- Email: [email protected]
Unusual cholinergic response of bullfrog sympathetic ganglion cells
European Journal of Pharmacology, 31 (1975) 281--286 © North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands
UNUSUAL CHOLINERGIC RESPONSE OF BULLFROG SYMPATHETIC GANGLION CELLS KYOZO KOKETSU and KEIKO YAMAMOTO Department of Physiology, Kurume University School of Medicine, 67 Asahi-machi, Kurume-shi, 830, Japan Received 25 October 1974, revised MS received 31 December 1974, accepted 7 January 1975
K. KOKETSU and K. YAMAMOTO, Unusual cholinergic response of bullfrog sympathetic ganglion cells, European J. Pharmacol. 31 (1975) 281--286. The membrane of bullfrog sympathetic ganglion cells was hyperpolarized by a direct action of ACh (more than 0.5 mM) in a solution containing both nicotine {0.24 mM) and atropine (0.14 mM). This ACh hyperpolarization could be imitated by neither carbachol nor bethanechol, suggesting that the ACh hyperpolarization was the response which was produced by a specific action of ACh, which appeared to be neither nicotinic nor muscarinic. The size of ACh hyperpolarization was increased during a conditioning hyperpolarization. The ACh hyperpolarization was completely blocked by ouabain (2 x 10 -3 mM) and eliminated in the Na-free lithium solution. These aspects of the ACh hyperpolarization suggested that generation of this hyperpolarization was associated with the sodium pump. The ACh hyperpolarization seemed to be partially responsible for the production of the slow IPSP, since a part of the slow IPSP remained occasionally in the presence of both nicotine and atropine. Electrogenic sodium pump
Sympathetic ganglion
1. I n t r o d u c t i o n Since c e r t a i n a c t i o n s o f a c e t y l c h o l i n e ( A C h ) c o u l d be i m i t a t e d b y n i c o t i n e a n d others by muscarine, the actions of ACh were originally classified as e i t h e r ' n i c o t i n i c ' o r ' m u s c a r i n i c ' . In general, t h e n i c o t i n i c a c t i o n o f ACh c a n b e selectively b l o c k e d b y e i t h e r n i c o t i n e or d - t u b o c u r a r i n e (d-TC), w h e r e a s t h e m u s c a r i n i c a c t i o n c a n b e selectively b l o c k ed b y e i t h e r m u s c a r i n e o r a t r o p i n e . T h u s , it can be e x p e c t e d t h a t all k i n d s o f cellular responses produced by ACh should be complet e l y e l i m i n a t e d b y n i c o t i n e ( o r d-TC) a n d muscarine (or atropine). T h e m e m b r a n e p o t e n t i a l changes o f bullfrog s y m p a t h e t i c ganglion cells, w h i c h w e r e produced by ACh, consist of nicotinic and m u s c a r i n i c c o m p o n e n t s (cf. K o k e t s u , 1 9 6 9 ) . In fact, t h e cholinergic r e s p o n s e s o f t h e s e cells are r e p o r t e d t o b e c o m p l e t e l y b l o c k e d in t h e presence of nicotine and atropine (Koketsu et al., 1 9 6 8 ) . D u r i n g r e c e n t e x p e r i m e n t s in o u r
Acetylcholine
Hyperpolarization
l a b o r a t o r y , h o w e v e r , it was f o u n d t h a t careful e x p e r i m e n t s c o u l d reveal an u n u s u a l cholinergic r e s p o n s e o f these cells, w h i c h was v e r y resistant t o t h e a c t i o n s o f b o t h n i c o t i n e a n d a t r o p i n e a n d r e m a i n e d in t h e p r e s e n c e s o f t h e s e drugs. P h a r m a c o l o g i c a l analyses o f this n e w t y p e o f cholinergic r e s p o n s e o f ganglion cells h a v e b e e n t h e r e f o r e m a d e in t h e p r e s e n t experiments.
2. Materials a n d m e t h o d s I s o l a t e d p a r a v e r t e b r a l s y m p a t h e t i c ganglion chains o f bullfrogs (Rana catesbeiana) were used. T h e m e m b r a n e p o t e n t i a l o f ganglion cells w e r e r e c o r d e d b y t h e sucrose-gap m e t h od, a n d extrinsic electrical c u r r e n t s were applied t o t h e ganglion t h r o u g h an i n p u t bridge circuit ( K o k e t s u a n d Nishi, 1 9 6 7 ; K o s t e r l i t z et al., 1968). A t r a i n o f t e t a n i c s t i m u l a t i o n ( 3 0 / s e c f o r a p p r o x i m a t e l y 4 sec) was a p p l i e d t o t h e preganglionic B n e r v e fibres t h r o u g h a
282
pair of platinum electrodes. ACh was directly applied to a preparation either adding a desired concentration of ACh chloride to the external solution (perfusate) or simply injecting 0.2 ml of ACh chloride solution (0.1 M) to the perfusate. Ionic composition (mM = millimol per 1000 ml H 2 0 ) of the Ringer solution is: NaC1 112, KC1 2, CaC12 1.8 and NaHCO3 2; and that of the Ca-deficient Mg solution is: NaC1 112, KCI 2, CaC12 0.1, MgC12 6 and NaHCO3 2. NaC1 in these solutions was totally replaced by equimolar LiC1 for the Na-free lithium solution. Isolated ganglia were continuously perfused with a solution flowing through a chamber (50 × 5 × 4 mm) at the rate of 0.2 ml/sec. Drugs added to the solutions were nicotine sulfate (Katayama), d-tubocurarine chloride (Wako), atropine sulfate (Nakarai), carbachol chloride (Merck), bethanechol chloride (Ezai), eserine sulfate (Merck) and ouabain (Merck).
3. Results 3.1. ACh responses of ganglion cells Orthodromic responses of bullfrog sympathetic ganglion cells to a single or a tetanic preganglionic B nerve stimulation consist of nicotinic and muscarinic components; these responses were reported to be completely eliminated in the presence of both nicotine and atropine in the previous experiments (Koketsu, 1969). In the present experiment, however, a small slow hyperpolarizing response of ganglia was found to be produced from some preparations under such an experimental condition. This response was observed only in small numbers of preparations (2 or 3 cases within 10 preparations). An example of experiments, in which a small and slow hyperpolarizing response of ganglion cells was observed in the presence of both nicotine and atropine in high concentrations, was demonstrated in A in fig. 1. A typical positive potential (P potential) which was followed b y a late negative potential (LN po-
K. KOKETSU, K. YAMAMOTO
tential) (Nishi and Koketsu, 1968a) was recorded from this preparation after an application of nicotine (0.24 mM) for more than 30 min, as seen in the record A-1. The P potential of this preparation, however, could not be completely eliminated, whereas the LN potential disappeared with the addition of atropine. As seen in the record A-2, a small and slow hyperpolarizing response persistently remained even 60 min after addition of atropine (0.14 mM). This hyperpolarizing response was unchanged even when the concentration of atropine was raised to 0.42 mM. As seen in the record A-3, however, the hyperpolarizing response was completely eliminated when the external solution was changed to the Ca-deficient Mg solution containing both nicotine (0.24 mM) and atropine (0.14 mM). This suggested that the observed response was produced by a synaptic action of some chemical transmitter in a ganglion. The response of ganglia to ACh which was directly applied to the preparation also consists of nicotinic and muscarinic components; this response has been reported to be eliminated completely in the presence of both nicotine and atropine (cf. Koketsu, 1969). The present experiment, however, disclosed the fact that ganglion cells could be hyperpolarized by a direct action of ACh in the presence of a high concentration of nicotine and atropine. Its amplitude, however, was very small compared with the nicotinic or muscarinic response, and it was detectable only when a relatively large concentration of ACh (more than 0.5 mM) was applied. When 5 mM ACh was used, this hyperpolarizing response was detected from two or three preparations o u t of 10 preparations tested. Although the amplitude of the hyperpolarizing response to ACh varied according to different preparations, its amplitude was apparently dependent on the ACh concentration (cf. fig. 2, A-2 and A-3). An example of the experiments, in which the hyperpolarizing response to directly applied ACh (5 mM) was detected in the presence of both nicotine and atropine in high
ACh RESPONSE IN GANGLION CELLS
283
concentrations, was demonstrated in B in fig. 1. A typical muscarinic hyperpolarizing response which was followed by muscarinic depolarization (Koketsu and Nishi, 1967; Nishi and Koketsu, 1968a) was recorded from this preparation after an application of nicotine (0.24 mM) for more than 30 min (record B-l). The muscarinic hyperpolarizing response, however, could n o t be completely eliminated even 60 min after an addition of atropine (0.14 mM). As seen in the record B-2, a small hyperpolarizing response persisted under such experimental conditions. This response was unchanged even when the concentration of atropine was raised up to 0.42 mM. The hyperpolarizing response remained unchanged or was occasionally enhanced when the external solution was changed to the Ca-deficient Mg solution containing both nicotine and atropine, as seen in the record B-3 in fig. 1. This suggested that the hyperpolarizing response was produced by a direct action of ACh.
2 t~ 3
3
t*
/2mv IOsec
t
~
J 1mY I min
Fig. 1. Membrane potential changes of ganglion cells, produced by tetanic preganglionic B nerve stimulations (A) and direct applications of ACh (5 raM) (B). Records 1 and 2 were taken 30 min after an application of nicotine (0.24 raM), and 60 min after an addition of atropine (0.14 mM), respectively. Solution was changed from Ringer to Ca-deficient Mg solution containing both nicotine and atropine, and records 3 were taken 50 min thereafter. Durations of electrical stimulations or addition of ACh are marked by arrows.
3.2. ACh response o f nerve axons Experiments were carried o u t in order to find whether the hyperpolarizing response, which was described in the previous section, could be observed in the axonal elements in the present preparation. In these experiments, the membrane potential of isolated preganglionic or postganglionic-nerve trunk was recorded by the sucrose-gap method. These preparations were previously perfused for more than 30 min with the Ca-deficient Mg solution containing both nicotine (0.24 mM) and atropine (0.14 mM). Under such experimental conditions no responses were obtained when ACh in high concentrations (0.5--5 mM) was directly applied to the preparations. 3.3. Analyses o f the ACh hyperpolarizing response (the A Ch h y perpolarization ) 3. 3.1. General Some analyses were made in order to clarify the nature of the ACh hyperpolarization. In these experiments, the nature of ACh hyperpolarization which was produced by direct application of ACh was analysed in the presence of both nicotine (0.24 mM) and atropine {0.14 mM). The ganglionic responses to directly applied ACh could be recorded when a small a m o u n t (0.2 ml) of ACh solution (0.1 M) was injected to the perfusate which was rapidly flowing. This injection m e t h o d for ACh application was useful, because the ACh hyperpolarization, whose amplitude and time course were fairly constant, could be repeatedly obtained when ACh injections were given at the constant interval (15 rain). Thus, the following experiments were carried out by use of this injection method. The ACh hyperpolarization recorded by this injection m e t h o d is shown in the record A-1 of fig. 2, its amplitude and time course were compared with the ACh hyperpolarizations which were produced by applications of k n o w n concentrations of ACh for a certain period in the records A-2 and A-3 of fig. 2. It
284
K. KOKETSU, K. YAMAMOTO
A 1
B 1
2
A
2
B
C
I
I
I
2
2
2
3
3
~
2rn¥
t
3
3
J 2rn~/ lmin
__J 1my Imin
Fig. 2. (A) The ACh hyperpolarization produced by the injection method (record 1) and the conventional method (records 2 and 3) for ACh application. 0.2 ml of ACh solution (0.1 M) was injected into the perfusate at the moment indicated by an arrow in record 1, and 0.5 mM and 5 mM ACh were applied during the periods indicated by upward and downward arrows in record 2 and 3, respectively. These records were taken from the same preparation. (B) The effects of eserine on the ACh hyperpolarization produced by injection of ACh. Records I and 2 were obtained, respectively, before and 15 min after an addition of eserine (3.6 × 10-2 mM). Record 3 was the response obtained 20 min after a withdrawal of eserine. Times of ACh injection indicated by arrows.
s h o u l d be m e n t i o n e d here t h a t the a m p l i t u d e o f n i c o t i n i c d e p o l a r i z a t i o n , w h i c h was prod u c e d in t h e p r e s e n t p r e p a r a t i o n s with A C h a p p l i e d b y this i n j e c t i o n m e t h o d in the presence o f o n l y a t r o p i n e ( 0 . 1 4 mM), was app r o x i m a t e l y 5--6 mV.
Fig. 3. (A) The effects of membrane hyperpolarization on the ACh hyperpolarization. Records 1 and 2 were obtained without and during conditioning hyperpolarization, respectively. (B) The effects of ouabain on the ACh hyperpolarization. Records 1 and 2 were obtained, respectively, before and 10 rain after an addition of ouabain (2 × 10 -3 mM). Records 3 was the response obtained 30 rain after a withdrawal of ouabain. (C) Disappearance of the ACh-hyperpolarization in the sodium-free lithium solution. Record 1 is the ACh hyperpolarization which disappeared in the sodium-free lithium solution (record 2), and the ACh hyperpolarization is restored in the sodium-containing solution. Times of ACh injection are marked by arrows.
3.3.4. Effects o f ouabain The A C h h y p e r p o l a r i z a t i o n was reversibly depressed b y the a c t i o n o f o u a b a i n in a conc e n t r a t i o n as low as 2 × 10 -3 mM (fig. 3, B). The A C h h y p e r p o l a r i z a t i o n was c o m p l e t e l y e l i m i n a t e d when o u a b a i n in this c o n c e n t r a t i o n was applied f o r 2 0 - - 3 0 min, and it was partially r e s t o r e d u p o n w i t h d r a w a l o f ouabain.
3.3.5. Effects o f sodium ions 3.3.2. Effects o f changes in the membrane potential The a m p l i t u d e o f A C h h y p e r p o l a r i z a t i o n was increased w h e n t h e m e m b r a n e o f ganglion cells was h y p e r p o l a r i z e d ( c o n d i t i o n i n g h y p e r polarization) by applying a constant anodal c u r r e n t t h r o u g h an i n p u t bridge circuit (fig. 3, A). On the o t h e r h a n d , it was d e c r e a s e d during a c o n d i t i o n i n g d e p o l a r i z a t i o n .
3.3.3. Effects o f an anti-cholinesterase The ACh h y p e r p o l a r i z a t i o n was p r o l o n g e d in the presence o f eserine (3.6 × 10 -2 mM), a n d t h e e f f e c t o f eserine was reversible (fig. 2, B).
When t h e p r e p a r a t i o n was per×used with t h e s o l u t i o n w h e r e the NaC1 was t o t a l l y replaced with e q u i m o l a r LiC1, the h y p e r p o l a r i z a t i o n was c o m p l e t e l y b l o c k e d . It was res t o r e d b y r e - i m m e r s i o n in the N a - c o n t a i n i n g s o l u t i o n (fig. 3, C).
3. 4. Effects o f cholinomimetic drugs The effects o f c a r b a c h o l and b e t h a n e c h o l o n isolated ganglia were t e s t e d a f t e r preparations were per×used with the Ca-deficient s o l u t i o n c o n t a i n i n g b o t h n i c o t i n e (0.24 mM) and a t r o p i n e ( 0 . 1 4 mM) f o r m o r e t h a n 30 min.
ACh RESPONSE IN GANGLION CELLS
A !
285
B 1
i~
J
2
~
: f
2
3
3 ----
-7"-
_~2mV 1rain
Fig. 4. The effects of carbachol and bethanechol on the ACh hyperpolarization. The ACh hyperpolarization was produced by an injection of ACh (record A-1 and B-l), but no response was elicited by an injection of carbachol (record A-2) or bethanechol (record B-2) in equivalent amounts. The ACh hyperpolarization was produced immediately after (5 min) an injection of carbachol (record A-3) or bethanechol (record B-3). Records A and B were obtained from two separate preparations. The times of injections of drugs are indicated by arrows in each record.
When a drop of carbachol (0.1 M) was injected to the perfusate, ganglion cells showed no detectable hyperpolarization, nevertheless the application of ACh to the same preparation resulted in the generation of the ACh hyperpolarization (fig. 4-A). The ACh hyperpolarization was obtained w i t h o u t diminution, even when ACh was applied immediately after an application of carbachol (fig. 4-A). Similarly, no detectable hyperpolarizing response of ganglia was obtained when a drop of bethanechol (0.1 M) was injected to the perfusate. As has been observed with carbachol, bethanechol showed no effect on the production of the ACh hyperpolarization.
4. Discussion The present experiments demonstrated an unusual cholinergic response of bullfrog sympathetic ganglia, which was very resistant to the actions of nicotine and atropine in high concentrations. Such an ACh response (the ACh hyperpolarization) has n o t been f o u n d in previous experiments (cf. Koketsu, 1969), be-
cause the amplitude of the response is very small compared with that of nicotinic or muscarinic response. It seems that this ACh hyperpolarization was produced by a specific ACh action and was neither a nicotinic nor a muscarinic response. Indeed, neither carbachol nor bethanechol in high concentrations produced hyperpolarization under experimental conditions where ACh in the same concentrations was able to produce the hyperpolarization. The cholinergic response which is blocked neither by d-TC nor by atropine has been observed in ganglion cells of Aplysia (Kehoe, 1972; Pinsker and Kandel, 1969). During the course of the present experiments, the effect of d-TC was tested, in order to clarify if the ACh hyperpolarization could be observed in the presence of this drug. However experiments were difficult, because the nicotinic response (the fast EPSP) was very resistant to d-TC; ganglia often showed depolarization but never exhibited the hyperpolarization by ACh in the presence o f d-TC in high concentration (1.4 mM) and atropine (0.14 mM). Thus, it appeared that the ACh hyperpolarization was observable only when the nicotinic response was blocked by nicotine. The possibility that the ACh hyperpolarization might be produced by some u n k n o w n transmitters, which were released from somewhere in a ganglion affected by ACh, could be discarded since the ACh hyperpolarization caused by a direct application of ACh remained in the Ca-deficient solution containing Mg. The fact that the ACh hyperpolarization was very sensitive to ouabain and eliminated in the Na-free lithium solution, suggested that the generation of ACh hyperpolarization was associated with the sodium pump. ACh might be able to accelerate the sodium pump by some u n k n o w n mechanism. In the present experiments, sodium ions may be accumulated in ganglion cells and consequently the electrogenic sodium pump may be accelerated in the presence of nicotine (Gebber and Volle, 1966; Jaramillo and Volle, 1968a,b; Kosterlitz et al., 1968; Pascoe, 1956). If ACh can actually ac-
286
celerate the sodium pump, the membrane hyperpolarization caused by this action of ACh may be enhanced under such an experimental condition. It must be noted that experimental results suggesting that ACh would be able to accelerate the sodium pump have b e e n reported in Aplysia neuron (Pinsker and Kandel, 1969), snail parietal ganglia (Kerkut et al., 1969a,b) and skeletal muscle (Dockry et al., 1966). The nature of the ACh hyperpolarization is very similar to that of the slow inhibitory postsynaptic potential (slow IPSP) and of the adrenaline hyperpolarization which have been suggested to be generated by an acceleration of the electrogenic sodium pump (Nishi and Koketsu, 1967, 1968b; Nakamura and Koketsu, 1972). Although the slow IPSP has been considered to be produced by adrenaline (Libet, 1970; Libet and Kobayashi, 1974; Nakamura and Koketsu, 1972), the present results suggested that a part of the slow IPSP recorded from nicotinized ganglia may be produced by a direct action of ACh. Indeed, the present experiments demonstrated that the slow IPSP could be occasionally observed even after addition of a high concentration of atropine (0.14 mM). It should be noted here that Weight and Padjen (1973) and Libet and Kobayashi (1974) have recently reported that a part of the muscarinic hyperpolarization, which was produced by a direct action of ACh in the presence of nicotine, remained in a Ca-deficient solution containing M g . . On the basis of these observations, they suggested that a part of the slow IPSP may be produced by a direct action of ACh. The hyperpolarization observed in these experiments, however, seems to be essentially different from the ACh hyperpolarization described in the present paper, since it was reported to be blocked by atropine (Libet and Kobayashi, 1974).
References Dockry, M., R.P. Kernan and A. Tangney, 1966, Active transport of sodium and potassium in mammalian skeletal muscle and its modification by nerve and by cholinergic and adrenergic agents, J. Physiol. 186, 187.
K. KOKETSU, K. YAMAMOTO Gebber, G.L. and R.L. Votle, 1966, Mechanisms in-
volved in ganglionic blockade induced by tetramethylammonium, J. Pharmacol. Exptl. Therap. 152, 18. Jaramillo, J. and R.L. Volle, 1968a, Effects of lithium on ganglionic hyperpolarization and blockade by dimethylphenylpiperazinium, J. Pharmacol. Exptl. Therap. 164, 166. Jarami]lo, J. and R.L. Volle, 1968b, A comparison of the ganglionic stimulating and blocking properties of some nicotinic drugs, Arch. Intern. Pharmacodyn. 174, 88. Kehoe, J., 1972, Three acetylcholine receptors in aplysia neurones, J. Physiol. 225, 115. Kerkut, G.A., L.C. Brown and R.J. Walker, 1969a, Post-synaptic stimulation of the electrogenic sodium pump, Life Sci. 8, 297. Kerkut, G.A., L.C. Brown and R.J. Walker, 1969b, Cholinergic IPSP by stimulation of the electrogenic sodium pump, Nature 223, 864. Koketsu, K., 1969, Cholinergic synaptic potentials and the underlying ionic mechanisms, Federation Proc. 28,101. Koketsu, K. and S. Nishi, 1967, Characteristics of the slow inhibitory postsynaptic potential of bullfrog sympathetic ganglion cells, Life Sci. 6, 1827. Koketsu, K., 8. Nishi and H. Soeda, 1968, Acetylcholine-potential of sympathetic ganglion cell membrane, Life Sci. 7, 741. Kosterlitz, H.W., G.M. Lees and D.I. Wallis, 1968, Resting and action potentials recorded by the sucrose-gap method in the superior cervical ganglion of the rabbit, J. Physiol. 195, 39. Libet, B., 1970, Generation of slow inhibitory and excitatory postsynaptic potentials, Federation Proc. 29, 1945. Libet, B. and H. Kobayashi, 1974, Adrenergic mediation of slow inhibitory postsynaptic potential in sympathetic ganglia of the frog, J. Neurophysiol. 37,805. Nakamura, M. and K. Koketsu, 1972, The effect of adrenaline on sympathetic ganglion cells of bullfrogs, Life Sci. 11, 1165. Nishi, S. and K. Koketsu, 1967, Origin of ganglionic inhibitory postsynaptic potential, Life Sci. 6, 2O49. Nishi, S. and K. Koketsu, 1968a, Early and late afterdischarges of amphibian sympathetic ganglion cells, J. Neurophysiol. 31, 109. Nishi, S. and K. Koketsu, 1968b, Analysis of slow inhibitory postsynaptic potential of bullfrog sympathetic ganglion, J. Neurophysiol. 31,717. Pascoe, J.E., 1956, The effects of acetylcholine and other drugs on the isolated superior cervical ganglion, J. Physiol. 132, 242. Pinsker, H. and E.R. Kandel, 1969, Synaptic activation of an electrogenic sodium pump, Science 163, 931. Weight, F.F. and A. Padjen, 1973, Acetylcholine and slow synaptic inhibition in frog sympathetic ganglion cells, Brain Res. 55, 225.
UNUSUAL CHOLINERGIC RESPONSE OF BULLFROG SYMPATHETIC GANGLION CELLS KYOZO KOKETSU and KEIKO YAMAMOTO Department of Physiology, Kurume University School of Medicine, 67 Asahi-machi, Kurume-shi, 830, Japan Received 25 October 1974, revised MS received 31 December 1974, accepted 7 January 1975
K. KOKETSU and K. YAMAMOTO, Unusual cholinergic response of bullfrog sympathetic ganglion cells, European J. Pharmacol. 31 (1975) 281--286. The membrane of bullfrog sympathetic ganglion cells was hyperpolarized by a direct action of ACh (more than 0.5 mM) in a solution containing both nicotine {0.24 mM) and atropine (0.14 mM). This ACh hyperpolarization could be imitated by neither carbachol nor bethanechol, suggesting that the ACh hyperpolarization was the response which was produced by a specific action of ACh, which appeared to be neither nicotinic nor muscarinic. The size of ACh hyperpolarization was increased during a conditioning hyperpolarization. The ACh hyperpolarization was completely blocked by ouabain (2 x 10 -3 mM) and eliminated in the Na-free lithium solution. These aspects of the ACh hyperpolarization suggested that generation of this hyperpolarization was associated with the sodium pump. The ACh hyperpolarization seemed to be partially responsible for the production of the slow IPSP, since a part of the slow IPSP remained occasionally in the presence of both nicotine and atropine. Electrogenic sodium pump
Sympathetic ganglion
1. I n t r o d u c t i o n Since c e r t a i n a c t i o n s o f a c e t y l c h o l i n e ( A C h ) c o u l d be i m i t a t e d b y n i c o t i n e a n d others by muscarine, the actions of ACh were originally classified as e i t h e r ' n i c o t i n i c ' o r ' m u s c a r i n i c ' . In general, t h e n i c o t i n i c a c t i o n o f ACh c a n b e selectively b l o c k e d b y e i t h e r n i c o t i n e or d - t u b o c u r a r i n e (d-TC), w h e r e a s t h e m u s c a r i n i c a c t i o n c a n b e selectively b l o c k ed b y e i t h e r m u s c a r i n e o r a t r o p i n e . T h u s , it can be e x p e c t e d t h a t all k i n d s o f cellular responses produced by ACh should be complet e l y e l i m i n a t e d b y n i c o t i n e ( o r d-TC) a n d muscarine (or atropine). T h e m e m b r a n e p o t e n t i a l changes o f bullfrog s y m p a t h e t i c ganglion cells, w h i c h w e r e produced by ACh, consist of nicotinic and m u s c a r i n i c c o m p o n e n t s (cf. K o k e t s u , 1 9 6 9 ) . In fact, t h e cholinergic r e s p o n s e s o f t h e s e cells are r e p o r t e d t o b e c o m p l e t e l y b l o c k e d in t h e presence of nicotine and atropine (Koketsu et al., 1 9 6 8 ) . D u r i n g r e c e n t e x p e r i m e n t s in o u r
Acetylcholine
Hyperpolarization
l a b o r a t o r y , h o w e v e r , it was f o u n d t h a t careful e x p e r i m e n t s c o u l d reveal an u n u s u a l cholinergic r e s p o n s e o f these cells, w h i c h was v e r y resistant t o t h e a c t i o n s o f b o t h n i c o t i n e a n d a t r o p i n e a n d r e m a i n e d in t h e p r e s e n c e s o f t h e s e drugs. P h a r m a c o l o g i c a l analyses o f this n e w t y p e o f cholinergic r e s p o n s e o f ganglion cells h a v e b e e n t h e r e f o r e m a d e in t h e p r e s e n t experiments.
2. Materials a n d m e t h o d s I s o l a t e d p a r a v e r t e b r a l s y m p a t h e t i c ganglion chains o f bullfrogs (Rana catesbeiana) were used. T h e m e m b r a n e p o t e n t i a l o f ganglion cells w e r e r e c o r d e d b y t h e sucrose-gap m e t h od, a n d extrinsic electrical c u r r e n t s were applied t o t h e ganglion t h r o u g h an i n p u t bridge circuit ( K o k e t s u a n d Nishi, 1 9 6 7 ; K o s t e r l i t z et al., 1968). A t r a i n o f t e t a n i c s t i m u l a t i o n ( 3 0 / s e c f o r a p p r o x i m a t e l y 4 sec) was a p p l i e d t o t h e preganglionic B n e r v e fibres t h r o u g h a
282
pair of platinum electrodes. ACh was directly applied to a preparation either adding a desired concentration of ACh chloride to the external solution (perfusate) or simply injecting 0.2 ml of ACh chloride solution (0.1 M) to the perfusate. Ionic composition (mM = millimol per 1000 ml H 2 0 ) of the Ringer solution is: NaC1 112, KC1 2, CaC12 1.8 and NaHCO3 2; and that of the Ca-deficient Mg solution is: NaC1 112, KCI 2, CaC12 0.1, MgC12 6 and NaHCO3 2. NaC1 in these solutions was totally replaced by equimolar LiC1 for the Na-free lithium solution. Isolated ganglia were continuously perfused with a solution flowing through a chamber (50 × 5 × 4 mm) at the rate of 0.2 ml/sec. Drugs added to the solutions were nicotine sulfate (Katayama), d-tubocurarine chloride (Wako), atropine sulfate (Nakarai), carbachol chloride (Merck), bethanechol chloride (Ezai), eserine sulfate (Merck) and ouabain (Merck).
3. Results 3.1. ACh responses of ganglion cells Orthodromic responses of bullfrog sympathetic ganglion cells to a single or a tetanic preganglionic B nerve stimulation consist of nicotinic and muscarinic components; these responses were reported to be completely eliminated in the presence of both nicotine and atropine in the previous experiments (Koketsu, 1969). In the present experiment, however, a small slow hyperpolarizing response of ganglia was found to be produced from some preparations under such an experimental condition. This response was observed only in small numbers of preparations (2 or 3 cases within 10 preparations). An example of experiments, in which a small and slow hyperpolarizing response of ganglion cells was observed in the presence of both nicotine and atropine in high concentrations, was demonstrated in A in fig. 1. A typical positive potential (P potential) which was followed b y a late negative potential (LN po-
K. KOKETSU, K. YAMAMOTO
tential) (Nishi and Koketsu, 1968a) was recorded from this preparation after an application of nicotine (0.24 mM) for more than 30 min, as seen in the record A-1. The P potential of this preparation, however, could not be completely eliminated, whereas the LN potential disappeared with the addition of atropine. As seen in the record A-2, a small and slow hyperpolarizing response persistently remained even 60 min after addition of atropine (0.14 mM). This hyperpolarizing response was unchanged even when the concentration of atropine was raised to 0.42 mM. As seen in the record A-3, however, the hyperpolarizing response was completely eliminated when the external solution was changed to the Ca-deficient Mg solution containing both nicotine (0.24 mM) and atropine (0.14 mM). This suggested that the observed response was produced by a synaptic action of some chemical transmitter in a ganglion. The response of ganglia to ACh which was directly applied to the preparation also consists of nicotinic and muscarinic components; this response has been reported to be eliminated completely in the presence of both nicotine and atropine (cf. Koketsu, 1969). The present experiment, however, disclosed the fact that ganglion cells could be hyperpolarized by a direct action of ACh in the presence of a high concentration of nicotine and atropine. Its amplitude, however, was very small compared with the nicotinic or muscarinic response, and it was detectable only when a relatively large concentration of ACh (more than 0.5 mM) was applied. When 5 mM ACh was used, this hyperpolarizing response was detected from two or three preparations o u t of 10 preparations tested. Although the amplitude of the hyperpolarizing response to ACh varied according to different preparations, its amplitude was apparently dependent on the ACh concentration (cf. fig. 2, A-2 and A-3). An example of the experiments, in which the hyperpolarizing response to directly applied ACh (5 mM) was detected in the presence of both nicotine and atropine in high
ACh RESPONSE IN GANGLION CELLS
283
concentrations, was demonstrated in B in fig. 1. A typical muscarinic hyperpolarizing response which was followed by muscarinic depolarization (Koketsu and Nishi, 1967; Nishi and Koketsu, 1968a) was recorded from this preparation after an application of nicotine (0.24 mM) for more than 30 min (record B-l). The muscarinic hyperpolarizing response, however, could n o t be completely eliminated even 60 min after an addition of atropine (0.14 mM). As seen in the record B-2, a small hyperpolarizing response persisted under such experimental conditions. This response was unchanged even when the concentration of atropine was raised up to 0.42 mM. The hyperpolarizing response remained unchanged or was occasionally enhanced when the external solution was changed to the Ca-deficient Mg solution containing both nicotine and atropine, as seen in the record B-3 in fig. 1. This suggested that the hyperpolarizing response was produced by a direct action of ACh.
2 t~ 3
3
t*
/2mv IOsec
t
~
J 1mY I min
Fig. 1. Membrane potential changes of ganglion cells, produced by tetanic preganglionic B nerve stimulations (A) and direct applications of ACh (5 raM) (B). Records 1 and 2 were taken 30 min after an application of nicotine (0.24 raM), and 60 min after an addition of atropine (0.14 mM), respectively. Solution was changed from Ringer to Ca-deficient Mg solution containing both nicotine and atropine, and records 3 were taken 50 min thereafter. Durations of electrical stimulations or addition of ACh are marked by arrows.
3.2. ACh response o f nerve axons Experiments were carried o u t in order to find whether the hyperpolarizing response, which was described in the previous section, could be observed in the axonal elements in the present preparation. In these experiments, the membrane potential of isolated preganglionic or postganglionic-nerve trunk was recorded by the sucrose-gap method. These preparations were previously perfused for more than 30 min with the Ca-deficient Mg solution containing both nicotine (0.24 mM) and atropine (0.14 mM). Under such experimental conditions no responses were obtained when ACh in high concentrations (0.5--5 mM) was directly applied to the preparations. 3.3. Analyses o f the ACh hyperpolarizing response (the A Ch h y perpolarization ) 3. 3.1. General Some analyses were made in order to clarify the nature of the ACh hyperpolarization. In these experiments, the nature of ACh hyperpolarization which was produced by direct application of ACh was analysed in the presence of both nicotine (0.24 mM) and atropine {0.14 mM). The ganglionic responses to directly applied ACh could be recorded when a small a m o u n t (0.2 ml) of ACh solution (0.1 M) was injected to the perfusate which was rapidly flowing. This injection m e t h o d for ACh application was useful, because the ACh hyperpolarization, whose amplitude and time course were fairly constant, could be repeatedly obtained when ACh injections were given at the constant interval (15 rain). Thus, the following experiments were carried out by use of this injection method. The ACh hyperpolarization recorded by this injection m e t h o d is shown in the record A-1 of fig. 2, its amplitude and time course were compared with the ACh hyperpolarizations which were produced by applications of k n o w n concentrations of ACh for a certain period in the records A-2 and A-3 of fig. 2. It
284
K. KOKETSU, K. YAMAMOTO
A 1
B 1
2
A
2
B
C
I
I
I
2
2
2
3
3
~
2rn¥
t
3
3
J 2rn~/ lmin
__J 1my Imin
Fig. 2. (A) The ACh hyperpolarization produced by the injection method (record 1) and the conventional method (records 2 and 3) for ACh application. 0.2 ml of ACh solution (0.1 M) was injected into the perfusate at the moment indicated by an arrow in record 1, and 0.5 mM and 5 mM ACh were applied during the periods indicated by upward and downward arrows in record 2 and 3, respectively. These records were taken from the same preparation. (B) The effects of eserine on the ACh hyperpolarization produced by injection of ACh. Records I and 2 were obtained, respectively, before and 15 min after an addition of eserine (3.6 × 10-2 mM). Record 3 was the response obtained 20 min after a withdrawal of eserine. Times of ACh injection indicated by arrows.
s h o u l d be m e n t i o n e d here t h a t the a m p l i t u d e o f n i c o t i n i c d e p o l a r i z a t i o n , w h i c h was prod u c e d in t h e p r e s e n t p r e p a r a t i o n s with A C h a p p l i e d b y this i n j e c t i o n m e t h o d in the presence o f o n l y a t r o p i n e ( 0 . 1 4 mM), was app r o x i m a t e l y 5--6 mV.
Fig. 3. (A) The effects of membrane hyperpolarization on the ACh hyperpolarization. Records 1 and 2 were obtained without and during conditioning hyperpolarization, respectively. (B) The effects of ouabain on the ACh hyperpolarization. Records 1 and 2 were obtained, respectively, before and 10 rain after an addition of ouabain (2 × 10 -3 mM). Records 3 was the response obtained 30 rain after a withdrawal of ouabain. (C) Disappearance of the ACh-hyperpolarization in the sodium-free lithium solution. Record 1 is the ACh hyperpolarization which disappeared in the sodium-free lithium solution (record 2), and the ACh hyperpolarization is restored in the sodium-containing solution. Times of ACh injection are marked by arrows.
3.3.4. Effects o f ouabain The A C h h y p e r p o l a r i z a t i o n was reversibly depressed b y the a c t i o n o f o u a b a i n in a conc e n t r a t i o n as low as 2 × 10 -3 mM (fig. 3, B). The A C h h y p e r p o l a r i z a t i o n was c o m p l e t e l y e l i m i n a t e d when o u a b a i n in this c o n c e n t r a t i o n was applied f o r 2 0 - - 3 0 min, and it was partially r e s t o r e d u p o n w i t h d r a w a l o f ouabain.
3.3.5. Effects o f sodium ions 3.3.2. Effects o f changes in the membrane potential The a m p l i t u d e o f A C h h y p e r p o l a r i z a t i o n was increased w h e n t h e m e m b r a n e o f ganglion cells was h y p e r p o l a r i z e d ( c o n d i t i o n i n g h y p e r polarization) by applying a constant anodal c u r r e n t t h r o u g h an i n p u t bridge circuit (fig. 3, A). On the o t h e r h a n d , it was d e c r e a s e d during a c o n d i t i o n i n g d e p o l a r i z a t i o n .
3.3.3. Effects o f an anti-cholinesterase The ACh h y p e r p o l a r i z a t i o n was p r o l o n g e d in the presence o f eserine (3.6 × 10 -2 mM), a n d t h e e f f e c t o f eserine was reversible (fig. 2, B).
When t h e p r e p a r a t i o n was per×used with t h e s o l u t i o n w h e r e the NaC1 was t o t a l l y replaced with e q u i m o l a r LiC1, the h y p e r p o l a r i z a t i o n was c o m p l e t e l y b l o c k e d . It was res t o r e d b y r e - i m m e r s i o n in the N a - c o n t a i n i n g s o l u t i o n (fig. 3, C).
3. 4. Effects o f cholinomimetic drugs The effects o f c a r b a c h o l and b e t h a n e c h o l o n isolated ganglia were t e s t e d a f t e r preparations were per×used with the Ca-deficient s o l u t i o n c o n t a i n i n g b o t h n i c o t i n e (0.24 mM) and a t r o p i n e ( 0 . 1 4 mM) f o r m o r e t h a n 30 min.
ACh RESPONSE IN GANGLION CELLS
A !
285
B 1
i~
J
2
~
: f
2
3
3 ----
-7"-
_~2mV 1rain
Fig. 4. The effects of carbachol and bethanechol on the ACh hyperpolarization. The ACh hyperpolarization was produced by an injection of ACh (record A-1 and B-l), but no response was elicited by an injection of carbachol (record A-2) or bethanechol (record B-2) in equivalent amounts. The ACh hyperpolarization was produced immediately after (5 min) an injection of carbachol (record A-3) or bethanechol (record B-3). Records A and B were obtained from two separate preparations. The times of injections of drugs are indicated by arrows in each record.
When a drop of carbachol (0.1 M) was injected to the perfusate, ganglion cells showed no detectable hyperpolarization, nevertheless the application of ACh to the same preparation resulted in the generation of the ACh hyperpolarization (fig. 4-A). The ACh hyperpolarization was obtained w i t h o u t diminution, even when ACh was applied immediately after an application of carbachol (fig. 4-A). Similarly, no detectable hyperpolarizing response of ganglia was obtained when a drop of bethanechol (0.1 M) was injected to the perfusate. As has been observed with carbachol, bethanechol showed no effect on the production of the ACh hyperpolarization.
4. Discussion The present experiments demonstrated an unusual cholinergic response of bullfrog sympathetic ganglia, which was very resistant to the actions of nicotine and atropine in high concentrations. Such an ACh response (the ACh hyperpolarization) has n o t been f o u n d in previous experiments (cf. Koketsu, 1969), be-
cause the amplitude of the response is very small compared with that of nicotinic or muscarinic response. It seems that this ACh hyperpolarization was produced by a specific ACh action and was neither a nicotinic nor a muscarinic response. Indeed, neither carbachol nor bethanechol in high concentrations produced hyperpolarization under experimental conditions where ACh in the same concentrations was able to produce the hyperpolarization. The cholinergic response which is blocked neither by d-TC nor by atropine has been observed in ganglion cells of Aplysia (Kehoe, 1972; Pinsker and Kandel, 1969). During the course of the present experiments, the effect of d-TC was tested, in order to clarify if the ACh hyperpolarization could be observed in the presence of this drug. However experiments were difficult, because the nicotinic response (the fast EPSP) was very resistant to d-TC; ganglia often showed depolarization but never exhibited the hyperpolarization by ACh in the presence o f d-TC in high concentration (1.4 mM) and atropine (0.14 mM). Thus, it appeared that the ACh hyperpolarization was observable only when the nicotinic response was blocked by nicotine. The possibility that the ACh hyperpolarization might be produced by some u n k n o w n transmitters, which were released from somewhere in a ganglion affected by ACh, could be discarded since the ACh hyperpolarization caused by a direct application of ACh remained in the Ca-deficient solution containing Mg. The fact that the ACh hyperpolarization was very sensitive to ouabain and eliminated in the Na-free lithium solution, suggested that the generation of ACh hyperpolarization was associated with the sodium pump. ACh might be able to accelerate the sodium pump by some u n k n o w n mechanism. In the present experiments, sodium ions may be accumulated in ganglion cells and consequently the electrogenic sodium pump may be accelerated in the presence of nicotine (Gebber and Volle, 1966; Jaramillo and Volle, 1968a,b; Kosterlitz et al., 1968; Pascoe, 1956). If ACh can actually ac-
286
celerate the sodium pump, the membrane hyperpolarization caused by this action of ACh may be enhanced under such an experimental condition. It must be noted that experimental results suggesting that ACh would be able to accelerate the sodium pump have b e e n reported in Aplysia neuron (Pinsker and Kandel, 1969), snail parietal ganglia (Kerkut et al., 1969a,b) and skeletal muscle (Dockry et al., 1966). The nature of the ACh hyperpolarization is very similar to that of the slow inhibitory postsynaptic potential (slow IPSP) and of the adrenaline hyperpolarization which have been suggested to be generated by an acceleration of the electrogenic sodium pump (Nishi and Koketsu, 1967, 1968b; Nakamura and Koketsu, 1972). Although the slow IPSP has been considered to be produced by adrenaline (Libet, 1970; Libet and Kobayashi, 1974; Nakamura and Koketsu, 1972), the present results suggested that a part of the slow IPSP recorded from nicotinized ganglia may be produced by a direct action of ACh. Indeed, the present experiments demonstrated that the slow IPSP could be occasionally observed even after addition of a high concentration of atropine (0.14 mM). It should be noted here that Weight and Padjen (1973) and Libet and Kobayashi (1974) have recently reported that a part of the muscarinic hyperpolarization, which was produced by a direct action of ACh in the presence of nicotine, remained in a Ca-deficient solution containing M g . . On the basis of these observations, they suggested that a part of the slow IPSP may be produced by a direct action of ACh. The hyperpolarization observed in these experiments, however, seems to be essentially different from the ACh hyperpolarization described in the present paper, since it was reported to be blocked by atropine (Libet and Kobayashi, 1974).
References Dockry, M., R.P. Kernan and A. Tangney, 1966, Active transport of sodium and potassium in mammalian skeletal muscle and its modification by nerve and by cholinergic and adrenergic agents, J. Physiol. 186, 187.
K. KOKETSU, K. YAMAMOTO Gebber, G.L. and R.L. Votle, 1966, Mechanisms in-
volved in ganglionic blockade induced by tetramethylammonium, J. Pharmacol. Exptl. Therap. 152, 18. Jaramillo, J. and R.L. Volle, 1968a, Effects of lithium on ganglionic hyperpolarization and blockade by dimethylphenylpiperazinium, J. Pharmacol. Exptl. Therap. 164, 166. Jarami]lo, J. and R.L. Volle, 1968b, A comparison of the ganglionic stimulating and blocking properties of some nicotinic drugs, Arch. Intern. Pharmacodyn. 174, 88. Kehoe, J., 1972, Three acetylcholine receptors in aplysia neurones, J. Physiol. 225, 115. Kerkut, G.A., L.C. Brown and R.J. Walker, 1969a, Post-synaptic stimulation of the electrogenic sodium pump, Life Sci. 8, 297. Kerkut, G.A., L.C. Brown and R.J. Walker, 1969b, Cholinergic IPSP by stimulation of the electrogenic sodium pump, Nature 223, 864. Koketsu, K., 1969, Cholinergic synaptic potentials and the underlying ionic mechanisms, Federation Proc. 28,101. Koketsu, K. and S. Nishi, 1967, Characteristics of the slow inhibitory postsynaptic potential of bullfrog sympathetic ganglion cells, Life Sci. 6, 1827. Koketsu, K., 8. Nishi and H. Soeda, 1968, Acetylcholine-potential of sympathetic ganglion cell membrane, Life Sci. 7, 741. Kosterlitz, H.W., G.M. Lees and D.I. Wallis, 1968, Resting and action potentials recorded by the sucrose-gap method in the superior cervical ganglion of the rabbit, J. Physiol. 195, 39. Libet, B., 1970, Generation of slow inhibitory and excitatory postsynaptic potentials, Federation Proc. 29, 1945. Libet, B. and H. Kobayashi, 1974, Adrenergic mediation of slow inhibitory postsynaptic potential in sympathetic ganglia of the frog, J. Neurophysiol. 37,805. Nakamura, M. and K. Koketsu, 1972, The effect of adrenaline on sympathetic ganglion cells of bullfrogs, Life Sci. 11, 1165. Nishi, S. and K. Koketsu, 1967, Origin of ganglionic inhibitory postsynaptic potential, Life Sci. 6, 2O49. Nishi, S. and K. Koketsu, 1968a, Early and late afterdischarges of amphibian sympathetic ganglion cells, J. Neurophysiol. 31, 109. Nishi, S. and K. Koketsu, 1968b, Analysis of slow inhibitory postsynaptic potential of bullfrog sympathetic ganglion, J. Neurophysiol. 31,717. Pascoe, J.E., 1956, The effects of acetylcholine and other drugs on the isolated superior cervical ganglion, J. Physiol. 132, 242. Pinsker, H. and E.R. Kandel, 1969, Synaptic activation of an electrogenic sodium pump, Science 163, 931. Weight, F.F. and A. Padjen, 1973, Acetylcholine and slow synaptic inhibition in frog sympathetic ganglion cells, Brain Res. 55, 225.