ KO mice, although the potency and E_max were both slightly lower [1.7-fold increase in EC₅₀ (P = 0.012) and a nonsignificant 11% decrease in E_max (P = 0.64)]. In contrast, the contractile response to oxotremorine-M was greatly inhibited in urinary bladder from M₃ KO mice. This effect was characterized by a highly significant (P = 4.3 x 10^-6) 85% reduction in the E_max without a significant change in EC₅₀. It was impossible to detect contractile responses to oxotremorine-M in urinary bladder from M₂/M₃ KO mice. The results in Fig. 1a are summarized in Table 1.

View larger version (22K): [in this window] [in a new window]   Fig. 1. The contractile effects of oxotremorine-M (a) and PGF2 (b) in urinary bladder from wild-type, M2 KO, M3 KO, and M2/M3 KO mice. The contractile responses are normalized relative to that elicited by 50 mM KCl. The data represent mean values ± S.E.M. The number of replicates for each experiment is indicated in Tables 1 and 2.

View this table: [in this window] [in a new window]   TABLE 2 Contractile activity of PGF2 in urinary bladder from wild-type, M2 KO, M3 KO, and M2/M3 KO mice The data were calculated from the experiments shown in Fig. 1B. Mean values ± S.E.M. are shown. The number of experiments is indicated in parentheses. Each experiment was done on urinary bladder from a different mouse.

We also investigated the effects of PGF₂ in the isolated urinary bladder (see Fig. 1b). In contrast to oxotremorine-M, PGF₂ was much less potent in urinary bladder from wild-type (pEC₅₀ = 5.48) and M₂ KO (pEC₅₀ = 5.23) mice (see Fig. 1b). Although we did not investigate high concentrations of PGF₂ that elicited clear maximal responses, the estimates of E_max by regression analysis in wild-type and M₂ KO mice were 89 and 86%, respectively. In urinary bladder from mice lacking M₃ receptors, PGF₂ was much more active. The pEC₅₀ and E_max values of PGF₂ in M₃ KO mice were 6.39 and 186%, respectively, and the corresponding values in M₂/M₃ KO mice were 6.60 and 138%. Thus, the loss of M₃ receptor function in the urinary bladder from male mice seems to trigger an increase in sensitivity to PGF₂. These results are summarized in Table 2.

Competitive Antagonism. We investigated the ability of M₂-selective (AF-DX 116) and M₃-selective (4-DAMP) muscarinic antagonists to inhibit the contractile response to oxotremorine-M in urinary bladder from wild-type and muscarinic receptor KO mice. At a concentration of 1 µM, AF-DX 116 only caused 3.0- and 2.1-fold shifts in the concentration-response curve to oxotremorine-M in urinary bladder from wild-type and M₂ KO mice. These data yield pK_B estimates of 6.28 and 6.01, respectively. In contrast, the same concentration of AF-DX 116 caused an 11-fold shift in the oxotremorine-M concentration response in urinary bladder from M₃ KO mice, yielding a pK_B estimate of 6.97. At a concentration of 10 nM, 4-DAMP shifted the oxotremorine-M concentration-response curve to the right 12- and 16-fold in urinary bladder from wild-type and M₂ KO mice, respectively, which yields pK_B estimates of 9.03 and 9.19. These data are summarized in Table 3 together with the binding affinities of AF-DX 116 and 4-DAMP at recombinant human M₂ and M₃ receptors. The pK_B values estimated in wild-type and M₂ KO mice agree with the binding affinity measured at M₃ receptors. In contrast, the pK_B value measured for AF-DX 116 in the M₃ KO mouse agrees with the binding affinity at M₂ receptors.

4-DAMP Mustard Treatment. It has been shown that treatment of cell lines and native tissues with the aziridinium ion of 4-DAMP mustard in combination with AF-DX 116 causes a selective, irreversible alkylation of the recognition site of M₃ receptors while having little effect on M₂ receptors (Thomas et al., 1992, 1993; Griffin et al., 2003). By itself, 4-DAMP mustard exhibits moderate selectivity for M₃ receptors over M₂, although it has the capacity to alkylate both receptors depending upon its concentration and length of incubation. By carrying out the incubation in the presence of the competitive, M₂-selective antagonist AF-DX 116, it is possible to increase the apparent selectivity of 4-DAMP mustard for M₃ receptors by protecting the M₂ with AF-DX 116. Consequently, we were interested in examining the influence of 4-DAMP mustard treatment on the muscarinic contractile response in urinary bladder from wild-type and muscarinic receptor KO mice. Because of the instability of the aziridinium ion, urinary bladders were given two consecutive 1-h treatments with 10 nM 4-DAMP mustard in combination with 1 µM AF-DX 116, with fresh drug solutions being used for the second treatment. The contractile activity of oxotremorine-M was assessed after each 1-h treatment. Treatment of urinary bladder from wild-type mice with 4-DAMP mustard caused a large shift to the right in the oxotremorine-M concentration-response curve after 1 h and a further shift to the right and decrease in E_max after 2 h of treatment (see Fig. 2). Similar effects were observed in experiments on urinary bladder from M₂ KO mice except that the inhibitory effects of 4-DAMP mustard were greater and characterized by a larger depression in the E_max of oxotremorine-M. In contrast, 4-DAMP mustard treatment had little influence on the contractile response to oxotremorine-M in the urinary bladder from M₃ KO mice. We did measure 2.4- and 2.7-fold shifts to the right in the oxotremorine-M concentration-response curve in these experiments after 1- and 2-h treatment with 4-DAMP mustard, respectively. However, control experiments showed that incubation of the mouse urinary bladder from M₃ KO mice with 1 µM AF-DX 116 only for 1 and 2 h followed by four washes over 30 min cause similar 3.3- and 4.7-fold shifts, respectively, in the concentration-response curves. In contrast, incubating the urinary bladder from M₃ KO mice in the absence of any drugs for several hours caused no change in its sensitivity to oxotremorine-M. Consequently, the small shift in the concentration-response curve to oxotremorine-M noted in urinary bladder from M₃ KO mice after 4-DAMP mustard treatment can be attributed to residual AF-DX 116 in the tissue and not to inactivation of muscarinic receptors with 4-DAMP mustard. The results in Fig. 2 are consistent with the postulate that the M₃ receptor mediates most of the direct contractile response in the urinary bladder from wild-type and M₂ KO mice, whereas the 4-DAMP mustard-insensitive M₂ receptor mediates contraction in the M₃ KO mouse urinary bladder. These results are summarized in Table 4.

View larger version (15K): [in this window] [in a new window]   Fig. 2. Effect of 4-DAMP mustard treatment on the contractile response to oxotremorine-M in urinary bladder from wild-type (a), M2 KO (b), and M3 KO (c) mice. Urinary bladder was treated with 10 nM 4-DAMP mustard in combination with 1 µM AF-DX 116 for 1 or 2 h, and the tissue was washed repeatedly as described under Materials and Methods. The contractile responses are normalized relative to that elicited by 50 mM KCl. The data represent mean values ± S.E.M. The number of replicates for each experiment is indicated in Table 4.

Although the contractile response to oxotremorine-M was greatly inhibited by 4-DAMP mustard in urinary bladder from both wild-type and M₂ KO mice, the response in M₂ KO mice was inhibited more. Figure 3a shows a plot of the concentration-response curves to oxotremorine-M in urinary bladder from wild-type and M₂ KO mice after 1- and 2-h treatment with 4-DAMP mustard. The larger size of the responses in wild-type bladder relative to those measured in M₂ KO bladder is readily apparent. These results suggest that the M₂ receptor partially rescues contraction in the urinary bladder from wild-type mice after most of the M₃ receptors have been inactivated with 4-DAMP mustard. To investigate the nature of the interaction between M₂ and M₃ receptors, we measured the competitive antagonism of contraction in wild-type urinary bladder after 2-h 4-DAMP mustard treatment. In these experiments, urinary bladder was first treated with 4-DAMP mustard for 2 h and washed, and then a concentration-response curve to oxotremorine-M was measured. The tissue was washed and incubated with AF-DX 116 or 4-DAMP for 30 min, and contractions were measured again in the presence of the antagonist. However, control experiments without the antagonist showed that there was some small recovery from 4-DAMP mustard treatment during the 30 min after measurement of the first concentration-response curve—the pEC₅₀ value of oxotremorine-M increased by 0.17 ± 0.071 log units (1.5-fold increase in potency), and the E_max increased by 33 ± 7%. Thus, in these experiments, the antagonist induced shifts were corrected for the slow recovery from 4-DAMP mustard blockade. Following 4-DAMP mustard treatment, 1 µM AF-DX 116 caused a 1.6-fold shift in the oxotremorine-M concentration response curve, whereas 10 nM 4-DAMP caused a 8.5-fold shift (see Fig. 3, b and c). When these shifts were corrected for recovery of the oxotremorine-M response, the calculated pK_B values of AF-DX 116 and 4-DAMP were 6.09 ± 0.098 and 9.06 ± 0.047, respectively. These values are in close agreement with the binding affinities of AF-DX 116 (pK_D = 6.10) and 4-DAMP (pK_D = 8.81) for recombinant human M₃ receptors but not M₂ receptors (i.e., 7.27 and 7.87, respectively). Thus, competitive antagonism experiments in wild-type urinary bladder after inactivation of a majority of the M₃ receptors with 4-DAMP mustard provides no evidence for a role of the M₂ receptor. Nevertheless, as described in the Introduction and under Discussion, the antagonist profile for a response mediated through a directly acting M₃ receptor and an indirectly acting M₂ receptor tends to resemble the profile of the M₃ receptor and not that of the M₂ receptor.

View larger version (19K): [in this window] [in a new window]   Fig. 3. Effect of 4-DAMP mustard treatment on the contractile response to oxotremorine-M in urinary bladder from wild-type and M2 KO mice. a, contractile response to oxotremorine-M measured in urinary bladder from wild-type and M2 KO mice after 1 and 2 h of 4-DAMP mustard treatment. The data are from Fig. 2. b, competitive antagonism of the contractile response to oxotremorine-M in wild-type mouse urinary bladder by 1 µM AF-DX 116 measured after 2 h of 4-DAMP mustard treatment. The data represent the mean contractile values ± S.E.M. from seven experiments, each done on urinary bladder from a different mouse. c, competitive antagonism of the contractile response to oxotremorine-M in wild-type mouse urinary bladder by 10 nM 4-DAMP measured after 2 h of 4-DAMP mustard treatment. The data represent the mean contractile values ± S.E.M. from four experiments, each done on urinary bladder from a different mouse. The contractile responses are normalized relative to that elicited by 50 mM KCl. For these experiments, 4-DAMP mustard treatment was accomplished by incubating urinary bladder with 10 nM 4-DAMP mustard in combination with 1 µM AF-DX 116 for the indicated times.

Effects of Isoproterenol and Forskolin. As described in the Introduction, it is possible to measure indirect, M₂ receptor-mediated contractions in guinea pig smooth muscle by first inactivating M₃ receptors with 4-DAMP mustard and then measuring contraction to a muscarinic agonist in the presence of both a contractile (e.g., histamine) and cAMP-generating relaxant agent (isoproterenol). Presumably, under these conditions, M₂ receptors mediate an inhibition of relaxation, thereby allowing histamine to elicit contraction. Consequently, we were interested in using urinary bladder from M₃ KO mice in this experimental paradigm. M₃ KO mice are devoid of M₃ receptors; thus, it should be possible to measure contractions mediated indirectly by the M₂ receptor without interference from the M₃ and without the necessity of 4-DAMP mustard treatment. The latter is never completely effective in eliminating the contractile response of the M₃ receptor. In these experiments, isolated urinary bladder from M₃ KO mice was first contracted with 1 µM PGF₂. After approximately 1 min, the contractile response to PGF₂ reached a stable plateau. At this time, 1 µM isoproterenol or 10 µM forskolin was added, which causes a complete relaxation of the PGF₂-induced contraction. Then, in the continued presence of PGF₂ and the relaxant agent, a cumulative concentration-response curve to oxotremorine-M was measured. Figure 4 shows the results of these experiments. In the presence of 1 µM PGF₂ and 1 µM isoproterenol, oxotremorine-M elicits a potent contractile response characterized by mean pEC₅₀ and E_max values ± S.E.M. of 6.87 ± 0.10 and 114 ± 10%, respectively (Fig. 4a). When forskolin was used as the relaxant agent, the pEC₅₀ and E_max values of oxotremorine-M were 6.58 ± 0.061 and 99 ± 18%, respectively. The E_max value of oxotremorine-M under these conditions was much greater than the E_max value of oxotremorine-M measured in the absence of PGF₂ and the relaxant agents (about 30–50%; Figs. 1a and 2c). Thus, most of the contraction in these experiments represents an oxotremorine-M-mediated inhibition of the relaxant effects of forskolin and isoproterenol on PGF₂-induced contractions and not simply a direct, M₂ receptor-mediated contraction. Oxotremorine-M was without effect in urinary bladder from M₂/M₃ KO mice under these conditions, indicating that the indirect contractile response in M₃ KO mouse urinary bladder was mediated by the M₂ receptor.

View larger version (18K): [in this window] [in a new window]   Fig. 4. Contractile effects of oxotremorine-M in the presence of PGF2 and isoproterenol (a) and forskolin (b) in urinary bladder from M3 KO and M2/M3 KO mice. The data represent the mean contractile values ± S.E.M. from five experiments on wild-type and three experiments on M2/M3 KO mice, each done on urinary bladder from a different mouse. The contractile responses are normalized relative to that elicited by 50 mM KCl.

Since the foregoing data demonstrate that the M₂ receptor in urinary bladder from M₃ KO mice mediates a substantial contraction, we predicted that M₂-selective antagonists should inhibit this response with high potency. To test this postulate, we measured the ability of 1 µM AF-DX 116 to inhibit the contractile response to oxotremorine-M in the presence of 0.6 to 1.0 µM PGF₂ and either 0.6 µM isoproterenol or 10 µM forskolin. When isoproterenol was used, AF-DX 116 caused a 6.9-fold shift in the concentration-response curve, whereas a 11.2-fold shift was noted when forskolin was present (Fig. 5). These shifts yield mean pK_B estimates ± S.E.M. of 6.77 ± 0.05 and 7.01 ± 0.076 for AF-DX 116 in experiments with isoproterenol and forskolin, respectively. These values are in closer agreement with the binding affinity of AF-DX 116 at M₂ muscarinic receptors (pK_D = 7.27) compared with that of M₃ receptors (pK_D = 6.10).

View larger version (19K): [in this window] [in a new window]   Fig. 5. Competitive antagonism of the contractile response to oxotremorine-M in M3 KO mouse urinary bladder by AF-DX 116 when measured in the presence of PGF2 and isoproterenol (a) or forskolin (b). The data represent the mean contractile values ± S.E.M. from four experiments with isoproterenol and three experiments with forskolin, each done on urinary bladder from a different mouse. The contractile responses are normalized relative to that elicited by 50 mM KCl.

We also investigated whether the M₂ receptor could mediate contractions under similar conditions in urinary bladder from wild-type mice that had been treated with 4-DAMP mustard to inactivate M₃ receptors. In these experiments, urinary bladder was incubated with 10 nM 4-DAMP mustard in combination with 1 µM AF-DX 116 for a total of 2 h as described under Materials and Methods. Contractions to oxotremorine-M were subsequently measured in the presence of KCl and either 1 µM isoproterenol (Fig. 6a) or 10 µM forskolin (Fig. 6b). Since PGF₂ exhibited low activity in urinary bladder from wild-type mice (see Fig. 1b), we used KCl as the heterologous contractile agent in these experiments. We found that isoproterenol and forskolin caused a complete inhibition of the KCl response when KCl was present at a concentration of 37.5 mM but no higher. Consequently, we used KCl at a concentration of 37.5 mM for these experiments. When measured after 4-DAMP mustard treatment and in the presence of KCl and isoproterenol, oxotremorine-M elicited contractions in urinary bladder from wild-type mice that were characterized by mean pEC₅₀ and E_max values ± S.E.M. of 6.19 ± 0.15 and 311 ± 21%, respectively. The corresponding values in urinary bladder from M₂ KO mice were smaller (5.57 ± 0.22 and 189 ± 45%, respectively). The reduction in agonist potency (4.2-fold increase in EC₅₀) in the M₂ KO mouse was statistically significant (P = 0.032), whereas the 39% decrease in E_max was not quite significant (P = 0.067). When measured under similar conditions in wild-type mice using forskolin as the relaxant agent, the mean pEC₅₀ and E_max values ± S.E.M. of oxotremorine-M were 5.89 ± 0.16 and 285 ± 37%, respectively. The corresponding values in urinary bladder from M₂ KO mice were smaller (5.55 ± 0.21 and 105 ± 27%, respectively). In these experiments, the 63% reduction in E_max in the M₂ KO mouse was statistically significant (P = 0.017), whereas the reduction in agonist potency (2.2-fold increase in EC₅₀) was not (P = 0.26).

View larger version (17K): [in this window] [in a new window]   Fig. 6. Contractile effects of oxotremorine-M in the presence of KCl and isoproterenol (a) and forskolin (b) in urinary bladder from wild-type and M2 KO mice following 2-h 4-DAMP mustard treatment. The data represent the mean contractile values ± S.E.M. from three experiments, each done on urinary bladder from a different mouse. The contractile responses are normalized relative to that elicited by 50 mM KCl. For these experiments, 4-DAMP mustard treatment was accomplished by incubating urinary bladder with 10 nM 4-DAMP mustard in combination with 1 µM AF-DX 116 for two 1-h time periods.

It is important to note that, with regard to the data in Fig. 6, the E_max values of oxotremorine-M measured in urinary bladder from wild-type mice greatly exceeded the initial contraction elicited by 37.5 mM KCl. This latter concentration of KCl elicited a contraction that was approximately 69 ± 8.7% of that elicited by the standard test concentration of 50 mM KCl. Thus, the oxotremorine-M-induced contractions in wild-type bladder shown in Fig. 6 cannot only be attributed to a simple reversal of relaxation but must also involve a substantial direct muscarinic contractile component, probably mediated in part through residual M₃ receptors not inactivated by 4-DAMP mustard.

We also measured the ability of AF-DX 116 and 4-DAMP to antagonize the contractile response to oxotremorine-M in wild-type mice under the conditions of the experiments shown in Fig. 6. When measured after 4-DAMP mustard treatment and in the presence of KCl and isoproterenol, 1 µM AF-DX 116 and 10 nM 4-DAMP shifted the oxotremorine-M concentration-response curve to the right 3.7- and 3.5-fold, respectively (Fig. 7a). These values represent shifts that were corrected for the time-dependent recovery of contraction after 4-DAMP mustard treatment as described above, in connection with the data shown in Fig. 3, b and c. The corresponding mean pK_B estimates ± S.E.M. of AF-DX 116 and 4-DAMP were 6.43 ± 0.10 and 8.40 ± 0.060, respectively. When these experiments were repeated under the same conditions, but with isoproterenol replaced with forskolin (see Fig. 7b), the mean pK_B values ± S.E.M. of AF-DX 116 and 4-DAMP were 6.45 ± 0.21 and 8.74 ± 0.090, respectively. The pK_B values of AF-DX 116 (approximately 6.4) and 4-DAMP (approximately 8.6) in these experiments were in closer agreement with their respective binding affinities (pK_D valules) for M₃ receptors (6.10 and 8.81) compared with M₂ receptors (7.27 and 7.87). Thus, although the data in Fig. 6 demonstrate a substantial loss of contractile function in the urinary bladder from M₂ KO mice, the pharmacological antagonism experiments provide little evidence for an M₂ response. As described under Discussion, these results can be rationalized by the nature of the interaction between M₂ and M₃ receptors.

View larger version (20K): [in this window] [in a new window]   Fig. 7. Competitive antagonism of the contractile response to oxotremorine-M in wild-type mouse urinary bladder by AF-DX 116 and 4-DAMP when measured following 2-h 4-DAMP mustard treatment in the presence of KCl and isoproterenol (a) or forskolin (b). The data represent the mean contractile values ± S.E.M. from three experiments, each done on urinary bladder from a different mouse. The contractile responses are normalized relative to that elicited by 50 mM KCl. For these experiments, 4-DAMP mustard treatment was accomplished by incubating urinary bladder with 10 M 4-DAMP mustard in combination with 1 µM AF-DX 116 for two 1-h time periods.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
Our results on the contractile effects of oxotremorine-M in urinary bladder are consistent with previous reports showing little loss of muscarinic contractile function in M₂ KO mice (Stengel et al., 2000; Matsui et al., 2002), a very large loss of function in M₃ KO mice (Matsui et al., 2000) and a complete loss of function in M₂/M₃ KO mice (Matsui et al., 2002). We also noted that male mice lacking M₃ muscarinic receptors exhibited a large increase in sensitivity to the contractile effects of PGF₂ in urinary bladder. Perhaps this increase in sensitivity may partially compensate for the substantial urinary bladder distension that occurs in male M₃ KO and M₂/M₃ KO mice (Matsui et al., 2000, 2002). In contrast, female mice lacking M₃ receptors do not exhibit urinary bladder distension (Matsui et al., 2000) nor an increased sensitivity to PGF₂ (data not shown). We have previously observed a modest increase in sensitivity to PGF₂ in ileum from M₂ KO and M₃ KO mice (Matsui et al., 2003).

As reported by others, we found that competitive antagonists inhibited the muscarinic contractile response in urinary bladder from wild-type and M₂ KO mice in a manner consistent with an M₃ response, whereas behavior consistent with an M₂ response was observed in tissue from the M₃ KO mouse (Matsui et al., 2000; Stengel et al., 2000). We also noted that the contractile response to oxotremorine-M in urinary bladder from wild-type and M₂ KO mice was greatly inhibited by 4-DAMP mustard treatment, whereas that observed in the M₃ KO mouse was unaffected. These results are consistent with the postulate that the M₃ receptor is the major muscarinic subtype generating the direct contractile response in wild-type and M₂ KO mice, because 4-DAMP mustard treatment (i.e., 10 nM 4-DAMP and 1 µM AF-DX 116) has been shown to inactivate M₃ receptors selectively while having little effect on M₂ receptors (Griffin et al., 2003).

Although 4-DAMP mustard treatment is effective in alkylating M₃ receptors with high selectivity, it is difficult to inactivate M₃ receptors completely with this agent. Furchgott analysis (Furchgott, 1966) of the contractile measurements in urinary bladder from M₂ KO mice after 1 and 2 h of 4-DAMP mustard treatment yields estimates of 8.5 and 1.5%, respectively, for the fraction of residual M₃ receptors mediating contraction. It may be impossible to inactive M₃ receptors much beyond this level using our 2-h treatment paradigm, because even a very small percentage of new receptors being transported to the sarcolemma during the wash period (30–40 min) could restore contractile function to the levels that we observe.

When measured after 4-DAMP mustard treatment, the contractile responses to oxotremorine-M were much greater in wild-type than in M₂ KO mice, particularly when oxotremorine-M was used in the concentration range of 0.05 to 1.5 µM (see Fig. 3a). This difference in contractile function between wild-type and M₂ KO mice amounted to 78 to 162% of the response to 50 mM KCl. It follows that the M₂ component of contraction in wild-type mice under these conditions is equivalent to 78 to 162% of the KCl-induced contraction. However, this M₂ component is much greater than the E_max value of oxotremorine-M in urinary bladder from M₃ KO mice, which was only 30 to 50% of the KCl response. Thus, the contractile mechanism of the M₂ receptor in wild-type urinary bladder seems much greater than the direct M₂ receptor-mediated contractions observed in M₃ KO mice. This situation suggests that the M₂ component in wild-type urinary bladder is not a direct contraction but rather an M₂ receptor-mediated enhancement in the contractile response of the M₃ receptor. If this M₂ mechanism is less potent than the direct contractile mechanism of the M₃ receptor, this condition could explain why this M₂ mechanism is more apparent after M₃ receptors in wild-type urinary bladder have been inactivated with 4-DAMP mustard. A related phenomenon has been reported in guinea pig colon. The muscarinic contractile response of this tissue exhibits an M₃ profile in competitive antagonism studies and is insensitive to pertussis toxin, which uncouples M₂ receptor-mediated responses (Sawyer and Ehlert, 1998, 1999b). However, after extensive inactivation of M₃ receptors, the residual muscarinic contractile response is greatly inhibited by pertussis toxin treatment, suggesting a role for the M₂ receptor. However, this residual contractile response exhibits an M₃ profile in competitive antagonism experiments, just like that of the wild-type mouse urinary bladder after 4-DAMP mustard treatment. As described above, this behavior is consistent with a model in which the M₂ receptor acts indirectly to enhance the direct contractile response of the M₃ receptor. Mathematical modeling shows that this interaction has a tendency to exhibit an M₃ profile in competitive antagonism studies (Sawyer and Ehlert, 1999b; Ehlert, 2003b).

Previous studies have demonstrated that it is possible to measure relatively pure M₂ muscarinic contractile responses in wild-type smooth muscle from guinea pigs by first inactivating M₃ receptors with 4-DAMP mustard and then measuring muscarinic agonist-induced contractions in the presence of histamine and isoproterenol or forskolin (Thomas et al., 1993). These contractions are pertussis toxin-sensitive (Thomas and Ehlert, 1994) and exhibit an M₂ profile in competitive antagonism experiments (Ehlert and Thomas, 1995). A limitation in this experiment is that it is difficult to eliminate the contractile response of the M₃ receptor completely. Consequently, we were interested in exploring this paradigm in M₃ KO mouse bladder, in which the problem of M₃ receptor-mediated contractions is obviously precluded. We found that oxotremorine-M mediated substantial contractions when measured in the presence of PGF₂ and either forskolin or isoproterenol in M₃ KO mouse urinary bladder. These contractions were completely eliminated in M₂/M₃ KO mouse bladder, indicating that they were mediated by the M₂ receptor. In addition, the M₂ selective antagonist AF-DX 116 potently antagonized these contractions. The contractile mechanism probably involves an M₂ receptor-mediated inhibition of the relaxant effect of isoproterenol and forskolin on PGF₂-mediated contractions. This M₂ mechanism should act to diminish relaxant effects on contractile receptors other than those for PGF₂ (i.e., prostaglandin FP). We have shown that the relaxant effects of isoproterenol and forskolin against muscarinic agonist-induced contractions are enhanced in M₂ KO mouse urinary bladder (Matsui et al., 2003). Collectively, these results demonstrate that muscarinic agonists activate both M₂ and M₃ receptors in urinary bladder and that activation of the M₂ receptor inhibits the relaxant effects of isoproterenol and forskolin on M₃ receptor-mediated contractions.

We were unable to demonstrate a role for the M₂ receptor in wild-type mouse urinary bladder in competitive antagonism studies after 4-DAMP mustard treatment, although there was a large loss of contractile function in M₂ KO mouse urinary bladder under the same conditions. These results can be explained by the nature of the interaction between M₂ and M₃ receptors under these conditions. Even in the presence of KCl and relaxant agents (Fig. 6), most of the M₂ contractile mechanism in our experiments on wild-type urinary bladder involves an enhancement in M₃ contractile function, either by inhibiting relaxation (Fig. 6) or directly enhancing M₃-mediated contractions (Figs. 2a and 3a). We have previously shown that this type of interaction exhibits an M₃ profile in competitive antagonism experiments (Sawyer and Ehlert, 1999b; Ehlert, 2003b). The paradigm used for the experiments shown in Fig. 6 has the capacity to reveal M₂ responses in competitive antagonism studies. However, to do so, it is necessary to inactivate M₃ receptors effectively and use a heterologous contractile agent that elicits a substantial contraction that is greatly inhibited by the relaxant agent. In our experiments, we were unable to generate sizable contractions to KCl that were sensitive to isoproterenol or forskolin. Moreover, we could not overcome this limitation through more effective inactivation of M₃ receptors. Our results show the utility of muscarinic KO mice for addressing muscarinic function in this situation.

Studies on human urinary bladder have shown an abundance of M₂ muscarinic receptors (Kories et al., 2003) and a large cholinergic component to the contractile response to electrical field stimulation (Sibley, 1984), suggesting a role for M₂ mechanisms in the contraction of human urinary bladder. Nevertheless, several investigators have suggested that cholinergic contractions are mediated exclusively by M₃ receptors, because the pharmacological antagonism of the muscarinic contractile response in human urinary bladder exhibits an M₃ profile (Fetscher et al., 2002). If one considers the possibility of two receptors, then the data are also not inconsistent with an M₂/M₃ interaction, in which the M₂ receptor enhances the direct contractile action of the M₃ receptor through a mechanism contingent upon M₃-receptor activation. As mentioned above, this model exhibits an M₃ profile in competitive antagonism studies (Ehlert, 2003a,b). Thus, our results showing a substantial indirect role for the M₂ receptor in mediating contraction of the urinary bladder suggest that muscarinic antagonists with high affinity for both M₂ and M₃ receptors may be more useful in the treatment of urinary incontinence in humans than antagonists with selectivity for the M₃ receptor only.

	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 a grant from Pfizer (F.J.E.); grants-in-aid for Scientific Research from the Ministry of Education, Science, Sports and Culture (M.M., M.M.T.); Industrial Technology Research Grant Program in 2000 and 2002 from the New Energy and Industrial Technology Development Organization of Japan (M.M.); Health and Labor Sciences Research grants on Research on Measures for Intractable Diseases from Ministry of Health, Labor and Welfare of Japan (M.M.); and a grant from the Organization for Pharmaceutical Safety and Research, Tokyo, Japan (M.M.T.).

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.104.077909.

ABBREVIATIONS: KO, knockout; 4-DAMP mustard, N-2-chloroethyl-4-piperidinyl diphenylacetate; 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; PGF₂, prostaglandin F₂; 4-DAMP, N,N-dimethyl-4-piperidinyl diphenylacetate.

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

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

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