Biology Bulletin, Vol. 29, No. 2, 2002, pp. 148–153. Translated from Izvestiya Akademii Nauk, Seriya Biologicheskaya, No. 2, 2002, pp. 186–191. Original Russian Text Copyright © 2002 by Manukhin, Boiko.
ANIMAL PHYSIOLOGY
Kinetics of b1-Adrenergic Reaction of the Chick Embryo Amnion B. N. Manukhin and O. V. Boiko Kol’tsov Institute of Developmental Biology, Russian Academy of Sciences, ul. Vavilova 26, Moscow, 119991 Russia e-mail:
[email protected] Received August 7, 2001
Abstract—Dose-dependent curves of the inhibitory β1-adrenergic reaction of the chick embryo amnion were analyzed on the basis of different mathematical models. Selection of the optimal model, closest to the experimental data, allows us to obtain the reaction parameters with the smallest statistical error. Two main characteristics of the physiological reaction, EC50 and Pm, sufficed for the description of the action of agonists nonselective for β1-adrenoreceptors: adrenaline (EC50 0.19 ± 0.02 µM, Pm 101.3 ± 1.9%) and salbutamol (EC50 0.58 ± 0.01 µM, Pm 42.7 ± 0.3%). The Hill coefficient n in these experiments was close to 1 (1.06 ± 0.14; 0.96 ± 0.02). It was necessary to introduce fractional values 1 < n < 2 to describe the effects of noradrenaline and isopropyl noradrenaline.
INTRODUCTION For quantitative analysis of the enzymatic, radioligand, and physiological reactions, the method of estimation of the kinetic parameters is used, which is common in its general features for all these systems. In the physiological experiment, the transmitter-receptor interaction is separated from the terminal reaction by a series of intracellular transformations. The mechanisms of signal transduction are complex and multivariate and, hence, the values characterizing the ligand-receptor interactions in these conditions were introduced with certain restrictions. Nevertheless, a definite mathematical model corresponds to each species' experimental dose-effect curve and, respectively, one mathematical and graphical expression which reflects this curve with a minimal error. Identification of this optimal model, closest to the experimental data, provides, finally, the values of parameters used for the analysis of physiological reactions, with the least possible statistical error (Manukhin et al., 1998). Here, we will consider specific features of the quantitative analysis of the β-adrenergic reaction of the chick embryo amnion, which is expressed in the inhibition of its contractile activity. β-Adrenoreceptors belong to a large group of receptors coupled with G-proteins (Ulloa-Aguirre et al., 1999). The pathways of transduction of the signal of exogenous ligands for β-adrenoreceptors include a receptor, Gs-protein, and adenylate cyclase cascade. It was shown using cloning that there is a structural homology among membrane receptors coupled with G-proteins. The order of the amino acids and receptor stereostructure are factors of the β-adrenoreceptor pharmacological profile, which also determines the type of interaction with β-agonists and β-blockers (Nagatomo and Koike, 2000). Although the chick embryo amniotic tissues is fully devoid of nerve ele-
ments, the amnion β-adrenoreceptors are quite comparable in their pharmacological and quantitative characteristics with the receptors of innervated tissues (Boiko and Manukhin, 2000; Manukhin and Boiko, 2000). Computer analysis of the experimental data allowed calculation of each does-dependent curve on the basis of different mathematical models. An analog of the Michaelis-Menten equation widely used in enzymatic kinetics is the base equation for all variants: p = (PmA)/(EC50 + A), (1) where p is the value of reaction to agonist at concentration A, EC50 is the concentration of ligand that induces a reaction equal to a half of the maximum. Correspondingly, equation (1) can be presented for two pools of receptors as p = [(Pm1A)/(EC501 + A)] + [(Pm2A)/(EC502 + A)]. (2) Since a specific receptor can have several binding sites, one receptor can bind two or more ligand molecules. Some models were proposed for analysis of such systems (Klotz and Hunston, 1971; Feldman, 1972; Dixon and Webb, 1982). One of the earliest models was introduced by Hill (Dixon and Webb, 1982). If n ligand molecules are bound to the receptor, equation (1) assumes the form (3) p = (PmAn)/(EC50n + An), and for two pools of receptors, p = [ ( Pm 1 A )/ ( EC50 1 + A ) ] n1
n1
n1
+ [ ( Pm 2 A )/ ( EC50 2 + A ) ]. n2
n2
n2
(4)
MATERIALS AND METHODS Studies were carried out on the amnion of 14-day chick embryos. An isolated band of amnion was placed in a thermostat (38°C) aerated 10-ml chamber with
1062-3590/02/2902-0148$27.00 © 2002 MAIK “Nauka /Interperiodica”
KINETICS OF β1-ADRENERGIC REACTION OF THE CHICK EMBRYO AMNION
Hanks solution, mM: NaCl 137, KCl, 5.4, CaCl2 1.26, MgSO4 0.41, MgCl2 0.49, Na2HPO4 0.34, KH2PO4 0.44, NaHCO3 4.2, and glucose 5.6. The initial load on the preparation amounted to 100 mg. Contractions were recorded using a 6MKh1B mechanotron on H-339 and H-399 self-recorders. The studied substances were introduced in the chamber at 100 µl after a 30-min preincubation of the tissue. Dose-dependent reactions of the amnion were recorded cumulatively. In order to assess the inhibitory effect of adrenomimetics, the contractile activity was stimulated by acetylcholine (Ach, 50 µM). The reaction to Ach was taken as 100%. The following effects were expressed as a percentage of this value. It was already shown that catecholamines also stimulated the amnion contractile activity, with α2adrenoreceptors as mediators (Boiko and Manukhin, 1996). In order to exclude the action on α-adrenoreceptors, specific antagonists phentolamine (10 µM) or idazoxan (2 µM) were preliminarily introduced. When analyzing the inhibitory adrenergic reaction, we used agonists noradrenaline (NA), adrenaline (A), isopropyl noradrenaline (Iso), and salbutamol (Sal) in a wide range of concentrations. The main parameters of the adrenergic reactions, such as an agonist concentration that induces a reaction equal to a half of maximum (EC50), maximum reaction (Pm), and Hill coefficient (n), were calculated using Sigmaplot 5.0 software (Manukhin et al., 1998). RESULTS AND DISCUSSION Against the background of Ach stimulation, all used agonists of β-adrenoreceptors, Iso, NA, A, and Sal, induced a dose-dependent decrease in the amnion tone and (or) frequency of its contractions. In some cases, the experimental data could be satisfactorily described by means of two main characteristics of the physiological reactions: EC590 and Pm. The effects of A (EC50 0.19 ± 0.02 µM, Pm 101.3 ± 1.9%) or Sal (EC50 0.58 ± 0.01 µM, Pm 42.7 ± 0.3%) on the amnion can serve as examples. These are nonselective agonists for β1-adrenoreceptors. The value of n in these experiments was close to 1: 1.06 ± 0.14 and 0.96 ± 0.02 for A and Sal, respectively. Therefore equation (1) is sufficient for their analysis. Correspondingly, a graphic presentation of the results in Sketchard standard coordinates gives a straight line (Fig. 1). This suggests that a homogenous population of β-adrenoreceptors responds uniformly to A and Sal. In the vast majority of cases, we cannot get by without introducing the power coefficient, which, by analogy with the enzymatic and radioligand reactions, is called the Hill coefficient. The possibility of using an integer value n = 2 is an exception, rather than a rule. Figure 2 shows how the experimental curve of NA effect on the amnion of a 14-day chick embryo as a function of the agonist concentration can be linearized in Sketchard coordinates, if n = 2. The most widespread BIOLOGY BULLETIN
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Fig. 1. Inhibition of cholinergic reaction of the amnion of a 14-day chick embryo by adrenaline (A) and salbutamol (Sal): a—abscissa: agonist concentration, A (µM); ordinate: value of reaction, p (% of reaction value to acetylcholine); b—the same in Sketchard coordinates, abscissa: value of reaction; ordinate: ratio of reaction value to agonist concentration.
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Fig. 2. Inhibition of cholinergic contractile reaction of the amnion of a 14-day chick embryo by noradrenaline (NA). Abscissa: value of reaction, p (%); ordinate: ratio of reaction value to agonist concentration: (a) p/A, (b) p/A2.
variant of the mathematical model illustrating the doseeffect relationship in physiological reactions and, specifically, in the experiments on activation of the amnion β-adrenoreceptors remains its expression through fractional n values, as in the case of the Iso effect on the amnion of a 14-day chick embryo (Fig. 3). The graph in standard Sketchard coordinates is nonlinear at n = 1 and n = 2 (Fig. 3b), which suggests a more complicated relationship between EC50 and Pm. Based on the calculations we made, the curve can be linearized by introduction of the corresponding fractional n value, in a given case equal to 1.37 ± 0.07 (Fig. 3c). Linearization is not an end in itself. In such a case, it is essential that the main parameters of the reaction are determined with the least error (Table 1). At the same time, under the conditions 1 < n < 2, another interpretation is possible. Analysis based on the two-component model at n = 2 showed the presence of
150
MANUKHIN, BOIKO p, % 100
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Fig. 3. Effect of isopropyl noradrenaline (Iso) on the amnion of a 14-day chick embryo against the background of cholinergic reaction: a—abscissa: agonist concentration, A (µM); ordinate: value of reaction, p (%); b–d—abscissa: value of reaction; ordinate: ratio of reaction value to agonist concentration: (1) p/A, (2) p/A2; (c) p/A1.4; d—graph was plotted by calculated parameters: EC50 = 0.036 µM, P1 = 47.0% at n = 1 and P2 = 35.5% at n = 2 (Table 1, model no. 5).
ulation of receptors homogenous in their affinity to the agonist (EC50 = 0.036 ± 0.001 µM). The resulting value of Pm in this case consists of two: Pm1 = 47.0% and Pm2 = 35.5% (Fig. 3d), while the obtained values maximally approach the experimental data (Norm = 1.36). The values of EC50 of variants no. 3 and no. 5 are identical and the sum of Pm1 and Pm2 equal to 82.5% reproduces the value of Pm = 812.2 ± 1.3 (Table 1). Model no. 6 practically duplicates no. 5, since the values of EC50 are within the same confidence interval.
two conventional pools of receptors with a high coincidence of calculated and experimental points: Norm = 2.58. This value practically coincides with that for the single-component model at n = 1.37: Norm = 2.51. It was proposed to consider the two-pool model only in those cases when the data was described much better in this way than in the case of the single-component model (De Lean et al., 1980). Further analysis of the results of the given experiment has shown that, if two power indices n = 1 and n = 2 are introduced in the calculation formula (Manukhin, 2000), reaction parameters can be obtained, which characterize a uniform pop-
The reaction to NA and Iso was characterized by n < 1 in one experiment of 12 (n = 0.88 ± 0.08) and in
Table 1. Calculation of parameters of the amnion reaction to isopropyl noradrenaline based on different models (see Fig. 3) Ordinal number of model Parameters 1
2
3
4
5
6
EC501, µM
0.043 ± 0.006
0.031 ± 0.003
0.035 ± 0.002
0.007 ± 0.003
0.036 ± 0.001
0.030 ± 0.005
EC502, µM
–
–
–
0.045 ± 0.004
–
0.040 ± 0.003
Pm1, %
89.1 ± 3.5
76.0 ± 2.6
81.2 ± 1.3
15.9 ± 4.8
47.0 ± 3.5
42.7 ± 5.3
Pm2, %
–
–
–
62.1 ± 4.6
35.5 ± 3.0
38.7 ± 4.3
n1 n2 Norm
1
2
1.37 ± 0.07
2
1
1
2
2
2
2.58
1.36
1.1
–
–
–
7.87
9.61
2.51
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Fig. 4. Inhibition of cholinergic reaction of the amnion of a 14-day chick embryo by isopropyl noradrenaline: a—abscissa: agonist concentration, A (µM); ordinate: value of reaction, p (%); b–d—abscissa: value of reaction; ordinate: ratio of reaction value to agonist concentration: (1) p/A, (2) p/A2, (c) p/A2 (Table 2, model no. 4), (d) Table 2, model no. 6.
two experiments of 10 (n = 0.65 ± 0.06 and 0.70 ± 0.11), respectively. The results of analysis of the amnion reaction to Iso based on six models of ligand-receptor interaction are given in Fig. 4 and Table 2. Mathematical analysis of the reaction has revealed two pools of receptors or two functional states with EC50 differing by more than an order of magnitude.
Since the value of n changes within very narrow limits and every subsequent introduction of the same substance or a new agent somewhat changes the picture of dose dependence, it is important to mathematically analyze every individual experiment, which allows us to follow the general trend of changes. The correspondence of the model to the experimental results can be
Table 2. Calculation of parameters of the amnion reaction to isopropyl noradrenaline based on different models (see Fig. 4) Ordinal number of model Parameters EC501, µM EC502, µM Pm1, % Pm2, % n1 n2 Norm
1
2
3
4
5
6
0.030 ± 0.005 – 91.4 ± 3.9 – 1 – 9.98
0.022 ± 0.006 – 78.2 ± 6.9 – 2 – 27.21
0.057 ± 0.014 – 112.4 ± 7.7 – 0.65 ± 0.06 – 3.44
0.005 ± 0.001 0.078 ± 0.012 36.1 ± 3.9 52.2 ± 3.9 2 2 4.73
– – – – – – –
0.008 ± 0.00 0.10 ± 0.02 50.4 ± 6.9 39.9 ± 6.1 1 2 3.38
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estimated from the errors of calculated parameters and coincidence of the calculated and experimental points (Norm). The parameters that characterize physiological reactions are not that ambiguous as in the case of enzymatic and radioligand reactions. The value of EC50 in physiological experiments significantly differs from the Kd of radioligand experiments on one object (Suslova et al., 1989). The empirical Hill coefficient is often considered as the number of binding centers on the receptor molecule or an index of coperativity. Since physiological reactions are mediated by a cascade of intracellular processes and the coupling of the receptor with the system of secondary messengers, unconditioned interpretation of the index n still awaits its comprehension. At the same time, it is practically impossible to avoid using this parameter in calculations, since it allows a maximal approximation of the calculated curves to the initial experimental curves. Then, value n < 1 is usually considered as an index of the mixed, heterogenous population of receptors. Several hypotheses have been proposed. (1) The presence of heterogenous independent, i.e., noninteracting, receptors. (2) Heterogeneity may be determined by interaction with G-proteins (the reversible state of a part of the homogenous population of receptors). The coupled and noncoupled receptors coexist in equilibrium. Decrease in the proportion of a high affinity pool to an indeterminable level in the presence of guanine nucleotides suggests interconvertibility of the two forms of receptors (De Lean et al., 1980; Birnbaumer et al., 1990). (3) The existence of cooperativity upon binding suggests that the receptors can form oligomeric structures. The cooperative properties of M-cholinoreceptors are often explained by the oligomeric nature of receptors (Wregget and Wells, 1995; Chidiac et al., 1997). In the experiments with expressed β2-adrenoreceptors, it was shown that incubation of membranes in the presence of Iso increases the number of dimeric receptors. A synthesized peptide that inhibits dimerization blocked the β-agonist-induced activation of adenylate cyclase. A dynamic equilibrium has been assumed between the monomeric and dimeric forms of the receptor (Hebert et al., 1996). The possibility of cooperative binding of Iso to β1-adrenoreceptors of the rat brain has been shown. The value n < 1 determined upon inhibition of binding of antagonist [3H](−)CGP−12177 by Iso changed to n > 1 in the presence of diamide, an oxidizer of SH-groups (Fowler et al., 1999). It may be that the results we present: the heterogeneity of receptors shown in individual experiments with agonists reflects the multistep process and the summation of the kinetic parameters of a number of successive reactions. In such a case, the key role belongs to endogenous factors. Differential sensitivity can also be explained by the different functional state of receptors. In addition to postreceptor mechanisms, the parameters of reaction to an agonist is also affected by the
presence and size of a pool of receptors and desensitization. In the case of desensitization, first of all a high affinity fraction of receptors is inactivated (Wessels et al., 1979). The value of agonist Pm and the resistance of receptor structures against desensitization are also affected by the size of a pool of receptors (Drury et al., 1998). This study should be followed by investigation of the effects on the stages of reaction development following the ligand-receptor interaction at the level of mechanisms providing for intracellular signal transduction: with the use of cholera toxin, forskolin, etc. In addition to studying the systems of secondary messengers coupled with the receptor and involved in realization of the final effect, it may be interesting to compare, for the same object, the data of physiological experiments and radioligand analysis of binding agonists and antagonists to receptor structures. ACKNOWLEDGMENTS This study was supported by the Russian Foundation for Basic Research, project no. 99-04-49141. REFERENCES Birnbaumer, L., Abramowitz, J., and Brown, A.M., ReceptorEffector Coupling by G Proteins, Biochim. Biophys. Acta, 1990, vol. 1031, no. 2, pp. 163–224. Boiko, O.V. and Manukhin, B.N., α2-Adrenoreceptors of the Chick Embryo Amnion, Dokl. Ross. Akad. Nauk, 1996, vol. 347, no. 5, pp. 704–706. Boiko, O.V. and Manukhin, B.N., Pharmacokinetic Analysis of α- and β-Adrenoreceptors of the Chick Embryo Amnion, Ros. Fiziol. Zh., 2000, vol. 86, no. 11, pp. 1237–1245. Chidiac, P., Green, M.A., Pawagy, A.B., and Wells, J.W., Cardiac Muscarinic Receptors. Cooperativity as the Basis for Multiple States of Affinity, Biochemistry, 1997, vol. 36, no. 24, pp. 7361–7379. De Lean, A., Stadel, J.M., and Lefkowitz, R.J., A Ternery Complex Model Explains the Agonist-Specific Binding Properties of the Adenylate Cyclase-Coupled β-Adrenergic Receptor, J. Biol. Chem., 1980, vol. 255, no. 15, pp. 7108–7117. Dixon, M. and Webb, E., Fermenty (Enzymes), Moscow: Mir, 1982. Drury, D.E.J., Chong, L.K., Ghahramani, P., and Peachell, P.T., Influence of Receptor Reserve on β-Adrenoreceptor-Mediated Responses in Human Lung Mast Cells, Brit. J. Pharmacol., 1998, vol. 124, no. 4, pp. 711–718. Feldman, H.A., Mathematical Theory of Complex LigandBinding Systems at Equilibrium: Some Methods for Parameter Fitting, Anal. Biochem., 1972, vol. 48, no. 2, pp. 317–338. Fowler, C.J., Vedin, V., and Sjöberg, E., Evidence for Cooperative Binding of (–) Isoproterenol to Rat Brain β-Adrenergic Receptors, Biochem. Biophys. Res. Commun., 1999, vol. 257, no. 2, pp. 629–634. Hebert, T.E., Moffett, S., Morello, J.-P., et al., A Peptide Derived from a β2-Adrenergic Receptor Transmembrane Domain Inhibits both Receptor Dimerisation and Activation, J. Biol. Chem., 1996, vol. 271, no. 27, pp. 16384–16392. BIOLOGY BULLETIN
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KINETICS OF β1-ADRENERGIC REACTION OF THE CHICK EMBRYO AMNION Klotz, I.M. and Hunston, D.L., Properties of Graphical Representation of Multiple Classes of Binding Sites, Biochemistry, 1971, vol. 10, no. 16, pp. 3065–3069. Manukhin, B.N., Analysis of Ligand-Receptor Interactions at the Levels from Molecules to Organisms, Ros. Fiziol. Zh., 2000, vol. 86, no. 9, pp. 1220–1232. Manukhin, B.N. and Boiko, O.V., Analysis of β-Adrenergic Reaction of the Chick Embryo Amnion, Dokl. Ross. Akad. Nauk, 2000, vol. 374, no. 5, pp. 702–705. Manukhin, B.N., Berdysheva, L.V., Boiko, O.V., et al., Quantitative Analysis of Ligand-Receptor Interaction in Physiological Experiments, Ros. Fiziol. Zh., 1998, vol. 84, no. 10, pp. 1049–1060. Nagatomo, T. and Koike, K., Recent Advances in Structure, Binding Sites with Ligands and Pharmacological Function of β-Adrenoreceptors Obtained by Molecular Biology and Molecular Modeling, Life Sci., 2000, vol. 66, no. 25, pp. 2419–2426.
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Suslova. I.Yu., Moskovkin, G.N., and Turpaev, T.M., Comparative Analysis of Interaction between Ligands and Muscarinic Receptors in Isolated Cardiac Muscle and in Myocardium Homogenate, Dokl. Akad. Nauk SSSR, 1989, vol. 309, pp. 252–255. Ulloa-Aguirre, A., Stanislaus, D., Janovick, J.A., and Conn, P.M., Structure-Activity Relationships of G ProteinCoupled Receptors, Arch. Med. Res., 1999, vol. 30, pp. 420–435. Wessels, M.R., Millikin, D., and Lefkowitz, R.J., Selective Alteration in High Affinity against Binding: A Mechanism of β-Adrenergic Receptor Desensitization, Mol. Pharmacol., 1979, vol. 16, pp. 10–20. Wregget, K.A. and Wells, J.W., Cooperativity Manifests in the Binding Properties of Purified Cardiac Muscarinic Receptors, J. Biol. Chem., 1995, vol. 270, no. 38, pp. 22488–22499.
