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Facilitation and inhibition of nicotinic transmission by eserine in the sympathetic ganglia of the rabbit
0028-3908/85$3.00+ 0.00 Copyright 0 1985Pergamon Press Ltd
Neuropharmacology Vol. 24, No. 1 I, pp. 1093-I 101, 1985 Great Britain. All rights reserved
Printed in
FACILITATION AND INHIBITION OF NICOTINIC TRANSMISSION BY ESERINE IN THE SYMPATHETIC GANGLIA OF THE RABBIT N. MO, N. J. DUN* and A. G. KARCZMAR Loyola University Medical Center, Department of Pharmacology, 2160 S. First Avenue, Maywood, IL 60153, U.S.A. (Accepted 7 March 1985)
Summary-The effects of eserine on neurons and on ganglionic transmission of the isolated superior cervical ganglia of the rabbit were investigated by means of intracellular recording techniques. At the concentration of 10pM or less, eserine reversibly increased the amplitude and duration of the fast excitatory postsynaptic potential (f-epsp) induced by preganglionic nerve stimulation and of the membrane depolarization evoked by iontophoretically-applied acetylcholine (ACh), but not carbachol. At the concentration of 50pM or more, eserine consistently and reversibly depressed the fast excitatory postsynaptic potential as well as the depolarization induced by iontophoretic application of either ACh or carbachol. Furthermore, depolarization by ACh evoked in a low Ca/high Mg solution, which blocked the liberation of transmitter was similarly reduced by eserine in greater concentrations. The passive membrane properties of the sympathetic neurons were not significantly altered by eserine in the majority of neurons studied. The results indicate that the facilitatory action of eserine on ganglionic transmission may be explained by its anticholinesterase activity, whereas eserine-induced block of transmission appears to be related to a direct interaction between the compound and the postsynaptic ACh receptor-channel complex. Key
words: nicotinic transmission, facilitation and inhibition, eserine, postsynaptic mechanism.
While phosphorylation of the active site of cholinesterases (ChE) and the resulting accumulation of acetylcholine (ACh) is clearly involved in many aspects of the pharmacological actions of anticholinesterase (antiChE) agents, including their ganglionic effects (for review, see Holmstedt, 1963; Karczmar, 1967; Voile, 1980), a number of studies suggest that these pharmacological or toxicological actions may not be entirely related to their antiChE activity. For example, antiChE, whether of carbamate or organophosphorus type, block ganglionic transmission (Koppanyi, Karczmar and King, 1947; Feldberg and Vartianen, 1935; Paton and Perry, 1953; Mason, 1962; Riker and Kosay, 1970; McIsaac and Albrachet, 1975), yet the concentrations of antiChE agents needed to block ganglionic transmission do not seem to be closeley related to the concentrations inhibition of ganglionic AChE that produce (Koppanyi and Karczmar, 1951; Holaday, Kamijo and Koelle, 1954). Accordingly, several investigators suggested that the ganglionic or neuromyal actions of antiChE depend on their direct actions (Koppanyi and Karczmar, 195 1; Volle and Koelle, 1961; Karczmar, 1970; Kuba, Albuquerque, Daly and Barnard, 1974). The large majority of ganglionic
*To
whom correspondence
should be addressed.
studies referred to above were carried out with relatively simple methods including extracellular or surface recording techniques; thus, the site and possible mechanism of action of antiChE agents on ganglia could not be demonstrated. The present study was carried out by means of microelectrode procedures in an attempt to better define the site and mechanism of the potentiating and blocking effects of eserine (physostigmine) on the sympathetic ganglia. METHODS Young white rabbits (1.5-2 kg) were used in this study. The animals were sacrificed by an overdose of pentobarbital sodium and the superior cervical ganglia, together with their cervical sympathetic nerve trunks, were rapidly excised and transferred to the recording chamber. The ganglia were superfused with a Krebs solution of the following composition (in millimolar): NaCl, 117; KCI, 4.7; CaCl,, 2.5; MgCl,, 1.2; NaHCO,, 25; NaH,PO,, 1.2; and glucose, 11.5. The solution was saturated with 95% 0, and 5% CO* and the temperature of the solution was kept at 34 f 0.5”C. Intracellular recordings were obtained from ganglion cells by means of fibercontaining glass capillaries filled with 3 M KC1 that had a resistance of 35-60 Ma, The cervical sympathetic (preganglionic) nerve trunk was drawn into 1093
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1094
CON
MO et al.
ESERINE,
1 pM
WASH
I IOmV 20 msec
Fig. 1. Enhancing effect of eserine (1 PM) on the fast excitatory postsynaptic potentials evoked by nerve stimulation in a cell of the superior cervical ganglion of the rabbit. Control: 4 consecutive subthreshold potentials elicted at 0.1 Hz. Application of eserine (1 PM) to the ganglion cell for 5 min resulted in a large increase of the amplitude as well as the duration of the potentials (middle panel). The amplitude of the potentials returned to the control level about 30 min after washing with drug-free Krebs solution (right panel).
a suction electrode for orthodromic stimulation. Depolarization by acetylcholine (ACh) or carbachol was evoked by iontophoresis of the agonist from micropipettes filled with 2 M of the agent and applied to the surface of ganglion cells from which intracellular recordings were made. The ACh or carbachol-containing electrodes had a d.c. resistance of 80-100 MR. Acetylcholine or carbachol was discharged from the pipette by a positive current pulse of brief duration (5-15 msec); a negative retaining current of l-3 nA was applied to the pipette to minimize the leakage of ACh or carbachol (for detailed description of these procedures see Dun and Nishi, 1974; Dun and Karczmar, 1977). Eserine and other pharmacological agents were dissolved in Krebs solution and applied to the ganglia in known concentrations by superfusion. The following compounds were used: acetylcholine chloride, atropine sulfate, carbamylcholine chloride (carbachol), eserine sulfate and d-tubocurarine chloride. All chemical agents were purchased from Sigma. RESULTS
Single electrical stimulation applied to the cervical sympathetic trunk elicits in the ganglion cells a fast excitatory postsynaptic potential (f-epsp); when this potential reaches the threshold, the cell fires an action potential (Nichi, 1974). The fast excitatory postsynaptic potential can be antagonized pharmacologically by d-tubocurarine (0.05 mM) or hexamethonium (0.5 mM) and is therefore mediated by the nicotonic action of acetylcholine (Nishi, 1974). The effects of eserine on the fast excitatory post-
synaptic potential of the sympathetic neurons of the rabbit were clearly concentration-dependent: eserine facilitated the potential at smaller concentrations (I 10pM) and depressed it at the concentration of 50pM or greater; variable results (slight facilitation or slight depression of the f-epsp) were obtained at intermediate concentrations. Facilitation of fast excitatory postsynaptic potentials
At the concentrations of 0.1-10 PM, when applied to the ganglia for 5-10 min, eserine consistently and reversibly increased the amplitude and duration of the fast excitatory postsynaptic potential evoked by low frequency stimulation of the preganglionic nerve (0.1-0.5 Hz). At the concentration of 0.05 PM eserine produced a negligible change of the amplitude and duration of the potentials in 4 cells tested. A representative experiment illustrating the potentiating effect of eserine (1 PM) on synaptic transmission is shown in Fig. 1. As can be seen, the facilitatory action reached its peak in about 5-10 min after the addition of eserine. A period of 3WtOmin washing with drug-free Krebs solution was needed before the response returned to control level. Table 1 summarizes the percentage increases in the amplitude and duration of fast excitatory postsynaptic potentials induced by eserine (1 and 10 PM). Depression of fast excitatory postsynaptic potentials
In contrast to the enhancing effect at small concentrations, eserine at the concentration of 50pM or greater reversibly depressed or in some instances completely blocked the fast excitatory postsynaptic potentials in 34 of the 40 cells examined. That the
Table 1. Effects of eserine on the amplitude and duration of fast excitatory postsynaptic of the superior cervical ganglion of the rabbit Concentration f-epsp
1pM
Amplitude (percentage change) Duration (percentage change) + and -denote *Statistically
(n=7)
+62+28* f82 + 19’
1OpM (n = 19) +150*47* +133+17*
increase and decrease over the control value, significant, P < 0.05, paired Students t-test.
respectively.
potentials
(f-epsp) evoked in cells
of eserine 50/1M (n = 12) -44 -27
IOOpM (n = 22)
f 18’ + 12*
The values
are expressed
-91 -57
* 22* * 14*
as mean + SD.
Eserine and ganglionic transmission CON
ESERINE,50pM
1095
WASH
I 1OmV 20msec
Fig. 2. Depressant effect of eserine (50 PM) on the fast excitatory postsynaptic potentials evoked by nerve stimulations in a cell of the superior cervical ganglion of the rabbit. Control: 4 consecutive potentials elicited at 0.1 Hz. Application of eserine (50 PM) which caused no significant change of the resting membrane potential markedly suppressed the potentials. In fact, a fast excitatory postsynaptic potential was not evoked after the first 2 nerve stimulations (middle panel). The potentials recovered to near control level more than 30min after washing with Krebs solution (right panel).
reduction of the potentials caused by eserine (50 and 100 p M) was dose-dependent is shown in Table 1. An experiment in which eserine (50 PM) effectively suppressed the fast excitatory postsynaptic potentials in a sympathetic neuron is demonstrated in Fig. 2. The depressant effect developed rapidly after the initiation of superfusion with eserine and reached its maximum in about 5-10min. The depressant effect of eserine was reversible by a 2&40 min washing with drug-free Krebs solution (Fig. 2). In some instances the synaptic depression after administration of eserine was followed by a facilitation during the washing period (see below and Fig. 7). Electrical properties
of the membrane
The resting membrane potential and input resistance as determined from the amplitude of the electrotonic potentials induced by hyperpolarizing current pulses (Fig. 3) were not significantly changed by either large or small concentrations of eserine, ranging from 1 to 100 PM, in all 22 cells investigated. The mean resting membrane potential and input resistance of these cells were -47 f 13 mV and
CON
53 MQ before and -44 f 16mV and 51 f 16 MR after superfusion with eserine. An experiment in which eserine (50 PM) depressed the fast excitatory postsynaptic potentials without affecting the membrane potential and input resistance of the cell is illustrated in Fig. 3.
Effects of eserine on nicotinic depolarization by ACh
induced
This series of experiments served to evalute the effects of eserine on the sensitivity of the postsynaptic receptors to ACh. As eserine consistently facilitated and depressed the fast excitatory postsynaptic potential when used in concentrations of 1 and 50pm, respectively, these two concentrations were selected for the study of the effects of eserine on the potential induced by ACh. When the micropipette containing ACh was manoeuvred close to the ganglion cell which was impaled for intracellular recording, ACh discharged from the pipette by a brief cathodal current pulse (I 15 msec) elicited a brisk depolarization that was completely blocked by d-tubocurarine
ESERINE,~OJJM
WASH
IlOmV IOmsec
Fig. 3. Effect of eserine on the membrane potential, membrane resistance and fast excitatory postsynaptic potential of a cell of the superior cervical ganglion of the rabbit. The upper trace of each pair of tracings represents the current pulse used to induce the hyperpolarizing electrotonic potential (lower trace). The current trace also represents the zero potential level. The synaptic potential elicited by orthodromic stimulation was superimposed on the electrotonic potential. Following the superfusion of eserine (50 PM) for 5 min, the amplitude of fast excitatory postsynaptic potential was reduced by 40% (middle panel), whereas the resting membrane potential and membrane resistance of the cell were not noticeably changed. After a period of 20 min wash with Krebs solution, the potential returned to control level (right recording).
N. Mo et al.
1096
WASH
ESERINE,lpM
3Osec
_//------1.,,
L~.I_X_--^----__~-
200msec
I
._.
IOmV
Fig. 4. Enhancement of nicotinic depolarization induced by iontophoretic application of ACh by eserine in a cell of the superior cervical ganglion of the rabbit. Depolarization was induced by ACh, applied iontophoretically (30 nA; 10msec pulse duration). After the application of eserine for 60 set, the depolarization by ACh reached threshold and the cell fired an action potential. The potentiating effect persisted for many minutes after the removal of the drug from the Krebs solution, as indicated by the recordings taken at 30 and 90 set after washing. The depolarization by ACh returned to the control level 30 min after washing. Note that the peak amplitude of the action potential was truncated because of the limited frequency response of the pen recorder used to record these tracings.
(50pM), indicating that the response was mediated by nicotinic receptors. Similar to the effects of eserine on synaptically evoked fast excitatory postsynaptic potentials, eserine increased and decreased depolarization induced by the nicotinic action of ACh in a concentrationdependent manner. An experiment in which eserine (1 PM) markedly increased the depolarization by ACh is shown in Fig. 4. Shortly after the superfusion of eserine, depolarization by ACh was sufficiently enhanced to reach the threshold for the initiation of the action potential. The facilitation outlasted the duration of the application of eserine by many minutes (Fig. 4). The quantification of the increase of deploarization by ACh, induced by eserine was difficult because of spiking (see for example, Fig. 4). Nevertheless, it was clearly apparent that eserine (1 PM) potentiated depolarizations by ACh in all 8 cells examined. On the other hand, when employed at the concentration of SOpM, eserine produced a marked and conspicuous depression of depolarization by ACh. One of the experiments in which eserine reversibly blocked the depolarization by ACh is shown in Fig. 5. What is quite noticeable is that the depressant effect of eserine was not preceded by facilitation. It must be added that eserine was more effective in blocking depolarization by ACh induced by iontophoretic application of ACh than the responses
elicited by nerve stimulation; indeed, at the concentration of 50 p M, eserine completely suppressed the fast excitatory postsynaptic potentials in only 3 of the 8 neurons, while it completely blocked the depolarization by ACh in all cells investigated (n = 5). Efects of eserine on depolarization bachol
induced by car-
This series of experiments served to evaluate the effects of eserine on membrane depolarization induced by carbachol, a cholinergic agonist resistant to cholinesterases. Carbachol was applied iontophoretically to the surface of the ganglion cells as described for ACh. At the concentration of 1 PM, eserine caused no significant change of the amplitude and duration of the depolarization induced by carbachol in all 5 cells tested; the mean amplitude and duration of the response to carbachol were 7.2 +_ 3.6 mV and 132 f 11 msec before, and 7.7 f 4.2 mV and 140 + 13 msec after superfusion of eserine, respectively. A representative experiment is shown in Fig. 6. (upper tracings). On the other hand, depolarizations induced by carbachol were reversibly blocked by eserine at the concentration of 50pM as shown in Fig. 6. (lower tracings). The effect was rapid in onset and was not preceded by facilitation (Fig. 6). A similar blocking effect was observed in another 3 cells.
Eserine and ganglionic transmission
h
CON
ZE +-_
ESERINE,
45sec
1097
50~~
+__
120sec
+__
I
1OmV
IOOmsec
Fig. 5. Depression of nicotinic depolarization induced by ACh by eserine in a cell of the superior cervical ganglion of the rabbit. Depolarization by ACh was induced by iontophoretic application of ACh (35 nA, 10 msec pulse duration). The depolarization by ACh was almost completely eliminated after 80 set superfusion of eserine 50 PM. The depolarization by ACh gradually recovered to control level 5 min after washing with Krebs solution. Comparison
7, upper a longer period of
As
of eserine
is
of ChE,
its
by eserine
of desensitization
of
ACh. To evaluate of the by superfusion of ACh by eserine. of ACh (1 mM) to the a brief period (30-60 set) caused rapid depolarization of a concomitant blockade of In this as the At
is characteristic is be stressed of ACh an a marked fall in
is that in both initial of transmission
lower
by eserine by ACh. by any 7,
in membrane or input
t ESERINE.
WASII
1uM
-
1OOmsec
__&f--------
2’
--p----
t ESERINE.
4’
+-----a5’
I
1OmV
-J-----
t SOti
WASH
Fig. 6. Effects of small and large concentrations of eserine on membrane depolarization induced by carbachol in a cell of the superior cervical ganglion of the rabbit. Depolarizations were induced by iontophoretic application of carbachol (30 nA, 12 msec pulse duration). At the concentration of 1 y M, eserine caused no discernible change of the amplitude and duration of the depolarizations induced by carbachol (top tracings). Whereas, at the concentration of SOpM, eserine reversibly blocked the depolarization induced by carbachol (lower tracing). Numerals in minutes indicate the time after application of eserine or wash. The recordings were taken from the same neuron.
N. MO et
1098
ACh
1mM
ESERINE
-
50~,M
40
9-c
I
10mv
Fig. 7. Blockade of fast excitatory postsynaptic potentials by ACh and eserine. Preganglionic nerve stimulation elicited subthreshold potentials (small upward deflections) as well as spike potentials (larger upward deflections). Superfusion of ACh (1 mM) to the ganglion cell caused a rapid depolarization and a concomitant blockade of synaptic responses. After perfusion with Krebs solution the synaptic responses reappeared shortly after the membrane potential had returned to the resting level. No apparent facilitation of the synaptic response was seen following the withdrawal of ACh. Application of eserine (50 PM), which did not cause a membrane depolarization, depressed the synaptic responses. There was a clear facilitation of the synaptic response after a period of washing as preganglionic nerve stimulation regularly elicted full spike potentials.
Fig. 3). Second, after washing the synaptic depression was followed by a period of facilitation as electrical stimulation of the preganglionic nerve regularly elicited full spikes instead of intermittent action potentials that were recorded prior to superfusion with eserine (Fig. 7, lower tracing). Efects
of atropine
It is well known that muscarinic receptors are present on the preganglionic nerve fibers and that the activation of these receptors may result in a reduction in release of ACh (Koketsu and Yamada, 1982; Morita, North and Tokimasa, 1982). To evaluate the contribution of this mechanism to the block in transmission caused by eserine, the magnitude of depression induced by eserine was compared before and after superfusion of atropine (1 PM) which has been demonstrated to block muscarinic receptors on nerve terminals (o.c.). In all 4 cells tested, the degree of depression of the fast excitatory postsynaptic potential was not altered by atropine; the mean reduction induced by eserine (50 PM) before and after atropine was 53 k 7 and 58 f 4%, respectively. Efsects of eserine (50@4) on depolarization evoked in a low calcium solution
by ACh
In this series of experiments the effects of eserine on depolarization induced by iontophoretic application of ACh were evaluated in a low Ca (0.12 mM)/high Mg (12mM) solution; in this solution, liberation of transmitter is blocked as evident from the failure to elicit a synaptic potential after nerve stimulation (Fig. 8). Under these conditions, eserine (50pM)
effectively and reversibly blocked depolarization ACh in all 4 cells tested (Fig. 8).
by
DISCUSSION
The principal observation reported here is that the carbamate antiChE, eserine, caused a concentrationdependent facilitation and depression of nicotinic transmission of the mammalian sympathetic ganglia. A biphasic action of antiChE agents, including eserine, on ganglionic transmission was noted early by Feldberg and Vartianen (1935) and subsequently by a number of investigators (Paton and Perry, 1953; Mason, 1962; Riker and Kosay, 1970). The facilitatory phase of the action of eserine was noticed at concentrations ranging from 0.1 to 10 PM. A marked effect was caused by concentrations of 1 to 10 PM; at these concentrations, eserine markedly enhanced the fast excitatory postsynaptic potential evoked synaptically, as well as the depolarization elicited by iontophoretic application of ACh. On the other hand, employed at these concentrations, eserine did not facilitate the response to carbachol. These findings are consistent with the long standing concept that eserine inhibits junctional ChE thereby protecting ACh from rapid hydrolysis, the result of which is an amplification of the duration and the extent of the action of ACh (Eccles, 1944; Koppanyi and Karczmar, 1951; Kamijo and Koelle, 1952; Long, 1963; Bornstein, 1974). In this context, it should be noted that at concentrations of I-lOpM, which produced a marked facilitation of ganglionic transmission, eserine was found to reduce markedly or
Eserine
LOW Ca l
and ganglionic
1099
transmission
r
ESERINE
IOOmsec
I
IOmV
200msec
I IOmV
Fig. 8. Suppression of depolarization induced by ACh by eserine (50 pM) in a cervical ganglion cell bathed with low Ca/high Mg solution. Recordings to the left are synaptic potentials evoked by stimulation of the preganglionic nerve trunk, and depolarizations induced by iontophoretic application of ACh (25 nA, 10 msec duration) are shown in the right hand column. Control synaptic potential and depolarization by ACh are shown in the top row. Superfusion of the ganglion with low Ca (0.12 mM)/high Mg (12 mM) solution completely blocked the synaptic potential (middle row). Under these conditions, eserine (50 PM) remained effective in reducing the depolarization by ACh to less than 20% of the control. Both the synaptic potential and depolarization by ACh returned to control levels after washing with Krebs solution (bottom recordings).
even inhibit completely tissue ChE (Long, 1963; Usdin, 1970; Karczmar, unpublished). Contrary to the facilitatory action at smaller coneserine depressed ganglionic transcentrations, mission when used at greater concentrations. The question that is of immediate interest is whether or not the blockade of ganglionic transmission by eserine was a result of accumulation of ACh, subsequent to inhibition of ganglionic ChE. If the blockade were a consequence of accumulation of ACh, it would be expected that the block should be preceded by a depolarization accompanied by a reduction in membrane resistance. Indeed, as shown in the present experiments, superfusion of ACh onto the ganglion cells caused a rapid block of fast excitatory postsynaptic potentials, accompanied by depolarization and a reduction in membrane resistance. However, failure of transmission induced by eserine was characteristically different from that produced by ACh, as the blocking action of eserine developed without a noticeable change of membrane potential or input resistance. Furthermore, as mentioned earlier, a complete inhibition of ganglionic ChE should result from 10 PM or smaller concentrations of eserine, if the block of transmission was due to accumulation of ACh, there is no reason why it should arise only at concentrations of eserine 5-fold greater than those capable of complete inhibition of ChE. In the same context, the block of the response
to carbachol by eserine was elicited in the quiescent (unstimulated) ganglion; under these conditions, there could be little or no accumulation of ACh that would contribute to the block. In a similar context, it could be argued that accumulation of ACh may lead to desensitization. However, it would be expected that the latter would be preceded by depolarization, which was not the case. Moreover, eserine blocked the response to ACh even in a low Ca/high Mg solution, i.e. when the release of the transmitter was blocked. It follows then that the synaptic blockade caused by eserine was not causally related to inhibition of ChE and accumulation of ACh. There is evidence that activation of muscarinic receptors, located on preganglionic nerve fibers, may exert a negative feedback inhibition on evoked release of ACh (Koketsu and Yamada, 1982; Morita et al., 1982). Accordingly, atropine has been found to increase release of ACh from the peripheral and central synapses (Szerb, 1977). It is conceivable that the amount of ACh in the junctional area may reach a sufficiently high level, following inhibition of ChE to accentuate the negative feedback mechanism and block release of ACh. The finding that the block of fast excitatory postsynaptic potentials caused by eserine was unchanged after pretreatment with atropine indicates that the presynaptic muscarinic mechanisms plays a minimal role, if any, in the synaptic blockade induced by this antiChE
N. MO et al.
1100
agent. The possibility that eserine blocks synaptic transmission by a postsynaptic mechanism should therefore be considered. Indeed, membrane depolarization, induced by iontophoretic application of either ACh or carbachol, was effectively and reversibly blocked by eserine. This finding indicates that a postsynaptic site of action is primarily involved in the depression of ganglionic transmission induced by eserine. It should be pointed out that block of fast excitatory postsynaptic potentials and ACh potentials developed rapidly after the initiation of perfusion of eserine and that this effect was not preceded by facilitation. This is again consistent with the concept that eserine exerts two actions at the ganglion, facilitatory and inhibitory, and that these actions are mediated by two separate sites or mechanisms. The precise postsynaptic mechanism involved in the inhibition of nicotinic transmission in the ganglion by eserine cannot be defined at present. In this regard, there is evidence that antiChE agents of both carbamate and organophosphorus type may block transmission of amphibian and mammalian skeletal neuromyal junctions by acting primarily on the AChion-channel complex rather than the receptor (Kuba et al., 1974; Shaw, Akaike and Albuquerque, 1983). Eserine, while blocking the fast excitatory postsynaptic potential and the response to ACh did not affect the membrane characteristics. This is consistent with either channel or receptor blockade and further work is needed to resolve which site is involved in the ganglionic blocking action of eserine. In summary, these results show that the action of eserine on sympathetic ganglia may not be limited to its well known antiChE activity; rather, eserine may interact directly with the nicotinic AChion-channel/receptor complex, thereby inhibiting ganglionic transmission, albeit in a relatively large concentration. Acknowledgement-This study was supported by U.S. Army Medical Research and Development Command Contract DAMD17-83-C-3133.
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Neuropharmacology Vol. 24, No. 1 I, pp. 1093-I 101, 1985 Great Britain. All rights reserved
Printed in
FACILITATION AND INHIBITION OF NICOTINIC TRANSMISSION BY ESERINE IN THE SYMPATHETIC GANGLIA OF THE RABBIT N. MO, N. J. DUN* and A. G. KARCZMAR Loyola University Medical Center, Department of Pharmacology, 2160 S. First Avenue, Maywood, IL 60153, U.S.A. (Accepted 7 March 1985)
Summary-The effects of eserine on neurons and on ganglionic transmission of the isolated superior cervical ganglia of the rabbit were investigated by means of intracellular recording techniques. At the concentration of 10pM or less, eserine reversibly increased the amplitude and duration of the fast excitatory postsynaptic potential (f-epsp) induced by preganglionic nerve stimulation and of the membrane depolarization evoked by iontophoretically-applied acetylcholine (ACh), but not carbachol. At the concentration of 50pM or more, eserine consistently and reversibly depressed the fast excitatory postsynaptic potential as well as the depolarization induced by iontophoretic application of either ACh or carbachol. Furthermore, depolarization by ACh evoked in a low Ca/high Mg solution, which blocked the liberation of transmitter was similarly reduced by eserine in greater concentrations. The passive membrane properties of the sympathetic neurons were not significantly altered by eserine in the majority of neurons studied. The results indicate that the facilitatory action of eserine on ganglionic transmission may be explained by its anticholinesterase activity, whereas eserine-induced block of transmission appears to be related to a direct interaction between the compound and the postsynaptic ACh receptor-channel complex. Key
words: nicotinic transmission, facilitation and inhibition, eserine, postsynaptic mechanism.
While phosphorylation of the active site of cholinesterases (ChE) and the resulting accumulation of acetylcholine (ACh) is clearly involved in many aspects of the pharmacological actions of anticholinesterase (antiChE) agents, including their ganglionic effects (for review, see Holmstedt, 1963; Karczmar, 1967; Voile, 1980), a number of studies suggest that these pharmacological or toxicological actions may not be entirely related to their antiChE activity. For example, antiChE, whether of carbamate or organophosphorus type, block ganglionic transmission (Koppanyi, Karczmar and King, 1947; Feldberg and Vartianen, 1935; Paton and Perry, 1953; Mason, 1962; Riker and Kosay, 1970; McIsaac and Albrachet, 1975), yet the concentrations of antiChE agents needed to block ganglionic transmission do not seem to be closeley related to the concentrations inhibition of ganglionic AChE that produce (Koppanyi and Karczmar, 1951; Holaday, Kamijo and Koelle, 1954). Accordingly, several investigators suggested that the ganglionic or neuromyal actions of antiChE depend on their direct actions (Koppanyi and Karczmar, 195 1; Volle and Koelle, 1961; Karczmar, 1970; Kuba, Albuquerque, Daly and Barnard, 1974). The large majority of ganglionic
*To
whom correspondence
should be addressed.
studies referred to above were carried out with relatively simple methods including extracellular or surface recording techniques; thus, the site and possible mechanism of action of antiChE agents on ganglia could not be demonstrated. The present study was carried out by means of microelectrode procedures in an attempt to better define the site and mechanism of the potentiating and blocking effects of eserine (physostigmine) on the sympathetic ganglia. METHODS Young white rabbits (1.5-2 kg) were used in this study. The animals were sacrificed by an overdose of pentobarbital sodium and the superior cervical ganglia, together with their cervical sympathetic nerve trunks, were rapidly excised and transferred to the recording chamber. The ganglia were superfused with a Krebs solution of the following composition (in millimolar): NaCl, 117; KCI, 4.7; CaCl,, 2.5; MgCl,, 1.2; NaHCO,, 25; NaH,PO,, 1.2; and glucose, 11.5. The solution was saturated with 95% 0, and 5% CO* and the temperature of the solution was kept at 34 f 0.5”C. Intracellular recordings were obtained from ganglion cells by means of fibercontaining glass capillaries filled with 3 M KC1 that had a resistance of 35-60 Ma, The cervical sympathetic (preganglionic) nerve trunk was drawn into 1093
N.
1094
CON
MO et al.
ESERINE,
1 pM
WASH
I IOmV 20 msec
Fig. 1. Enhancing effect of eserine (1 PM) on the fast excitatory postsynaptic potentials evoked by nerve stimulation in a cell of the superior cervical ganglion of the rabbit. Control: 4 consecutive subthreshold potentials elicted at 0.1 Hz. Application of eserine (1 PM) to the ganglion cell for 5 min resulted in a large increase of the amplitude as well as the duration of the potentials (middle panel). The amplitude of the potentials returned to the control level about 30 min after washing with drug-free Krebs solution (right panel).
a suction electrode for orthodromic stimulation. Depolarization by acetylcholine (ACh) or carbachol was evoked by iontophoresis of the agonist from micropipettes filled with 2 M of the agent and applied to the surface of ganglion cells from which intracellular recordings were made. The ACh or carbachol-containing electrodes had a d.c. resistance of 80-100 MR. Acetylcholine or carbachol was discharged from the pipette by a positive current pulse of brief duration (5-15 msec); a negative retaining current of l-3 nA was applied to the pipette to minimize the leakage of ACh or carbachol (for detailed description of these procedures see Dun and Nishi, 1974; Dun and Karczmar, 1977). Eserine and other pharmacological agents were dissolved in Krebs solution and applied to the ganglia in known concentrations by superfusion. The following compounds were used: acetylcholine chloride, atropine sulfate, carbamylcholine chloride (carbachol), eserine sulfate and d-tubocurarine chloride. All chemical agents were purchased from Sigma. RESULTS
Single electrical stimulation applied to the cervical sympathetic trunk elicits in the ganglion cells a fast excitatory postsynaptic potential (f-epsp); when this potential reaches the threshold, the cell fires an action potential (Nichi, 1974). The fast excitatory postsynaptic potential can be antagonized pharmacologically by d-tubocurarine (0.05 mM) or hexamethonium (0.5 mM) and is therefore mediated by the nicotonic action of acetylcholine (Nishi, 1974). The effects of eserine on the fast excitatory post-
synaptic potential of the sympathetic neurons of the rabbit were clearly concentration-dependent: eserine facilitated the potential at smaller concentrations (I 10pM) and depressed it at the concentration of 50pM or greater; variable results (slight facilitation or slight depression of the f-epsp) were obtained at intermediate concentrations. Facilitation of fast excitatory postsynaptic potentials
At the concentrations of 0.1-10 PM, when applied to the ganglia for 5-10 min, eserine consistently and reversibly increased the amplitude and duration of the fast excitatory postsynaptic potential evoked by low frequency stimulation of the preganglionic nerve (0.1-0.5 Hz). At the concentration of 0.05 PM eserine produced a negligible change of the amplitude and duration of the potentials in 4 cells tested. A representative experiment illustrating the potentiating effect of eserine (1 PM) on synaptic transmission is shown in Fig. 1. As can be seen, the facilitatory action reached its peak in about 5-10 min after the addition of eserine. A period of 3WtOmin washing with drug-free Krebs solution was needed before the response returned to control level. Table 1 summarizes the percentage increases in the amplitude and duration of fast excitatory postsynaptic potentials induced by eserine (1 and 10 PM). Depression of fast excitatory postsynaptic potentials
In contrast to the enhancing effect at small concentrations, eserine at the concentration of 50pM or greater reversibly depressed or in some instances completely blocked the fast excitatory postsynaptic potentials in 34 of the 40 cells examined. That the
Table 1. Effects of eserine on the amplitude and duration of fast excitatory postsynaptic of the superior cervical ganglion of the rabbit Concentration f-epsp
1pM
Amplitude (percentage change) Duration (percentage change) + and -denote *Statistically
(n=7)
+62+28* f82 + 19’
1OpM (n = 19) +150*47* +133+17*
increase and decrease over the control value, significant, P < 0.05, paired Students t-test.
respectively.
potentials
(f-epsp) evoked in cells
of eserine 50/1M (n = 12) -44 -27
IOOpM (n = 22)
f 18’ + 12*
The values
are expressed
-91 -57
* 22* * 14*
as mean + SD.
Eserine and ganglionic transmission CON
ESERINE,50pM
1095
WASH
I 1OmV 20msec
Fig. 2. Depressant effect of eserine (50 PM) on the fast excitatory postsynaptic potentials evoked by nerve stimulations in a cell of the superior cervical ganglion of the rabbit. Control: 4 consecutive potentials elicited at 0.1 Hz. Application of eserine (50 PM) which caused no significant change of the resting membrane potential markedly suppressed the potentials. In fact, a fast excitatory postsynaptic potential was not evoked after the first 2 nerve stimulations (middle panel). The potentials recovered to near control level more than 30min after washing with Krebs solution (right panel).
reduction of the potentials caused by eserine (50 and 100 p M) was dose-dependent is shown in Table 1. An experiment in which eserine (50 PM) effectively suppressed the fast excitatory postsynaptic potentials in a sympathetic neuron is demonstrated in Fig. 2. The depressant effect developed rapidly after the initiation of superfusion with eserine and reached its maximum in about 5-10min. The depressant effect of eserine was reversible by a 2&40 min washing with drug-free Krebs solution (Fig. 2). In some instances the synaptic depression after administration of eserine was followed by a facilitation during the washing period (see below and Fig. 7). Electrical properties
of the membrane
The resting membrane potential and input resistance as determined from the amplitude of the electrotonic potentials induced by hyperpolarizing current pulses (Fig. 3) were not significantly changed by either large or small concentrations of eserine, ranging from 1 to 100 PM, in all 22 cells investigated. The mean resting membrane potential and input resistance of these cells were -47 f 13 mV and
CON
53 MQ before and -44 f 16mV and 51 f 16 MR after superfusion with eserine. An experiment in which eserine (50 PM) depressed the fast excitatory postsynaptic potentials without affecting the membrane potential and input resistance of the cell is illustrated in Fig. 3.
Effects of eserine on nicotinic depolarization by ACh
induced
This series of experiments served to evalute the effects of eserine on the sensitivity of the postsynaptic receptors to ACh. As eserine consistently facilitated and depressed the fast excitatory postsynaptic potential when used in concentrations of 1 and 50pm, respectively, these two concentrations were selected for the study of the effects of eserine on the potential induced by ACh. When the micropipette containing ACh was manoeuvred close to the ganglion cell which was impaled for intracellular recording, ACh discharged from the pipette by a brief cathodal current pulse (I 15 msec) elicited a brisk depolarization that was completely blocked by d-tubocurarine
ESERINE,~OJJM
WASH
IlOmV IOmsec
Fig. 3. Effect of eserine on the membrane potential, membrane resistance and fast excitatory postsynaptic potential of a cell of the superior cervical ganglion of the rabbit. The upper trace of each pair of tracings represents the current pulse used to induce the hyperpolarizing electrotonic potential (lower trace). The current trace also represents the zero potential level. The synaptic potential elicited by orthodromic stimulation was superimposed on the electrotonic potential. Following the superfusion of eserine (50 PM) for 5 min, the amplitude of fast excitatory postsynaptic potential was reduced by 40% (middle panel), whereas the resting membrane potential and membrane resistance of the cell were not noticeably changed. After a period of 20 min wash with Krebs solution, the potential returned to control level (right recording).
N. Mo et al.
1096
WASH
ESERINE,lpM
3Osec
_//------1.,,
L~.I_X_--^----__~-
200msec
I
._.
IOmV
Fig. 4. Enhancement of nicotinic depolarization induced by iontophoretic application of ACh by eserine in a cell of the superior cervical ganglion of the rabbit. Depolarization was induced by ACh, applied iontophoretically (30 nA; 10msec pulse duration). After the application of eserine for 60 set, the depolarization by ACh reached threshold and the cell fired an action potential. The potentiating effect persisted for many minutes after the removal of the drug from the Krebs solution, as indicated by the recordings taken at 30 and 90 set after washing. The depolarization by ACh returned to the control level 30 min after washing. Note that the peak amplitude of the action potential was truncated because of the limited frequency response of the pen recorder used to record these tracings.
(50pM), indicating that the response was mediated by nicotinic receptors. Similar to the effects of eserine on synaptically evoked fast excitatory postsynaptic potentials, eserine increased and decreased depolarization induced by the nicotinic action of ACh in a concentrationdependent manner. An experiment in which eserine (1 PM) markedly increased the depolarization by ACh is shown in Fig. 4. Shortly after the superfusion of eserine, depolarization by ACh was sufficiently enhanced to reach the threshold for the initiation of the action potential. The facilitation outlasted the duration of the application of eserine by many minutes (Fig. 4). The quantification of the increase of deploarization by ACh, induced by eserine was difficult because of spiking (see for example, Fig. 4). Nevertheless, it was clearly apparent that eserine (1 PM) potentiated depolarizations by ACh in all 8 cells examined. On the other hand, when employed at the concentration of SOpM, eserine produced a marked and conspicuous depression of depolarization by ACh. One of the experiments in which eserine reversibly blocked the depolarization by ACh is shown in Fig. 5. What is quite noticeable is that the depressant effect of eserine was not preceded by facilitation. It must be added that eserine was more effective in blocking depolarization by ACh induced by iontophoretic application of ACh than the responses
elicited by nerve stimulation; indeed, at the concentration of 50 p M, eserine completely suppressed the fast excitatory postsynaptic potentials in only 3 of the 8 neurons, while it completely blocked the depolarization by ACh in all cells investigated (n = 5). Efects of eserine on depolarization bachol
induced by car-
This series of experiments served to evaluate the effects of eserine on membrane depolarization induced by carbachol, a cholinergic agonist resistant to cholinesterases. Carbachol was applied iontophoretically to the surface of the ganglion cells as described for ACh. At the concentration of 1 PM, eserine caused no significant change of the amplitude and duration of the depolarization induced by carbachol in all 5 cells tested; the mean amplitude and duration of the response to carbachol were 7.2 +_ 3.6 mV and 132 f 11 msec before, and 7.7 f 4.2 mV and 140 + 13 msec after superfusion of eserine, respectively. A representative experiment is shown in Fig. 6. (upper tracings). On the other hand, depolarizations induced by carbachol were reversibly blocked by eserine at the concentration of 50pM as shown in Fig. 6. (lower tracings). The effect was rapid in onset and was not preceded by facilitation (Fig. 6). A similar blocking effect was observed in another 3 cells.
Eserine and ganglionic transmission
h
CON
ZE +-_
ESERINE,
45sec
1097
50~~
+__
120sec
+__
I
1OmV
IOOmsec
Fig. 5. Depression of nicotinic depolarization induced by ACh by eserine in a cell of the superior cervical ganglion of the rabbit. Depolarization by ACh was induced by iontophoretic application of ACh (35 nA, 10 msec pulse duration). The depolarization by ACh was almost completely eliminated after 80 set superfusion of eserine 50 PM. The depolarization by ACh gradually recovered to control level 5 min after washing with Krebs solution. Comparison
7, upper a longer period of
As
of eserine
is
of ChE,
its
by eserine
of desensitization
of
ACh. To evaluate of the by superfusion of ACh by eserine. of ACh (1 mM) to the a brief period (30-60 set) caused rapid depolarization of a concomitant blockade of In this as the At
is characteristic is be stressed of ACh an a marked fall in
is that in both initial of transmission
lower
by eserine by ACh. by any 7,
in membrane or input
t ESERINE.
WASII
1uM
-
1OOmsec
__&f--------
2’
--p----
t ESERINE.
4’
+-----a5’
I
1OmV
-J-----
t SOti
WASH
Fig. 6. Effects of small and large concentrations of eserine on membrane depolarization induced by carbachol in a cell of the superior cervical ganglion of the rabbit. Depolarizations were induced by iontophoretic application of carbachol (30 nA, 12 msec pulse duration). At the concentration of 1 y M, eserine caused no discernible change of the amplitude and duration of the depolarizations induced by carbachol (top tracings). Whereas, at the concentration of SOpM, eserine reversibly blocked the depolarization induced by carbachol (lower tracing). Numerals in minutes indicate the time after application of eserine or wash. The recordings were taken from the same neuron.
N. MO et
1098
ACh
1mM
ESERINE
-
50~,M
40
9-c
I
10mv
Fig. 7. Blockade of fast excitatory postsynaptic potentials by ACh and eserine. Preganglionic nerve stimulation elicited subthreshold potentials (small upward deflections) as well as spike potentials (larger upward deflections). Superfusion of ACh (1 mM) to the ganglion cell caused a rapid depolarization and a concomitant blockade of synaptic responses. After perfusion with Krebs solution the synaptic responses reappeared shortly after the membrane potential had returned to the resting level. No apparent facilitation of the synaptic response was seen following the withdrawal of ACh. Application of eserine (50 PM), which did not cause a membrane depolarization, depressed the synaptic responses. There was a clear facilitation of the synaptic response after a period of washing as preganglionic nerve stimulation regularly elicted full spike potentials.
Fig. 3). Second, after washing the synaptic depression was followed by a period of facilitation as electrical stimulation of the preganglionic nerve regularly elicited full spikes instead of intermittent action potentials that were recorded prior to superfusion with eserine (Fig. 7, lower tracing). Efects
of atropine
It is well known that muscarinic receptors are present on the preganglionic nerve fibers and that the activation of these receptors may result in a reduction in release of ACh (Koketsu and Yamada, 1982; Morita, North and Tokimasa, 1982). To evaluate the contribution of this mechanism to the block in transmission caused by eserine, the magnitude of depression induced by eserine was compared before and after superfusion of atropine (1 PM) which has been demonstrated to block muscarinic receptors on nerve terminals (o.c.). In all 4 cells tested, the degree of depression of the fast excitatory postsynaptic potential was not altered by atropine; the mean reduction induced by eserine (50 PM) before and after atropine was 53 k 7 and 58 f 4%, respectively. Efsects of eserine (50@4) on depolarization evoked in a low calcium solution
by ACh
In this series of experiments the effects of eserine on depolarization induced by iontophoretic application of ACh were evaluated in a low Ca (0.12 mM)/high Mg (12mM) solution; in this solution, liberation of transmitter is blocked as evident from the failure to elicit a synaptic potential after nerve stimulation (Fig. 8). Under these conditions, eserine (50pM)
effectively and reversibly blocked depolarization ACh in all 4 cells tested (Fig. 8).
by
DISCUSSION
The principal observation reported here is that the carbamate antiChE, eserine, caused a concentrationdependent facilitation and depression of nicotinic transmission of the mammalian sympathetic ganglia. A biphasic action of antiChE agents, including eserine, on ganglionic transmission was noted early by Feldberg and Vartianen (1935) and subsequently by a number of investigators (Paton and Perry, 1953; Mason, 1962; Riker and Kosay, 1970). The facilitatory phase of the action of eserine was noticed at concentrations ranging from 0.1 to 10 PM. A marked effect was caused by concentrations of 1 to 10 PM; at these concentrations, eserine markedly enhanced the fast excitatory postsynaptic potential evoked synaptically, as well as the depolarization elicited by iontophoretic application of ACh. On the other hand, employed at these concentrations, eserine did not facilitate the response to carbachol. These findings are consistent with the long standing concept that eserine inhibits junctional ChE thereby protecting ACh from rapid hydrolysis, the result of which is an amplification of the duration and the extent of the action of ACh (Eccles, 1944; Koppanyi and Karczmar, 1951; Kamijo and Koelle, 1952; Long, 1963; Bornstein, 1974). In this context, it should be noted that at concentrations of I-lOpM, which produced a marked facilitation of ganglionic transmission, eserine was found to reduce markedly or
Eserine
LOW Ca l
and ganglionic
1099
transmission
r
ESERINE
IOOmsec
I
IOmV
200msec
I IOmV
Fig. 8. Suppression of depolarization induced by ACh by eserine (50 pM) in a cervical ganglion cell bathed with low Ca/high Mg solution. Recordings to the left are synaptic potentials evoked by stimulation of the preganglionic nerve trunk, and depolarizations induced by iontophoretic application of ACh (25 nA, 10 msec duration) are shown in the right hand column. Control synaptic potential and depolarization by ACh are shown in the top row. Superfusion of the ganglion with low Ca (0.12 mM)/high Mg (12 mM) solution completely blocked the synaptic potential (middle row). Under these conditions, eserine (50 PM) remained effective in reducing the depolarization by ACh to less than 20% of the control. Both the synaptic potential and depolarization by ACh returned to control levels after washing with Krebs solution (bottom recordings).
even inhibit completely tissue ChE (Long, 1963; Usdin, 1970; Karczmar, unpublished). Contrary to the facilitatory action at smaller coneserine depressed ganglionic transcentrations, mission when used at greater concentrations. The question that is of immediate interest is whether or not the blockade of ganglionic transmission by eserine was a result of accumulation of ACh, subsequent to inhibition of ganglionic ChE. If the blockade were a consequence of accumulation of ACh, it would be expected that the block should be preceded by a depolarization accompanied by a reduction in membrane resistance. Indeed, as shown in the present experiments, superfusion of ACh onto the ganglion cells caused a rapid block of fast excitatory postsynaptic potentials, accompanied by depolarization and a reduction in membrane resistance. However, failure of transmission induced by eserine was characteristically different from that produced by ACh, as the blocking action of eserine developed without a noticeable change of membrane potential or input resistance. Furthermore, as mentioned earlier, a complete inhibition of ganglionic ChE should result from 10 PM or smaller concentrations of eserine, if the block of transmission was due to accumulation of ACh, there is no reason why it should arise only at concentrations of eserine 5-fold greater than those capable of complete inhibition of ChE. In the same context, the block of the response
to carbachol by eserine was elicited in the quiescent (unstimulated) ganglion; under these conditions, there could be little or no accumulation of ACh that would contribute to the block. In a similar context, it could be argued that accumulation of ACh may lead to desensitization. However, it would be expected that the latter would be preceded by depolarization, which was not the case. Moreover, eserine blocked the response to ACh even in a low Ca/high Mg solution, i.e. when the release of the transmitter was blocked. It follows then that the synaptic blockade caused by eserine was not causally related to inhibition of ChE and accumulation of ACh. There is evidence that activation of muscarinic receptors, located on preganglionic nerve fibers, may exert a negative feedback inhibition on evoked release of ACh (Koketsu and Yamada, 1982; Morita et al., 1982). Accordingly, atropine has been found to increase release of ACh from the peripheral and central synapses (Szerb, 1977). It is conceivable that the amount of ACh in the junctional area may reach a sufficiently high level, following inhibition of ChE to accentuate the negative feedback mechanism and block release of ACh. The finding that the block of fast excitatory postsynaptic potentials caused by eserine was unchanged after pretreatment with atropine indicates that the presynaptic muscarinic mechanisms plays a minimal role, if any, in the synaptic blockade induced by this antiChE
N. MO et al.
1100
agent. The possibility that eserine blocks synaptic transmission by a postsynaptic mechanism should therefore be considered. Indeed, membrane depolarization, induced by iontophoretic application of either ACh or carbachol, was effectively and reversibly blocked by eserine. This finding indicates that a postsynaptic site of action is primarily involved in the depression of ganglionic transmission induced by eserine. It should be pointed out that block of fast excitatory postsynaptic potentials and ACh potentials developed rapidly after the initiation of perfusion of eserine and that this effect was not preceded by facilitation. This is again consistent with the concept that eserine exerts two actions at the ganglion, facilitatory and inhibitory, and that these actions are mediated by two separate sites or mechanisms. The precise postsynaptic mechanism involved in the inhibition of nicotinic transmission in the ganglion by eserine cannot be defined at present. In this regard, there is evidence that antiChE agents of both carbamate and organophosphorus type may block transmission of amphibian and mammalian skeletal neuromyal junctions by acting primarily on the AChion-channel complex rather than the receptor (Kuba et al., 1974; Shaw, Akaike and Albuquerque, 1983). Eserine, while blocking the fast excitatory postsynaptic potential and the response to ACh did not affect the membrane characteristics. This is consistent with either channel or receptor blockade and further work is needed to resolve which site is involved in the ganglionic blocking action of eserine. In summary, these results show that the action of eserine on sympathetic ganglia may not be limited to its well known antiChE activity; rather, eserine may interact directly with the nicotinic AChion-channel/receptor complex, thereby inhibiting ganglionic transmission, albeit in a relatively large concentration. Acknowledgement-This study was supported by U.S. Army Medical Research and Development Command Contract DAMD17-83-C-3133.
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