ISSN 1062-3590, Biology Bulletin, 2008, Vol. 35, No. 2, pp. 187–193. © Pleiades Publishing, Inc., 2008. Original Russian Text © B.N. Manukhin, O.V. Boiko, 2008, published in Izvestiya Akademii Nauk, Seriya Biologicheskaya, 2008, No. 2, pp. 215–222.
ANIMAL AND HUMAN PHYSIOLOGY
Muscarinic Acetylcholine Receptors of the Chick Amnion B. N. Manukhin and O. V. Boiko Kol’tsov Institute of Developmental Biology, Russian Academy of Sciences, ul. Vavilova 26, Moscow, 119991 Russia email:
[email protected],
[email protected] Received June 18, 2007
Abstract—The presence of muscarinic (M) acetylcholine receptors in the noninnervated chick amnion makes it possible to analyze their functioning with presynaptic effects excluded. The M receptors of the amnion mediating its contraction were identified by testing with selective antagonists: pirenzepine for M1, methoctramine for M2, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) for M3, and tropicamide for M4 receptor subtype. All antagonists acted as competitive inhibitors of M-acetylcholine receptors. With respect to cholinolytic activity estimated from the response to carbacholine (CBC) (–logIC50), the antagonists could be arranged in the following series: 4-DAMP (8.29) > tropicamide (6.97) > pirenzepine (5.85) > methoctramine (5.63). In addition, the effect of forskolin (5 µM), activator of adenylate cyclase (AC), was unidirectional with ≅-adrenergic agonists; it blocked CBC-induced contractile activity of the amnion, whereas phospholipase C (1.25 U/ml) stimulated this activity. These data suggest that CBC- or acetylcholine (ACh)-induced contractile activity of the amnion is mediated by M3 acetylcholine receptors. Evaluation of contractile response to ACh by the tonic component usually revealed one pool of M3 acetylcholine receptors. One pool was also revealed after treatment with 4-DAMP, with the Hill coefficient being increased (ACh, n = 1.07; ACh against the 4-DAMP background, n = 1.48). It is possible to detect two pools of M3-acetylcholine receptors on the basis of either phase-frequency or tonic response, i.e., independently of the test parameter. DOI: 10.1134/S1062359008020131
INTRODUCTION The muscarinic (M) acetylcholine receptors are divided into five subtypes, M1–M5 (Caulfield and Birdsall, 1998). Experiments with mice with knocked-out M-cholinergic receptors showed that each mutant strain has specific functional disturbances (Wess et al., 2003). This means that each M subtype mediates certain physiological functions. The majority of tissues express two or more subtypes of M-cholinergic receptors (Eglen et al., 1996; Ehlert, 2003). The peripheral M-cholinergic receptors play a key role in the control of heart contraction, secretion by glands, and activity of smooth muscles. In the smooth muscles, they may mediate contraction and relaxation, and regulate acetylcholine (Ach) release. Both presynaptic and postsynaptic receptors are involved in the development of these responses (Eglen, 2005). A specific feature of chick amnion—the presence of the receptors in the noninnervated smooth muscle tissue—may be used to analyze receptor functions with presynaptic effects excluded. An acetylcholine-like agonist induces activation of contractions and increases the tone of amnion. These responses are blocked by the nonselective antagonist of M-cholinergic receptors atropine but not d-tubocurarin. Hence, they are mediated by muscarinic receptors (Boiko and Manukhin, 1989a; Bowers, 1989; Nechaeva and Turpaev, 1995). In this study, we identified the subtype of muscarinic
receptors in the amnion, characterized them, and analyzed their involvement in the regulation of contraction frequency and tone of amnion. MATERIALS AND METHODS We used the amnion of 11- to 14-day chick embryos. Contractions of two amnion fragments were recorded in parallel with mechanotrons 6MKh1B (Russia) and recorders N-339 and N-399 (Russia). The fragments were placed in 10 ml thermostated (38°ë) aerated chambers with Hanks’ solution, which contained 137 mM NaCl, 5.4 mM KCl, 1.26 mM CaCl2, 0.41 mM MgSO4, 0.49 mM MgCl2, 0.34 mM Na2HPO4, 0.44 mM KH2PO4, 4.2 mM NaHCO3, and 5.6 mM glucose. The initial load on the preparation was 100 mg. The 100-µl aliquots of test substances were introduced into the chamber after 30-min preincubation. To calculate the dose–effect relationship, we evaluated the magnitude of either tonic component or contraction frequency. The contractile activity was stimulated with the use of cholinergic agonist ACh or carbacholine (CBC) at a concentration of 50 µM, because the agonist in this concentration induces the development of maximum contraction frequency response and submaximum tonic response. Three applications of the agonist were repeated at 10-min recovery intervals, and the magnitude of the response was taken as 100%. The subsequent effects of antagonists were expressed as percent-
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ages of this value. To analyze subtypes of the muscarinic cholinergic receptors, we used selective M-cholinergic antagonists in a wide range of concentrations: 0.1–10 µM pirenzepine for M1, 0.1–10 µM methoctramine for å2, 0.1–100 nM 4-DAMP for å3, and 0.01–1.0 µM tropicamide for å4. The dose-dependent responses to inhibitors were recorded cumulatively during evoked contractions. The effect of 4-DAMP was also studied in experiments with cumulative recording of response to ACh in the control and, then, in the presence of an inhibitor 10 min after its introduction. To analyze metabolic pathways of realization of muscarinic response, we used forskolin and phospholipase C from Bacillus cereus. The reagents used in the study were from Sigma (United States). All compounds were dissolved in distilled water, except for tropicamide and forskolin dissolved in ethyl alcohol. The main parameters of cholinergic response were calculated with the Sigma Plot program using the equation p = ( P m A )/ ( EC 50 + A ) n
p = ( Pm A
n
n
n )/ ( IC 50
n
or
+ A ), n
(1)
where p is the response to agonist at concentration Ä, Pm is the maximum response, Öë50 is the agonist concentration that induces half-maximum response, IC50 is the antagonist concentration that causes a twofold decrease in the maximum response, and n is the Hill coefficient (Manukhin et al., 1998). RESULTS AND DISCUSSION The cholinergic system of chick amnion is involved in the regulation of its contractions. The spontaneous contractile activity of the amnion correlates with ACh content and activity of choline esterase in the tissue (Bunkina, 1963; Cuthbert, 1963). Inhibition of choline esterase enhances the amnion response to exogenous ACh by a factor of three or stronger (Turpaev and Putintseva, 1957; Boiko and Manukhin, 1989a, 1989b). The choline esterase activity was found in the muscle but not epithelial cells of the amnion (Müller et al., 1983). Cholinergic agonists ACh and CBC (0.5−1000 µM) induce contractions of the amnion in a dose-dependent mode with respect to tone, amplitude, and frequency. A purely tonic response may sometimes appear at later stages of development (the amnion of 14-day chick embryo). Antagonist of muscarinic receptors atropine blocks response to ACh ( – log IC 50 = 8.57) whereas d-tubocurarine has no effect (Boiko and Manukhin, 1989a). The sensitivity of the amnion to CBC and ACh does not differ significantly, with the respective – log EC 50 being 4.88 ± 0.12 and 5.05 ± 0.07. The muscarinic receptors mediating amnion contraction were identified pharmacologically by testing
the activity of four subtype-specific antagonists. We evaluated inhibitory effect of pirenzepine, methoctramine, 4-DAMP, and tropicamide on the contractile responses induced by cholinergic agonist. All these antagonists inhibited contractions activated by CBC or ACh and caused a dose-dependent decrease in the amnion tone and the amplitude and frequency of contractions. A comparison of concentrations of antagonists that induced the maximum response (Fig. 1) showed that the inhibitor of å2-cholinergic receptors methoctramine had the weakest effect. Methoctramine at a concentration of 10 µM did not eliminate a CBCinduced increase in the tone, and the contractile activity of the amnion remained the same. Pirenzepine, a blocker of å1 receptors, at a concentration of 10 µM caused 80–100% inhibition of contractions. This effect was achieved in the presence of 1 µM tropicamide, an antagonist of å4 receptors. The å3 antagonist 4-DAMP completely inhibited the CBC-induced response when taken at a concentration of 50 nM. Thus, the selective antagonist of å3 4-DAMP had the strongest effect. With respect to cholinolytic activity during CBC-induced response, which was evaluated using – log IC 50 , the antagonists can be arranged in the following descending series: 4-DAMP (8.29 ± 0.09) > tropicamide (6.97 ± 0.05) > pirenzepine (5.85 ± 0.09) > methoctramine (5.63 ± 0.08) (Fig. 2), which indicates that the amnion has muscarinic receptors mainly of the å3 subtype (Boiko and Manukhin, 2007). The above values coincide with those determined for å3 cholinergic receptors in the smooth muscles of isolated mouse urinary bladder, where they were 7.6–8.4 for 4-DAMP, 6.1–6.4 for pirenzepine, and 5.6–6.1 for methoctramine (Choppin, 2002). A low sensitivity to the å1 inhibitor pirenzepine (the limits for – log IC 50 for å1 receptors were 7.8–8.5) suggests that the amnion tissue had neither å1 nor å4 receptors, because these receptors have high affinity to pirenzepine (7.1–8.1). Pirenzepine has similar affinity to å2- and å3 receptors, which differs from that to å1 cholinergic receptors by one or two orders of magnitude (Caulfield and Birdsall, 1998). A low inhibitory activity of tropicamide in our experiments was comparable to the activity determined for å3 cholinergic receptors of the pig myometrium (7.07), which also excludes the involvement of å4 receptors (Kitazawa et al., 1999). The selective antagonist of å2 cholinergic receptors methoctramine was a weak inhibitor of contractile activity of amnion: – log IC 50 differed by two orders of magnitude from the values accepted for the å2 receptors (7.8–8.3) (Caulfield and Birdsall, 1998). This means that the muscarinic receptors of the chick amnion are of the å3 subtype. To further analyze the inhibitory effect of M-cholinergic receptor antagonists, the results were processed mathematically and graphically (Manukhin et al., 1998). The kinetic parameters of the CBC-induced BIOLOGY BULLETIN
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MUSCARINIC ACETYLCHOLINE RECEPTORS
CBC
Methoctramine
CBC
Tropicamide
CBC
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4-DAMP
CBC
Pirenzepine
Fig. 1. Inhibition of the contractile response of 14-day chick embryo amnion to CBC (50 µM) by antagonists of muscarinic cholinergic receptors: methoctramine (10 µM), 4-DAMP (0.05 µM), tropicamide (1 µM), and pirenzepine (10 µM). Calibration: 1 min, 50 mg.
response of the chick amnion are as follows: EC50 – 13.98 ± 4.35 µM, Pm – 118.3 ± 12.3%, n – 0.90 ± 0.14, and the standard error of estimate (SEE) = 4.4%. The inhibitory activity of antagonists (IC50) with respect to the muscarinic receptors was higher than EC50 of CBC (table); i.e. cholinergic receptors of the chick amnion are more sensitive to antagonists than to specific agonist CBC. The maximum inhibitory response did not significantly differ from the maximum response to CBC. Hence, all studied antagonists of the muscarinic receptors act as competitive inhibitors. 4-DAMP in the concentrations of 1, 10, and 100 nM competitively blocked the effect of ACh, with – log IC 50 = 8.95 + 0.15. The dose–effect curve in the presence of the inhibitor shifted to the right of the control curve, being parallel to it, with the maximum response remaining unchanged (Fig. 3a). As a rule, one pool of cholinergic receptors can be detected during the tonic response to the agonist. One pool was also revealed after treatment with 4-DAMP, and the Hill coefficient during reaction in the presence of the antagonist was increased (ACh, n = 1.07 ± 0.16; ACh in the presence of 4-DAMP, n = 1.48 ± 0.24) (Fig. 3b). The dynamics of the cholinergic effect may be described using parameters of the tonic component or the frequency component of the response. It is not always possible to analyze both parameters for the same dose-dependent response to ACh. The range of changes in the contraction frequency is limited; hence, it is difficult to obtain enough data for mathematical analysis by the iteration method. Figure 4 shows the BIOLOGY BULLETIN
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results of the of experiment in which we performed both graphic and computer-aided quantitative analysis of the kinetics of dose-dependent response estimated from changes in the tone and contraction frequency of the amnion in response to ACh application in the control and in the presence of 10 nM 4-DAMP. An analysis of the response to ACh in the Scatchard plot revealed two pools of cholinergic receptors, regardless of the p, % 100
1
2
3 4
80 60 40 20 0 10
9
8
7
6
5 –logA, M
Fig. 2. Dose-dependent inhibition of CBC-induced tonic contractile response of the amnion by antagonists of muscarinic cholinergic receptors (1) 4-DAMP, (2) tropicamide, (3) pirenzepine, and (4) methoctramine. Abscissa shows antagonist concentration (–logA, M); ordinate shows the magnitude of response, p (% of the maximum response).
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Effect of muscarinic receptor antagonists on the CBC-induced cholinergic response of chick amnion Parameter/Antagonist Pirenzepine, µM Methoctramine, µM 4-DAMP, nM Tropicamide, µM
IC50
Pm, %
n
1.44 ± 0.09 3.14 ± 0.46 4.94 ± 0.40 0.095 ± 0.004
107.5 ± 2.8 109.9 ± 7.5 112.4 ± 4.6 104.2 ± 1.7
1.27 ± 0.07 1.19 ± 0.10 1.31 ± 0.09 1.87 ± 0.11
Standard error of estimate, % 1.49 (10) 1.87 (8) 2.05 (10) 1.86 (8)
Note: Figures in parentheses show the number of experiments.
parameter used to evaluate the response (Figs. 4b, 4e). The calculations also showed the presence of high- and low-affinity pools. This means that effects on both contraction frequency and tone of the amnion are mediated by the same population of cholinergic receptors. The EC50 of the high-affinity receptor pool was 0.61 ± 0.17 nM for the tonic response of the amnion and 0.013 ± 0.009 nM p, % 100
(a)
80
1
2
60 40 20 0
6
5
6 5
4
3 –logA, M
(b)
1.0 0.8
1
4 0.4 2 0.2
1
2
0
20
40
60
80
100
0
p Fig. 3. Tonic response of 12-day chick embryo amnion to acetylcholine (1) in the control and (2) in the presence of 10 nM 4-DAMP: (a) abscissa shows acetylcholine concentration (–logA, M), and ordinate shows magnitude of response p, %; (b) the same plotted in the Scatchard coordinates. Abscissa shows the magnitude of response p, %; ordinate shows the ratio of response to acetylcholine concentration p/An, %/µM; (1) p/A, (2) p/A1.5.
p/A1.5
p/A
0.6 3
for the response evaluated by its contraction frequency; the respective EC50 values for the low-affinity pool were 26.75 ± 7.49 and 0.68 ± 0.04 nM. After treatment with 4-DAMP, both graphic and mathematical analyses of the ACh-induced response of the amnion revealed only one receptor pool, whether the tonic or the frequency component was considered (Fig. 4c, 4f). The respective EC50 values were 674.31 ± 74.89 and 35.33 ± 0.35 nM; i.e., the affinity of the receptors mediate the amnion tone and contraction frequency decreased by factors of 25 and 51. The maximum response remained unchanged. The main parameters EC50 and êm characterize the affinity of receptors and their number in the effector system. The interpretation of parameter n (in enzymology, cooperativity coefficient, or the Hill coefficient) is less definite. Supposedly, n shows the number of ligand molecules binding to receptor and characterizes cooperativity of the ligand–receptor interaction and heterogeneity of the pool of receptors with the same affinity. The value n = 2 in the equation of ligand–receptor binding nominally indicates the binding of two ligand molecules to one receptor. Physically, this is possible when one receptor molecule has two specific sites for ligand binding or receptors are dimeric. It was recently shown using a quantitative bioluminescent method that different seven-domain receptors coupled with G proteins may form stable homo- and heterodimers (Terrillon and Bouvier, 2004; Avdonin, 2005). Dimerization is essential for the functioning of serotonin receptors (HerrickDavis et al., 2005). Heterodimerization of β1 and β2 adrenergic receptors in cardiomyocytes gives rise to a new population of β-adrenergic receptors with differing pharmacological properties (Zhu et al., 2005). It was shown that α-adrenergic receptors may form homoand heterodimers (Small et al., 2006). The muscarinic receptors are not an exception. Different subtypes of M-cholinergic receptors exist as dimers and oligomers (Zeng and Wess, 1999; Park and Wess, 2004) and exhibit positive cooperativity upon binding with ligands (Huang and Ellis, 2007). Recently obtained chimeric heterodimers α2/å3 make it possible to analyze the interaction of muscarinic and adrenergic ligands. Comparative studies on the functional properties of homo- and heterodimers of muscarinic receptor subtypes are underway, and their results, in particular, BIOLOGY BULLETIN
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MUSCARINIC ACETYLCHOLINE RECEPTORS p, % 120
(‡) 1
100
1
100
80
2
80
60
60
40 20 0
(d)
120
2
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40 7
6
5
p/A2 800
20
4 3 –logA, M
7
6
p/A1.5 2500
(b) 1
2000
600
5
4 3 –logA, M
(e) 1
1500 400 1000 200
500 0
0 p/A 0.18 0.16 0.14 0.12 0.10 0.08 0.06 0.04 0.02 0
p/A2 0.06 0.05 0.04 0.03 0.02
(c) 2
20
40
60
80
0.01 0 –0.01 100 20 p, %
(f) 2
40
60
80
100 p, %
Fig. 4. Acetylcholine-induced response of 11-day chick embryo amnion (1) in the control and (2) in the presence of 10 nmol 4-DAMP: (a, b, c) tone; (d, e, f) contraction frequency. Abscissa and ordinate in (a) and (d) show acetylcholine concentration and the magnitude of response, respectively; abscissa and ordinate in (b, c, e) and (f) show the magnitude of response and the ratio between the magnitude of response and acetylcholine concentration, p/An.
show the necessity of paired activation of two components within the receptor dimer (Novi et al., 2005). The possibility of the binding of two ligand molecules to one receptor or dimer is indirectly confirmed by the results of experiments in which the Hill coefficient greater than unity was obtained. It was shown that [3H]-quinuclidinylbenzylate and other ligands can cooperatively bind with M-cholinergic receptors (Nesterova et al., 1995; Wreggett and Wells, 1995; Chidiac et al., 1997; Huang and Ellis, 2007). The available experimental and theoretical data are not yet sufficient for exactly determining the n value in physiological reactions. However, this parameter, along with EC50 and êm, is an objective quantitative index of the kinetics of ligand-receptor interaction and a functional characteristic of physiological reactions. The Hill coefficient of 1 and 2 presumably characterizes the monomeric or BIOLOGY BULLETIN
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dimeric state of receptors. If so, ACh can activate the cholinergic response of the amnion through both monoand dimeric muscarinic receptors, and 4-DAMP changes the ratio of monomers and dimers involved in the reaction, depending on the initial state of the system. Cholinergic receptors of å1 subtype were initially revealed in the neuronal tissue; the å2 subtype was found in the heart, smooth muscles, glands, and the CNS; å3 was found in the glands and smooth muscles of the intestine, urinary bladder, bronchi, and blood vessels; å4 was detected in the striatum. Subsequently it was shown that a tissue-specific population of muscarinic receptors may include several functionally active M subtypes. The majority of smooth muscles have muscarinic receptors of å2- and å3 subtypes (Ehlert,
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2003; Unno et al., 2006). We showed that forskolin (5 µM), activator of adenylate cyclase, acted unidirectionally with β-adrenergic agonists and blocked CBCinduced contractile activity of the amnion, whereas phospholipase C (1.25 U/ml) stimulated this activity. This is additional evidence for the involvement of å3 receptors in realization of cholinergic response in the amnion. A signal from the muscarinic receptors, which belong to the protein superfamily with seven transmembrane domains, is transmitted by GTP-binding G-proteins. Stimulation of å2 cholinergic receptors activates Gi proteins sensitive to the pertussis toxin, which results in inhibition of adenylate cyclase. In contrast, å3 cholinergic receptors act via Gq proteins (insensitive to this toxin) and activate the phosphoinositide system, with the consequent formation of inositol triphosphate and diacylglycerol. The signal pathways of å3-cholinergic receptors include activation of phospholipase C (Caulfield and Birdsall, 1998; Eglen et al., 1996; Eglen, 2005). It is considered that, in the heterogeneous postsynaptic population of å2- and å3 cholinergic receptors of smooth muscles, å3 receptors directly mediate contractile responses to acetylcholine, whereas å2 receptors modulate contractile activity by potentiating contractions induced by activation of å3 receptors or by inhibiting relaxation caused by activation of β-adrenergic receptors (Ehlert et al., 2005). This is attributed to å2 receptor coupling with Gi proteins, which mediate inhibition of adenylate cyclase (Eglen et al., 1996; Ehlert, 2003). Evidence for the contractile role of å2 cholinergic receptors was obtained by inactivating the signal pathways by pertussis toxin or recording contractile responses to muscarinic agonists after inactivation of å3 receptors. The mice with knocked-out genes for different receptor subtypes are used to study interactions between å2- and å3 receptors and their physiological role (Unno et al., 2006; Kitazawa et al., 2007). Studies performed with transgenic mice confirm the hypothesis that å3 receptors play the main role in realization of smooth muscle contraction (Stengel et al., 2002; Unno et al., 2006). Our data indicate that the contractile activity of the chick amnion is induced by cholinergic agonists and mediated by å3 cholinergic receptors. These receptors of the amnion mediate activation of its contractions and regulation of smooth muscle tone and contraction frequency. ACKNOWLEDGMENTS This study was supported by the Russian Foundation for Basic Research, project no. 05-04-48340.
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