Introduction

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Messenger RNA for M₁, M₂, M₃, and M₄ receptors has been identified in gallbladder (Heinig et al., 1997), raising the possibility that one or more of these receptors may be responsible for cholinergically mediated contractility in this tissue. In fact, multiple studies using pharmacological tools have implicated a role for M₃ receptors on gallbladder contractility in guinea pig (von Schrenck et al., 1993; Takahashi et al., 1994; Eltze et al., 1997) and cat (Chen et al., 1995). In addition, M₂ receptor activation has been linked to contractility in the cat (Chen et al., 1995) and guinea pig gallbladder (Oktay et al., 1998), although this receptor may serve as an inhibitory presynaptic autoreceptor in guinea pig gallbladder (Parkman et al., 1999) rather than possessing a direct role in gallbladder smooth muscle contractility (Akici et al., 1999). The possibility of a prominent role for M₃ and a more modest role for M₂ receptors in muscarinic-induced contraction of gallbladder is consistent with previous observations documenting a similar role for these receptors in carbamylcholine-induced contraction of other smooth muscles such as mouse trachea, stomach fundus, and urinary bladder (Stengel et al., 2000). In addition to these muscarinic receptors, the M₄ receptor has also been proposed to play an important role in contractility of the guinea pig gallbladder (Ozkutlu et al., 1993; Karaalp et al., 1999; Akici et al., 2000). The possibility that M₄ receptors may be involved in gallbladder contraction deserves careful consideration because it markedly contrasts with the lack of M₄ receptor involvement in the contraction of other smooth muscle preparations (Stengel et al., 2000).

The availability of muscarinic M₂, M₃, and M₄ receptor knockout mice coupled to the use of pharmacological tools selective for these receptors has prompted the present study to evaluate definitively the role of each of these receptors in gallbladder contractility. Furthermore, unlike the guinea pig gallbladder that possesses an extensive intramural neuronal network (Sutherland, 1967; Yoshida and Tsuruta, 1988; Parkman et al., 1999), gallbladder from the mouse does not possess such an extensive neuronal network (Yoshida and Koeda, 1991, 1992). Thus, use of the M₂, M₃, and M₄ muscarinic knockout mice can permit a detailed evaluation of the role of these muscarinic receptors in gallbladder smooth muscle contraction.

	Materials and Methods

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Animals. The generation of M₂, M₃, and M₄ muscarinic receptor knockout mice has been described previously (Gomeza et al., 1999a,b; Matsui et al., 2000; Yamada et al., 2001). M₂ receptor knockout mice (129J1/CF-1 hybrids) and M₃ and M₄ receptor knockout mice (129SvEv/CF-1 hybrids) were obtained from either the National Institute of Diabetes and Digestive and Kidney Diseases (Bethesda, MD) or Taconic (Germantown, NY). Wild-type mice of the same genetic background as M₂, M₃, and M₄ receptor knockout mice served as controls. Animals were housed in polycarbonate-ventilated cages. The animal room was maintained at 22-24°C with a relative humidity of 35 to 70% and daily light/dark cycle (6:00 AM-6:00 PM light). Food (Laboratory Rodent Diet 5001; PMI Feeds, St. Louis, MO) and water were supplied ad libitum. Mice (33-58 g) were killed by cervical dislocation, and the gallbladder was quickly excised and placed in modified Krebs-bicarbonate buffer solution of the following composition: 4.6 mM KCl, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄, 118.2 mM NaCl, 10.0 mM glucose, 1.6 mM CaCl₂·2H₂O, and 24.8 mM NaHCO₃. Experimental protocols and procedures were approved by the Eli Lilly and Company Animal Care and Use Committee.

Smooth Muscle Preparation. The gallbladder body (cut from the cystic duct to the neck) was prepared for in vitro examination. One end of the gallbladder was attached with thread to a stationary glass rod, whereas the other end was tied with thread to the transducer. In some experiments, urinary bladder from wild-type mice was prepared as previously described (Stengel et al., 2000). Tissues were placed in organ baths containing 10 ml of Krebs-bicarbonate buffer (see the above-mentioned description for composition). The organ bath solution was maintained at 37°C and aerated with a 95:5% mixture of O₂/CO₂. Gallbladders and urinary bladders were placed under an initial optimal force of 1.0 and 4.0g, respectively, and equilibrated for 1 h during which time the tissues were washed at 15-min intervals. Isometric force in grams was measured with transducers (Sensotec, Columbus, OH). Tissues were initially challenged with 67 mM KCl to confirm viability of the preparation. No significant differences in contractile responses to 67 mM KCl occurred among tissues from M₂, M₃, or M₄ receptor knockout and wild-type mice. Cumulative contractile concentration-response curves to carbamylcholine (10⁷-10⁴ M) were generated and expressed as a percentage of the 67 mM KCl maximum contraction determined for each tissue. For experiments comparing responses of gallbladder from muscarinic receptor knockout and wild-type mice, tissues from muscarinic receptor knockout and wild-type mice were used on each day to avoid the possibility of any daily systematic effect.

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Statistical Analyses. Results were expressed as the mean ± S.E. Agonist concentration-response curves were analyzed by a three-parameter logistic nonlinear model (De Lean et al., 1978). The three modeled parameters included the maximal response of the tissue, the EC₅₀, and the slope of the curves. Each curve was fitted using SAS (SAS Institute, Cary, NC) on a Deskpro 5133 personal computer (Compaq Computer Corporation, Houston, TX). Unpaired Student's t test was used to compare mean KCl contractile responses and pEC₅₀ (the negative logarithm of the EC₅₀) values between two groups. Analyses were run using SigmaStat for Windows (version 2.03; SPSS, Inc., Chicago, IL) on a Compaq personal computer (Deskpro 5133). Comparisons were considered significant for P values of 0.05 or less.

Drugs. Carbamylcholine chloride, pirenzepine dihydrochloride, neostigmine bromide, indomethacin, and atropine sulfate were purchased from Sigma-Aldrich (St. Louis, MO). AF-DX 116 was provided by the Lilly Research Laboratories.

	Results

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Carbamylcholine-Induced Contractility in Gallbladder from Wild-Type, M₂, M₃, and M₄ Receptor Knockout Mice. Carbamylcholine produced marked contraction in gallbladder from wild-type mice with a pEC₅₀ of 5.77 ± 0.06 (n = 25) (Fig. 1). In fact, the maximum contractile force to carbamylcholine (10⁵ M) was approximately 2.5-fold greater than the response to 67 mM KCl, using a concentration of KCl that produced a near maximal contractile response in smooth muscle.

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Effect of carbamylcholine to contract gallbladder from wild-type mice and M2 (top), M3 (middle), and M4 (bottom) receptor knockout mice. Values are mean ± S.E.M. of the number of tissues in parentheses. Asterisks denote a significant difference between the contraction observed in gallbladder from muscarinic receptor knockout and wild-type mice. Absence of error bars indicates that the magnitude of error was less than the symbol size.

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Carbamylcholine-Induced Gallbladder Contraction after Repeated Administration. Before initiating studies with muscarinic receptor knockout mice and pharmacological antagonists, we examined the ability of carbamylcholine to produce a response in the gallbladder from wild-type mice after previous exposure to a contractile concentration response to carbamylcholine. Surprisingly, carbamylcholine-induced contraction in the gallbladder was markedly reduced after previous exposure to carbamylcholine (Fig. 2). Both the potency of carbamylcholine and its maximal response were reduced in each tissue after previous exposure to carbamylcholine (10⁶-10⁴ M), indicating the rapid development of tachyphylaxis. For this reason, only a single carbamylcholine concentration-response curve was generated in all subsequent tissues studied.

View larger version (23K): [in this window] [in a new window]   Fig. 2.   Contractile concentration-response curves in gallbladder from wild-type mice to exposures to carbamylcholine separated by 45 min. Values are mean ± S.E.M. of the number of tissues in parentheses. Asterisks denote a significant difference between the values of the second generated curve and the values obtained on initial exposure to carbamylcholine. Absence of error bars indicates that the magnitude of error was less than the symbol size.

Effect of Neostigmine on Carbamylcholine-Induced Contraction in Gallbladder from Wild-Type Mice. Because tachyphylaxis to the contractile response to carbamylcholine occurred, we considered the possibility that carbamylcholine might, in part, activate presynaptic cholinergic receptors to release acetylcholine, which might contribute to the contractile response. We reasoned that if carbamylcholine were exerting a modulating effect on acetylcholine release from presynaptic cholinergic nerves then neostigmine, an inhibitor of acetylcholinesterase, should alter carbamylcholine-induced contraction in gallbladder from wild-type mice. However, neostigmine (3.0 × 10⁷ M) pretreatment did not significantly affect carbamylcholine-induced contraction in gallbladder from wild-type mice (Fig. 3), suggesting that carbamylcholine did not exert an effect on neuronal acetylcholine release.

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Contractile concentration-response curves to carbamylcholine after either 3.0 × 107 M vehicle or neostigmine in gallbladder from wild-type mice. Values are mean ± S.E.M. of the number of tissues in parentheses. Absence of error bars indicates that the magnitude of error was less than the symbol size.

Effect of Indomethacin on Carbamylcholine-Induced Contraction in Gallbladder from Wild-Type Mice. In an effort to understand further the tachyphylaxis to the contractile response to carbamylcholine, we considered the possibility that carbamylcholine was activating the release of an endogenous cyclooxygenase product that could be depleted by excessive stimulation. For this reason, we evaluated the effect of indomethacin (10⁶ M), a cyclooxygenase inhibitor, on the contractile response to carbamylcholine in gallbladder from wild-type mice. Indeed, indomethacin (10⁶ M) produced a dramatic inhibition of the contractile response to carbamylcholine (Fig. 4, top).

View larger version (23K): [in this window] [in a new window]   Fig. 4.   Contractile concentration-response curves to carbamylcholine after either vehicle or 106 M indomethacin in gallbladder (top) and urinary bladder (bottom) from wild-type mice. Values are mean ± S.E.M. of the number of tissues in parentheses. Asterisks indicate those values that significantly differ from vehicle. Absence of error bars indicates that the magnitude of error was less than the symbol size.

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Effect of AF-DX 116 and Pirenzepine on Carbamylcholine-Induced Contraction in Gallbladder from Wild-Type Mice. AF-DX 116 is a muscarinic receptor antagonist relatively selective for the M₂ receptor, in contrast to pirenzepine, which possesses approximately 3-fold lower affinity than AF-DX 116 at the M₂ receptor and has highest affinity at M₁ and M₄ receptors (Table 1). AF-DX 116 (3 × 10⁶ M) dextrally shifted the contractile response to carbamylcholine in gallbladder from wild-type mice (Fig. 5), resulting in a pK_B value of 6.03 ± 0.16 (n = 4) consistent with the affinity of AF-DX 116 for M₃ receptors (Table 1). Pirenzepine (10⁷ M) modestly inhibited the contractile response to carbamylcholine (Fig. 5) with a pK_B value of 7.05 ± 0.16 (n = 5), also consistent with the affinity of pirenzepine at M₃ receptors (Table 1).

View larger version (29K): [in this window] [in a new window]   Fig. 5.   Effect of 3.0 × 106 M AF-DX 116 and 107 M pirenzepine on the contractile response to carbamylcholine in gallbladder from wild-type mice. Values are mean ± S.E.M. of the number of tissues in parentheses. Absence of error bars indicates that the magnitude of error was less than the symbol size.

Effect of AF-DX 116 and Pirenzepine on Carbamylcholine-Induced Contraction in Gallbladder from M₂ Receptor Knockout Mice. As in wild-type mice, 3 × 10⁶ M AF-DX 116 produced a marked dextral shift in the contractile response to carbamylcholine in gallbladder from M₂ receptor knockout mice (Fig. 6) with a resulting pK_B value of 5.88 ± 0.08 (n = 4), consistent again with the affinity of AF-DX 116 for M₃ receptors. Furthermore, 10⁷ M pirenzepine produced a small dextral shift of the contractile response to carbamylcholine in gallbladder from the M₂ receptor knockout mice (Fig. 6) with a resultant pK_B value of 6.96 ± 0.19 (n = 4), again consistent with the affinity of pirenzepine for M₃ receptors (Table 1).

View larger version (29K): [in this window] [in a new window]   Fig. 6.   Effect of 3.0 × 106 M AF-DX 116 and 107 M pirenzepine on contractile response to carbamylcholine in gallbladder from M2 receptor knockout mice Values are mean ± S.E.M. of the number of tissues in parentheses. Absence of error bars indicates that the magnitude of error was less than the symbol size.

Effect of Atropine, AF-DX 116, and Pirenzepine on Carbamylcholine-Induced Contraction in Gallbladder from M₃ Receptor Knockout Mice. Because the residual contractile response to carbamylcholine in M₃ receptor knockout mice was small, we first established whether this effect was mediated by activation of muscarinic receptors. For this reason, we evaluated the effect of the nonselective muscarinic receptor antagonist atropine (10⁶ M). Atropine (10⁶ M) abolished the contractile response to carbamylcholine in gallbladder from M₃ receptor knockout mice (Fig. 7). In addition, 3.0 × 10⁶ M AF-DX 116 dextrally shifted the contractile response to carbamylcholine in gallbladder from M₃ receptor knockout mice, resulting in a pK_B value of 6.83 ± 0.10 (n = 6), a value consistent with the affinity of AF-DX 116 for M₂ receptors (Table 1). Furthermore, 10⁷ M pirenzepine did not alter the contraction to carbamylcholine in gallbladder from M₃ receptor knockout mice (Fig. 7). However, 10⁶ M pirenzepine produced a dramatic inhibition of the contractile response to carbamylcholine, inhibiting both the EC₅₀ and maximal response to carbamylcholine. Calculation of a noncompetitive antagonist dissociation constant for pirenzepine resulted in a pK_B value of 6.58 ± 0.13 (n = 4), consistent with affinity of pirenzepine at M₂ receptors (Table 1).

View larger version (25K): [in this window] [in a new window]   Fig. 7.   Effect of 106 M atropine, 3.0 × 106 M AF-DX 116, and 107 and 106 M pirenzepine on the contractile response to carbamylcholine in gallbladder from the M3 receptor knockout mice. Values are mean ± S.E.M. of the number of tissues in parentheses. Absence of error bars indicates that the magnitude of error was less than the symbol size.

Effect of AF-DX 116 and Pirenzepine on Carbamylcholine-Induced Contraction in Gallbladder from M₄ Receptor Knockout Mice. AF-DX 116 (3 × 10⁶ M) produced a dextral shift in the contractile response to carbamylcholine in gallbladder from the M₄ receptor knockout mice (Fig. 8), with a pK_B value of 5.83 ± 0.10 (n = 5), consistent again with the affinity of AF-DX 116 for M₃ receptors (Table 1). Pirenzepine (10⁷ M) did not alter the contractile response to carbamylcholine in gallbladder from the M₄ receptor knockout mice (Fig. 8), consistent with the contention that M₃ receptors are mediating the contractile response to carbamylcholine in the gallbladder from M₄ receptor knockout mice.

View larger version (27K): [in this window] [in a new window]   Fig. 8.   Effect of 3.0 × 106 M AF-DX 116 and 107 M pirenzepine on the contractile response to carbamylcholine in gallbladder from M4 receptor knockout mice. Values are mean ± S.E.M. of the number of tissues in parentheses. Absence of error bars indicates that the magnitude of error was less than the symbol size.

	Discussion

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The receptors responsible for muscarinic-induced contraction of gallbladder have been the subject of intense experimentation and debate. M₁, M₂, M₃, and M₄ muscarinic receptors have been implicated in the contractile response to muscarinic agonists in gallbladder (Parkman et al., 1999). In addition, M₁, M₂, M₃, and M₄ receptor mRNA exists in human gallbladder, again raising the possibility that each of these receptors may be involved in the cholinergically mediated contractile response of gallbladder (Heinig et al., 1997). Previous studies examining the role of multiple muscarinic receptors in the contractile response of gallbladder smooth muscle relied heavily upon the use of relatively nonselective pharmacological tools. With the development of muscarinic knockout mice, it became possible to examine definitively the role of M₂, M₃, and M₄ receptors in carbamylcholine-induced gallbladder contraction.

Carbamylcholine produced a contractile concentration-response curve in gallbladder from wild-type mice with a pEC₅₀ (5.77 ± 0.06, n = 25) close to that described for carbamylcholine-induced contraction in guinea pig gallbladder (von Schrenck et al., 1993; Takahashi et al., 1994; Eltze et al., 1997). However, the contractile potency of carbamylcholine was approximately 5- to 10-fold lower in gallbladder compared with other smooth muscle preparations (Stengel et al., 2000). Furthermore, like the stomach fundus (Stengel et al., 2000), the maximal gallbladder response to carbamylcholine was 3-fold greater than the maximal response to 67 mM KCl. Maximal carbamylcholine contraction in gallbladder was greater than the maximal contractile response in trachea or urinary bladder (Stengel et al., 2000). These differences in potency and maximal contractility might reflect a lack of receptor reserve in this tissue or more likely a difference in signal transduction mechanisms.

The possibility of a unique signaling mechanism for carbamylcholine in gallbladder is consistent with the observation that contraction was associated with tachyphylaxis upon a second exposure to carbamylcholine, an effect that did not occur in other smooth muscle preparations (Stengel et al., 2000). Several possible explanations could account for the tachyphylaxis observed in mouse gallbladder. First, carbamylcholine could be increasing presynaptic neuronal release of acetylcholine, a possibility supported by the fact that certain peptides such as cholecystokinin can release acetylcholine in gallbladder (Garrigues et al., 1992), that nicotinic receptor activation can alter acetylcholine release in gallbladder (Parkman et al., 1998), and that presynaptic M₁ inhibitory autoreceptors and M₂ excitatory autoreceptors have been reported in guinea pig gallbladder (Parkman et al., 1999). However, neostigmine, an inhibitor of acetylcholinesterase, in concentrations that dramatically potentiated smooth muscle responses to acetylcholine (unpublished observations), did not affect carbamylcholine-induced gallbladder contraction. Thus, carbamylcholine did not exert a functional presynaptic effect to alter acetylcholine release in mouse gallbladder.

Second, carbamylcholine could be releasing inhibitory transmitters that may impact subsequent contraction to cause tachyphylaxis. Nicotinic receptor activation can both activate (Parkman et al., 1998) and inhibit gallbladder contractility (Pozo et al., 1989; Parkman et al., 1998) and nicotinic receptor-induced smooth muscle responses have been associated with tachyphylaxis (Rand and Li, 1992). The fact that neostigmine did not alter carbamylcholine-induced contraction suggests that carbamylcholine was not interacting with nicotinic receptors to alter acetylcholine release, but does not rule out the possibility that carbamylcholine could be interacting directly with nicotinic receptors to alter release of other neurotransmitters.

Last, tachyphylaxis to carbamylcholine may be occurring because carbamylcholine released other contractile mediators in the mouse gallbladder, which become depleted. Although gallbladder M₃ receptors have been linked to phosphoinositide hydrolysis and adenylate cyclase inhibition (Takahashi et al., 1994), M₁, M₃, and M₅ receptor activation has also been linked to stimulation of phospholipase A₂ (Felder, 1995), which could stimulate arachidonic acid release and formation of cyclooxygenase contractile products. In addition, carbamylcholine stimulated arachidonic acid and eicosanoid release from brain synaptosomes (Strosznajder and Samochocki, 1992). Consistent with these reports, 10⁶ M indomethacin markedly inhibited the contractile response to carbamylcholine in the mouse gallbladder, an effect that did not occur in urinary bladder, a tissue with a muscarinic receptor profile (Stengel et al., 2002) similar to the gallbladder. Thus, unlike other M₃ receptor-mediated responses, the cholinergic signaling mechanism in mouse gallbladder appears to involve release of a cyclooxygenase product for contraction to carbamylcholine.

A marked inhibition of the contractile response to carbamylcholine occurred in gallbladder from the M₃ receptor knockout mice, clearly indicating that M₃ receptors play a predominant role in the contraction to carbamylcholine. This conclusion was further supported by pharmacological observations demonstrating that AF-DX 116 inhibited carbamylcholine-induced contraction in the wild-type mouse with an antagonist dissociation constant similar to the antagonist dissociation constant reported for AF-DX 116 at M₃ receptors (Table 1). Furthermore, the antagonist dissociation constant for AF-DX 116 was similar in gallbladder from wild-type (pK_B = 6.03), M₂ (pK_B = 5.88), and M₄ (pK_B = 5.87) knockout mice, again consistent with M₃ receptors mediating the contractile response to carbamylcholine in gallbladder from M₂ and M₄ receptor knockout mice. Also, 10⁷ M pirenzepine produced only a modest inhibition of the contractile response to carbamylcholine in gallbladder from the wild-type mice, ruling out a role for M₁ receptors. The antagonist dissociation constant (pK_B = 7.05 ± 0.46) for pirenzepine in gallbladder was similar to the antagonist dissociation constant for pirenzepine at M₃ rather than M₁, M₂, or M₄ receptors (Table 1). Thus, M₃ receptors are the predominant receptors mediating the contractile response to carbamylcholine in mouse gallbladder.

However, M₂ receptors have been demonstrated in gallbladder (Oktay et al., 1998) and our data along with others (Chen et al., 1995) suggest that M₂ receptors also play a modest role in carbamylcholine-induced contraction of mouse gallbladder. This conclusion is based on 1) a modest inhibition of the maximum contractile response to carbamylcholine in M₂ receptor knockout mice compared with wild-type mice (Fig. 1); 2) a small residual contraction to carbamylcholine in tissues from M₃ receptor knockout mice; and 3) the demonstration that AF-DX 116 and pirenzepine both inhibited the residual contractile response to carbamylcholine in gallbladder from M₃ receptor knockout mice with affinities similar to that for M₂ receptors (Table 1). The future availability of M_2/3 double receptor knockout mice will be important to confirm the lack of involvement of other cholinergic receptors in carbamylcholine-induced gallbladder contraction.

The gallbladder was a uniquely interesting smooth muscle based on the proposed presence of M₄ receptors and their role in mediating smooth muscle contractility (Ozkutlu et al., 1993; Karaalp et al., 1999; Akici et al., 2000). Carbamylcholine-induced contraction in gallbladder from M₄ receptor knockout mice was dextrally shifted compared with the response in gallbladder from wild-type mice, suggesting that the M₄ receptor participated in the contraction to carbamylcholine. However, antagonism of carbamylcholine-induced contraction by AF-DX 116 or pirenzepine was not consistent with an interaction with M₄ receptors. It is possible that an intact M₄ receptor is required for optimal functioning of M₂ and M₃ receptor-mediated responses to carbamylcholine as proposed for isolated atria (Stengel et al., 2000). We speculate that activation of the M₄ receptor may be permissive to the intracellular events necessary for optimal efficacy of M₂ and/or M₃ receptor activation. For example, activation of phospholipases and protein kinases (Chen et al., 1995) along with activation of G protein-coupled receptor kinases (Wess, 2000) has been linked to M₂ and M₃ receptor activation and their possible desensitization. We speculate that M₄ receptor activation may synergize with these G protein-coupled transduction events. Further studies will be required to understand more fully the precise role of M₄ receptors in permitting optimal expression of functional responsiveness to muscarinic responses.

In conclusion, these data using gallbladder from M₂, M₃, and M₄ receptor knockout mice have unequivocally demonstrated that both M₂ and M₃ receptors are involved in carbamylcholine-induced contractility of gallbladder smooth muscle. In addition, gallbladder contractility to carbamylcholine is dependent for optimal efficacy on the presence of the M₄ receptor. Last, carbamylcholine-induced gallbladder contraction is associated with the release of a cyclooxygenase product that mediates gallbladder contractility consistent with previous observations that M₃ receptors can couple to phospholipase A₂ activation (Conklin et al., 1988). Because cholinergic innervation and muscarinic receptor activation regulate gallbladder contractility, these studies may provide information useful to our understanding of the postprandial responses that regulate biliary excretion and dynamics.