J. Mol. Biol. (1976) 106, 457467
Studies on the Electrogenic Action of Acetylcholine with Torpedo marmorata Electric Organ I. Pharmacological
Properties of the Electroplaque
MA~CMOREAU*ANDJE~-PIERRECHAN~EUX~~ Wahkm Biobgique, 29211 Roswff and bNeurobiologie Molbulaire Institut Pasteur, 28 rue a’u Dr Roux, 75015 Paris, France (Received 19 November 1975) The membrane potential (average = - 62 mV) of a freely exposed electroplaque from a dissected prism of Torpedo naarmora&z electric organ is recorded with an intracellular glass microelectrode. The resting potential decreases with external potassium concentration. Acetylcholine (in the presence of O,O’-diethyl S-(p-diethylamino)ethyl phosphorothiolete), decamethonium, phenyltrimethylammonium and carbamylcholine added to the bath cause a decrease of membrane potential, i.e. behave as agonists. Their effect is blocked in a competitive manner by d-tubocurarine, gallamine and hexamethonium, and in a non-competitive way by prilocaine; 1 pg Erabutoxin/ml completely abolishes the response to carbamylcholine. The upvent dissociation constants for seven oholinergic ligands are determined from the dose-response curves, and found to be closely related to those previously determined with Electrophome ele&kus electroplaque with, however, a few differences. During these experiments it was noticed that potassium ions affect, in a differential manner, the response of T. rnarmaorata electroplaque to carbamylcholine and decamethonium.
1. Introduction One of the recent achievements of the research on the mechanisms by which acetylcholine regulates the cationic permeability of an excitable membrane has been the characterization, isolation and purification of the receptor protein which mediates this important physiological interaction (for reviews see Karlin, 1974; Rang, 1975;
Changeux, 1976). Among the several factors which made this approach successful, a critical one was the deliberate choice of an original biochemical material exceptionally rich in one class of cholinergic synapse : the electric organ from fish (Nachmansohn, 1959,1976) such as Electrophmu-s electricus or several species of the genus TorpecEo (Nachmansohn, 1959; Nschmansohn & Neumann, 1975). However, the dissection t-d isolation of single electroplaques, which permits a quantitative analysis of the physiological response to cholinergio agonists, was achieved only with Electrophorus (Schoffeniels t Nachmansohn, 1957). It is therefore only with this fish that a close comparison
between
the response to cholinergic
ligands in vivo and the actual binding
of the radioactive ligands to the isolated receptor protein (Meunier & Changeux, 1973; Meunier et al., 1974) could be established. t To whom correspondence should be addressed. 31
467
458
M. MOREAU
AND
J.-P.
CHANGEUX
Several characteristic structural features of the purified protein being recognized, becomes of primary interest to understand the modalities of its integration to the membrane phase and its regulatory properties in situ. A number of recent technical achievements render this approach feasible: in particular the isolation from Torpedo electric organ of membrane fragments extremely rich in cholinergic receptor protein (Cohen et al., 1972; Cohen & Changeux, 1975), and the demonstration that these fragments still preserve in the test tube the ability to respond to cholinergic agonists by an increase of 22Na+ permeability (Hazelbauer & Changeux, 1974). The aim of this series of papers is the study of some functional properties of the receptor protein present in these receptor-rich membrane fragments. Continuing with the method used for Electrophorw (Changeux et al., 1970; Changeux, 1975), a close comparison is systematically made between the phenomena observed at the three levels of reduction of the system: the living cell, the membrane-bound receptor, and the solubilized and purified protein. Surprisingly little is known of the pharmacology of the live Torpedo electroplaque (Bennett et al., 1961), possibly because of the difficulty of handling this particularly fragile cell ; this is why the first paper of this series deals with this question. Subsequently, the main features of the permeability response of the receptorrich membranes and its “desensitization” in vitro are established (Popot et al., 1976; Sugiyama et al., 1976). Finally, it is shown that conformational transitions accompany the binding of cholinergic ligands to the receptor-rich membranes labelled by a fluorescent local anaesthetic, quinacrine (Grimhagen & Changeux, 1976a,b). These structural transitions are correlated with charaoteristic changes of membrane permeability . As already mentioned, the present knowledge of the pharmacology of Torpedo electroplaque appears rather fragmentary. In the 195Os,Fessard & Taut (1952) made the first intracellular recordings of the electroplaque resting potential with the electric organ of T. wuzrmorata. At the same time it was also established that the elementary discharge of Torpedo electroplaque is a large, neurally evoked, postsynaptic potential (see review by Grundfest, 1967) and not an action potential, as in the case of Electrophorus and other fresh-water fish. Subsequently, Bennett et al. (1961) demonstrated with Torpedo nobiliana electroplaque that acetylcholine and carbamylcholine act as depolarizing agonists, and that d-tubocurarine is a nondepolarizing blocking agent. They also presented the first evidence for pharmacological desensitization. However, in these studies, the final concentration of agonist at equilibrium was not under control and the results were strictly qualitative. Finally, Miledi et al. (1971) have recorded miniature end-plate potentials from T. mrmorata electroplaque and mentioned that these disappear after preincubation with a snake venom a-toxin. This paper deals with the effect of several cholinergic ligands on T. mxwmorata electroplaque. Only measurements of electrical potential have been carried out with freely exposed cells dissected from isolated prisms of electroplaques. Dose-response curves have been established for a variety of cholinergic agonists and antagonists, and apparent dissociation constants determined from these curves. it
2. Materials and Methods The specimens of T. marmorata were fished off the French coast of the Atlantic near Arcachon or Concarneau, sent by train to Roseoff and kept for a few days
ocean in the
!aboratory before the experiments were started. During the summer, the sea-water of
ACETYLCHOLINE
RECEPTOR:
RESPONSE
IN
VIVO
459
the aquaria wss constantly renewed. In winter, a closed circulation of water was established. In both instances, the temperature of the water was kept close to 15°C. Only female animals of adult size were studied. After immobilization of the fish by cooling with crushed ice, both electric organs were removed and rapidly transferred to an oxygenated physiological solution at 14°C. The ionic composition of the physiological solution used in all the experiments was established from that of the blood serum determined in 100 different animals (Table 1) : 275 mu-NaCl, 7.6 mM-KCl, 4.3 mM-C&1,, 1.4 m&r-MgCl,. The saline solution was supplemented with urea (415 rnx final concn) and buffered with 1.5 mM-phosphate (pH 7.3). When urea was replaced by sucrose, the dissected organs could be preserved in excellent conditions at 4°C for more than 24 h.
TABLET Analytical
data for Torpedo marmorata serum
Ion
Concn (mn)
Na+ K+ ClC&P+ Mg=+ Lipids
and oholesterol
Normal
animals
Abnormal animals (do not respond to decamethonium)
282 7.3 303 4.4 1.3
S.D.
4.3 2.1 5.7 0.8 0.1
Concn (g/l)
S.D.
Cholesterol Lipids
1.06 3.75
0.1 0.15
Cholesterol Lipids
2.27 7.06
-
Sodium and potassium were measured by flame spectrophotometry, ohloride by complexometry, magnesium by the method of Javillier & Lavollay (1934), total lipids by gravimetry after extraction with chloroform/methanol, and cholesterol by the method of Polonovski (1947). Values are from 100 individuals, except for the abnormal animals, where 12 = 8.
The electrical measurements were carried out on 4-mm thick prisms of electroplaques freshly dissected from the electric organ. The dissected prism was oriented with the innervated (ventral) face of the electroplaques on top, then the first superficial, often injured, cells were removed. The preparation was immobilized with small needles inside a 2-ml Plexiglass chamber and maintained under a constant flow (1 l/h) of physiological solution for approximately 20 min. Intracellular recordings of membrane potential were then started with standard glass microelectrodes filled with 3 ~-Kc1 and with a resistance always larger than 20 M s1 against a Ag/AgCl bath electrode ; all the experiments were carried out at room temperature (20°C f 1 deg. C). The choline& effecters were added to the bath in less than 10 s (mixing time of the chamber), and as a continuous flow of solution (in general 100 ml). For each concentration of effector tested, 4 recordings were made on different cells from the same organ, then the same experiment was repeated 4 times with different animals. Each point shown in the Figures is therefore the average of 16 independent measurements. Experiments which, with the same animal and under the same experimental conditions, gave differences of more than 10 mV between different recordings of membrane potential were disregarded. When the effect of acetylcholine was tested, acetylcholinestersse was blocked by pre incubating the preparation with 0.1 mm-O,O’-diethyl S-(/3-diethylamino) ethyl phos phorothiolate for 10 min. Acetylcholine was added in the presence of 6.1 mm-T&ram?. t Abbreviation
used: Tetram,
O,O’-diethyl
S-(j%diethylamino)ethyl
phosphorothiolate.
M. MOREAU
460
AND
J.-P.
CHANGEUX
a control experiment, it was noticed that extensive exposure to 0.1 rnrd-Tetram (300 ml of solution) in the absenceof acetylcholine caused, by itself, a alight depolarization (approx.
In
10% of the resting potential). The blocking effect of d-tubocurarine, gallamine, hexamethonium or prilocaine was tested in the presence of carbamylcholine after lo-min equilibration of the preparation with the solution of antagonist. Carbamylcholine chloride and decamethonium bromide were purchaeed from K & K laboratoriea; phenyltrimethylammonium chloride from Eastman Kodak; d-tubocurarine chloride and gallamine triethiodide (Flaxedil) from Sigma; hexamethonium chloride and acetylcholine chloride from Merck; prilocaine hydrochloride was a gift from Laboratoire Roger Bellon, Neuilly, France and Erabutoxin a gift from Professor N. Tamiya, Tohoku University, Sendai, Japan.
3. ResuIts (a) Redng potentiub Figure 1 shows records of membrane potentials from dissected T. mwmor& electroplaquea. Under our experimental conditions (see Materials and Methods), the membrane potential remains stable for several minutes but, because of mechanical instability, recordings from a given electroplaque could never be continued for more than 15 to 20 minutes. The value of the resting potential measured with different electroplaques appears rather constant (fluctuation of only a few mV) within a given electric organ. It varies within much larger limits from -40 mV up to -70 mV, from one animal to another. The reasons for these tluctuations are not understood. The average value of the resting potential from 500 different cells was -52 mV (negative
-4SmV
I
0
I
I
Time
I
(min)
1
f
’
FIQ. 1. Changes of membrane potential recorded with T. marmoraka electropleque upon addition of aholinergic egoniete. The value of the resting potential is given at the beginning of the trace. PTA, phenyltrimethylammonium ; Dean, deoaC&b, carbamylcholine; ACh, acetylcholine; methonium; R, physiologiael solution.
ACETYLCHOLINE
RECEPTOR:
RESPONSE
IN
VIVO
461
inside) with u = 7.2. This value falls in the range of those reported by Fessard & TauE (1952) with T. murnwrata and by Bennett et al. (1961) with T. nobilianu. The resting potential varies with the concentration of K+ in the outside medium. Exchange of Na + by K + does not influence the resting potential below 40 rnrd-K + , but at higher concentrations the membrane potential decreases by approximately 30 mV for a tenfold increase of the extrscellular concentration of K+ (Fig. 2). Potassium ions are therefore involved in the establishment of the membrane potential.
c -5o-
-0-o 0
Y r P \ “\
OL”“” 0
IO [K’]
FIG. 2. Effect
of the external
I
L
too
IO00
1 meqwv
concentrations
1
of K+ on the resting potential.
(b) Effect of cholinergic agonists and antagonists on the rnernbrane potential As shown in Figure 1, the addition of acetylcholine (in the presence of O-1 mruTetram), carbamylcholine, decamethonium or phenyltrimethylammonium causes a decrease of membrane potential. These four compounds behave as agonists. With all of them, the time-course of the change of membrane potential looks the same. After a fast exponential decrease the membrane potential reaches a steady state, which lasts for a few minutes. At very high concentrations of agonists (e.g. 10 mM-carbamylcholine), the depolarization becomes transient. After a fast decrease the membrane potential progressively returns to its resting value. Desensitization might take place. The depolarization caused by all the agonists tested appears reversible. However, with several of them (acetylchohne, phenyltrimethylammonium and, to a lesser extent, carbamylcholine) it was noticed that after an extensive wash with the physiological solution, the membrane potential did not return to its resting va.lue but remained significantly lower. Each point on the dose-response curves is the average of 16 independent recordings (4 different electroplaques from 4 different animals). For each measurement, the amplitude of the depolarization was normalized to the value of the resting potential where E was the membrane potential (E,) and the response taken m (E-E,)/E,, at the plateau in the presence of the tested concentration of effector. Figure 3 shows a semi-logarithmic plot of the dose-response curves for the four agonists tested. A double reciprocal plot of the same data (Fig. 4) gives a straight line which extmpalates to the same maximal response: 72 to 75% of the resting potential (approx. - 14 mV when the membrane potential has its average value of -62 mV). The apparent dissociation constants estimated from these graphs are given in Table 2.
462
M. MOREAU
AND
J.-P.
CHANGEUX
ACh
0.6 0.5 0.4 0.3 0.2 0.1 I
I
I
I
I
I o-7
I o-6
to-5
I o-4
[AgonistI FIQ.
of 2’. murmorata electroplaque to several cholinergic agonists. and E the steady-state potential at any given concentration of agonist. Deca, decamethonium; PTA, phenyltrimethylammoniium; Garb, carbamyl-
3. Dose-response
curves
E, is the resting potential ACh, acetyloholine; choline.
TABLE
2
Apparertt dissociation constants estimated from the dose-response curves of T. marmorata eledroplaque to several chdinergic agonists and antagonists Torpedo K am (PM) Agonists
Antagonists (agonist carbamylcholine)
Aoetylaholine (0.1 mM-Tetram) Decamethonium Carbamyloholine Phenyltrimethylammonium d-Tubocurarine Gallamine Hexamethonium
The K,,, values were estimated from the double reciprocal of the data shown in Fig. 2. t From Changeux & Podleski (1969). $ From Higman et aE. (1963). $ From Karlin & Winnik (1988).
0.13AO.02
2.6 *0+3 30 f6 11 &3
0.2*0-04 ti.2*0+3 l.Qf0.3
Electrophorus K aw (PM) 1.1 1.27
3.0$ 12t
0.16$ 0.37 308
plots (Fig. 3) and from a Dixon
plot
The slopes of the Hill plots of the same data are close to unity, sometimes slightly lower, but never higher. In the absence of agonist, d-tubocurarine, gallamine and hexamethonium do not cause any change of membrane potential (pig. 1). As illustrated by Figure 5, all of them block the response to carbamyloholine by decreasing its apparent affinity without significantly changing its maximal response. They behave as competitive blocking agents. On the other hand, as expected for a typical local anaesthetic, prilocaine causes a significant decrease of the maximum response to carbamylcholine (Fig. 0).
ACETYLCHOLINE
RECEPTOR:
I
RESPONSE
IO XM-l
I06
IN
VIVO
463
20
CAChl 2*5*104 [Deco] FIQ. 4. Double-reciprocal
xM-,
2.5~10~ [PTA]
x ,o-, M
plot of the data of Fig. 3.
20
I
I I.0
I 0.5 I05
xM -1
[Garb]
FIQ. 6. Competitive blocking of the response to carbamylcholine by various nicotinic antagonists. Double reoiprooal plot. (0) 1Om8 M-d-tubocurarine; (A) 1Om6 ar-f%bxedil; (0) 1.6~ 1Om6 M-hexamethonium; ( x ) lo-’ M-d-tubocurarine; (0) control.
464
M. MOREAU
AND
J.-P.
CHANGEUX
2
I
I05
I M-1
[Carb]
FIG. 6. Non-competitive blocking of the response to carbamylcholine by a local anaesthetic, prilocaine. Double reciprocal plot. (0) 5 x 10e6M-prilooaine; (0) control.
During these experiments it was noticed that a change in the external concentration of K+ differentially affects the response to carbamylcholine and decamethonium. Increasing the extracellular concentration of K+ from 76 to 40 mM does not significantly modify the membrane potential and the amplitude of the response to 10 ,u~carbamylcholine; under the same circumstances, however, the response to 1 PMdecamethonium becomes negligible (Fig. 1). In the presence of 40 mM-K+, 2 j&Mdecamethonium does not affect the membrane potential and does not block the response to 20 PM-carbamylcholine. A similar, although distinct, effect of K+ on the frog motor end-plate has been reported by Dunin-Barkovskii et al. (1969). A rather unexpected observation was made with eight of the more than 100 animals examined. The electroplaques from these particular animals responded to carbamylcholine or acetylcholine but were never depolarized by decamethonium at any of the concentrations tested. All the electroplaques dissected from the electric organ of these Torpedo exhibited the same properties. Interestingly, their serum contained twice as much cholesterol and lipids as that of normal animals. Exposure of the preparation to a solution of Erabutoxin (1 pg/ml) does not alter the membrane potential but causes a 100% blockade of the depolarization caused by 20 mnr-carbamylcholine (Fig. 7) or 2 PM’-decamethonium. The same blocking effect takes place in the presence of 40 m&r-K+. 4. Discussion The thinness and fragility of T. marmorata electroplaque renders this preparation much more difficult to study by electrophysiological methods than the electroplaque from E. electricus (Schoffeniels & Nachmansohn, 1957). Nevertheless, a method has
ACETYLCHOLINE
RECEPTOR:
t 2x 1o-5 M- Carb
RESPONSE
IN
V’IVO
465
t R
2 min -
t R 1 P9 Erabutoxin FIG. 7. Blockade solution.
/ml
by Erabutoxin
I of the response to carbamylcholine
(Carb). R, Physiological
been developed which, from a dissected prism of electric tissue, gives reproducible and quantitative intracellular recordings of membrane potential from a single electroplaque upon addition of a cholinergic ligand. Only measurements of membrane potential have yet been obtained and we are aware of the possibility that with this preparation, as with Electropiaon*l electroplaque (Lester et al., 1975), some of the results might have to be reinterpreted when conductance measurements become available. The value of the resting potential is, on average, significantly lower (-52 mV) than that of Electropkww electroplaque (-430 mV). In both cases, changes of K+ concentration in the bathing fluid significantly affect the resting potential. The application of an agonist like carbamylcholine to T. murmorata electroplaque causes a decrease of membrane potential, which rapidly reaches a steady-state value. A similar finding was reported with Eleckrophorus eleotroplaque. It has been shown with this fish (Lester et al., 1975) that, if a fast increase of conductance parallels the early fast decrease of membrane potential, the conductance subsequently docreases while the membrane potential remains at its steady-state value. Such a phenomenon was interpreted as revealing the “pharmacological desensitization” of the response to the agonist. It might occur as well with T. marwwrata electroplaque, although direct experimental evidence for it is still lacking. The observation that, at very high concentrations of carbamylcholine the agonist-induced depolarization becomes transient suggests that, with T. murmorata as well, desensitization might take place. Similarly, Bennett et al. (1961) have reported with T. nobiltinu electroplaque that the amplitude of the end-plate potential decreases during the application of acetylcholine. Acetylcholine (in the presence of Tetram), decamethonium, carbamylcholine and phenyltrimethylammonium behave as agonists with T. marmorata electroplaque, and d-tubocurarine, gallamine, hexamethonium and a snake a-toxin block their effect. The pharmacological specificity of the response of this electroplaque is, therefore, as for Electro$orus, that of a nicotinic receptor site. Since the electrical potential is the only parameter yet measurable with Torpedo
466
M. MOREAU
AND
J.-P.
CHANGEUX
electroplaque, dose-response curves were constructed by plotting the amplitude of the steady-state depolarization as a function of agonist concentration. In contrast with what was found with Electrophorw electroplaque (Higman et al., 1963 ; Karlin, 1967; Changeux & Podleski, 1968; Lester et al., 1975), the shape of these curves does not deviate signifioantly from that of an hyperbola; still in contrast with the observations made with Electrophorus, the maximal responses given by saturating levels of the four agonists tested appear close to each other. The reason for these differences is not known. One possibility is that the reversal potential (Bennett et al., 1961) limits the amplitude of the depolarization reached at the saturating level of all the agonists tested. The apparent dissociation constants determined from the dose-response curves (Table 2) for the four agonists tested appear closely related to those found with Electrophorus electroplaque. Significant differences exist, however, in the case of acetylcholine and some of the antagonists. The apparent affinities of acetylcholine and gallamine are significantly smaller with Torpedo than with Electrophorus, while that of hexamethonium is about one order of magnitude higher. The apparent afiinity for d-tubocurarine is the same for both fish. With Electrophorus electroplaque, as for the amphibian and mammalian neuromuscular junction (ref. in Rang, 1975), the permeability change caused by carbamylcholine does not differ from that initiated by decamethonium. On the other hand, with T. marmerata electroplaque, the response to decamethonium seems more vulnerable to changes of potassium concentration in the bathing medium than that caused by carbamylcholine. Despite these few significant differences, the pharmacological properties of T. marmorata and E. electricus electroplaques appear basically similar: those expected from the presence of a typical nicotinic receptor. We thank J. L. Popot and M. Weber for aid and fruitful discussions in this work. One author (M. M.) was supported by a Roussel-Uolaf fellowship. We are grateful to Professors Boisseau and Cazaux from the Station Biologique d’ilrcachon for their constant help in the supply of Torpedo. This research was supported by grants from the National Institutes of Health, United States Public Health Service, the Centre National de la Recherche Scientifique, the Delegation G&&ale it la Recherche Scientifique et Technique, the Fondation pour la Recherche Medicale Franpaise, the College de France and the Commissariat & I’Energie Atomique. REFERENCES Bennett, M. V. L., Wurzel, M. & Grundfest, H. (1961). J. Gen. Physiol. 44, 757-804. Changeux, J. P. (1975). In Handbook of Psychopharmacology (Snyder, S. & Iversen, L., eds), pp. 235301, Plenum Publ. Co. New-York. Changeux, J. P. & Podleski, T. R. (1968). Proc. Nat. Acad. Sci., U.S.A. 59, 944-950. Changeux, J. P., Kasai, M. & Lee, C. V. (1970). PTOC. Nat. Acad. Sci., U.S.A. 67, 12411247. Cohen, J. B. & Changeux, J. P. (1975). Annu. Rev. Phurmacol. 15, 83-103. Cohen, J. B., Weber, M., Huchet, M. t Changeux, J. P. (1972). FEBS Letters, 26, 43-47. Dunin-Barkovskii, V. L., Kovalev, S. A., Magazanik, L. G., Potapova, T. V. &, Chaylakhyan, L. M. (1969). Biojzika, 14, 485-494. Fessard, A. t Tar& L. (1952). C.R.H. Acad. Sci. 233, 1228-1231. Grundfest, H. (1967). In Sharks, Skates and Raye (Gilbert, P. W., Mathewson, R. F. & Rall, D. P., eds), pp. 399-432, The Johns Hopkins Press, Baltimore. Grtinhagen, H. t Changeux, J. P. (1976a). J. Mol. Biol. 106, 497-516. Grtinhagen, H. & Changeux, J. P. (19766). J. Mol. Biol. 196, 517-535. Hazelbauer, G. L. & Changeux, J. P. (1974). Proc. Nat. Acad. Sci., U.S.A. 71, 1479-1483.
ACETYLCHOLINE
RECEPTOR:
RESPONSE
IN
VIVO
467
Higman, H., Podleski, T. R. BEBartels, E. (1963). B&him. Biophys. Actu, 75, 187-193. Javillier & Lavollay (1934). BUZZ.Sot. Chim. Biol. 16, 1531. Karlin, A. (1967). Biochim. Biophys. Acta, 139, 358-362. Karlin, A. (1974). Life Sci. 14, 1385-1415. Karlin, A. & Winnik, M. (1968). Proc. Nat. Acad. Sci., U.S.A. 60, 668-674. Lester, H. A., Changeux, J. P. & Sheridan, R. E. (1975). J. Gen. Physiol. 65, 797-816. Meunier, J. C. & Changeux, J. P. (1973). FEBS Letters, 32, 143-148. Meunier, J. C., Sealock, R., Olsen, R. & Changeux, J. P. (1974). Eur. J. Biochem. 45, 371394. Miledi, R., Molinoff, P. & Potter, L. T. (1971). Nature (London), 229, 554-557. Nachmansohn, D. (1959). Chemical and Molecular Bask of Nerve Activity, Academic Press, New York and London. Nachmansohn, D. & Neumann, E. (1975). Chemical and Molecular basis of Nerve Activity, Academic Press, New York and London. Polonovski, M. (1947). Biochimie MtWicaZe, Masson and Cie, Paris. Popot, J. L., Sugiyama, H. & Changeux, J. P. (1976). J. MOE. Biol. 106, 469-483. Rang, H. P. (1975). Quart. Rev. Biophys. 7, 283-399. Schoffeniels, E. & Naohmansohn, D. (1957) Biochim. Biophys. Acta, 26, 1-15. Sugiyama, H., Popot, J. L. & Changeux, J. P. (1976), J. Mol. BioZ. 106, 485-496.
Studies on the Electrogenic Action of Acetylcholine with Torpedo marmorata Electric Organ I. Pharmacological
Properties of the Electroplaque
MA~CMOREAU*ANDJE~-PIERRECHAN~EUX~~ Wahkm Biobgique, 29211 Roswff and bNeurobiologie Molbulaire Institut Pasteur, 28 rue a’u Dr Roux, 75015 Paris, France (Received 19 November 1975) The membrane potential (average = - 62 mV) of a freely exposed electroplaque from a dissected prism of Torpedo naarmora&z electric organ is recorded with an intracellular glass microelectrode. The resting potential decreases with external potassium concentration. Acetylcholine (in the presence of O,O’-diethyl S-(p-diethylamino)ethyl phosphorothiolete), decamethonium, phenyltrimethylammonium and carbamylcholine added to the bath cause a decrease of membrane potential, i.e. behave as agonists. Their effect is blocked in a competitive manner by d-tubocurarine, gallamine and hexamethonium, and in a non-competitive way by prilocaine; 1 pg Erabutoxin/ml completely abolishes the response to carbamylcholine. The upvent dissociation constants for seven oholinergic ligands are determined from the dose-response curves, and found to be closely related to those previously determined with Electrophome ele&kus electroplaque with, however, a few differences. During these experiments it was noticed that potassium ions affect, in a differential manner, the response of T. rnarmaorata electroplaque to carbamylcholine and decamethonium.
1. Introduction One of the recent achievements of the research on the mechanisms by which acetylcholine regulates the cationic permeability of an excitable membrane has been the characterization, isolation and purification of the receptor protein which mediates this important physiological interaction (for reviews see Karlin, 1974; Rang, 1975;
Changeux, 1976). Among the several factors which made this approach successful, a critical one was the deliberate choice of an original biochemical material exceptionally rich in one class of cholinergic synapse : the electric organ from fish (Nachmansohn, 1959,1976) such as Electrophmu-s electricus or several species of the genus TorpecEo (Nachmansohn, 1959; Nschmansohn & Neumann, 1975). However, the dissection t-d isolation of single electroplaques, which permits a quantitative analysis of the physiological response to cholinergio agonists, was achieved only with Electrophorus (Schoffeniels t Nachmansohn, 1957). It is therefore only with this fish that a close comparison
between
the response to cholinergic
ligands in vivo and the actual binding
of the radioactive ligands to the isolated receptor protein (Meunier & Changeux, 1973; Meunier et al., 1974) could be established. t To whom correspondence should be addressed. 31
467
458
M. MOREAU
AND
J.-P.
CHANGEUX
Several characteristic structural features of the purified protein being recognized, becomes of primary interest to understand the modalities of its integration to the membrane phase and its regulatory properties in situ. A number of recent technical achievements render this approach feasible: in particular the isolation from Torpedo electric organ of membrane fragments extremely rich in cholinergic receptor protein (Cohen et al., 1972; Cohen & Changeux, 1975), and the demonstration that these fragments still preserve in the test tube the ability to respond to cholinergic agonists by an increase of 22Na+ permeability (Hazelbauer & Changeux, 1974). The aim of this series of papers is the study of some functional properties of the receptor protein present in these receptor-rich membrane fragments. Continuing with the method used for Electrophorw (Changeux et al., 1970; Changeux, 1975), a close comparison is systematically made between the phenomena observed at the three levels of reduction of the system: the living cell, the membrane-bound receptor, and the solubilized and purified protein. Surprisingly little is known of the pharmacology of the live Torpedo electroplaque (Bennett et al., 1961), possibly because of the difficulty of handling this particularly fragile cell ; this is why the first paper of this series deals with this question. Subsequently, the main features of the permeability response of the receptorrich membranes and its “desensitization” in vitro are established (Popot et al., 1976; Sugiyama et al., 1976). Finally, it is shown that conformational transitions accompany the binding of cholinergic ligands to the receptor-rich membranes labelled by a fluorescent local anaesthetic, quinacrine (Grimhagen & Changeux, 1976a,b). These structural transitions are correlated with charaoteristic changes of membrane permeability . As already mentioned, the present knowledge of the pharmacology of Torpedo electroplaque appears rather fragmentary. In the 195Os,Fessard & Taut (1952) made the first intracellular recordings of the electroplaque resting potential with the electric organ of T. wuzrmorata. At the same time it was also established that the elementary discharge of Torpedo electroplaque is a large, neurally evoked, postsynaptic potential (see review by Grundfest, 1967) and not an action potential, as in the case of Electrophorus and other fresh-water fish. Subsequently, Bennett et al. (1961) demonstrated with Torpedo nobiliana electroplaque that acetylcholine and carbamylcholine act as depolarizing agonists, and that d-tubocurarine is a nondepolarizing blocking agent. They also presented the first evidence for pharmacological desensitization. However, in these studies, the final concentration of agonist at equilibrium was not under control and the results were strictly qualitative. Finally, Miledi et al. (1971) have recorded miniature end-plate potentials from T. mrmorata electroplaque and mentioned that these disappear after preincubation with a snake venom a-toxin. This paper deals with the effect of several cholinergic ligands on T. mxwmorata electroplaque. Only measurements of electrical potential have been carried out with freely exposed cells dissected from isolated prisms of electroplaques. Dose-response curves have been established for a variety of cholinergic agonists and antagonists, and apparent dissociation constants determined from these curves. it
2. Materials and Methods The specimens of T. marmorata were fished off the French coast of the Atlantic near Arcachon or Concarneau, sent by train to Roseoff and kept for a few days
ocean in the
!aboratory before the experiments were started. During the summer, the sea-water of
ACETYLCHOLINE
RECEPTOR:
RESPONSE
IN
VIVO
459
the aquaria wss constantly renewed. In winter, a closed circulation of water was established. In both instances, the temperature of the water was kept close to 15°C. Only female animals of adult size were studied. After immobilization of the fish by cooling with crushed ice, both electric organs were removed and rapidly transferred to an oxygenated physiological solution at 14°C. The ionic composition of the physiological solution used in all the experiments was established from that of the blood serum determined in 100 different animals (Table 1) : 275 mu-NaCl, 7.6 mM-KCl, 4.3 mM-C&1,, 1.4 m&r-MgCl,. The saline solution was supplemented with urea (415 rnx final concn) and buffered with 1.5 mM-phosphate (pH 7.3). When urea was replaced by sucrose, the dissected organs could be preserved in excellent conditions at 4°C for more than 24 h.
TABLET Analytical
data for Torpedo marmorata serum
Ion
Concn (mn)
Na+ K+ ClC&P+ Mg=+ Lipids
and oholesterol
Normal
animals
Abnormal animals (do not respond to decamethonium)
282 7.3 303 4.4 1.3
S.D.
4.3 2.1 5.7 0.8 0.1
Concn (g/l)
S.D.
Cholesterol Lipids
1.06 3.75
0.1 0.15
Cholesterol Lipids
2.27 7.06
-
Sodium and potassium were measured by flame spectrophotometry, ohloride by complexometry, magnesium by the method of Javillier & Lavollay (1934), total lipids by gravimetry after extraction with chloroform/methanol, and cholesterol by the method of Polonovski (1947). Values are from 100 individuals, except for the abnormal animals, where 12 = 8.
The electrical measurements were carried out on 4-mm thick prisms of electroplaques freshly dissected from the electric organ. The dissected prism was oriented with the innervated (ventral) face of the electroplaques on top, then the first superficial, often injured, cells were removed. The preparation was immobilized with small needles inside a 2-ml Plexiglass chamber and maintained under a constant flow (1 l/h) of physiological solution for approximately 20 min. Intracellular recordings of membrane potential were then started with standard glass microelectrodes filled with 3 ~-Kc1 and with a resistance always larger than 20 M s1 against a Ag/AgCl bath electrode ; all the experiments were carried out at room temperature (20°C f 1 deg. C). The choline& effecters were added to the bath in less than 10 s (mixing time of the chamber), and as a continuous flow of solution (in general 100 ml). For each concentration of effector tested, 4 recordings were made on different cells from the same organ, then the same experiment was repeated 4 times with different animals. Each point shown in the Figures is therefore the average of 16 independent measurements. Experiments which, with the same animal and under the same experimental conditions, gave differences of more than 10 mV between different recordings of membrane potential were disregarded. When the effect of acetylcholine was tested, acetylcholinestersse was blocked by pre incubating the preparation with 0.1 mm-O,O’-diethyl S-(/3-diethylamino) ethyl phos phorothiolate for 10 min. Acetylcholine was added in the presence of 6.1 mm-T&ram?. t Abbreviation
used: Tetram,
O,O’-diethyl
S-(j%diethylamino)ethyl
phosphorothiolate.
M. MOREAU
460
AND
J.-P.
CHANGEUX
a control experiment, it was noticed that extensive exposure to 0.1 rnrd-Tetram (300 ml of solution) in the absenceof acetylcholine caused, by itself, a alight depolarization (approx.
In
10% of the resting potential). The blocking effect of d-tubocurarine, gallamine, hexamethonium or prilocaine was tested in the presence of carbamylcholine after lo-min equilibration of the preparation with the solution of antagonist. Carbamylcholine chloride and decamethonium bromide were purchaeed from K & K laboratoriea; phenyltrimethylammonium chloride from Eastman Kodak; d-tubocurarine chloride and gallamine triethiodide (Flaxedil) from Sigma; hexamethonium chloride and acetylcholine chloride from Merck; prilocaine hydrochloride was a gift from Laboratoire Roger Bellon, Neuilly, France and Erabutoxin a gift from Professor N. Tamiya, Tohoku University, Sendai, Japan.
3. ResuIts (a) Redng potentiub Figure 1 shows records of membrane potentials from dissected T. mwmor& electroplaquea. Under our experimental conditions (see Materials and Methods), the membrane potential remains stable for several minutes but, because of mechanical instability, recordings from a given electroplaque could never be continued for more than 15 to 20 minutes. The value of the resting potential measured with different electroplaques appears rather constant (fluctuation of only a few mV) within a given electric organ. It varies within much larger limits from -40 mV up to -70 mV, from one animal to another. The reasons for these tluctuations are not understood. The average value of the resting potential from 500 different cells was -52 mV (negative
-4SmV
I
0
I
I
Time
I
(min)
1
f
’
FIQ. 1. Changes of membrane potential recorded with T. marmoraka electropleque upon addition of aholinergic egoniete. The value of the resting potential is given at the beginning of the trace. PTA, phenyltrimethylammonium ; Dean, deoaC&b, carbamylcholine; ACh, acetylcholine; methonium; R, physiologiael solution.
ACETYLCHOLINE
RECEPTOR:
RESPONSE
IN
VIVO
461
inside) with u = 7.2. This value falls in the range of those reported by Fessard & TauE (1952) with T. murnwrata and by Bennett et al. (1961) with T. nobilianu. The resting potential varies with the concentration of K+ in the outside medium. Exchange of Na + by K + does not influence the resting potential below 40 rnrd-K + , but at higher concentrations the membrane potential decreases by approximately 30 mV for a tenfold increase of the extrscellular concentration of K+ (Fig. 2). Potassium ions are therefore involved in the establishment of the membrane potential.
c -5o-
-0-o 0
Y r P \ “\
OL”“” 0
IO [K’]
FIG. 2. Effect
of the external
I
L
too
IO00
1 meqwv
concentrations
1
of K+ on the resting potential.
(b) Effect of cholinergic agonists and antagonists on the rnernbrane potential As shown in Figure 1, the addition of acetylcholine (in the presence of O-1 mruTetram), carbamylcholine, decamethonium or phenyltrimethylammonium causes a decrease of membrane potential. These four compounds behave as agonists. With all of them, the time-course of the change of membrane potential looks the same. After a fast exponential decrease the membrane potential reaches a steady state, which lasts for a few minutes. At very high concentrations of agonists (e.g. 10 mM-carbamylcholine), the depolarization becomes transient. After a fast decrease the membrane potential progressively returns to its resting value. Desensitization might take place. The depolarization caused by all the agonists tested appears reversible. However, with several of them (acetylchohne, phenyltrimethylammonium and, to a lesser extent, carbamylcholine) it was noticed that after an extensive wash with the physiological solution, the membrane potential did not return to its resting va.lue but remained significantly lower. Each point on the dose-response curves is the average of 16 independent recordings (4 different electroplaques from 4 different animals). For each measurement, the amplitude of the depolarization was normalized to the value of the resting potential where E was the membrane potential (E,) and the response taken m (E-E,)/E,, at the plateau in the presence of the tested concentration of effector. Figure 3 shows a semi-logarithmic plot of the dose-response curves for the four agonists tested. A double reciprocal plot of the same data (Fig. 4) gives a straight line which extmpalates to the same maximal response: 72 to 75% of the resting potential (approx. - 14 mV when the membrane potential has its average value of -62 mV). The apparent dissociation constants estimated from these graphs are given in Table 2.
462
M. MOREAU
AND
J.-P.
CHANGEUX
ACh
0.6 0.5 0.4 0.3 0.2 0.1 I
I
I
I
I
I o-7
I o-6
to-5
I o-4
[AgonistI FIQ.
of 2’. murmorata electroplaque to several cholinergic agonists. and E the steady-state potential at any given concentration of agonist. Deca, decamethonium; PTA, phenyltrimethylammoniium; Garb, carbamyl-
3. Dose-response
curves
E, is the resting potential ACh, acetyloholine; choline.
TABLE
2
Apparertt dissociation constants estimated from the dose-response curves of T. marmorata eledroplaque to several chdinergic agonists and antagonists Torpedo K am (PM) Agonists
Antagonists (agonist carbamylcholine)
Aoetylaholine (0.1 mM-Tetram) Decamethonium Carbamyloholine Phenyltrimethylammonium d-Tubocurarine Gallamine Hexamethonium
The K,,, values were estimated from the double reciprocal of the data shown in Fig. 2. t From Changeux & Podleski (1969). $ From Higman et aE. (1963). $ From Karlin & Winnik (1988).
0.13AO.02
2.6 *0+3 30 f6 11 &3
0.2*0-04 ti.2*0+3 l.Qf0.3
Electrophorus K aw (PM) 1.1 1.27
3.0$ 12t
0.16$ 0.37 308
plots (Fig. 3) and from a Dixon
plot
The slopes of the Hill plots of the same data are close to unity, sometimes slightly lower, but never higher. In the absence of agonist, d-tubocurarine, gallamine and hexamethonium do not cause any change of membrane potential (pig. 1). As illustrated by Figure 5, all of them block the response to carbamyloholine by decreasing its apparent affinity without significantly changing its maximal response. They behave as competitive blocking agents. On the other hand, as expected for a typical local anaesthetic, prilocaine causes a significant decrease of the maximum response to carbamylcholine (Fig. 0).
ACETYLCHOLINE
RECEPTOR:
I
RESPONSE
IO XM-l
I06
IN
VIVO
463
20
CAChl 2*5*104 [Deco] FIQ. 4. Double-reciprocal
xM-,
2.5~10~ [PTA]
x ,o-, M
plot of the data of Fig. 3.
20
I
I I.0
I 0.5 I05
xM -1
[Garb]
FIQ. 6. Competitive blocking of the response to carbamylcholine by various nicotinic antagonists. Double reoiprooal plot. (0) 1Om8 M-d-tubocurarine; (A) 1Om6 ar-f%bxedil; (0) 1.6~ 1Om6 M-hexamethonium; ( x ) lo-’ M-d-tubocurarine; (0) control.
464
M. MOREAU
AND
J.-P.
CHANGEUX
2
I
I05
I M-1
[Carb]
FIG. 6. Non-competitive blocking of the response to carbamylcholine by a local anaesthetic, prilocaine. Double reciprocal plot. (0) 5 x 10e6M-prilooaine; (0) control.
During these experiments it was noticed that a change in the external concentration of K+ differentially affects the response to carbamylcholine and decamethonium. Increasing the extracellular concentration of K+ from 76 to 40 mM does not significantly modify the membrane potential and the amplitude of the response to 10 ,u~carbamylcholine; under the same circumstances, however, the response to 1 PMdecamethonium becomes negligible (Fig. 1). In the presence of 40 mM-K+, 2 j&Mdecamethonium does not affect the membrane potential and does not block the response to 20 PM-carbamylcholine. A similar, although distinct, effect of K+ on the frog motor end-plate has been reported by Dunin-Barkovskii et al. (1969). A rather unexpected observation was made with eight of the more than 100 animals examined. The electroplaques from these particular animals responded to carbamylcholine or acetylcholine but were never depolarized by decamethonium at any of the concentrations tested. All the electroplaques dissected from the electric organ of these Torpedo exhibited the same properties. Interestingly, their serum contained twice as much cholesterol and lipids as that of normal animals. Exposure of the preparation to a solution of Erabutoxin (1 pg/ml) does not alter the membrane potential but causes a 100% blockade of the depolarization caused by 20 mnr-carbamylcholine (Fig. 7) or 2 PM’-decamethonium. The same blocking effect takes place in the presence of 40 m&r-K+. 4. Discussion The thinness and fragility of T. marmorata electroplaque renders this preparation much more difficult to study by electrophysiological methods than the electroplaque from E. electricus (Schoffeniels & Nachmansohn, 1957). Nevertheless, a method has
ACETYLCHOLINE
RECEPTOR:
t 2x 1o-5 M- Carb
RESPONSE
IN
V’IVO
465
t R
2 min -
t R 1 P9 Erabutoxin FIG. 7. Blockade solution.
/ml
by Erabutoxin
I of the response to carbamylcholine
(Carb). R, Physiological
been developed which, from a dissected prism of electric tissue, gives reproducible and quantitative intracellular recordings of membrane potential from a single electroplaque upon addition of a cholinergic ligand. Only measurements of membrane potential have yet been obtained and we are aware of the possibility that with this preparation, as with Electropiaon*l electroplaque (Lester et al., 1975), some of the results might have to be reinterpreted when conductance measurements become available. The value of the resting potential is, on average, significantly lower (-52 mV) than that of Electropkww electroplaque (-430 mV). In both cases, changes of K+ concentration in the bathing fluid significantly affect the resting potential. The application of an agonist like carbamylcholine to T. murmorata electroplaque causes a decrease of membrane potential, which rapidly reaches a steady-state value. A similar finding was reported with Eleckrophorus eleotroplaque. It has been shown with this fish (Lester et al., 1975) that, if a fast increase of conductance parallels the early fast decrease of membrane potential, the conductance subsequently docreases while the membrane potential remains at its steady-state value. Such a phenomenon was interpreted as revealing the “pharmacological desensitization” of the response to the agonist. It might occur as well with T. marwwrata electroplaque, although direct experimental evidence for it is still lacking. The observation that, at very high concentrations of carbamylcholine the agonist-induced depolarization becomes transient suggests that, with T. murmorata as well, desensitization might take place. Similarly, Bennett et al. (1961) have reported with T. nobiltinu electroplaque that the amplitude of the end-plate potential decreases during the application of acetylcholine. Acetylcholine (in the presence of Tetram), decamethonium, carbamylcholine and phenyltrimethylammonium behave as agonists with T. marmorata electroplaque, and d-tubocurarine, gallamine, hexamethonium and a snake a-toxin block their effect. The pharmacological specificity of the response of this electroplaque is, therefore, as for Electro$orus, that of a nicotinic receptor site. Since the electrical potential is the only parameter yet measurable with Torpedo
466
M. MOREAU
AND
J.-P.
CHANGEUX
electroplaque, dose-response curves were constructed by plotting the amplitude of the steady-state depolarization as a function of agonist concentration. In contrast with what was found with Electrophorw electroplaque (Higman et al., 1963 ; Karlin, 1967; Changeux & Podleski, 1968; Lester et al., 1975), the shape of these curves does not deviate signifioantly from that of an hyperbola; still in contrast with the observations made with Electrophorus, the maximal responses given by saturating levels of the four agonists tested appear close to each other. The reason for these differences is not known. One possibility is that the reversal potential (Bennett et al., 1961) limits the amplitude of the depolarization reached at the saturating level of all the agonists tested. The apparent dissociation constants determined from the dose-response curves (Table 2) for the four agonists tested appear closely related to those found with Electrophorus electroplaque. Significant differences exist, however, in the case of acetylcholine and some of the antagonists. The apparent affinities of acetylcholine and gallamine are significantly smaller with Torpedo than with Electrophorus, while that of hexamethonium is about one order of magnitude higher. The apparent afiinity for d-tubocurarine is the same for both fish. With Electrophorus electroplaque, as for the amphibian and mammalian neuromuscular junction (ref. in Rang, 1975), the permeability change caused by carbamylcholine does not differ from that initiated by decamethonium. On the other hand, with T. marmerata electroplaque, the response to decamethonium seems more vulnerable to changes of potassium concentration in the bathing medium than that caused by carbamylcholine. Despite these few significant differences, the pharmacological properties of T. marmorata and E. electricus electroplaques appear basically similar: those expected from the presence of a typical nicotinic receptor. We thank J. L. Popot and M. Weber for aid and fruitful discussions in this work. One author (M. M.) was supported by a Roussel-Uolaf fellowship. We are grateful to Professors Boisseau and Cazaux from the Station Biologique d’ilrcachon for their constant help in the supply of Torpedo. This research was supported by grants from the National Institutes of Health, United States Public Health Service, the Centre National de la Recherche Scientifique, the Delegation G&&ale it la Recherche Scientifique et Technique, the Fondation pour la Recherche Medicale Franpaise, the College de France and the Commissariat & I’Energie Atomique. REFERENCES Bennett, M. V. L., Wurzel, M. & Grundfest, H. (1961). J. Gen. Physiol. 44, 757-804. Changeux, J. P. (1975). In Handbook of Psychopharmacology (Snyder, S. & Iversen, L., eds), pp. 235301, Plenum Publ. Co. New-York. Changeux, J. P. & Podleski, T. R. (1968). Proc. Nat. Acad. Sci., U.S.A. 59, 944-950. Changeux, J. P., Kasai, M. & Lee, C. V. (1970). PTOC. Nat. Acad. Sci., U.S.A. 67, 12411247. Cohen, J. B. & Changeux, J. P. (1975). Annu. Rev. Phurmacol. 15, 83-103. Cohen, J. B., Weber, M., Huchet, M. t Changeux, J. P. (1972). FEBS Letters, 26, 43-47. Dunin-Barkovskii, V. L., Kovalev, S. A., Magazanik, L. G., Potapova, T. V. &, Chaylakhyan, L. M. (1969). Biojzika, 14, 485-494. Fessard, A. t Tar& L. (1952). C.R.H. Acad. Sci. 233, 1228-1231. Grundfest, H. (1967). In Sharks, Skates and Raye (Gilbert, P. W., Mathewson, R. F. & Rall, D. P., eds), pp. 399-432, The Johns Hopkins Press, Baltimore. Grtinhagen, H. t Changeux, J. P. (1976a). J. Mol. Biol. 106, 497-516. Grtinhagen, H. & Changeux, J. P. (19766). J. Mol. Biol. 196, 517-535. Hazelbauer, G. L. & Changeux, J. P. (1974). Proc. Nat. Acad. Sci., U.S.A. 71, 1479-1483.
ACETYLCHOLINE
RECEPTOR:
RESPONSE
IN
VIVO
467
Higman, H., Podleski, T. R. BEBartels, E. (1963). B&him. Biophys. Actu, 75, 187-193. Javillier & Lavollay (1934). BUZZ.Sot. Chim. Biol. 16, 1531. Karlin, A. (1967). Biochim. Biophys. Acta, 139, 358-362. Karlin, A. (1974). Life Sci. 14, 1385-1415. Karlin, A. & Winnik, M. (1968). Proc. Nat. Acad. Sci., U.S.A. 60, 668-674. Lester, H. A., Changeux, J. P. & Sheridan, R. E. (1975). J. Gen. Physiol. 65, 797-816. Meunier, J. C. & Changeux, J. P. (1973). FEBS Letters, 32, 143-148. Meunier, J. C., Sealock, R., Olsen, R. & Changeux, J. P. (1974). Eur. J. Biochem. 45, 371394. Miledi, R., Molinoff, P. & Potter, L. T. (1971). Nature (London), 229, 554-557. Nachmansohn, D. (1959). Chemical and Molecular Bask of Nerve Activity, Academic Press, New York and London. Nachmansohn, D. & Neumann, E. (1975). Chemical and Molecular basis of Nerve Activity, Academic Press, New York and London. Polonovski, M. (1947). Biochimie MtWicaZe, Masson and Cie, Paris. Popot, J. L., Sugiyama, H. & Changeux, J. P. (1976). J. MOE. Biol. 106, 469-483. Rang, H. P. (1975). Quart. Rev. Biophys. 7, 283-399. Schoffeniels, E. & Naohmansohn, D. (1957) Biochim. Biophys. Acta, 26, 1-15. Sugiyama, H., Popot, J. L. & Changeux, J. P. (1976), J. Mol. BioZ. 106, 485-496.
