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The effect of dimethylaminoadamantane on neuronal membranes
European Journal of Pharmacology, 35 (1976) 379--388 © North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands
THE EFFECT OF DIMETHYLAMINOADAMANTANE ON NEURONAL MEMBRANES* ANDREA GROSSMANN, WERNER GROSSMANN and ILMAR JURNA
Pharmakologisches lnstitut der UniversitKt des Saarlandes, D-665 Homburg/Saar, German Federal Republic Received 30 January 1975, revised MS received 11 July 1975, accepted 29 September 1975
A. GROSSMANN, W. GROSSMANN and I. JURNA, The effect of dimethylaminoadamantane on neuronal
membranes, European J. Pharmacol. 35 (1976)379--388. The effect of dimethylaminoadamantane (DMAA), an amantadine derivative with an anti-Parkinson property, on rat sensory nerve fibres was studied with the sucrose gap method. DMAA 10 -4 M in normal Locke solution reduced the spike amplitude without changing the resting potential, increased the membrane resistance and depressed repetitive spike activity elicited by depolarizing currents. From experiments performed with changed concentrations of sodium, potassium, calcium and chloride ions in the suspension medium it appears that the permeability of sodium, potassium and chloride ions is reduced by DMAA. The possible implication of the membrane effects of the drug in its action on dopaminergic transmission in the brain is discussed. Dimethylaminoadamantane
Neuronal membrane
I. Introduction Amantadine is used as an anti-Parkinson agent which, either by stimulating the formation and release of dopamine or by inhibiting the uptake of the transmitter into dopaminergic terminals (Scatton et al., 1970; Von Voigtlander and Moore, 1971a, b; Heimans et al., 1972), increases the availability of the monoamine at dopaminergic receptor sites in the brain. Dimethylaminoadamantane (DMAA)~, an amantadine derivative also successfully applied in Parkinson's disease, is assumed to act both indirectly, as does amantadine, and directly on dopaminergic receptors (Svensson, 1973; Maj et al., 1974), as does apomorphine (And~n, 1967; Ernst, 1967). However, DMAA reduced the rigidity in decerebrate cats (Sontag and Wand, 1973), which has been ascribed either to an effect on dopaminergic transmission in nigro-reticular pathways or to a depressant action on neuro* Supported by 'Membranen'.
the
Sonderforschungsbereich 38
Ion permeability
nal membranes. The latter assumption is supported by the observation that the drug inhibited a-motoneurones activated monosynaptically by muscle vibration (Miihlberg and Sontag, 1973). Amantadine has been shown to exert an effect on isolated neuronal membranes which is independent of transmitter action (Grossmann, 1973). In the present investigation it was aimed to determine whether DMAA also influences the membrane properties of isolated nerve fibre bundles, which might account for the results of Sontag and coworkers (Miihlberg and Sontag, 1973; Sontag and Wand, 1973), and if its membrane effects might also contribute to its action at dopaminergic receptor sites.
2. Materials and methods
2.1. General procedure The experiments were performed on sensory nerve fibre bundles of the rat (250--300 g
380
b o d y weight; Wistar Strain). The rats were anesthetized b y an i.p. injection of pentobarbital 50 mg]kg. Sensory nerve fibre bundles were obtained b y isolating the dorsal roots of L 6 or S 1. The dorsal roots were cooled to 4 ° C in Locke solution immediately after removal, cut to a length of 22--25 mm and introduced into the sucrose gap apparatus. After having been m o u n t e d the preparation was superfused with oxygenated Locke and sucrose solutions respectively at a temperature of 36 ° C. A period of at least 1 hr was allowed for the preparation to stabilize in the perfusion medium before starting the experiment. The sucrose gap apparatus employed has been described in detail b y Schmidt (1962). The mean diameter o f the dorsal roots e m p l o y e d was 0.5 ram, and the diameter of the channel into which the nerve was inserted 0.65 mm. 2. 2. Stimulation and recording Stimulation was performed by means of sintered Ag/AgC1 electrodes (Bionetic Instruments, Santa Ana, California). Spike potentials were elicited by applying single rectangular pulses of 10 psec duration and a strength that was twice the intensity required to obtain a maximal spike response. To determine the membrane resistance, rectangular pulses of 200 msec duration and various strengths were applied. The polarizing currents were applied through a 50 M~2 resistor. Membrane potentials were led off with calomel electrodes. The electrodes were connected to a differential amplifier through cathode followers arranged in parallel and the signals recorded on a cathode ray oscilloscope. The resting potential was displayed on a physiograph. The membrane resistance was determined according to Ohm's law from the intensity of the current applied and the membrane polarization produced. The absolute membrane resistance can be calculated o n l y when the shortcircuiting factor of the sucrose gap is k n o w n (Schmidt, 1962). However, an exact determination of this factor cannot be achieved si-
A. G R O S S M A N N E T AL.
multaneously with t h e measurement membrane potential under current tion. Therefore, relative values only, ues disregarding the short~circuiting will be given in the text.
of the applicai.e. valfactor,
2. 3. Solutions The unmodified Locke solution consisted of NaCI 154.0, KC1 5.6, CaC12 5.0, tris 2.5, glucose 5.0 mM/l. The modified solutions are presented in table 1. In all the different suspension mediums listed, the concentration of tris and glucose was the same as in the unmodified Locke solution. The pH-value of the solutions was adjusted to 7.3 by adding adequate amounts of sulphuric acid. The sucrose solution was prepared b y dissolving 100 g sucrose (Saccharose p.a.; Merck, Darmstadt) in 100 ml double distilled water. The specific resistance of the sucrose solution under atmospheric conditions was 1.5 M ~ . To further reduce the conductance the solution was boiled under vacuum for 3 min and afterwards oxygenated. The solutions were supplied to the compartments of the sucrose gap apparatus by means of PVC tubes fitted to a stopcock with a minimal deadspace (Kilb and S~mpfli, 1974) which allowed a rapid change from one solution to another. The perfusion ~ate was controlled b y flow meters (Rotameter; Rota KG, S~ickingen). The perfusion rate of the Locke solution in b o t h outer compartments was 2 ml/min, and in the inner c o m p a r t m e n t 3.5 ml/min; the perfusion rate of the sucrose solution in the gap compartments was 0.8 ml/min. DMAA was administered in a concentration of 10 -4 M/1. 2. 4. Drugs The drugs used were DMAA (1,3-dimethyl5-aminoadamantane; Merz & Co, Frankfurt/ Main), pentobarbital (Nembutal ® ; Abbot, Ingelheim/Rhein) and TEA (tetraethylammoniumchlozide; EGA-Chemie, Steinheim/Albuch).
381
MEMBRANE EFFECTS OF DIMETHYLADAMANTANE TABLE 1 Modified Locke solutions.
NaCI Na+-free K+-free High K + (I) High K + (II) High K ÷(III) High K + (IV) High K + and Na+-free Ca2+-free Ca2+.free and Na+-free Cl--free Na+-free and K+-free
KCI 5.6
154.0 142.8 126.0 92.4
154.0
16.8 33.6 67.2 163.6 67.2
CaCl2 5.0 5.0 5.0 5.0 5.0 5.0 5.0
5.6 5.6
Mannitol Choline
3. Results 3.1. General The results were obtained in sensory nerve fibre bundles (dorsal roots) in which the amplitude of the spike potential ranged between 40--90 mV and a Locke solution containing an elevated potassium concentration (16.8 raM; table 1, solution with high K÷ (I)) depolarized the-resting potential by at least 15 mV. The preparation of nerve fibre bundles used may be considered as desheathed, because the dura and arachnoidea, which correspond to the peri. and epineureum of the peripheral nerve, had to be taken away when removing the dorsal roots from the spinal channel. Changes in the resting potential similar to those recorded in the present experiments have been found when investigating desheathed nerve fibre bundles (St~mpfli, 1954; S~mpfli and Nishie, 1956; Straub, 1956; Schmidt and S~mpfli, 1957, 1959). 3. 2. Spike and resting potential The amplitude of the spike potentials elicited by electrical stimulation was determined
K2S04
CaS04
77.2
2.8
5.0
154.0 10.0
92.4 15.0 15.0
5.0
Na2S04
154.0
159.6
in 12 experiments carried out in normal Locke solution. DMAA reduced the amplitude by 19.2 + 3.4% (mean value + S.D.) of the controls 15 rain after having started the administration. In 3 pilot experiments it was found that the effect developed rather slowly and did not reach its maximum until after 30--45 rein'of drug administration; at that time the reduction of the amplitude amounted to 48.0 ± 7.5% of the controls. It took the effect even more time to disappear after switching over to normal Locke solution than to develop, and the recovery of the preparation was the more delayed the longer its exposition to the drug had lasted. Therefore, in all experiments the drug effect was determined 15 rain after the beginning of the superfusion. The resting potential was not changed by D M A A in the concentration employed. 3. 3. Membrane resistance The membrane resistance in normal Locke solution was determined in 8 experiments before and during the administration of DMAA. The drug increased the membrane resistance (fig. 1), the effect being more pronounced
382
A. GROSSMANN ET AL. ,mp/sec 600.
/uA 121086" 4. 2"
500' 400" 300.
• ~o' 4"o mv
o//-64 ,,/o/
a
Fig. 1. The effect of DMAA on the membrane resistance of sensory nerve fibres. Ordinate: current in pA. Abscissa: electrotonic potentials in inV. Circles: control values. Triangles: values obtained 15 rain after starting the superfusion with Locke solution containing DMAA in a concentration of 10 -4 M. The points are the mean values of 8 determinations each.
during the application of depolarizing (25.8 + 4.8% o f t h e c o n t r o l s ; m e a n value + S.D.) t h a n o f h y p e r p o l a r i z i n g {15.4 + 3.5% o f t h e controls} pulses.
200, 100O.
b
½ '~
6
8
'rb 1"2 14 16 18 20 22 24 2'6 msec
Fig. 2. The frequency of spike discharges induced by rectangular depolarizing pulses in sensory nerve fibres before and during the administration of DMAA. Ordinate: number of spikes per sec calculated from the interval between successive spike discharges. Abscissa: time in msec after starting the depolarization; the current strength employed was 5--6 pA and produced a maximal response. The pulse duration was 40 msec. The points on the curves are the mean values of 5 determinations each; the vertical lines in each point show the standard deviation. Circles: control values. Triangles: values obtained 15 rain after starting the superfusion with a Locke solution containing DMAA in a concentration of 10 -4 M.
3.4. Repetitive activity In 5 e x p e r i m e n t s , r e p e t i t i v e spike discharges w e r e elicited b y a p p l y i n g long-lasting d e p o l a r i z i n g r e c t a n g u l a r pulses. A t a c u r r e n t s t r e n g t h b e t w e e n 5 and 6 # A r e p e t i t i v e activit y was m a x i m a l , t h e n u m b e r o f spikes e v o k e d a m o u n t i n g t o 3 - - 5 . T h e a m p l i t u d e o f successive spikes s h o w e d a t e n d e n c y t o decrease, a n d t h e interval b e t w e e n t h e spikes increased. DMAA reduced both the amplitude and the n u m b e r o f spikes d i s c h a r g e d a n d f u r t h e r increased t h e s p i k e intervals. Fig. 2 s h o w s t h e c h a n g e in t h e f r e q u e n c y o f spike discharges c a l c u l a t e d f r o m t h e intervals b e t w e e n t w o successive spikes to be d e p e n d e n t o n t h e t i m e a f t e r s w i t c h i n g o n the. d e p o l a r i z i n g c u r r e n t b e f o r e a n d during t h e a d m i n i s t r a t i o n o f D M A A . ~A s p i k e was a l w a y s e v o k e d w i t h t h e rising p h a s e o f t h e r e c t a n g u l a r d e p o l a r i z i n g pulse. I n t h e c o n t r o l a s e c o n d spike f o l l o w e d t h e first a t a m e a n interval o f 1.9 m s e c , w h i c h y i e l d e d a f r e q u e n c y o f 530 impulses/sec. Dur-
ing t h e s u p e r f u s i o n w i t h D M A A , t h e interval b e t w e e n t h e first and t h e s e c o n d s p i k e was increased t o 2.1 msec, w h i c h c o r r e s p o n d s to a discharge f r e q u e n c y of 4 8 0 i m p u l s e s / s e c . T h e last s p i k e a p p e a r e d in t h e c o n t r o l s a t 24 m s e c a f t e r starting t h e d e p o l a r i z a t i o n , w h e r e a s under t h e i n f l u e n c e o f D M A A n o spike was discharged b e y o n d a depolarization of 18.2 msec duration. The difference between the values o b t a i n e d b e f o r e a n d d u r i n g t h e a d m i n i s t r a t i o n o f D M A A are statistically significant (p < 0.005; c a l c u l a t e d a c c o r d i n g t o S t u d e n t ' s t-test}.
3. 5. Sodi um inactivation T h e r e d u c t i o n o f t h e spike a m p l i t u d e observed d u r i n g t h e a d m i n i s t r a t i o n o f D M A A m a y be t h e result o f an e n h a n c e d s o d i u m i n a c t i v a t i o n b e c a u s e o f w h i c h a smaller a m o u n t o f s o d i u m ions w o u l d b e available f o r t h e g e n e r a t i o n of a spike p o t e n t i a l . T h e
MEMBRANE EFFECTS OF DIMETHYLADAMANTANE
A
B
'1
E
0o
20
0
,o0
°
20
40
60
80
membran~ p o l e n h o l in mV
20
0
20
40
c~O
membrane potenfJol n mV
Fig. 3. The absolute (A) and relative (B) spike amplitude at a given membrane potential before and during the administration of DMAA. Ordinate in A: spike amplitude in mV. Ordinate in B: spike amplitude in per cent of the maximal spike (spike evoked during membrane hyperpolarization). Abscissa: polarization of the nerve fibre membranes (hyperpolarization is indicated by the negative sign). Circles: control values; identical values were obtained during the administration of TEA. Open triangles: values obtained 15 rain after starting the superfusion with a Locke solution containing DMAA in a concentration of 10 -4 M. Filled triangles: values" obtained after starting the superfusion with a Locke solution containing DMAA 10 -4 M and TEA 10 -3 M.
amplitude of the spike potential in dependence of the membrane potential may be considered as an indicator of the sodium inactivation (Schmidt and Stiimpfli, 1966). Fig. 3 shows that the spike amplitude is maximal when the membrane is hyperpolarized. Most probably, under this condition no sodium inactivation is present (Hodgkin and Huxley, 1952; Frankenhiiuser, 1960). Depolarization of the membrane reduced the spike amplitude along an S-shaped curve which, on the basis of the made assumption, signals an enhanced sodium inactivation. Under the influence of DMAA the spike amplitude in the hyperpolarizing and in the depolarizing range was smaller than in the respective control (fig. 3A), the effect of the drug on the amplitude being more pronounced in the hyperpolarizing than in the depolarizing range (fig. 3A). In fig. 3B the amplitude of the spike potentials was plotted in percent of the maximal amplitude (i.e. the amplitude of the spike elicited during membrane hyperpolarization) either in a normal Locke solution or in a Locke solution containing DMAA. This shows that under
383
the influence of DMAA the slope of the Sshaped curve is flattened. To assess the dependence of this effect of DMAA on changes in the potassium conductance, TEA was administered to the preparations. TEA selectively blocks the permeation of potassium ions through the neuronal membrane (Hille, 1967; KoppenhSfer, 1967). The values determined without and during the administration of TEA 10 -3 M did not differ; the curve plotted from the values obtained during superfusion with a Locke solution containing TEA was identical with the control curves of fig. 3. However, when TEA was administered in a concentration of 10 -3 M in the Locke solution containing DMAA, the amplitude of the spike potential during membrane depolarization was more reduced than under the influence of DMAA alone. Plotting the values of the amplitudes in per cent of the maximal amplitude yielded a curve which did not differ from that obtained in normal Locke solution without drug administration (fig. 3B). These results suggest that the effects produced by DMAA are not due to an interference of the drug with the sodium inactivation but to a change in the potassium current. Experiments performed with DMAA in voltage clamp experiments on single nerve fibres of the frog also indicate that DMAA does not influence the sodium inactivation (Schwarz, personal communication).
3.6. The effect of DMAA in dependence of ion changes The following experiments were performed to evaluate the ions required by the drug to exert its membrane effects.
3. 6.1. Sodium-free suspension medium Replacement of sodium by choline ions, which do not penetrate the neuronal membrane, caused a hyperpolarization and an increase in the membrane resistance (table 2). These changes are in accord with the observations made in single nerve fibres (Huxley and St~impfli, 1951) and nerve fibre bundles
384
A. GROSSMANN ET AL.
TABLE 2 Changes in the resting potential and membrane resistance induced by modified Locke solutions and DMAA. The underlined values are significantly different from the corresponding values obtained in normal Locke solution or in the modified l_~cke solutions without DMAA (p < 0.01, calculated according to Student's t-test). The number of determinations in each series of experiments was 6. Modified Locke solution
Change of Resting potential in mV
Na+-free K+-free High K + (IV) High K +, Na+-free CaZ+-free Low Ca2+, Na-free C]--free Na+-free andK+-free
--8.2 --4.6 44.2 41.9 3.5 --5.9 12.1
+ 0.6 + 0.9 -+ 4.7 -+ 3.2 -+ 0.8 +- 1.1 + 3.9
--15.8+- 1.2
Change induced by DMAA (10 -4) of Membrane resistance in % of control in normal Locke* 19.4 52,4 --17,5 --2.9 --20,8 21.9 14.6
+- 6.1 +- 12.0 -+ 6.2 + 1.5 + 3.3 -+ 5.8 + 2.5
11.8-+
4.3
Resting potential in mV
Membrane resistance in % of control in normal Locke*
--6.6 + 1.8 --0.5 +- 0.3 38.7 -+ 1.9 3_3~_.7+ 1.2 0.__66-+ 0.4 --6.7 -+ 1.2 22.4 + 4.5
39.1 + 12.9 64.6 + 16.2 14.5 -+ 4.5 14_ .___33+ 4.9 4!~9 -+ 17.0 40.___!1 + 8.9 2.0.9 +- 4.6
--12.1 +2.3
32.9 + 11.7
* Mean value + S.D.
( S c h m i d t and St~mpfli, 1959}. D M A A did n o t p r o d u c e a p e r c e p t i b l e c h a n g e in t h e p o t e n t i a l o f t h e m e m b r a n e h y p e r p o l a r i z e d b y t h e sodiu m - f r e e suspension m e d i u m , whilst the m e m brane resistance was significantly increased (table 2).
3.6.2. High and low potassium concentration An increase o f t h e p o t a s s i u m c o n c e n t r a t i o n in the s u s p e n s i o n m e d i u m depolarized, and a reduction of the concentration hyperpolarized t h e n e u r o n a l m e m b r a n e s (fig. 4 and table 2). A t a c o n c e n t r a t i o n range a b o v e 10 mM/1 t h e relationship b e t w e e n the l o g a r i t h m o f the p o t a s s i u m c o n c e n t r a t i o n and t h e m e m b r a n e p o t e n t i a l was linear; t h e curve h a d a slope o f 43.5 m V per 10 times c h a n g e in t h e external p o t a s s i u m c o n c e n t r a t i o n . D M A A abolished the membrane hyperpolarization induced by a low potassium concentration and reduced the m e m b r a n e d e p o l a r i z a t i o n f o u n d with a high p o t a s s i u m c o n c e n t r a t i o n (fig. 4 and table 2).
The slope o f t h e curve in t h e linear range was d i m i n i s h e d t o 39.5 m V per 10 times c h a n g e in the external p o t a s s i u m c o n c e n t r a t i o n . Changes in the external p o t a s s i u m c o n c e n t r a t i o n p r o d u c e changes in t h e p o t a s s i u m per> gO" E E
~
60,
40-
~ 20" .Q E 0 0
5
,0
,0
,0o 200 log ( K )e in mM
Fig. 4. The membrane potential of sensory nerve fibres in relation to potassium concentration in the suspension medium before and during the administration of DMAA. Ordinate: membrane potential in mV. Abscissa: logarithm of the potassium concentration in mM. Circles: control values. Triangles: values obtained 15 min after starting the superfusion with the suspension media containing DMAA 10 -4 M.
MEMBRANE EFFECTS OF DIMETHYLADAMANTANE meability and thus in the membrane resistance (Schmidt, 1962). At a potassium concentration of 163.6 mM]l the membrane resistance was significantly lower than in normal Locke solution, and DMAA produced a significant increase of the resistance {table 2). 3.6.3. High potassium concentration in sodium-free suspension medium The increase in the membrane resistance produced by DMAA in normal Locke solution as well as in high external potassium concentration may be due to a diminished conductance of sodium or potassium ions, or both. Moreover, DMAA hyperpolarized the membrane in high external potassium concentrations; this can be ascribed to either a decreased sodium or an enhanced potassium permeability. Thus, the change in the membrane potential and resistance will be prompted by one and the same change in sodium conductance, whereas opposite changes in the potassium conductance are required to bring a b o u t these membrane effects. To test whether the potassium conductance is affected by DMAA, experiments were carried out with a sodium-free suspension medium containing a high potassium concentration. Replacement of sodium by choline ions did n o t influence the depolarization produced by a high external potassium concentration (table 2) as compared with the effect of a high concentration of potassium ions in the presence of sodium ions. However, the membrane resistance was much less decreased b y a high potassium concentration in the sodium-free medium than in normal Locke solution. DMAA depolarized the membrane and increased the membrane resistance significantly (table 2). This result indicates that DMAA does not increase b u t diminishes the potassium conductance. 3. 6. 4. Low calcium concentration Lowering the external concentration of calcium ions depolarizes neuronal membranes because of an increase in the sodium permeability (Schmidt, 1960). In accord with an increased
385 sodium permeability the membrane resistance was reduced by a low external calcium concentration (table 2). DMAA repolarized the membranes and increased the membrane resistance (table 2), possibly in consequence of a reduced sodium permeability. These effects of DMAA were statistically significant. 3.6.5. Low calcium concentration in sodium-free suspension medium Since the increase in the sodium permeability induced by a low calcium concentration cannot manifest itself in a sodium-free suspension medium, no depolarization of the neuronal membrane was observed under this experimental condition b u t rather a hyperpolarization (table 2). At the same time the membrane resistance had increased. DMAA did not change the potential of the hyperpolarized membrane b u t further increased the membrane resistance (table 2). 3. 6. 6. L o w chloride concentration Replacement of chloride by sulfate ions depolarized the membranes and increased the membrane resistance (table 2). These changes agree with those observed in frog nerve fibre bundles (Straub, 1956}. DMAA further depolarized the membranes and increased the membrane resistance (table 2). 3. 6. Z Sodium- and potassium-free suspension medium The results obtained in the experiments carried out with a chloride-free suspension medium suggested that chloride ions are involved in the membrane action of DMAA. To provide further evidence that the drug interferes with chloride permeability, nerve fibre bundles were suspended in a medium devoid of all ions important for the maintenance of the membrane potential except chloride ions. In such a medium the neuronal membrane showed a considerable hyperpolarization, and the membrane resistance was increased (table 2). DMAA repolarized the membrane potential and further increased the membrane resistance (table 2). These effects were statistically significant.
386
4. Discussion DMAA depressed the activity in bundles of rat sensory nerve fibres. The decrease in the amplitude of the spike potentials on the background of an unchanged resting potential and the elimination of the depolarization due to calcium deficiency strongly suggest that DMAA reduces the sodium permeability of the neuronal membrane. Moreover, the results obtained in a potassium-free suspension medium as well as in a sodium-free suspension medium with high potassium concentrations indicate that the potassium permeability is also decreased by the drug. The hyperpolarization by DMAA of the membrane depolarized by a high external potassium concentration does n o t invalidate this assumption. In connection with the increased membrane resistance observed, such hyperpolarization could have resulted from either a decreased sodium or an increased potassium conductance. If the drug decreased the potassium conductance, and exerted this effect exclusively, the membrane should have further depolarized because of a decreased outward current of potassium ions. However, this was not the case. Therefore, the hyperpolarization observed must be due to a reduction of the sodium permeability which is so powerful that it surpasses the effect on the membrane potential of a reduced potassium permeability. From the results obtained in a sodium- and chloride-free suspension medium it may be concluded t h a t chloride ions also participate in the action of the drug and that their permeability is reduced. The diminished permeability of sodium, potassium and chloride ions accounts for the increase in the membrane resistance under the influence of DMAA in normal Locke solution. Since the resting potential in normal Locke solution was n o t perceptibly changed by the drug, it is tempting to assume that the decrease of the inward sodium current is of the same size as that of the inward chloride and outward potassium currents taken together. However, this remains an assumption as long
A. G R O S S M A N N ET AL.
as the influence of the drug on the total leak-current, which includes chloride currents, has not been determined. DMAA reduced the spike amplitude and depressed repetitive spike discharges elicited by long lasting depolarizing pulses. These effects might account for the observation that the drug reduced decerebrate rigidity (Sontag and Wand, 1973) and the activity of alpha motoneurones in the vibration reflex {Miihlberg and Sontag, 1973), because the m o t o r responses under both experimental conditions are built up by a repetitive impulse input to the motoneurones. DMAA has been suggested to act directly on dopaminergic receptor sites in the brain apart from exerting an indirect effect on dopaminergic synapses (Svensson, 1973; Maj et al., 1974). The drug differs in this respect from its chemical homologue amantadine, which is considered to act exclusively in an indirect way by increasing the concentration of dopamine at the receptor sites. Amantadine tested in a small number of expdriments performed on sensory nerve fibre bundles was n o t found to exert membrane effects similar to those of DMAA. It is conceivable that DMAA mimicks the transmitter substance by eliciting an inhibitory postsynaptic potential which spreads along the somadendritic membrane. The dose of DMAA administered by Svensson (1973) to demonstrate a direct dopamine receptor activity was 50 mg/kg. Provided that DMAA is equally distributed in all organs, including the brain and the spinal cord, this dose would establish a tissue concentration of about 5 X 10 -3, which is double t h a t employed in this investigation. It is evident t h a t the present results do by no means allow anything to be said about the effect of DMAA on the dopaminergic receptor site. However, if the drug were assumed to exert an influence on the postsynaptic membrane similar to t h a t observed in the membranes of sensory nerve fibres, it might increase the inhibitory postsynaptic potential on account of its action on the potassium and chloride permeability. During the generation of an in-
MEMBRANE EFFECTS OF DIMETHYLADAMANTANE hibitory postsynaptic potential the permeability o f t h e n e u r o n a l m e m b r a n e f o r p o t a s s i u m and c h l o r i d e ions is t e m p o r a r i l y increased (Eccles, 1964). I f D M A A , like a m a n t a d i n e (Velkov, 1 9 7 4 ) , d o e s n o t i n h i b i t t h e s o d i u m - p o t a s s i u m d e p e n d e n t A T P a s e b u t diminishes t h e m e m b r a n e p e r m e a b i l i t y o f p o t a s s i u m and c h l o r i d e ions, a higher c o n c e n t r a t i o n g r a d i e n t o f these ions s h o u l d ensue. A t c o n c e n t r a t i o n gradients higher t h a n n o r m a l , a larger a m o u n t o f ions is available t o pass t h e n e u r o n a l m e m b r a n e w h e n t h e i r p e r m e a b i l i t y is t e m p o r a r i l y increased b y t h e i n h i b i t o r y t r a n s m i t t e r . T h u s , a small a m o u n t o f i n h i b i t o r y t r a n s m i t t e r released in t h e p r e s e n c e o f D M A A c o u l d e v o k e an i n h i b i t o r y p o s t s y n a p t i c p o t e n t i a l o f t h e s a m e size as t h a t g e n e r a t e d b y a large a m o u n t o f t h e t r a w s m i t t e r in t h e a b s e n c e o f t h e drug. Studies p e r f o r m e d with t h e m i c r o e l e c t r o d e technique on inhibitory postsynaptic potentials, r e c o r d e d in p a r t i c u l a r f r o m c a u d a t e neurones, will be n e c e s s a r y t o p r o v e t h e validity o f t h e a s s u m p t i o n . An a l t e r n a t i v e e x p l a n a t i o n for the abolition by DMAA of the motor disturbance due to d o p a m i n e deficiency could b e t h a t t h e d r u g r e d u c e s t h e repetitiv.e a c t i v i t y in d e s c e n d i n g spinal p a t h w a y s w h i c h s e e m s t o be prerequisite for the maintenance of the rigidity f o l l o w i n g c e n t r a l m o n o a m i n e deplet i o n ( J u r n a a n d Theres, 1969).
Acknowledgement The authors are indebted to Dr. Scherm of Merz & Co, Frankfurt/Main, for the generous supply of dimethylaminoadamantane.
References
And~n, N.-E., A. Rubenson, K. Fuxe and T. HSkfelt, 1967, Evidence for dopamine receptor stimulation by apomorphine, J. Pharm. Pharmacol. 19,627. Eccies, J.D., 1964, The Physiology of Synapses (Springer-Verlag, Berlin--GSttingen--Heidelberg ). Ernst, A.M., 1967, Mode of action of apomorphine and dexamphetamine on gnawing compulsion in rats, Psychopharmacol. 10, 316.
387 Frankenhiiuser, B., 1960, Quantitative description of sodium currents in myelinated nerve fibres of Xenopus laevis, J. Physiol. (London) 151,491. Grossmann, W., 1973, Studies on the effect of amantadine on neuronal membranes, Naunyn-Schmiedeb. Arch. Pharmacol., Suppl. to 277, R 23. Heimans, R.L.H., M.J. Rand and M.R. Fennessy, 1972, Effects of amantadine on uptake and release of dopamine by a particulate fraction of rat basal ganglia, J. Pharm. Pharmacol. 24, 875. Hille, B., 1967, The selective inhibition of delayed potassium currents in nerve by tetraethylammonium ion, J. Gen. Physiol. 50, 1287. Hodgkin, A.L. and A.F. Huxley, 1952, The dual effect of membrane potential on sodium conductance in the giant axon of Loligo, J. Physiol. (London) 116, 497. Huxley, A.F. and R. Stiimpfli, 1951, Effect of potassium and sodium on resting and action potentials of single myelinated nerve fibres, J. Physiol. (London) 112, 496. Jurna, L and C. Theres, 1969, The effect of phenytoin and metamphetamine on spinal motor activity, Naunyn-Schmiedeb. Arch. Pharmacol. 265, 244. Kilb, K.H. and R. St~mpfli, 1974, A new stopcock for pharmacological purposes, Naunyn-Schmiedeb. Arch. Pharmacol. 285, 293. Koppenh~fer, E., 1967, Die Wirkung yon Tetra~ithylammoniumchlorid auf die Membranstr~me Ranvierscher Schni~rringe von Xenopus laevis, Pflllgers Arch. 293, 34. Maj, J., H. Sowinska, L. Baran and J. Sarnek, 1974, Pharmacological effects of 1,3-dimethyl-5-aminoadamantane, a new adamantane derivative, European J. Pharmacol. 26, 9. M~hlberg, B. and K.-H. Sontag, 1973, The depression of monosynaptically excited ~-motoneurons during vibration reflex by dimethylaminoadamantan (DMAA), Naunyn-Schmiedeb. Arch. Pharmacol. 280, 113. Scatton, B., A. Cheramy, M. Besson and J. Glowinsky, 1970, Increased synthesis and release of dopamine in the striatum of the rat after amantadine treatment, European J. Pharmacol. 13, 131. Schmidt, H., 1960, Die Wirkung von Calcium-Ionen auf das Membranpotential markhaltiger Nervenfasern, Pfliigers Arch. 271,634. Schmidt, H., 1962, Messungen von Anderungen des Membranwiderstandes markhaltiger Nervenfasern mit der Saccharose-Trennwand-Methode, Pfliigers Arch. 274, 632. Schmidt, H. and R. St~impfli, 1957, Die Depolarisation durch Calciummangel und ihre Abh~/ngigkeit yon der Kalium-Konzentration, Helv. Physiol. Acta 15, 200. Schmidt, H. and R. St~impfli, 1959, Das Ruhepoten-
388 tial markhaltiger Nervenfasern in natrium- und chlorarmen Ringer-I.~sungen, Helv. Physiol. Acta 17, 62. Schmidt, H. and R. Stiimpfli, 1966, Die Wirkung yon Tetraa'thylammonium auf den einzelnen Ranvierschen Schniirring, Pfliigers Arch. 2 8 7 , 3 1 1 . St~impfli, R., 1954, A new method for measuring membrane potentials with external electrodes, Experientia 10, 509. St~impfli, R. and K. Nishie, 19~6, Effects of calciumfree solutions on membrane potential of myelinated nerve fibres of the Brazilian frog Leptodactylus oceUatus, Helv. Physiol. Acta 14, 93. Sontag, K.-H. and P. Wand, 1973, Decrease of muscle rigidity by dimethylaminoadamantan (DMAA) in intercollicularly decerebrated cats, Arzneim. Forsch. 23, 1737.
A. GROSSMANN ET AL. Straub, R.W., 1956, Die Wirkung yon Verhtridin und Ionen auf das Ruhepotential markhaltiger Nervenfasern des Frosches, Helv. Physiol. Acta 14, 1. Svensson, T.H., 1973, Dopamine release and direct dopamine receptor activation in the central nervous system by dimethylaminoadamantan, an amantadine derivative, European J. Pharmacol. 23, 232. Velkov, V.A., 1974, O n the effect of amantadine on A T P content and ATPase activity in brain and blood of rats, Experientia 30, 395. V o n Voigtlander, P.F. and K.E. Moore, 1971a, In vivo release of 3H-dopamine from cat brain by amantadine, Pharmacologist 13, 202. .. V o n Voigtlander, P.F. and K.E. Moore, 1971b, Dopamine: release from the brain in vivo by amantadine, Science 174, 408.
THE EFFECT OF DIMETHYLAMINOADAMANTANE ON NEURONAL MEMBRANES* ANDREA GROSSMANN, WERNER GROSSMANN and ILMAR JURNA
Pharmakologisches lnstitut der UniversitKt des Saarlandes, D-665 Homburg/Saar, German Federal Republic Received 30 January 1975, revised MS received 11 July 1975, accepted 29 September 1975
A. GROSSMANN, W. GROSSMANN and I. JURNA, The effect of dimethylaminoadamantane on neuronal
membranes, European J. Pharmacol. 35 (1976)379--388. The effect of dimethylaminoadamantane (DMAA), an amantadine derivative with an anti-Parkinson property, on rat sensory nerve fibres was studied with the sucrose gap method. DMAA 10 -4 M in normal Locke solution reduced the spike amplitude without changing the resting potential, increased the membrane resistance and depressed repetitive spike activity elicited by depolarizing currents. From experiments performed with changed concentrations of sodium, potassium, calcium and chloride ions in the suspension medium it appears that the permeability of sodium, potassium and chloride ions is reduced by DMAA. The possible implication of the membrane effects of the drug in its action on dopaminergic transmission in the brain is discussed. Dimethylaminoadamantane
Neuronal membrane
I. Introduction Amantadine is used as an anti-Parkinson agent which, either by stimulating the formation and release of dopamine or by inhibiting the uptake of the transmitter into dopaminergic terminals (Scatton et al., 1970; Von Voigtlander and Moore, 1971a, b; Heimans et al., 1972), increases the availability of the monoamine at dopaminergic receptor sites in the brain. Dimethylaminoadamantane (DMAA)~, an amantadine derivative also successfully applied in Parkinson's disease, is assumed to act both indirectly, as does amantadine, and directly on dopaminergic receptors (Svensson, 1973; Maj et al., 1974), as does apomorphine (And~n, 1967; Ernst, 1967). However, DMAA reduced the rigidity in decerebrate cats (Sontag and Wand, 1973), which has been ascribed either to an effect on dopaminergic transmission in nigro-reticular pathways or to a depressant action on neuro* Supported by 'Membranen'.
the
Sonderforschungsbereich 38
Ion permeability
nal membranes. The latter assumption is supported by the observation that the drug inhibited a-motoneurones activated monosynaptically by muscle vibration (Miihlberg and Sontag, 1973). Amantadine has been shown to exert an effect on isolated neuronal membranes which is independent of transmitter action (Grossmann, 1973). In the present investigation it was aimed to determine whether DMAA also influences the membrane properties of isolated nerve fibre bundles, which might account for the results of Sontag and coworkers (Miihlberg and Sontag, 1973; Sontag and Wand, 1973), and if its membrane effects might also contribute to its action at dopaminergic receptor sites.
2. Materials and methods
2.1. General procedure The experiments were performed on sensory nerve fibre bundles of the rat (250--300 g
380
b o d y weight; Wistar Strain). The rats were anesthetized b y an i.p. injection of pentobarbital 50 mg]kg. Sensory nerve fibre bundles were obtained b y isolating the dorsal roots of L 6 or S 1. The dorsal roots were cooled to 4 ° C in Locke solution immediately after removal, cut to a length of 22--25 mm and introduced into the sucrose gap apparatus. After having been m o u n t e d the preparation was superfused with oxygenated Locke and sucrose solutions respectively at a temperature of 36 ° C. A period of at least 1 hr was allowed for the preparation to stabilize in the perfusion medium before starting the experiment. The sucrose gap apparatus employed has been described in detail b y Schmidt (1962). The mean diameter o f the dorsal roots e m p l o y e d was 0.5 ram, and the diameter of the channel into which the nerve was inserted 0.65 mm. 2. 2. Stimulation and recording Stimulation was performed by means of sintered Ag/AgC1 electrodes (Bionetic Instruments, Santa Ana, California). Spike potentials were elicited by applying single rectangular pulses of 10 psec duration and a strength that was twice the intensity required to obtain a maximal spike response. To determine the membrane resistance, rectangular pulses of 200 msec duration and various strengths were applied. The polarizing currents were applied through a 50 M~2 resistor. Membrane potentials were led off with calomel electrodes. The electrodes were connected to a differential amplifier through cathode followers arranged in parallel and the signals recorded on a cathode ray oscilloscope. The resting potential was displayed on a physiograph. The membrane resistance was determined according to Ohm's law from the intensity of the current applied and the membrane polarization produced. The absolute membrane resistance can be calculated o n l y when the shortcircuiting factor of the sucrose gap is k n o w n (Schmidt, 1962). However, an exact determination of this factor cannot be achieved si-
A. G R O S S M A N N E T AL.
multaneously with t h e measurement membrane potential under current tion. Therefore, relative values only, ues disregarding the short~circuiting will be given in the text.
of the applicai.e. valfactor,
2. 3. Solutions The unmodified Locke solution consisted of NaCI 154.0, KC1 5.6, CaC12 5.0, tris 2.5, glucose 5.0 mM/l. The modified solutions are presented in table 1. In all the different suspension mediums listed, the concentration of tris and glucose was the same as in the unmodified Locke solution. The pH-value of the solutions was adjusted to 7.3 by adding adequate amounts of sulphuric acid. The sucrose solution was prepared b y dissolving 100 g sucrose (Saccharose p.a.; Merck, Darmstadt) in 100 ml double distilled water. The specific resistance of the sucrose solution under atmospheric conditions was 1.5 M ~ . To further reduce the conductance the solution was boiled under vacuum for 3 min and afterwards oxygenated. The solutions were supplied to the compartments of the sucrose gap apparatus by means of PVC tubes fitted to a stopcock with a minimal deadspace (Kilb and S~mpfli, 1974) which allowed a rapid change from one solution to another. The perfusion ~ate was controlled b y flow meters (Rotameter; Rota KG, S~ickingen). The perfusion rate of the Locke solution in b o t h outer compartments was 2 ml/min, and in the inner c o m p a r t m e n t 3.5 ml/min; the perfusion rate of the sucrose solution in the gap compartments was 0.8 ml/min. DMAA was administered in a concentration of 10 -4 M/1. 2. 4. Drugs The drugs used were DMAA (1,3-dimethyl5-aminoadamantane; Merz & Co, Frankfurt/ Main), pentobarbital (Nembutal ® ; Abbot, Ingelheim/Rhein) and TEA (tetraethylammoniumchlozide; EGA-Chemie, Steinheim/Albuch).
381
MEMBRANE EFFECTS OF DIMETHYLADAMANTANE TABLE 1 Modified Locke solutions.
NaCI Na+-free K+-free High K + (I) High K + (II) High K ÷(III) High K + (IV) High K + and Na+-free Ca2+-free Ca2+.free and Na+-free Cl--free Na+-free and K+-free
KCI 5.6
154.0 142.8 126.0 92.4
154.0
16.8 33.6 67.2 163.6 67.2
CaCl2 5.0 5.0 5.0 5.0 5.0 5.0 5.0
5.6 5.6
Mannitol Choline
3. Results 3.1. General The results were obtained in sensory nerve fibre bundles (dorsal roots) in which the amplitude of the spike potential ranged between 40--90 mV and a Locke solution containing an elevated potassium concentration (16.8 raM; table 1, solution with high K÷ (I)) depolarized the-resting potential by at least 15 mV. The preparation of nerve fibre bundles used may be considered as desheathed, because the dura and arachnoidea, which correspond to the peri. and epineureum of the peripheral nerve, had to be taken away when removing the dorsal roots from the spinal channel. Changes in the resting potential similar to those recorded in the present experiments have been found when investigating desheathed nerve fibre bundles (St~mpfli, 1954; S~mpfli and Nishie, 1956; Straub, 1956; Schmidt and S~mpfli, 1957, 1959). 3. 2. Spike and resting potential The amplitude of the spike potentials elicited by electrical stimulation was determined
K2S04
CaS04
77.2
2.8
5.0
154.0 10.0
92.4 15.0 15.0
5.0
Na2S04
154.0
159.6
in 12 experiments carried out in normal Locke solution. DMAA reduced the amplitude by 19.2 + 3.4% (mean value + S.D.) of the controls 15 rain after having started the administration. In 3 pilot experiments it was found that the effect developed rather slowly and did not reach its maximum until after 30--45 rein'of drug administration; at that time the reduction of the amplitude amounted to 48.0 ± 7.5% of the controls. It took the effect even more time to disappear after switching over to normal Locke solution than to develop, and the recovery of the preparation was the more delayed the longer its exposition to the drug had lasted. Therefore, in all experiments the drug effect was determined 15 rain after the beginning of the superfusion. The resting potential was not changed by D M A A in the concentration employed. 3. 3. Membrane resistance The membrane resistance in normal Locke solution was determined in 8 experiments before and during the administration of DMAA. The drug increased the membrane resistance (fig. 1), the effect being more pronounced
382
A. GROSSMANN ET AL. ,mp/sec 600.
/uA 121086" 4. 2"
500' 400" 300.
• ~o' 4"o mv
o//-64 ,,/o/
a
Fig. 1. The effect of DMAA on the membrane resistance of sensory nerve fibres. Ordinate: current in pA. Abscissa: electrotonic potentials in inV. Circles: control values. Triangles: values obtained 15 rain after starting the superfusion with Locke solution containing DMAA in a concentration of 10 -4 M. The points are the mean values of 8 determinations each.
during the application of depolarizing (25.8 + 4.8% o f t h e c o n t r o l s ; m e a n value + S.D.) t h a n o f h y p e r p o l a r i z i n g {15.4 + 3.5% o f t h e controls} pulses.
200, 100O.
b
½ '~
6
8
'rb 1"2 14 16 18 20 22 24 2'6 msec
Fig. 2. The frequency of spike discharges induced by rectangular depolarizing pulses in sensory nerve fibres before and during the administration of DMAA. Ordinate: number of spikes per sec calculated from the interval between successive spike discharges. Abscissa: time in msec after starting the depolarization; the current strength employed was 5--6 pA and produced a maximal response. The pulse duration was 40 msec. The points on the curves are the mean values of 5 determinations each; the vertical lines in each point show the standard deviation. Circles: control values. Triangles: values obtained 15 rain after starting the superfusion with a Locke solution containing DMAA in a concentration of 10 -4 M.
3.4. Repetitive activity In 5 e x p e r i m e n t s , r e p e t i t i v e spike discharges w e r e elicited b y a p p l y i n g long-lasting d e p o l a r i z i n g r e c t a n g u l a r pulses. A t a c u r r e n t s t r e n g t h b e t w e e n 5 and 6 # A r e p e t i t i v e activit y was m a x i m a l , t h e n u m b e r o f spikes e v o k e d a m o u n t i n g t o 3 - - 5 . T h e a m p l i t u d e o f successive spikes s h o w e d a t e n d e n c y t o decrease, a n d t h e interval b e t w e e n t h e spikes increased. DMAA reduced both the amplitude and the n u m b e r o f spikes d i s c h a r g e d a n d f u r t h e r increased t h e s p i k e intervals. Fig. 2 s h o w s t h e c h a n g e in t h e f r e q u e n c y o f spike discharges c a l c u l a t e d f r o m t h e intervals b e t w e e n t w o successive spikes to be d e p e n d e n t o n t h e t i m e a f t e r s w i t c h i n g o n the. d e p o l a r i z i n g c u r r e n t b e f o r e a n d during t h e a d m i n i s t r a t i o n o f D M A A . ~A s p i k e was a l w a y s e v o k e d w i t h t h e rising p h a s e o f t h e r e c t a n g u l a r d e p o l a r i z i n g pulse. I n t h e c o n t r o l a s e c o n d spike f o l l o w e d t h e first a t a m e a n interval o f 1.9 m s e c , w h i c h y i e l d e d a f r e q u e n c y o f 530 impulses/sec. Dur-
ing t h e s u p e r f u s i o n w i t h D M A A , t h e interval b e t w e e n t h e first and t h e s e c o n d s p i k e was increased t o 2.1 msec, w h i c h c o r r e s p o n d s to a discharge f r e q u e n c y of 4 8 0 i m p u l s e s / s e c . T h e last s p i k e a p p e a r e d in t h e c o n t r o l s a t 24 m s e c a f t e r starting t h e d e p o l a r i z a t i o n , w h e r e a s under t h e i n f l u e n c e o f D M A A n o spike was discharged b e y o n d a depolarization of 18.2 msec duration. The difference between the values o b t a i n e d b e f o r e a n d d u r i n g t h e a d m i n i s t r a t i o n o f D M A A are statistically significant (p < 0.005; c a l c u l a t e d a c c o r d i n g t o S t u d e n t ' s t-test}.
3. 5. Sodi um inactivation T h e r e d u c t i o n o f t h e spike a m p l i t u d e observed d u r i n g t h e a d m i n i s t r a t i o n o f D M A A m a y be t h e result o f an e n h a n c e d s o d i u m i n a c t i v a t i o n b e c a u s e o f w h i c h a smaller a m o u n t o f s o d i u m ions w o u l d b e available f o r t h e g e n e r a t i o n of a spike p o t e n t i a l . T h e
MEMBRANE EFFECTS OF DIMETHYLADAMANTANE
A
B
'1
E
0o
20
0
,o0
°
20
40
60
80
membran~ p o l e n h o l in mV
20
0
20
40
c~O
membrane potenfJol n mV
Fig. 3. The absolute (A) and relative (B) spike amplitude at a given membrane potential before and during the administration of DMAA. Ordinate in A: spike amplitude in mV. Ordinate in B: spike amplitude in per cent of the maximal spike (spike evoked during membrane hyperpolarization). Abscissa: polarization of the nerve fibre membranes (hyperpolarization is indicated by the negative sign). Circles: control values; identical values were obtained during the administration of TEA. Open triangles: values obtained 15 rain after starting the superfusion with a Locke solution containing DMAA in a concentration of 10 -4 M. Filled triangles: values" obtained after starting the superfusion with a Locke solution containing DMAA 10 -4 M and TEA 10 -3 M.
amplitude of the spike potential in dependence of the membrane potential may be considered as an indicator of the sodium inactivation (Schmidt and Stiimpfli, 1966). Fig. 3 shows that the spike amplitude is maximal when the membrane is hyperpolarized. Most probably, under this condition no sodium inactivation is present (Hodgkin and Huxley, 1952; Frankenhiiuser, 1960). Depolarization of the membrane reduced the spike amplitude along an S-shaped curve which, on the basis of the made assumption, signals an enhanced sodium inactivation. Under the influence of DMAA the spike amplitude in the hyperpolarizing and in the depolarizing range was smaller than in the respective control (fig. 3A), the effect of the drug on the amplitude being more pronounced in the hyperpolarizing than in the depolarizing range (fig. 3A). In fig. 3B the amplitude of the spike potentials was plotted in percent of the maximal amplitude (i.e. the amplitude of the spike elicited during membrane hyperpolarization) either in a normal Locke solution or in a Locke solution containing DMAA. This shows that under
383
the influence of DMAA the slope of the Sshaped curve is flattened. To assess the dependence of this effect of DMAA on changes in the potassium conductance, TEA was administered to the preparations. TEA selectively blocks the permeation of potassium ions through the neuronal membrane (Hille, 1967; KoppenhSfer, 1967). The values determined without and during the administration of TEA 10 -3 M did not differ; the curve plotted from the values obtained during superfusion with a Locke solution containing TEA was identical with the control curves of fig. 3. However, when TEA was administered in a concentration of 10 -3 M in the Locke solution containing DMAA, the amplitude of the spike potential during membrane depolarization was more reduced than under the influence of DMAA alone. Plotting the values of the amplitudes in per cent of the maximal amplitude yielded a curve which did not differ from that obtained in normal Locke solution without drug administration (fig. 3B). These results suggest that the effects produced by DMAA are not due to an interference of the drug with the sodium inactivation but to a change in the potassium current. Experiments performed with DMAA in voltage clamp experiments on single nerve fibres of the frog also indicate that DMAA does not influence the sodium inactivation (Schwarz, personal communication).
3.6. The effect of DMAA in dependence of ion changes The following experiments were performed to evaluate the ions required by the drug to exert its membrane effects.
3. 6.1. Sodium-free suspension medium Replacement of sodium by choline ions, which do not penetrate the neuronal membrane, caused a hyperpolarization and an increase in the membrane resistance (table 2). These changes are in accord with the observations made in single nerve fibres (Huxley and St~impfli, 1951) and nerve fibre bundles
384
A. GROSSMANN ET AL.
TABLE 2 Changes in the resting potential and membrane resistance induced by modified Locke solutions and DMAA. The underlined values are significantly different from the corresponding values obtained in normal Locke solution or in the modified l_~cke solutions without DMAA (p < 0.01, calculated according to Student's t-test). The number of determinations in each series of experiments was 6. Modified Locke solution
Change of Resting potential in mV
Na+-free K+-free High K + (IV) High K +, Na+-free CaZ+-free Low Ca2+, Na-free C]--free Na+-free andK+-free
--8.2 --4.6 44.2 41.9 3.5 --5.9 12.1
+ 0.6 + 0.9 -+ 4.7 -+ 3.2 -+ 0.8 +- 1.1 + 3.9
--15.8+- 1.2
Change induced by DMAA (10 -4) of Membrane resistance in % of control in normal Locke* 19.4 52,4 --17,5 --2.9 --20,8 21.9 14.6
+- 6.1 +- 12.0 -+ 6.2 + 1.5 + 3.3 -+ 5.8 + 2.5
11.8-+
4.3
Resting potential in mV
Membrane resistance in % of control in normal Locke*
--6.6 + 1.8 --0.5 +- 0.3 38.7 -+ 1.9 3_3~_.7+ 1.2 0.__66-+ 0.4 --6.7 -+ 1.2 22.4 + 4.5
39.1 + 12.9 64.6 + 16.2 14.5 -+ 4.5 14_ .___33+ 4.9 4!~9 -+ 17.0 40.___!1 + 8.9 2.0.9 +- 4.6
--12.1 +2.3
32.9 + 11.7
* Mean value + S.D.
( S c h m i d t and St~mpfli, 1959}. D M A A did n o t p r o d u c e a p e r c e p t i b l e c h a n g e in t h e p o t e n t i a l o f t h e m e m b r a n e h y p e r p o l a r i z e d b y t h e sodiu m - f r e e suspension m e d i u m , whilst the m e m brane resistance was significantly increased (table 2).
3.6.2. High and low potassium concentration An increase o f t h e p o t a s s i u m c o n c e n t r a t i o n in the s u s p e n s i o n m e d i u m depolarized, and a reduction of the concentration hyperpolarized t h e n e u r o n a l m e m b r a n e s (fig. 4 and table 2). A t a c o n c e n t r a t i o n range a b o v e 10 mM/1 t h e relationship b e t w e e n the l o g a r i t h m o f the p o t a s s i u m c o n c e n t r a t i o n and t h e m e m b r a n e p o t e n t i a l was linear; t h e curve h a d a slope o f 43.5 m V per 10 times c h a n g e in t h e external p o t a s s i u m c o n c e n t r a t i o n . D M A A abolished the membrane hyperpolarization induced by a low potassium concentration and reduced the m e m b r a n e d e p o l a r i z a t i o n f o u n d with a high p o t a s s i u m c o n c e n t r a t i o n (fig. 4 and table 2).
The slope o f t h e curve in t h e linear range was d i m i n i s h e d t o 39.5 m V per 10 times c h a n g e in the external p o t a s s i u m c o n c e n t r a t i o n . Changes in the external p o t a s s i u m c o n c e n t r a t i o n p r o d u c e changes in t h e p o t a s s i u m per> gO" E E
~
60,
40-
~ 20" .Q E 0 0
5
,0
,0
,0o 200 log ( K )e in mM
Fig. 4. The membrane potential of sensory nerve fibres in relation to potassium concentration in the suspension medium before and during the administration of DMAA. Ordinate: membrane potential in mV. Abscissa: logarithm of the potassium concentration in mM. Circles: control values. Triangles: values obtained 15 min after starting the superfusion with the suspension media containing DMAA 10 -4 M.
MEMBRANE EFFECTS OF DIMETHYLADAMANTANE meability and thus in the membrane resistance (Schmidt, 1962). At a potassium concentration of 163.6 mM]l the membrane resistance was significantly lower than in normal Locke solution, and DMAA produced a significant increase of the resistance {table 2). 3.6.3. High potassium concentration in sodium-free suspension medium The increase in the membrane resistance produced by DMAA in normal Locke solution as well as in high external potassium concentration may be due to a diminished conductance of sodium or potassium ions, or both. Moreover, DMAA hyperpolarized the membrane in high external potassium concentrations; this can be ascribed to either a decreased sodium or an enhanced potassium permeability. Thus, the change in the membrane potential and resistance will be prompted by one and the same change in sodium conductance, whereas opposite changes in the potassium conductance are required to bring a b o u t these membrane effects. To test whether the potassium conductance is affected by DMAA, experiments were carried out with a sodium-free suspension medium containing a high potassium concentration. Replacement of sodium by choline ions did n o t influence the depolarization produced by a high external potassium concentration (table 2) as compared with the effect of a high concentration of potassium ions in the presence of sodium ions. However, the membrane resistance was much less decreased b y a high potassium concentration in the sodium-free medium than in normal Locke solution. DMAA depolarized the membrane and increased the membrane resistance significantly (table 2). This result indicates that DMAA does not increase b u t diminishes the potassium conductance. 3. 6. 4. Low calcium concentration Lowering the external concentration of calcium ions depolarizes neuronal membranes because of an increase in the sodium permeability (Schmidt, 1960). In accord with an increased
385 sodium permeability the membrane resistance was reduced by a low external calcium concentration (table 2). DMAA repolarized the membranes and increased the membrane resistance (table 2), possibly in consequence of a reduced sodium permeability. These effects of DMAA were statistically significant. 3.6.5. Low calcium concentration in sodium-free suspension medium Since the increase in the sodium permeability induced by a low calcium concentration cannot manifest itself in a sodium-free suspension medium, no depolarization of the neuronal membrane was observed under this experimental condition b u t rather a hyperpolarization (table 2). At the same time the membrane resistance had increased. DMAA did not change the potential of the hyperpolarized membrane b u t further increased the membrane resistance (table 2). 3. 6. 6. L o w chloride concentration Replacement of chloride by sulfate ions depolarized the membranes and increased the membrane resistance (table 2). These changes agree with those observed in frog nerve fibre bundles (Straub, 1956}. DMAA further depolarized the membranes and increased the membrane resistance (table 2). 3. 6. Z Sodium- and potassium-free suspension medium The results obtained in the experiments carried out with a chloride-free suspension medium suggested that chloride ions are involved in the membrane action of DMAA. To provide further evidence that the drug interferes with chloride permeability, nerve fibre bundles were suspended in a medium devoid of all ions important for the maintenance of the membrane potential except chloride ions. In such a medium the neuronal membrane showed a considerable hyperpolarization, and the membrane resistance was increased (table 2). DMAA repolarized the membrane potential and further increased the membrane resistance (table 2). These effects were statistically significant.
386
4. Discussion DMAA depressed the activity in bundles of rat sensory nerve fibres. The decrease in the amplitude of the spike potentials on the background of an unchanged resting potential and the elimination of the depolarization due to calcium deficiency strongly suggest that DMAA reduces the sodium permeability of the neuronal membrane. Moreover, the results obtained in a potassium-free suspension medium as well as in a sodium-free suspension medium with high potassium concentrations indicate that the potassium permeability is also decreased by the drug. The hyperpolarization by DMAA of the membrane depolarized by a high external potassium concentration does n o t invalidate this assumption. In connection with the increased membrane resistance observed, such hyperpolarization could have resulted from either a decreased sodium or an increased potassium conductance. If the drug decreased the potassium conductance, and exerted this effect exclusively, the membrane should have further depolarized because of a decreased outward current of potassium ions. However, this was not the case. Therefore, the hyperpolarization observed must be due to a reduction of the sodium permeability which is so powerful that it surpasses the effect on the membrane potential of a reduced potassium permeability. From the results obtained in a sodium- and chloride-free suspension medium it may be concluded t h a t chloride ions also participate in the action of the drug and that their permeability is reduced. The diminished permeability of sodium, potassium and chloride ions accounts for the increase in the membrane resistance under the influence of DMAA in normal Locke solution. Since the resting potential in normal Locke solution was n o t perceptibly changed by the drug, it is tempting to assume that the decrease of the inward sodium current is of the same size as that of the inward chloride and outward potassium currents taken together. However, this remains an assumption as long
A. G R O S S M A N N ET AL.
as the influence of the drug on the total leak-current, which includes chloride currents, has not been determined. DMAA reduced the spike amplitude and depressed repetitive spike discharges elicited by long lasting depolarizing pulses. These effects might account for the observation that the drug reduced decerebrate rigidity (Sontag and Wand, 1973) and the activity of alpha motoneurones in the vibration reflex {Miihlberg and Sontag, 1973), because the m o t o r responses under both experimental conditions are built up by a repetitive impulse input to the motoneurones. DMAA has been suggested to act directly on dopaminergic receptor sites in the brain apart from exerting an indirect effect on dopaminergic synapses (Svensson, 1973; Maj et al., 1974). The drug differs in this respect from its chemical homologue amantadine, which is considered to act exclusively in an indirect way by increasing the concentration of dopamine at the receptor sites. Amantadine tested in a small number of expdriments performed on sensory nerve fibre bundles was n o t found to exert membrane effects similar to those of DMAA. It is conceivable that DMAA mimicks the transmitter substance by eliciting an inhibitory postsynaptic potential which spreads along the somadendritic membrane. The dose of DMAA administered by Svensson (1973) to demonstrate a direct dopamine receptor activity was 50 mg/kg. Provided that DMAA is equally distributed in all organs, including the brain and the spinal cord, this dose would establish a tissue concentration of about 5 X 10 -3, which is double t h a t employed in this investigation. It is evident t h a t the present results do by no means allow anything to be said about the effect of DMAA on the dopaminergic receptor site. However, if the drug were assumed to exert an influence on the postsynaptic membrane similar to t h a t observed in the membranes of sensory nerve fibres, it might increase the inhibitory postsynaptic potential on account of its action on the potassium and chloride permeability. During the generation of an in-
MEMBRANE EFFECTS OF DIMETHYLADAMANTANE hibitory postsynaptic potential the permeability o f t h e n e u r o n a l m e m b r a n e f o r p o t a s s i u m and c h l o r i d e ions is t e m p o r a r i l y increased (Eccles, 1964). I f D M A A , like a m a n t a d i n e (Velkov, 1 9 7 4 ) , d o e s n o t i n h i b i t t h e s o d i u m - p o t a s s i u m d e p e n d e n t A T P a s e b u t diminishes t h e m e m b r a n e p e r m e a b i l i t y o f p o t a s s i u m and c h l o r i d e ions, a higher c o n c e n t r a t i o n g r a d i e n t o f these ions s h o u l d ensue. A t c o n c e n t r a t i o n gradients higher t h a n n o r m a l , a larger a m o u n t o f ions is available t o pass t h e n e u r o n a l m e m b r a n e w h e n t h e i r p e r m e a b i l i t y is t e m p o r a r i l y increased b y t h e i n h i b i t o r y t r a n s m i t t e r . T h u s , a small a m o u n t o f i n h i b i t o r y t r a n s m i t t e r released in t h e p r e s e n c e o f D M A A c o u l d e v o k e an i n h i b i t o r y p o s t s y n a p t i c p o t e n t i a l o f t h e s a m e size as t h a t g e n e r a t e d b y a large a m o u n t o f t h e t r a w s m i t t e r in t h e a b s e n c e o f t h e drug. Studies p e r f o r m e d with t h e m i c r o e l e c t r o d e technique on inhibitory postsynaptic potentials, r e c o r d e d in p a r t i c u l a r f r o m c a u d a t e neurones, will be n e c e s s a r y t o p r o v e t h e validity o f t h e a s s u m p t i o n . An a l t e r n a t i v e e x p l a n a t i o n for the abolition by DMAA of the motor disturbance due to d o p a m i n e deficiency could b e t h a t t h e d r u g r e d u c e s t h e repetitiv.e a c t i v i t y in d e s c e n d i n g spinal p a t h w a y s w h i c h s e e m s t o be prerequisite for the maintenance of the rigidity f o l l o w i n g c e n t r a l m o n o a m i n e deplet i o n ( J u r n a a n d Theres, 1969).
Acknowledgement The authors are indebted to Dr. Scherm of Merz & Co, Frankfurt/Main, for the generous supply of dimethylaminoadamantane.
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