QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.106.103093v1 318/2/649    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Tran, J. A. Articles by Ehlert, F. J. Search for Related Content PubMed PubMed Citation Articles by Tran, J. A. Articles by Ehlert, F. J.

CELLULAR AND MOLECULAR

Differential Coupling of Muscarinic M₁, M₂, and M₃ Receptors to Phosphoinositide Hydrolysis in Urinary Bladder and Longitudinal Muscle of the Ileum of the Mouse

Department of Pharmacology, College of Medicine, University of California, Irvine, California (J.A.T., F.J.E.); and Division of Neuronal Network, Department of Basic Medical Sciences, the Institute of Medical Science, the University of Tokyo, Minato-ku, Tokyo, Japan (M.M.)

Received February 16, 2006; accepted May 3, 2006.

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
We investigated the coupling of muscarinic receptor (M) subtypes to phosphoinositide hydrolysis in ileum and urinary bladder using muscarinic receptor knockout mice. In urinary bladder from wild-type mice, the muscarinic agonist oxotremorine-M, elicited a robust phosphoinositide response characterized by an EC₅₀ value of 0.22 µM and a maximal response (E_max) of 32.8% conversion of [³H]inositol-labeled phosphoinositides into [³H]inositol phosphates. A similar response was observed in urinary bladder from M₂ knockout mice, whereas no measurable response was observed in urinary bladder from M₃ and M₂/M₃ knockout mice. In ilea from wild-type and M₂ knockout mice, substantial phosphoinositide responses to oxotremorine-M were measured, characterized by EC₅₀ values of 0.37 and 0.52 µM and E_max values of 35.8 and 34.7%, respectively. Oxotremorine-M also elicited phosphoinositide hydrolysis in ilea from M₃ and M₂/M₃ knockout mice, although these responses were less sensitive (EC₅₀ values of 1.6 and 1.4 µM; E_max values of 31.2 and 20.8%, respectively). The response in ileum from the M₂/M₃ knockout was significantly smaller than that from the M₃ knockout. The muscarinic phosphoinositide response in ilea from M₂/M₃ knockout mice originated in the smooth muscle and exhibited a profile for competitive antagonism consistent with an M₁ mechanism. These data suggest a major role for the M₃ receptor in eliciting phosphoinositide hydrolysis in the ileum and urinary bladder and minor roles for the M₁ and M₂ in ileum.

Muscarinic receptors belong to the G protein-coupled receptor family, which elicit cellular responses by interacting with heterotrimeric G proteins (Stryer and Bourne, 1986). When transfected into cell lines, the M₁, M₃, and M₅ receptors couple to G_q to mediate phosphoinositide hydrolysis, whereas the M₂ and M₄ receptors couple to G_i/o to mediate an inhibition of adenylyl cyclase (Bonner et al., 1988; Peralta et al., 1988). A similar picture emerges from pharmacological studies on smooth muscle. The muscarinic phosphoinositide response is antagonized in a manner consistent with an M₃ receptor mechanism (Noronha-Blob et al., 1989; Candell et al., 1990), whereas muscarinic inhibition of adenylyl cyclase exhibits an M₂ profile in competitive antagonism studies (Noronha-Blob et al., 1989; Candell et al., 1990). The coupling of the M₃ receptor to phosphoinositide hydrolysis in smooth muscle is consistent with its direct role in mediating contraction, presumably through mobilization of Ca²⁺. Likewise, the coupling of M₂ receptors to inhibition of adenylyl cyclase is consistent with its role in mediating an inhibition of the relaxant effect of agents that increase cAMP levels (Thomas et al., 1993; Sawyer and Ehlert, 1998). Activation of M₂ receptor has also been shown to mediate an increase in a nonselective cationic conductance (Bolton, 1979) and an inhibition of Ca²⁺-activated K⁺ channels (Cole et al., 1989), which might explain how M₂ receptors mediate an enhancement in contractions elicited by the M₃ receptor.

In most prior studies on muscarinic signaling mechanism in smooth muscle, a pharmacological approach has been used to identify the coupling of receptor subtypes to specific coupling mechanisms. However, the pharmacological approach has limitations in situations where more than one receptor contributes to the response. Consequently, we used muscarinic receptor knockout mice to investigate the coupling of muscarinic receptor subtypes to phosphoinositide hydrolysis in the smooth muscle of mouse ileum and urinary bladder. Our findings demonstrate that, in the urinary bladder, only the M₃ receptor couples to phosphoinositide hydrolysis, whereas in the longitudinal muscle of the mouse ileum, significant contributions of the M₁ and M₂ receptor are apparent in addition to a major role for the M₃ receptor.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Mice. M₂ receptor knockout (M₂^-/-; M₂ KO), M₃ receptor knockout (M₃^-/-; M₃ KO), and M₂/M₃ receptor double knockout (M₂^-/- and M₃^-/-; M₂/M₃ KO) mice were generated as described in Matsui et al. (2000, 2002). 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. Only male knockout mice were used as well as male wild-type (M₂^+/+, M₃^+/+) C57BL/6 mice.

Phosphoinositide Hydrolysis. Our method is based on the [³H]inositol-labeling method of Berridge et al. (1982) and the tissue extraction method of Kendall and Hill (1990). Segments of ileum (10 cm) from one to two mice were removed and washed with Krebs' Ringer bicarbonate buffer (KRB; 124 mM NaCl, 5 mM KCl, 1.3 mM MgCl₂, 26 mM NaHCO₃, 1.2 mM KH₂PO₄, 1.8 mM CaCl₂, and 10 mM glucose). The longitudinal muscle was isolated by rubbing off the outer layer of the ileum with a cotton swab. The muscle was cut into small strips (0.5 cm). The urinary bladders from two mice were removed, washed with KRB buffer, and trimmed of excess fat. Each bladder was cut into nine to 10 strips. The pooled strips of tissue from ileum and urinary bladder were incubated separately in 5 ml of KRB buffer containing myo-[³H]inositol (100 µCi) in a 50-ml Erlenmeyer flask. The flask was gassed with O₂/CO₂ (19:1), capped with a rubber stopper, and incubated at 37°C for 90 min. The strips were washed with fresh KRB buffer and put into conical tubes containing KRB buffer, 10 mM LiCl, and 0.1 µM tetrodotoxin in a final volume of 0.3 ml. One strip of tissue was added to each tube, except in initial experiments on the ileum where two strips were used. The reaction was started with the addition of oxotremorine-M, and the mixture was gassed with O₂/CO₂, capped with a rubber stopper, and incubated at 37°C for 30 min. The reaction was stopped with 5% perchloric acid (200 µl), and the tubes were placed on ice. [³H]Inositol phosphates were isolated as described previously (Ehlert et al., 2001).

When the competitive effects of pirenzepine (0.2 µM) and AF-DX 116 (2 µM; [[2-[(diethylamino) methyl]-1-piperidinyl]acetyl]-5,11-dihydro-6H-pyrido[2,3b][1,4]-benzodiazepine-6-one) were investigated, these antagonists were preincubated with the oxotremorine-M in the absence of LiCl for 30 min at 37°C to allow equilibration. LiCl (final concentration of 10 mM) was added, and the incubation was allowed to proceed for 30 min at 37°C. The reaction was then stopped with 5% perchloric acid (200 µl) as described above.

Radioligand Binding. The binding of the muscarinic antagonist [³H]N-methylscopolamine ([³H]NMS) was measured as described in Thomas and Ehlert (1994). The urinary bladder and longitudinal muscle of the ileum were isolated as described under Phosphoinositide Hydrolysis. The tissue was minced with scissors and homogenized with a Polytron homogenizer at setting 5 in 20 ml of a binding buffer containing 100 mM NaCl, 30 mM NaHEPES, pH 7.4, 0.5 mM EGTA, and 0.5 mM dithiothreitol The homogenates were centrifuged 30,000g for 10 min. The pellet was resuspended to a concentration of 5 mg (original wet weight/milliliter in fresh binding buffer). An aliquot (0.7 ml) of the tissue homogenate was added to glass tubes containing the binding buffer, 0.5 nM [³H]NMS, and other drugs in a final volume of 1.0 ml. [³H]NMS bound to the membrane of the homogenates was trapped on glass-fiber filters (Whatman Inc., Clifton, NJ) by vacuum filtration on a cell harvester (Brandel, Gaithersburg, MD). The filters were washed four times with aliquots (4 ml) of ice-cold saline. Nonspecific binding was defined as binding in the presence of atropine (10 µM). When hypotonic buffer was used, it contained 1.5 mM MgSO₄, 20 mM HEPES/Tris, pH 7.5, and 0.5 mM dithiothreitol.

In Vivo Pertussis Toxin Treatment. C57BL/6 male mice (20-40 g) were injected i.p. with a dose of pertussis toxin (islet-activating protein; 70-100 µg/kg body weight) 3 days prior to experimentation.

Dissociated Smooth Muscle. Ileal longitudinal muscle was dissociated by a method similar to Yang et al. (1991), with adaptations. The longitudinal muscle was obtained as described above. The strips were incubated in Medium 199 (5 ml; Invitrogen, Carlsbad, CA) containing 0.2% collagenase II and 0.01% DNase I in a 50-ml centrifuge tube for 90 min at 37°C and gassed with O₂/CO₂ (19:1). The tissue was also incubated with myo-[³H]inositol (100 µCi) for phosphoinositide hydrolysis assays. The tissue was triturated with a disposable Pasteur pipette to release smooth muscle cells (5 min). The mixture was filtered through a 500-µm Nitex mesh to remove connective tissue and undissociated tissue. The dissociated muscle cells were washed twice by centrifugation at 300g for 5 min. The cells were incubated in enzyme-free KRB for 1 h at 37°C between the first and second centrifugation. Afterward, the cells were suspended in a volume of 2 ml, and aliquots (0.3 ml) of the cell suspension were added to assay tubes. An aliquot (3 µl) of LiCl (1 M) was added followed by an aliquot (3 µl) of oxotremorine-M (10 mM), and phosphoinositide hydrolysis was measured as described earlier.

Analysis of the Loss of Function in Knockout Mice. We developed a simple method to estimate the contribution of muscarinic receptor subtypes to the phosphoinositide hydrolysis in smooth muscle. This analysis is based on the assumption that there is no compensatory change in responses elicited by residual muscarinic receptors in tissue from knockout mice. We observed that the phosphoinositide response to oxotremorine-M exhibited a logistic concentration-response curve with a Hill slope similar to one in ileum and urinary bladder. Consequently, we used the operational model to describe the stimulus-response function (Black and Leff, 1983):

(1)

in which S denotes the total stimulus, M_sys denotes the maximal response of the system, and K_E denotes a constant representing the sensitivity of the stimulus-response function. We assume that the degree of receptor activation is proportional to the stimulus as defined by Furchgott (1966). The total stimulus is equivalent to the summation of the stimuli generated by each receptor contributing to the response and is given by,

(2)

in which _i and K_i denote the intrinsic efficacy and dissociation constant of oxotremorine-M at receptor subtype R_i, respectively, and n denotes the number of receptor subtypes contributing to the response. When used in this context, _i denotes the ability of oxotremorine-M to activate the receptor as well as the ability of the receptor to trigger phosphoinositide hydrolysis in smooth muscle. We assume that each receptor acts independently to generate a stimulus but that the independent stimuli converge on the same effector system. As described under Results, our data suggest that the M₁, M₂, and M₃ receptor subtypes contribute to the phosphoinositide response in ileal smooth muscle. It has been shown that the dissociation constants of oxotremorine-M for M₁ (2.5 µM; Mei et al., 1991), M₂ (3.9 µM; Ehlert, 1985), and M₃ (2.9 µM; Ringdahl, 1985) receptors are approximately the same. Consequently, we assumed that the dissociation constant of oxotremorine-M for these subtypes was the same, which leads to the following simplification of eq. 2,

(3)

in which, K_X denotes the dissociation constant of oxotremorine-M for muscarinic receptor subtypes eliciting phosphoinositide hydrolysis. Substitution of eq. 3 into 1 yields

(4)

This equation simplifies to

(5)

in which

(6)

(7)

(8)

In the analysis of our data, we assume that the product _iR_i is a measure of the contribution of receptor R_i to the total phosphoinositide response. This product is essentially equal to the maximal stimulus elicited by the receptor. In our study, there are three discernable populations of muscarinic receptors (n = 3): M₂ receptors (R₂), M₃ receptors (R₃), and the residual receptors (R₁). It is possible to obtain an estimate of the parameter _iR_i for a given receptor relative to the total contribution of all receptors using the following equation, which specifically applies to the M₃ receptor,

(9)

in which RC₃ denotes the relative contribution of the M₃ receptor to the phosphoinositide response, E_max-KO3 and E_max-WT denote the maximal phosphoinositide responses, and EC_50-KO3 and EC_50-WT denote the EC₅₀ values for oxotremorine-M in M₃ KO and wild-type mice, respectively. Substitution of eqs. 6 and 7 for the EC₅₀ and E_max values in eq. 9 followed by simplification yields

(10)

Using an analogous approach, the relative contribution of the M₂ receptor to the phosphoinositide response (RC₂) can be estimated as

(11)

in which E_max-KO2 and EC_50-KO2 denote the E_max and EC₅₀ values of oxotremorine-M in M₂ KO mice. Equation 11 simplifies to

(12)

Likewise, the contribution of the residual receptors (CE₁) can be estimated from the following equation:

(13)

in which E_max-KO23 and EC_50-KO23 denote the E_max and EC₅₀ values of oxotremorine-M in M₂/M₃ KO mice. Equation 13 simplifies to

(14)

Analysis of Competitive Antagonism. The dissociation constants of antagonists (K_B), based on competitive antagonism of the phosphoinositide response, were estimated using the following equation:

(15)

in which [B] denotes the concentration of the 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 the absence of the antagonist.

Statistical Analysis of Concentration-Response Curves. Global nonlinear regression analysis was used to determine the significance of difference between the EC₅₀ and E_max values determined in experiments on phosphoinositide hydrolysis in ilea from wild-type and muscarinic receptor knockout mice. Using this approach, the data of the concentration-response curves are fitted simultaneously, allowing the parameter of interest to be shared among the curves for the different mouse strains or estimated independently for each strain. The significance of the reduction in the residual sum of squares that occurs when the parameter is fitted independently was determined using an F test.

Materials. The reagents used in this study were obtained from the following sources. myo-[³H]Inositol (PerkinElmer Life and Analytical Sciences (Boston, MA), tetrodotoxin and DNase I (Sigma-Aldrich, St. Louis, MO), AF-DX 116 (Boehringer Ingelheim Pharmaceuticals, Ridgefield, CT), pirenzepine (Research Biochemicals, Natick, MA), pertussis toxin (List Biological Laboratories, Campbell, CA), and collagenase type II (Worthington Biochemical, Lakewood, NJ).

	Results

Top Abstract Materials and Methods Results Discussion References

 
Phosphoinositide Hydrolysis in the Urinary Bladder and Ileum. Oxotremorine-M elicited a robust muscarinic phosphoinositide response in urinary bladder from wild-type and M₂ KO mice (Fig. 1a). In contrast, little or no significant muscarinic response was detected in urinary bladder from M₃ KO and M₂/M₃ KO mice. These results indicate that the muscarinic phosphoinositide response in mouse urinary bladder is mediated by the M₃ receptor. Table 1 summarizes the EC₅₀ and E_max values of oxotremorine-M for eliciting phosphoinositide hydrolysis in urinary bladder from wild-type and M₂ KO mice.

View larger version (24K): [in this window] [in a new window]   Fig. 1. a and b, oxotremorine-M-induced phosphoinositide hydrolysis in wild-type, M2 KO, M3 KO, and M2/M3 KO mouse urinary bladder strips (a) and longitudinal muscle strips of the ileum (b). Each point represents the mean values ± S.E.M. from four to 10 separate experiments.

We also investigated the effects of oxotremorine-M on phosphoinositide hydrolysis in the longitudinal muscle of the mouse ileum (Fig. 1b). Oxotremorine-M mediated an accumulation of inositol phosphates in all of the knockout mouse strains studied, with the more potent responses measured in wild-type and M₂ KO mice (pEC₅₀ = 6.44 ± 0.16 and 6.28 ± 0.10, respectively) and less potent responses in M₃ KO and M₂/M₃ KO mice (pEC₅₀ = 5.79 ± 0.12 and 5.83 ± 0.18, respectively). The responses observed in M₃ KO and M₂/M₃ KO mice also exhibited a diminished maximal response relative to wild type. A summary of these results is shown in Table 1. A substantial loss of function associated with the lack of the M₃ receptor is apparent when the data from the M₃ KO mouse are compared with those of the wild type as well as when the data from the M₂/M₃ KO mouse are compared with those of the M₂ KO mouse. A small role for the M₂ receptor in phosphoinositide hydrolysis is suggested by the nonsignificant loss of function in the M₂ KO relative to wild type, which was confirmed by the significant loss of function in the M₂/M₃ KO relative to M₃ KO. The contribution of muscarinic receptor subtypes to the phosphoinositide response was estimated using the approach described under Materials and Methods. Using eqs. 9 and 13, we estimated that the M₃ receptor contributes to 80% of the response, whereas the residual muscarinic receptors in the M₂/M₃ KO mouse contribute to 15% of the response. If the contributions of the M₂, M₃, and residual receptors equal to 100%, then the contribution of the M₂ receptor is approximately 5%. It is difficult to estimate the contribution of the M₂ receptor by comparison of muscarinic phosphoinositide response in wild-type and M₂ KO mice, because a contribution of 5% would be manifest as only a 1.054-fold increase in the EC₅₀ value of oxotremorine-M. The observed increase (1.42-fold) was not significantly different from this value. Consequently, it is more reliable to estimate the contribution of the M₂ receptor by the loss of function in the M₂/M₃ KO mouse relative to the M₃ KO mouse.

If the dissociation constants of oxotremorine-M for M₁, M₂, and M₃ receptor subtypes are approximately the same, then the concentration-response curves in wild-type and muscarinic receptor knockout mice can be compared with those obtained when the method of partial receptor inactivation (Furchgott, 1966) is used to estimate the dissociation constant of an agonist. To test this postulate, we used Furchgott's method to estimate the dissociation constant of oxotremorine-M by comparing equiactive concentrations of oxotremorine-M in wild-type, M₃ KO, and M₂/M₃ KO mice. The overall estimate of the pK_D value of oxotremorine-M (5.23 ± 0.17) did not differ significantly when estimated by comparing the wild type data with those obtained in M₃ KO or M₂/M₃ KO mice. Consequently, these results are consistent with the postulate that the dissociation constants of oxotremorine-M are similar for M₁, M₂, and M₃ receptors. This result is consistent with the assumption made in eq. 3 of the mathematical derivation of our method for estimation of the RC value.

Competitive Antagonism of the Phosphoinositide Response in M₂/M₃ KO Ileum. We measured the competitive antagonism of the phosphoinositide response to oxotremorine-M in the ileum of the M₂/M₃ KO mouse to characterize the nature of the muscarinic receptor mediating this response (see Fig. 2a). At a concentration of 2 µM, the M₂-selective muscarinic antagonist AF-DX 116 caused a 6.3-fold dextral shift in the concentration-response curve to oxotremorine-M, which yields a pK_B estimate of 6.42 ± 0.42 (see eq. 15). When present at a concentration of 0.2 µM, the M₁-selective antagonist pirenzepine shifted the concentration-response curve to oxotremorine-M 22.9-fold, yielding a pK_B estimate of 8.04 ± 0.47. The pK_B values for AF-DX 116 and pirenzepine are in close agreement with their corresponding binding affinities for the human M₁ receptor (6.2 and 7.8, respectively), but not the M₂ (7.3 and 6.0), the M₃ (6.1 and 6.6), the M₄ (6.0 and 7.2), or the M₅ (5.3 and 6.6) (Esqueda et al., 1996). These results suggest that the M₁ receptor is primarily responsible for mediating the muscarinic phosphoinositide response in M₂/M₃ KO mouse ileum.

View larger version (10K): [in this window] [in a new window]   Fig. 2. Oxotremorine-M-mediated phosphoinositide in ileum from M2/M3 KO mice. The competitive antagonism of the response by AF-DX 116 and pirenzepine is shown in a, and the effect of oxotremorine-M (0.1 mM) on the response in dissociated smooth muscle cells is shown in b. Data are expressed as the percentage conversion of [3H]inositol-labeled phospholipid into [3H]inositol phosphates. The mean values ± S.E.M. of three experiments are shown.

Dissociated Smooth Muscle. To determine whether M₁ receptor-mediated phosphoinositide hydrolysis in the M₂/M₃ KO mouse originated from smooth muscle and not from other cell types, such as myenteric neurons, dissociated smooth muscle cells of the ileum were prepared. Because the protocol yielded a small amount of cells, the phosphoinositide response to only a single concentration of oxotremorine-M (0.1 mM) was measured. In the wild-type mouse, the phosphoinositide response to oxotremorine-M was 11.0 ± 0.98% when expressed as conversion of [³H]phosphoinositides into [³H]inositol phosphates (Fig. 2b). Oxotremorine-M also caused a significant phosphoinositide response in the M₂/M₃ KO mouse ileum of 6.1 ± 0.34%. The size of the response in smooth muscle cells from the M₂/M₃ KO mouse ileum relative to that measured in wild-type cells is similar to the relative sizes observed in intact tissue. We conclude that most, if not all, of the response in intact smooth muscle is contributed by smooth muscle cells.

Effects of Pertussis Toxin. Pertussis toxin catalyzes the ADP-ribosylation of G_i/o proteins, which prevents the coupling of receptors to these G proteins (Kurose et al., 1983). M₂ receptors are known to signal through G_i/o (Ashkenazi et al., 1987); therefore, pertussis toxin should be a useful probe for determining the extent to which muscarinic receptor signaling through G_i/o is involved in the phosphoinositide response in smooth muscle. In wild-type urinary bladder, pertussis toxin treatment had little significant effect on the phosphoinositide response to oxotremorine-M (Fig. 3a). Likewise, in ilea from wild-type (Fig. 3b) and M₃ KO mice (Fig. 3c), pertussis toxin had little or no inhibitory effect on oxotremorine-M-stimulated phosphoinositide hydrolysis. We also observed no significant effect of pertussis toxin on the pEC₅₀ and E_max values of oxotremorine-M in ilea from M₂ KO (6.25 ± 0.11 and 33.6 ± 1.2%, respectively) and M₂/M₃ KO mice (5.91 ± 0.17 and 21.3 ± 1.4%, respectively). It can be seen that the latter estimates are approximately the same as the corresponding values for the control data listed in Table 1. Collectively, these results provide no evidence for the role of G_i/o in mediating phosphoinositide hydrolysis in mouse ileum and urinary bladder.

View larger version (13K): [in this window] [in a new window]   Fig. 3. a-c, effects of pertussis toxin on muscarinic receptor-mediated phosphoinositide hydrolysis in wild-type mouse urinary bladder (a), wild-type ileum (b), and M3 KO ileum (c). Pertussis toxin (70-100 µg/kg body weight) was injected intraperitoneally 3 days before experiments. Mean values ± S.E.M. from three to 10 experiments are shown. The control concentration-response curves for the ileum shown in b and c are the same as those shown in Fig. 1b.

View larger version (10K): [in this window] [in a new window]   Fig. 4. a and b, the effects of pertussis toxin treatment on [3H]NMS binding in the longitudinal muscle of the ileum (a) and urinary bladder of the wild-type mouse (b). a, the competitive inhibition of [3H]NMS binding by oxotremorine-M was measured in the presence and absence of GTP. The concentration of [3H]NMS was 0.5 nM. b, the percentage enhancement of [3H]NMS binding by GTP was measured. In these experiments, the binding of [3H]NMS was measured at a single concentration of 0.25 nM. Mean values ± S.E.M. are shown in a and b.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
In our experiments, we focused on the contribution of M₂ and M₃ receptors to the phosphoinositide response because these are the two main muscarinic receptor subtypes expressed in smooth muscle. Our results show that the M₃ receptor mediates the muscarinic phosphoinositide response in urinary bladder because the response is completely lost in mice lacking M₃ receptors (Fig. 1a). These results agree with previous pharmacological studies demonstrating an M₃-mediated mechanism for the muscarinic phosphoinositide response in guinea pig urinary bladder (Noronha-Blob et al., 1989).

Our results on the ileum show a complex picture, suggesting that more than one muscarinic subtype mediates phosphoinositide hydrolysis. We observed a small loss of function in the M₂ KO, a large loss of function in the M₃ KO, and a substantial response that still persisted in the M₂/M₃ KO mouse (Fig. 1b) that exhibited an M₁ profile in competitive antagonism experiments (Fig. 2a). In situations where more than one receptor mediates a response and there is a receptor reserve, it is not a trivial matter to estimate the contribution of each receptor to the response. Nevertheless, we developed a parameter called RC that accurately estimates the contribution of each receptor to the response assuming that the observed dissociation constant of oxotremorine-M for each receptor is the same (see Materials and Methods). Accordingly, we estimated that the M₁, M₂, and M₃ receptor contributed 15, 5, and 80% respectively, to the phosphoinositide response in mouse ileum. These results are generally consistent with a prior pharmacological study demonstrating that the M₃ receptor mediates phosphoinositide hydrolysis in the longitudinal muscle of the rat ileum (Candell et al., 1990). The M₁ and M₂ effects on phosphoinositide response were most likely too low to be detected in prior pharmacological studies on rat ileum. Moreover, the M₁ effect would have been masked because the only so-called M₃ selective antagonists employed also exhibited high affinity for M₁ receptors. In a situation where a response exhibits a receptor reserve and is mediated by both M₁ and M₃ receptors, an M₁-selective antagonist would block the response with low M₃ potency, and an M₃ selective antagonist would block with low M₁ potency. An antagonist with high affinity for both M₁ and M₃ receptors would block the response with high potency. This phenomenon occurs because the M₁ receptor can rescue the M₃ receptor and vice versa.

Although the contribution of the M₂ receptor to phosphoinositide hydrolysis is small in the ileum, it was detected as a substantial loss of function in the M₂/M₃ KO relative to the M₃ KO (Fig. 1b). Evidence for recombinant M₂ receptors coupling to phosphoinositide hydrolysis through a pertussis toxin-sensitive mechanism has been noted in murine fibroblasts (Lai et al., 1991) and CHO cells (Ashkenazi et al., 1989). In rabbit intestinal smooth muscle, M₂ receptors have been shown to mediate phosphoinositide hydrolysis through a pertussis toxin-sensitive mechanism (Murthy et al., 2003). The authors suggest that the mechanism involves activation of PLC-₃ by the _i3 subunits of G_i (Murthy et al., 2003). In several prior studies on the longitudinal muscle of the guinea pig ileum, we have consistently found that pertussis toxin treatment causes a small increase in muscarinic agonist-mediated and histamine-mediated phosphoinositide hydrolysis (Thomas and Ehlert, 1994; Ehlert et al., 2001; Shehnaz et al., 2001). This result implies that pertussis toxin treatment (intraperitoneal injection in vivo, 3 days before) causes a small increase in agonist-mediated phosphoinositide hydrolysis in the ileum. Perhaps this enhancing effect prevented us from detecting a small loss of M₂ receptor G_I-mediated phosphoinositide hydrolysis in mouse strains expressing M₂ receptors (Fig. 3a,b). Consequently, we cannot discern whether the M₂ response is pertussis toxin-sensitive.

Our results on dissociated smooth muscle cells indicate that the M₁ response is in the muscle itself (Fig. 2b). If the M₁ response resided in a different cell type (e.g., neurons), the concentration-response curve for oxotremorine-M in the wild-type mouse should be equivalent to the summation of that for the M₂-M₃ response in smooth muscle plus the M₁ response in neurons. Because the maximal response in the M₂/M₃ KO was 57% that of wild type, this neuronal M₁ hypothesis would imply that the maximal response in smooth muscle only accounted for 43% of the total response. This situation seems unlikely. It is also possible that the M₁ response in smooth muscle is compensatory, caused by the lack of expression of M₂ and M₃ receptors. This hypothesis is difficult to test. A small amount of M₁ receptors (Wall et al., 1991) and mRNA (Maeda et al., 1988) has been detected in wild-type intestinal smooth muscle from rats, which could account for the muscarinic phosphoinositide response observed here. No compensatory increases in muscarinic receptor expression have been detected in muscarinic receptor knockout mice (Gautam et al., 2005), and evidence for a decrease in expression has actually been observed in brain (Oki et al., 2005). It would be difficult to ascribe potential changes in other signaling proteins in M₂/M₃ knockout mice to the small M₁ response that we observed.

The prominent coupling of M₃ receptors to phosphoinositide hydrolysis in mouse smooth muscle is consistent with prior pharmacological studies on other animals (Noronha-Blob et al., 1989; Candell et al., 1990) and is consistent with the direct role that the M₃ receptor has in contraction of smooth muscle. Many receptors that couple to G_q elicit contraction of smooth muscle. However, the details of the mechanism are unclear because the source of Ca²⁺ for contraction is mainly extracellular (Bolger et al., 1983) and not through inositol-1,4,5-trisphosphate-mediated release of intracellular Ca²⁺. M₂ receptors have been shown to trigger signaling mechanisms that are contingent upon M₃ receptor activation, including activation of a nonselective cation conductance (Bolton, 1979) and inhibition of Ca²⁺-activated K⁺ channels (Cole et al., 1989). Activation of M₂ receptors has been shown to enhance M₃ receptor-mediated contractions and inhibit the relaxant effects of isoproterenol and forskolin on contractions mediated by G_q-linked receptors, including the M₃ (Thomas et al., 1993; Sawyer and Ehlert, 1998). All of these M₂ responses can be attributed to G_i-mediated effects. The relationship between the M₂ phosphoinositide response observed in ileum here and the contractile response is unclear. M₂ receptors have been shown to mediate a weak, direct contractile response in both the ileum and urinary bladder of M₃ KO mouse, and this weak response is comparatively greater in the ileum. One unresolved question is that the ileum from M₂/M₃ KO mice does not exhibit a muscarinic agonist-induced contractile response (Matsui et al., 2002), yet as described herein, it does exhibit a significant phosphoinositide response elicited through activation of M₁ receptors. One possible explanation is that there is a differential cellular compartmentalization of M₁ and M₃ receptors in smooth muscle of the ileum. It has been shown that G_q, downstream effectors, and receptors that couple to G_q exhibit a differential distribution in lipid rafts (see review by Pike, 2003). Perhaps M₃ receptors, G_q, PLC-, and a downstream effector protein that initiates contraction are colocalized in lipid rafts, whereas M₁ receptors are localized elsewhere with G_q and PLC- but not with the relevant proteins involved in initiating contraction. Our results suggest that M₁ receptors may have a role in smooth muscle distinct from contraction that is mediated through phosphoinositide hydrolysis.

	Footnotes

 
This work was supported by Pfizer (to F.J.E.), National Institutes of Health Grant NS 30882 (to F.J.E.), a grant from Pharmacia (to M.M.) and the Detrol LA Research Grant Program from Pfizer (to M.M.), the Industrial Technology Research Grant 02A09001a from The New Energy and Industrial Technology Development Organization (NEDO) of Japan (to M.M.), and Grant-in-aid for Scientific Research on Priority Areas 16067101 from The Ministry of Education, Culture, Sports, Science and Technology (MEXT) (to M.M.).

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

doi:10.1124/jpet.106.103093.

ABBREVIATIONS: M, muscarinic receptor; KO, knockout; 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; [³H]NMS, [³H]N-methylscopolamine; PLC-, phospholipase-C.

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

	References

Top Abstract Materials and Methods Results Discussion References

 

Ashkenazi A, Peralta EG, Winslow JW, Ramachandran J, and Capon DJ (1989) Functionally distinct G proteins selectively couple different receptors to PI hydrolysis in the same cell. Cell 56: 487-493.[CrossRef][Medline]

Ashkenazi A, Winslow JW, Peralta EG, Peterson GL, Schimerlik MI, Capon DJ, and Ramachandran J (1987) An M₂ muscarinic receptor subtype coupled to both adenylyl cyclase and phosphoinositide turnover. Science (Wash DC) 238: 672-675.[Abstract/Free Full Text]

Berridge MJ, Downes CP, and Hanley MR (1982) Lithium amplifies agonist-dependent phosphatidylinositol responses in brain and salivary glands. Biochem J 206: 587-595.[Medline]

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

Bolger GT, Gengo P, Klockowski R, Luchowski E, Siegel H, Janis RA, Triggle AM, and Triggle DJ (1983) Characterization of binding of the Ca²⁺ channel antagonist, [³H]nitrendipine, to guinea pig ileal smooth muscle. J Pharmacol Exp Ther 225: 291-309.[Abstract/Free Full Text]

Bolton TB (1979) Mechanisms of action of transmitters and other substances on smooth muscle. Physiol Rev 59: 606-718.[Free Full Text]

Bonner TI, Young AC, Brann MR, and Buckley NJ (1988) Cloning and expression of the human and rat m5 muscarinic acetylcholine receptor genes. Neuron 1: 403-410.[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]

Caulfield MP (1993) Muscarinic receptors-characterization, coupling, and function. Pharmacol Ther 58: 319-379.[CrossRef][Medline]

Choppin A and Eglen RM (2001) Pharmacological characterization of muscarinic receptors in mouse isolated urinary bladder smooth muscle. Br J Pharmacol 133: 1035-1040.[CrossRef][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.[Medline]

Ehlert FJ (1985) The relationship between muscarinic receptor occupancy and adenylate cyclase inhibition in the rabbit myocardium. Mol Pharmacol 28: 410-421.[Abstract]

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, Abe DM, Vo TH, Taketo MM, Manabe T, and Matsui M (2005) The M₂ muscarinic receptor mediates contraction through indirect mechanisms in mouse urinary bladder. J Pharmacol Exp Ther 313: 368-378.[Abstract/Free Full Text]

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

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.[Medline]

Gautam D, Han SJ, Heard TS, Cui Y, Miller G, Bloodworth L, and Wess J (2005) Cholinergic stimulation of amylase secretion from pancreatic acinar cells studied with muscarinic acetylcholine receptor mutant mice. J Pharmacol Exp Ther 313: 995-1002.[Abstract/Free Full Text]

Hulme EC, Berrie CP, Birdsall NJ, and Burgen AS (1981) Two populations of binding sites for muscarinic antagonists in the rat heart. Eur J Pharmacol 73: 137-142.[CrossRef][Medline]

Kendall DA and Hill SJ (1990) Measurement of [³H]inositol phospholipid turnover, in Methods in Neurotransmitter Analysis (Yamamura HI, Enna SJ, and Kuhar MJ eds) pp 69-87, Raven Press, New York.

Kurose H, Katada T, Amano T, and Ui M (1983) Specific uncoupling by islet-activating protein, pertussis toxin, of negative signal transduction via -adrenergic, cholinergic and opiate receptors in neuroblastoma x glioma hybrid cells. J Biol Chem 258: 4870-4875.[Abstract/Free Full Text]

Lai J, Waite SL, Bloom JW, Yamamura HI, and Roeske WR (1991) The m2 muscarinic acetylcholine receptors are coupled to multiple signaling pathways via pertussis toxin-sensitive guanine nucleotide regulatory proteins. J Pharmacol Exp Ther 258: 938-944.[Abstract/Free Full Text]

Maeda A, Kubo T, Mishina M, and Numa S (1988) Tissue distribution of mRNAs encoding muscarinic acetylcholine receptor subtypes. FEBS Lett 239: 339-342.[CrossRef][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]

Mei L, Lai J, Yamamura H, and Roeske W (1991) Pharmacologic comparison of selected agonists for the M₁ muscarinic receptor in transfected murine fibroblast cells (B82). J Pharmacol Exp Ther 256: 689-694.[Abstract/Free Full Text]

Murthy KS, Zhou H, Grider JR, Brautigan DL, Eto M, and Makhlouf GM (2003) Differential signalling by muscarinic receptors in smooth muscle: m2-mediated inactivation of myosin light chain kinase via Gi3, Cdc42/Rac1 and p21-activated kinase 1 pathway and m3-mediated MLC20 (20-kDa regulatory light chain of myosin II) phosphorylation via Rho-associated kinase/myosin phosphatase targeting subunit 1 and protein kinase C/CPI-17 pathway. Biochem J 374: 145-155.[CrossRef][Medline]

Noronha-Blob L, Lowe V, Patton A, Canning B, Costello D, and Kinnier WJ (1989) Muscarinic receptors: relationships among phosphoinositide breakdown, adenylate cyclase inhibition, in vitro detrusor muscle contractions and in vivo cystometrogram studies in guinea pig bladder. J Pharmacol Exp Ther 249: 843-851.[Abstract/Free Full Text]

Oki T, Takagi Y, Inagaki S, Taketo MM, Manabe T, Matsui M, and Yamada S (2005) Quantitative analysis of binding parameters of [³H]N-methylscopolamine in central nervous system of muscarinic acetylcholine receptor knockout mice. Brain Res Mol Brain Res 133: 6-11.[Medline]

Peralta EG, Ashkenazi A, Winslow JW, Ramachandran J, and Capon DJ (1988) Differential regulation of PI hydrolysis and adenylyl cyclase by muscarinic receptor subtypes. Nature (Lond) 334: 434-437.[CrossRef][Medline]

Pike LJ (2003) Lipid rafts: bringing order to chaos. J Lipid Res 44: 655-667.[Abstract/Free Full Text]

Ringdahl B (1985) Structural requirements for muscarinic receptor occupation and receptor activation by oxotremorine analogs in the guinea-pig ileum. J Pharmacol Exp Ther 232: 67-73.[Abstract/Free Full Text]

Sawyer GW and Ehlert FJ (1998) Contractile roles of the M₂ and M₃ muscarinic receptors in the guinea pig colon. J Pharmacol Exp Ther 284: 269-277.[Abstract/Free Full Text]

Shehnaz D, Ansari KZ, and Ehlert FJ (2001) Acetylcholine-induced desensitization of the contractile response to histamine in guinea pig ileum is prevented by either pertussis toxin-treatment or by selective inactivation of muscarinic M₃ receptors. J Pharmacol Exp Ther 297: 1152-1159.[Abstract/Free Full Text]

Stryer L and Bourne HR (1986) G proteins: A family of signal transducers. Annu Rev Cell Biol 2: 391-419.[Medline]

Thomas EA, Baker SA, and Ehlert FJ (1993) Functional role for the M₂ muscarinic receptor in smooth muscle of guinea pig ileum. Mol Pharmacol 44: 102-110.[Abstract]

Thomas EA and Ehlert FJ (1994) Pertussis toxin blocks M₂ muscarinic receptor-mediated effects on contraction and cyclic AMP in the guinea pig ileum, but not M₃-mediated contractions and phosphoinositide hydrolysis. J Pharmacol Exp Ther 271: 1042-1050.[Abstract/Free Full Text]

Wall SJ, Yasuda RP, Li M, and Wolfe BB (1991) Development of an antiserum against m3 muscarinic receptors: distribution of m3 receptors in rat tissues and clonal cell lines. Mol Pharmacol 40: 783-789.[Abstract]

Yang CM, Chou SP, and Sung TC (1991) Muscarinic receptor subtypes coupled to generation of different second messengers in isolated tracheal smooth muscle cells. Br J Pharmacol 104: 613-618.[Medline]

This article has been cited by other articles:

				M. T. Griffin, K. W. Figueroa, S. Liller, and F. J. Ehlert Estimation of Agonist Activity at G Protein-Coupled Receptors: Analysis of M2 Muscarinic Receptor Signaling through Gi/o,Gs, and G15 J. Pharmacol. Exp. Ther., June 1, 2007; 321(3): 1193 - 1207. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.106.103093v1 318/2/649    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Tran, J. A. Articles by Ehlert, F. J. Search for Related Content PubMed PubMed Citation Articles by Tran, J. A. Articles by Ehlert, F. J.