Abstract
Muscarinic receptors play a major role in gallbladder function, although the muscarinic receptor(s) mediating smooth muscle contractility is unclear. This study compared smooth muscle contractile responses to carbamylcholine (10−7-10−3 M) in isolated gallbladder from wild-type and M2, M3, and M4 receptor knockout mice. Carbamylcholine-induced contraction in gallbladder was associated with tachyphylaxis and the release of a cyclooxygenase product because indomethacin (10−6 M) inhibited carbamylcholine-induced contraction. The M3 receptor was the major muscarinic receptor involved in contraction because carbamylcholine-induced contractility was inhibited in gallbladder from M3 receptor knockout mice. Furthermore, the muscarinic receptor antagonists 11-[[[2-diethylamino-O-methyl]-1-piperidinyl]acetyl]-5,11-dihydrol-6H-pyridol[2,3-b][1,4]benzodiazepine-6-one (AF-DX 116) and pirenzepine dextrally shifted contraction to carbamylcholine in gallbladder from wild-type, M2, and M4 receptor knockout mice, with affinities consistent with M3 receptor interaction. In addition, maximal contraction to carbamylcholine was reduced in gallbladder from M2receptor knockout mice and affinities for AF-DX 116 and pirenzepine in gallbladder from M3 receptor knockout mice were consistent with their affinities at M2 receptors. In M4receptor knockout mice, contraction to carbamylcholine was dextrally shifted, although the affinities for AF-DX 116 and pirenzepine in gallbladder from M2 or M3 knockout mice were not similar to their affinities at M4 receptors. The M4 receptor may serve as an accessory protein necessary for optimal potency of M2 and M3 receptor-mediated responses. Thus, muscarinic receptor knockout mice provided direct and unambiguous evidence that M3, and to a lesser extent, M2 receptors are the predominant muscarinic receptors mediating gallbladder contractility, and M4 receptors appear necessary for optimal potency of carbamylcholine in gallbladder contraction.
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 M4receptor 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
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 (10−7-10−4 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.
In some experiments, gallbladder from wild-type mice was exposed to an initial contractile concentration-response curve to carbamylcholine. Tissues were washed, returned to baseline, and 45 min later, the contractile concentration-response curve to carbamylcholine was repeated. Because tachyphylaxis was observed (see Results), subsequent studies used only a single concentration-response curve to carbamylcholine per tissue.
In other experiments, tissues were incubated with 3.0 × 10−7 M neostigmine, 10−6M indomethacin, 10−6 M atropine, 3.0 × 10−6 M AF-DX 116, 10−7 or 10−6 M pirenzepine, or vehicle for 20 min, and contractile concentration-response curves to 10−7 to 10−3 M carbamylcholine were generated. Only one antagonist or vehicle was examined in each tissue.
The equilibrium dissociation constants (KB) for antagonists versus carbamylcholine were determined according to the following equation (Furchgott, 1972): KB = [B]/[dose ratio − 1], where [B] is the concentration of the antagonist and the dose ratio is the EC50 of the agonist in the presence of the antagonist divided by the control EC50. EC50 was the concentration of agonist required to elicit 50% of the maximal response.
In some experiments where maximal carbamylcholine-induced contraction was markedly reduced in the presence of antagonists, antagonist equilibrium dissociation constants were determined as described for noncompetitive receptor antagonists according to the following equation: KB = [B]/[slope − 1], where [B] equals the antagonist concentration and slope is determined from a double reciprocal plot of 1/x1 versus 1/x, where x1 andx are the equieffective concentrations of carbamylcholine in the presence (x1) and absence (x) of inhibitor (Kenakin, 1993; Cohen et al., 1998). The antagonist equilibrium dissociation constant was expressed as the negative logarithm of the KB (i.e., pKB).
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
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 (10−5 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.
In the M2 receptor knockout mice, low concentrations of carbamylcholine (≤3.0 × 10−6 M) produced a contractile concentration response similar to the response in gallbladder from wild-type mice. However, as the concentration of carbamylcholine increased (10−5-10−4 M), the maximal contractile force to carbamylcholine tended to be lower in gallbladder from M2 receptor knockout mice compared with the response in wild-type mice, an effect that reached significance only at 3.0 × 10−5 M carbamylcholine (Fig. 1, top).
Carbamylcholine-induced contraction of the mouse gallbladder was most affected in the M3 receptor knockout mice (Fig.1, middle), where the maximal response to carbamylcholine was dramatically reduced relative to the maximal response in gallbladder from wild-type mice. Nevertheless, even in the M3receptor knockout mice, a small contraction to high concentrations of carbamylcholine remained.
In contrast to these changes in the contractile response to carbamylcholine in gallbladder from the M2 and M3 receptor knockout mice, the maximal response to carbamylcholine in gallbladder from M4receptor knockout mice was similar to the response in gallbladder from wild-type mice (Fig. 1, bottom). However, the EC50 for carbamylcholine-induced contraction was higher in gallbladder from M4 receptor knockout than from wild-type mice. Thus, removal of the M4receptor did not affect maximal contraction to carbamylcholine, but did reduce the sensitivity of the gallbladder to carbamylcholine.
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 (10−6-10−4 M), indicating the rapid development of tachyphylaxis. For this reason, only a single carbamylcholine concentration-response curve was generated in all subsequent tissues studied.
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 × 10−7 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.
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 (10−6 M), a cyclooxygenase inhibitor, on the contractile response to carbamylcholine in gallbladder from wild-type mice. Indeed, indomethacin (10−6 M) produced a dramatic inhibition of the contractile response to carbamylcholine (Fig.4, top).
To determine whether the effect of indomethacin on carbamylcholine-induced contractility was unique to the gallbladder, indomethacin was also examined for its effect in mouse urinary bladder, a tissue possessing a cholinergic receptor profile similar to the gallbladder (Stengel et al., 2002). Indomethacin (10−6 M) did not alter carbamylcholine-induced contraction in mouse urinary bladder (Fig. 4, bottom). These results indicate that 1) indomethacin was not a cholinergic receptor antagonist at 10−6 M and 2) the effect of indomethacin was restricted to the gallbladder and did not generalize to all M3 receptor-mediated contractile responses. These data were consistent with the contention that carbamylcholine was activating release of a cyclooxygenase product that mediated contraction in the gallbladder.
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 (Table1). AF-DX 116 (3 × 10−6 M) dextrally shifted the contractile response to carbamylcholine in gallbladder from wild-type mice (Fig.5), resulting in a pKB value of 6.03 ± 0.16 (n = 4) consistent with the affinity of AF-DX 116 for M3 receptors (Table 1). Pirenzepine (10−7 M) modestly inhibited the contractile response to carbamylcholine (Fig. 5) with a pKB value of 7.05 ± 0.16 (n = 5), also consistent with the affinity of pirenzepine at M3 receptors (Table 1).
Effect of AF-DX 116 and Pirenzepine on Carbamylcholine-Induced Contraction in Gallbladder from M2 Receptor Knockout Mice.
As in wild-type mice, 3 × 10−6M AF-DX 116 produced a marked dextral shift in the contractile response to carbamylcholine in gallbladder from M2receptor knockout mice (Fig. 6) with a resulting pKB value of 5.88 ± 0.08 (n = 4), consistent again with the affinity of AF-DX 116 for M3 receptors. Furthermore, 10−7 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 pKB value of 6.96 ± 0.19 (n = 4), again consistent with the affinity of pirenzepine for M3 receptors (Table 1).
Effect of Atropine, AF-DX 116, and Pirenzepine on Carbamylcholine-Induced Contraction in Gallbladder from M3Receptor 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 (10−6 M). Atropine (10−6M) abolished the contractile response to carbamylcholine in gallbladder from M3 receptor knockout mice (Fig.7). In addition, 3.0 × 10−6 M AF-DX 116 dextrally shifted the contractile response to carbamylcholine in gallbladder from M3 receptor knockout mice, resulting in a pKB value of 6.83 ± 0.10 (n = 6), a value consistent with the affinity of AF-DX 116 for M2 receptors (Table 1). Furthermore, 10−7 M pirenzepine did not alter the contraction to carbamylcholine in gallbladder from M3receptor knockout mice (Fig. 7). However, 10−6 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 pKB value of 6.58 ± 0.13 (n = 4), consistent with affinity of pirenzepine at M2 receptors (Table 1).
Effect of AF-DX 116 and Pirenzepine on Carbamylcholine-Induced Contraction in Gallbladder from M4 Receptor Knockout Mice.
AF-DX 116 (3 × 10−6 M) produced a dextral shift in the contractile response to carbamylcholine in gallbladder from the M4 receptor knockout mice (Fig. 8), with a pKB value of 5.83 ± 0.10 (n = 5), consistent again with the affinity of AF-DX 116 for M3 receptors (Table 1). Pirenzepine (10−7 M) did not alter the contractile response to carbamylcholine in gallbladder from the M4receptor 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.
Discussion
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, 10−6 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 M3receptor-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 M3receptors 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 (pKB = 6.03), M2(pKB = 5.88), and M4 (pKB = 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, 10−7 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 (pKB = 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 M2/3 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 M4receptors. It is possible that an intact M4receptor 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 M4receptors 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 M4receptor. 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.
Acknowledgments
We are grateful for the expert administrative assistance of Priscilla Kirsch. We also acknowledge Drs. Jesus Gomeza, Masahisa Yamada, and Jürgen Wess for the initial generation of the muscarinic receptor knockout mice and global collaboration in understanding muscarinic receptor responses.
Footnotes
- Abbreviations:
- M1 to M5 receptors
- muscarinic acetylcholine receptors
- AF-DX 116
- 11-[[[2-diethylamino-O-methyl]-1-piperidinyl]acetyl]-5,11-dihydrol-6H-pyridol[2,3-b][1,4]benzodiazepine-6-one
- Received October 2, 2001.
- Accepted January 21, 2002.
- The American Society for Pharmacology and Experimental Therapeutics