Department of Pharmacology and Experimental Therapeutics,
University of Maryland School of Medicine, Baltimore, Maryland (H.M.,
K.L.S., E.X.A.), and
Laboratory of Molecular Pharmacology II, Institute
of Biophysics "Carlos Chagas Filho," Federal University of Rio de
Janeiro, Rio de Janeiro, Brazil (E.X.A.)
The effects of amantadine on nicotinic acetylcholine receptors (nAChRs)
of hippocampal neurons were studied by recording three types of
acetylcholine (ACh)-evoked currents, using the whole-cell patch-clamp
technique. The rapidly desensitizing type IA nicotinic current, which
is 
-bungarotoxin-sensitive and is mediated by nAChRs bearing 7
subunits, was inhibited by application of amantadine to neurons for 10 min (IC50 = 6.5 µM), but the potency of ACh (EC50 = 0.27 mM) was not affected by the drug. Amantadine
(30-50 µM) attenuated the peak current amplitude in a
voltage-dependent manner, with greater effect at negative than at
positive membrane potentials. In contrast, the decay phase of the
currents was shortened in a voltage-independent manner. When amantadine
was coapplied briefly with ACh, the drug was markedly less potent
(IC50 = 130 µM). Thus, the noncompetitive effects of
amantadine on the type IA nicotinic current are complex, involving
actions on the closed and desensitized states of the 7 nAChR. The
slowly desensitizing, -bungarotoxin-insensitive nicotinic currents
of type II, which is inhibited by dihydro-
-erythroidine and is
mediated by 42 nAChRs, and of type III, which is inhibited by
mecamylamine and is mediated by 34 nAChRs, were also sensitive to
inhibition by amantadine. The peak amplitude of type II current was
reduced only slightly by 10 µM amantadine coapplied with ACh, but the decay-time constant and amplitude of the sustained current were markedly reduced. Type III current was also inhibited when amantadine was briefly coapplied with ACh. In contrast to its effects on nicotinic
currents, amantadine at 10 µM did not affect currents evoked by
N-methyl-D-aspartate plus glycine,
-aminobutyric acid, glycine or kainate. Thus, on cultured
hippocampal neurons, amantadine preferentially inhibits nicotinic
currents.
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Introduction |
Amantadine hydrochloride is
recommended for the treatment of influenza and Parkinson's disease
(Davies et al., 1964
; Schwab et al., 1972;
Standaert and Young, 1996). The effectiveness of the drug against
influenza A virus appears to be via the blockade of
pH-sensitive cation channels formed by the M2 integral membrane protein
(Duff et al., 1994; Wang et al., 1994). In
comparison, the mechanisms accounting for the mild efficacy of
amantadine against symptoms of Parkinson's disease are not clear.
Amantadine enhances the side effects of anticholinergic agents used in
treating parkinsonism (Franz, 1975), and a direct inhibition of
neurotransmission mediated by NMDA receptors in the caudate nucleus may
be beneficial (Lupp et al., 1992; Stoof et al.,
1992). In contrast, the NMDA-activated currents in superior colliculus
neurons and the experimental seizures in mice were antagonized only by
doses of amantadine well above the usual therapeutic levels (Parsons
et al., 1995). Nevertheless, evidence that amantadine may
favorably affect the limbic system in some cases was shown by the
response of several individuals with refractory absence epilepsy to
treatment with this compound (Shahar and Brand, 1992). Although large
groups of elderly patients are treated with amantadine, raising concern
about potential clinical effects on cognitive processes, amantadine has
been found not to impair cognition (McEvoy, 1987; Hitri et
al., 1987; Van Putten et al., 1987).
The question of whether amantadine alters the function of nAChRs in the
brain has not been addressed, even though amantadine has been known for
many years to modulate the function of nAChRs in muscle. In
nerve-muscle preparations, amantadine decreases the amplitude of the
EPC. The effect on the decay-time constant of the EPC is more complex,
with shortening at negative membrane potentials but lengthening at
positive potentials (Albuquerque et al., 1978; Tsai et
al., 1978). This shortening of the decay-time constant of the EPC
led to the suggestion that amantadine is a noncompetitive blocker at
the muscle-type nAChR. Furthermore, amantadine was found to activate
nAChR single-channel currents in isolated muscle fibers (Dumbill and
Albuquerque, 1987), leading to the suggestion that the compound also
may activate opening of the nAChR channel by acting as a weak
noncompetitive agonist, as does physostigmine (Shaw et al.,
1985; Pereira et al., 1993; Schrattenholz et al.,
1993; Albuquerque et al., 1997).
Several types of nAChRs present in the mammalian brain differ
sufficiently in their molecular structures, from each other and from
nAChRs in muscle, to yield diverse functional and pharmacological properties (Alkondon and Albuquerque, 1993; Sargent, 1993; Albuquerque et al., 1995a,b; Lindstrom, 1995). The neuronal nAChRs in
rat hippocampal neurons mediate three main types of current (Alkondon and Albuquerque, 1993). A fast desensitizing nicotinic current, named
type IA current, is the most frequently observed type of nicotinic
current in hippocampal neurons and is mediated by a nAChR that is
highly permeable to Ca++ and seems to bear 7 subunits
(Alkondon and Albuquerque, 1993; Alkondon et al., 1994;
Castro and Albuquerque, 1995; Bonfante-Cabarcas et al.,
1996). MLA, -BGT or -conotoxin-ImI potently inhibits the
activation of type IA current (Alkondon et al., 1992;
Pereira et al., 1996). The nicotinic currents of types II
and III are observed infrequently in cultured hippocampal neurons and
desensitize more slowly (Alkondon and Albuquerque, 1993). The type II
current is inhibited by DHE and seems to be carried by
42-bearing nAChRs. On the other hand, the type III current is
inhibited by mecamylamine and seems to be mediated by 34-bearing
nAChRs.
The possibility that amantadine has an action on neuronal nAChR ion
channels is supported by homology between the critical amino acids of
transmembrane fragments that are proposed to line the ion channel pore
of nAChRs (Séguéla et al., 1993; Unwin, 1995)
and the amantadine-sensitive channel of the influenza virus M2 protein
(Duff et al., 1994; Wang et al., 1994). In each
case, a critical serine or threonine residue may be present where the open channel is most constricted, and hydrophobic amino acids are
likely to line the inner wall of the channel in the closed state. The
present study evaluated the effects of amantadine on neuronal nAChRs
and a few other ligand-gated (GABA, glycine, kainate and NMDA) ion
channels. The results demonstrate that amantadine has a weak antagonist
effect that may signify ion channel blockade of the open state and a
potent noncompetitive action at closed and/or desensitized states of
the nAChRs in the mammalian central nervous system.
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Materials and Methods |
Neuron culture.
Hippocampal neurons in culture were prepared
by a procedure similar to that described previously (Aracava et
al., 1987; Alkondon and Albuquerque, 1993). Briefly, the
hippocampi of 16- to 18-day-old rat fetuses (Sprague-Dawley) were
dissected out, minced and incubated with 0.25% trypsin (Gibco-BRL,
Grand Island, NY) for 30 min at 36°C. Using a sterile Pasteur
pipette, neurons were dispersed and then plated at a density of
700,000 cells/35-mm culture dish (precoated with Vitrogen 100 collagen; Celtrix Laboratories, Palo Alto, CA). The cells were
incubated at 36°C in a water-saturated, 10% CO2/90% air
atmosphere. The medium surrounding the cells was replaced twice each
week. On the seventh day after plating, uridine and
5-fluoro-2
-deoxyuridine (final concentrations, 6.7 and 13.3 µg/ml,
respectively) were added to the culture medium for 24 hr to inhibit the
proliferation of non-neuronal cells. For this study, the neurons were
cultured for 14 to 30 days.
Whole-cell current recording.
Whole-cell currents were
recorded from hippocampal neurons with the standard patch-clamp
technique (Hamill et al., 1981), using an LM-EPC-7
patch-clamp system (List Electronics, Darmstadt, Germany). The patch
pipettes were pulled from borosilicate capillary glass, and their
resistance when filled with internal solution was 2 to 5 M
. The
series resistance of the patches was 8 to 20 M and was not
compensated. The signals were filtered at 3 kHz and either were
recorded on tape for later analysis or were sampled directly by a
microcomputer using the program pCLAMP (Axon Instruments, Foster City,
CA). All experiments were performed at room temperature (20-22°C).
The external bath solution (340 mOsm) consisted of 165 mM NaCl, 5 mM
KCl, 2 mM CaCl2, 10 mM glucose, 5 mM HEPES, 0.001 mM atropine and 0.0003 mM tetrodotoxin, and the pH of the solution was
adjusted to 7.3 with NaOH. The neurons were superfused continuously with external solution flowing at 1.5 to 2.0 ml/min.
In many studies, the pipettes were filled with an ATP-RS to reduce the
rate of rundown of type IA currents (Alkondon et al., 1994).
The ATP-RS was prepared daily from a stock solution of 60 mM CsCl, 60 mM CsF, 10 mM ethylene glycol bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid, 10 mM HEPES
and 2 mM MgCl2 by addition of 5 mM ATP (Tris salt), 20 mM
phosphocreatine (di-Tris salt), 50 U/ml creatine phosphokinase and a
small amount (5 µl) of 1 N CsOH (to adjust the solution to pH 7.3 and the concentration of Cs+ to 155 mM). The final
osmolarity was 340 mOsm. In other studies, the standard solution inside
the pipette consisted of 80 mM CsCl, 80 mM CsF, 10 mM ethylene glycol
bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid and 10 mM HEPES, and the pH of the solution was adjusted with CsOH
to pH 7.3 (the final concentration of Cs+ was 187 mM, and
the osmolarity was 340 mOsm). Studies of type IA current that used the
standard solution in the pipette were begun 20 min after establishment
of the whole-cell patch, when the rundown of type IA currents is
considerably smaller than immediately after patch formation, and the
studies were completed within 10 min (Alkondon and Albuquerque, 1993).
The standard pipette solution was also used in studies of the slowly
decaying type II and type III nicotinic currents (Alkondon and
Albuquerque, 1993) and of non-nicotinic currents that do not run down
within the time of the experiments.
Drug applications.
External solutions with ACh or other
agonists, with or without amantadine, were applied to neurons using the
U-tube system (Albuquerque et al., 1991). A U-shaped tube,
which had a 250- to 400-µm pore at the apex, was positioned 50
µm above the neuronal soma. A drug solution, which was selected by a
manual switch valve, was delivered to one end of the U-tube and was
withdrawn by suction through a polyethylene tube at the opposite end. A
small amount of bath solution was also removed to prevent leakage of
drug solution out of the U-tube. When a solenoid valve was closed
briefly (
2 sec) by an electric pulse, a rapid flow of drug solution
was forced out through the pore, completely displacing the bath
solution surrounding the neuronal soma and dendrites.
Two protocols were used to apply amantadine hydrochloride (1-500 µM)
to the neurons. In one procedure, a neuron was superfused for several
minutes with an external bath solution containing amantadine, and the
agonist solution delivered from the U-tube always contained the same
concentration of the drug. In the other procedure, amantadine was
applied to the target neuron only briefly (2 sec) when a mixture of
agonist and drug were delivered through the U-tube system.
Amantadine · HCl, ACh · HCl, NMDA, glycine · HCl, kainic acid,
GABA, tetrodotoxin, atropine sulfate, ATP (Tris salt), phosphocreatine (di-Tris salt) and creatine phosphokinase (type I) were obtained from
Sigma Chemical Co. (St. Louis, MO). DHE · HBr was a gift from
Merck, Sharp & Dohme Research Laboratories (Rahway, NJ).
Data analysis.
The peak amplitude and the decay-time
constants of the whole-cell currents were determined using the program
pCLAMP. The IC50 and Hill coefficient
(nH) values were determined with the program Sigma Plot by the best fit of the data to the equation:
where I, Imax, [amantadine],
IC50 and nH are the peak whole-cell
current at the test concentration of amantadine, the maximum peak
current in the absence of amantadine, the concentration of amantadine
applied, the calculated concentration of amantadine that would reduce
the current by 50% and the Hill coefficient, respectively. Values are
expressed as the mean ± S.E. A P value of 0.05 in the Students'
t test was taken to be statistically significant.
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Results |
Brief applications of ACh (0.5-1 sec, 1 mM) to cultured
hippocampal neurons evoked one of three types of nicotinic current. Each current was classified according to its kinetic and
pharmacological characteristics, in accordance with the previous
description; type IA current decays rapidly and is blocked by the
competitive antagonist MLA (1 nM), and the slower decaying type II and
type III currents are blocked by DHE (100 nM) and mecamylamine (1 µM), respectively (Alkondon and Albuquerque, 1993, 1995; Albuquerque et al., 1995a). A current that is a combination of type IA
and type II currents, called type IB current, could also be recorded from some neurons (Alkondon and Albuquerque, 1993, 1995).
Effects of amantadine on type IA nicotinic current.
Type IA
currents were isolated by using an external solution containing DHE
(100 nM) to block any type II current. Under this condition, rapidly
decaying type IA currents were found in 66 of 70 neurons tested in this
phase of the study. To prevent rundown of the type IA currents during
long protocol procedures, patch pipettes were filled with ATP-RS.
After the type IA current evoked by ACh (0.3 mM) was recorded several
times to ensure a stable response, amantadine (1-30 µM) was applied
to each neuron for 10 min and was found to reduce the peak amplitude of
this current in a concentration-dependent manner (fig.
1A). The amplitude of the small, slowly decaying component recorded in some of the type IA currents was also decreased by amantadine (fig. 1B). Nine neurons exhibiting a type IA current in
response to ACh were treated in this manner with one or more concentrations of amantadine for 10 min each, and the results were
combined by expressing the peak amplitude of each current evoked by ACh
plus amantadine as a percentage of the peak amplitude before exposure
to the drug. The inhibition of the type IA current by amantadine was
concentration dependent, with potency reflected by the IC50
of 6.5 µM and the nH of 0.96 (fig. 1C).
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Fig. 1.
Inhibition by amantadine, applied
via bath and U-tube, of type IA current. A, the traces
in the upper, center and lower rows were obtained from independent
experiments with different neurons. Left, a 1-sec pulse of ACh (0.3 mM), applied to the cell as a control, is shown. In this figure and all
other figures, the bar above the current trace indicates the period of
agonist application. Right, a 1-sec pulse of ACh (0.3 mM) plus
amantadine (1, 10 or 30 µM) was applied 10 min after changing to the
bath solution containing the same concentration of amantadine. Holding
potential was  60 mV. B, the current trace in the presence of
amantadine at each concentration was scaled to match the amplitude of
the corresponding control current trace. C, the relationship between peak amplitude of type IA current, as percentage of control, and the
concentration of amantadine is shown. Symbols represent the mean ± S.E. for three to nine neurons. The IC50 and
nH values were 6.5 ± 0.7 µM and
0.96 ± 0.08, respectively.
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To determine the mechanism by which amantadine inhibits type IA
current, several concentrations of ACh (0.05, 0.1, 0.3, 1 or 3 mM) were
applied at 2-min intervals before and after each neuron was superfused
for 10 min with an external solution containing the drug (10 µM). As
expected, the peak amplitude of type IA currents increased with
increasing ACh concentration (fig. 2A). The peak current
amplitude elicited by each concentration of ACh was considerably smaller during exposure of the neuron to amantadine (fig. 2A, center).
For each neuron, the peak current amplitudes were normalized relative
to the response to 3 mM ACh in the absence of amantadine. Linear
regression analysis of the normalized responses in a double-reciprocal plot revealed that the maximum response to ACh in the presence of
amantadine was reduced to 53% of control (fig. 2B). The apparent affinity of the receptor for ACh was, however, unchanged by treatment with the drug (fig. 2B). Superfusion of the neurons with
amantadine-free external solution reversed the inhibitory effect of
amantadine (fig. 2A); the small differences in amplitude noted during
the recovery phase could be accounted for by the expected rundown of
control responses over the 20-min interval. These results indicated that amantadine does not compete for the ACh binding site of the 7
nAChR that subserves the type IA current in hippocampal neurons, suggesting that the drug acts at an allosteric site on the receptor. Also, the impairment of mAChR function is reversible.
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Fig. 2.
Noncompetitive blockade by amantadine of type IA
current. A, type IA currents were evoked by 1-sec pulses of ACh at
various concentrations (left). Then amantadine was applied
via bath and U-tube for 10 min before various test
concentrations of ACh were reapplied (center). Finally, the preparation
was washed with drug-free solution for 10 min and the test
concentrations of ACh were reapplied (right). All traces were obtained
from a single neuron held at 60 mV. B, a series of experiments was
summarized by normalizing the amplitude of each response to the peak
current amplitude elicited by ACh (3 mM) in the absence of amantadine
in the same neuron. The data (mean ± S.E.) from three to eight
neurons are shown in a double-reciprocal plot. Linear regressions of
each group of data gave the affinity for ACh as 0.28 mM in the control
condition and 0.27 mM in the amantadine (10 µM)-treated condition.
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The effect of membrane potential on the inhibition caused by amantadine
was studied by recording type IA currents at negative and positive
potentials before and after superfusion of the neuron with
amantadine-containing external solution from the U-tube and through the
bath. Pulses of ACh (1 mM, 0.5 sec) were initially applied to five
neurons at holding potentials from 120 to 60 mV in 20-mV steps. The
peak current-voltage relationships of the type IA currents in the
control condition revealed a significant inward rectification (fig.
3B), which could be accounted for by the
Mg++ in the ATP-RS solution (Alkondon et al.,
1994; Bonfante-Cabarcas et al., 1996). Ten minutes after
bath superfusion of the neurons with amantadine (50 µM), pulses of
ACh plus the drug (50 µM) were applied while the neurons were held at
the same range of potentials (fig. 3A). Amantadine reduced the
normalized peak amplitudes at negative potentials. However, the peak
amplitude of the type IA current at positive potentials was decreased
to a lesser degree by amantadine, such that the current-voltage
relationship was linear from 120 to 60 mV (fig. 3B). Thus,
rectification was apparently reduced. For each holding potential, the
means of the ratios of the peak amplitudes in the presence of
amantadine to the amplitudes under the control condition were plotted
(fig. 3C). The ratio was small (0.2) at negative potentials and
increased to up to 0.4 with depolarization to 50 mV.
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Fig. 3.
Voltage-dependent inhibition by amantadine (50 µM), applied via bath and U-tube, of type IA current
evoked by ACh (1 mM). A, pulses of ACh (1 mM, 0.5 sec) were first
applied while holding potential was increased in 20-mV steps from 120
to 60 mV; sample current traces are shown for three holding potentials
(left). Ten minutes after changing to the bath solution containing
amantadine (50 µM), pulses of ACh plus amantadine (50 µM) were
applied at the same potentials (right). All traces shown were obtained
from one neuron. B, the peak amplitude measured under all conditions was normalized to the peak amplitude at 120 mV induced by ACh in the
absence of amantadine. The plotted data represent the mean ± S.E.
from experiments in five neurons. The current-voltage relationships below 0 mV were used to fit the lines. C, the relationship of the ratio
of the amplitude in the presence of amantadine (50 µM) to the
corresponding amplitude in control solution vs. holding potential is shown.
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The protocol used for figure 3 was repeated with a lower concentration
of amantadine (30 µM), again applied to the neurons via
bath and U-tube perfusion, and a lower concentration of ACh (0.3 mM).
Under these conditions, as at higher concentrations, amantadine reduced
the peak amplitude of the type IA current (fig. 4, A and
B) (n = 7 neurons). The current-voltage relationship appeared to confirm the loss of rectification (fig. 4B), and the ratio
of peak amplitude in the presence of amantadine to the corresponding amplitude under the control condition was small (0.2) at negative potentials (fig. 4C). At 40 mV, 30 µM amantadine was somewhat less
effective at reducing the peak amplitude of the current elicited by 0.3 mM ACh (40% inhibition) (fig. 4C) than was 50 µM amantadine at
reducing the peak amplitude of the current evoked by 1 mM ACh (60%
inhibition) (fig. 3C). This suggests a lack of competition between ACh
and amantadine, because the amount of inhibition was reduced when the
drug concentration/agonist concentration ratio was increased from 0.05 (fig. 3) to 0.1 (fig. 4).
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Fig. 4.
Voltage-dependent inhibition by amantadine (30 µM), applied via bath and U-tube, on the type IA
current evoked by ACh (0.3 mM). A, the procedures for these experiments
were the same as in figure 3, except that the concentrations were
reduced to 0.3 mM ACh and 30 µM amantadine. B, the peak current
amplitudes of each neuron under all conditions were normalized to the
peak current amplitude at 100 mV in the absence of amantadine. Data
represent the mean ± S.E. from seven neurons. The current-voltage
relationships below 0 mV were used to fit the lines. C, the
relationship between the ratio of peak current amplitude in the
presence of amantadine (30 µM) to the corresponding amplitude in
control solution and the holding potential is shown.
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In addition to the effects of amantadine on peak amplitude illustrated
in figures 1, 2, 3, 4, amantadine appeared to alter the kinetics of the
type IA current. The decay phases of the type IA currents elicited by
ACh (0.3 mM, 1 sec) before and 10 min after changing to the bath
solution containing amantadine (30 µM) (as summarized in figure 4)
were subjected to kinetic analysis. The type IA currents elicited by
ACh (0.3 mM) from six neurons were found to comprise a large (80%),
fast-desensitizing component and a small (20%), slow-desensitizing
component (fig. 5A) (Alkondon and Albuquerque, 1993).
Therefore, the decay phases of control type IA currents (ACh, 0.3 mM)
were each fitted by a double-exponential function. The two decay-time
constants (
values) at each holding potential averaged in the range
of 24 to 33 msec and 254 to 426 msec and were unaffected by changes in
voltage (120 to 20 mV) under the control condition (fig. 5B). For
comparison, the current traces obtained at 60 and 100 mV in
the presence of amantadine (30 µM) were scaled to match the peak
amplitude of the corresponding control traces (fig. 5A). Analysis
revealed that the drug had reduced the decay phase of the type IA
current to a single-exponential function with a of 31 to 36 msec,
which was voltage insensitive (fig. 5B). Furthermore, the decay rate of
ACh-evoked type IA currents in the presence of amantadine was similar
to the fast decay rate of the currents under the control condition.
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Fig. 5.
Effect of amantadine (30 µM) applied
via bath and U-tube on the decay phase of the type IA
current. A 1-sec pulse of ACh (0.3 mM) was applied to the neuron at
each holding potential. At 10 min, after changing to the bath solution
containing amantadine (30 µM), 1-sec pulses of ACh (0.3 mM) plus
amantadine (30 µM) were applied. A, the current traces in the
presence of amantadine (30 µM) at 60 and 100 mV were normalized
to the corresponding control traces. The decay phase of the current in
the presence of amantadine (30 µM) was shorter than that of control.
B, the voltage dependence of the decay phase of the currents is shown. The decay phase of the type IA current induced by ACh (0.3 mM) had a
fast desensitizing component ( ) and a slow desensitizing component
( ). Neither component changed with voltage. In the presence of
amantadine (30 µM), the decay phase of the type IA current induced by
ACh (0.3 mM) contained only the fast desensitizing component ( ), and
its decay-time constant did not depend on voltage. Data represent the
mean ± S.E. from six neurons.
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The effect of pre-exposure to amantadine on resting nAChRs was compared
with the effect of short-pulse exposure of neurons to amantadine.
Because these experiments used only short-pulse applications of
amantadine coapplied with ACh (1 mM) and could be performed in <10
min, a time frame with little rundown of current amplitude without the
use of ATP-RS, the internal pipette solution contained CsF and was
nominally Mg++-free. Under this condition, the inhibitory
effect of amantadine on the type IA current was concentration
dependent, with an IC50 of 130 ± 10 µM and a
nH of 1.1 ± 0.1 (n = 6)
(fig. 6). Whereas the application of 10 µM
amantadine via U-tube alone decreased the peak amplitude of
the type IA current elicited by ACh (1 mM) only to 94 ± 3% of
control (fig. 6), the application of 10 µM amantadine via
bath and U-tube reduced the current amplitude to 39 ± 8% of
control (n = 7) (fig. 1B), and the difference was
highly significant.
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Fig. 6.
Concentration-dependent inhibition of the type IA
current by amantadine applied via U-tube. A, pulses of
ACh (1 mM, 0.5 sec) alone or with amantadine at each concentration were
applied to a single hippocampal neuron at 60-mV holding potential at
1 min after the control pulse. The patch pipette contained the
nominally Mg++-free internal solution without
ATP-generating compounds. B, the peak amplitude induced by ACh (1 mM)
alone was used to normalize the responses of six neurons. The
relationship between the percentage of control peak amplitude of type
IA current and the concentration of amantadine is shown for four
concentrations of amantadine. The symbols represent mean ± S.E.
IC50 and nH values were 130 µM
and 1.07, respectively.
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Effects of amantadine on type II nicotinic currents.
Some
hippocampal neurons respond to ACh application with the slowly decaying
type II nicotinic current, which is sensitive to blockade by DHE
(Alkondon and Albuquerque, 1993). Because type II nicotinic current is
not subject to rundown, these currents were elicited from six neurons
by using patch pipettes filled with nominally Mg++-free
internal solution including CsF. In contrast to type IA currents, which
showed inward rectification with Mg++-containing ATP-RS
(figs. 3B and 4B) but not with the CsF-based, nominally
Mg++-free internal solution (fig. 7A)
(Alkondon and Albuquerque, 1993; Alkondon et al., 1994), the
type II current showed inward rectification at positive potentials with
internal solutions with or without Mg++ (fig. 7B) (Alkondon
and Albuquerque, 1993; Alkondon et al., 1994).
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Fig. 7.
Comparison of control current traces and
current-voltage plots for three types of nicotinic current found in
hippocampal neurons. Sample whole-cell currents illustrate the kinetics
of type IA (A), type II (B) and type III (C) currents. All traces in
each column were from a single hippocampal neuron and were evoked by a
brief pulse of ACh (1 mM).
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Four neurons that responded to pulses of ACh (1 mM, 2 sec)
delivered through the U-tube with type II currents were subjected to
coapplication of amantadine (10 µM) with ACh. Amantadine tended to
decrease the peak amplitude of the type II current (fig.
8), lowering the average peak amplitude to 90 ± 8% of control (n = 4). The decay phase of each current
was fitted to a single-exponential decay function with a baseline
offset, revealing that coapplication of 10 µM amantadine decreased
the decay-time constants to 77 ± 10% of their control values.
Whereas under the control condition the current at the end of the ACh
pulse (1 mM, 2 sec) was 59 ± 6% of peak amplitude, with
coapplication of amantadine and agonist the current amplitude decayed
to 26 ± 9% of the peak amplitude. Thus, amantadine (10 µM) had
greater effects on the decay phase and on the amplitude of the
steady-state current than on the peak amplitude of the type II current.
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Fig. 8.
Inhibitory effect of amantadine, applied
via U-tube, on type II current. Currents were elicited
by pulse application of 1 mM ACh in the control condition, with
amantadine (10 µM) in the U-tube solution and after washing
(recovery).
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Because of the small number of neurons expressing receptors for type II
current, it was not possible to perform complete dose-response or
time-dependent studies. Coapplication of 500 µM amantadine with ACh
reduced the peak response to 50% of control in one neuron. Exposure of
another neuron for 10 min to 10 µM amantadine also decreased the peak
amplitude to about 50% of control. Thus, the effect of amantadine on
type II currents appeared to be concentration and time dependent.
Comparison of the effects of short applications of amantadine on
three types of neuronal nAChR currents.
The effects of a
high concentration of amantadine (500 µM) on type IA, type II
and type III nicotinic currents of hippocampal neurons are compared in
figure 9. Each neuron was held at 60 mV, a nicotinic
current was evoked by a pulse of ACh (1 mM, 0.5-1 sec) and 1 or 2 min
later a pulse of ACh (1 mM) plus amantadine (500 µM) was applied to
the neuron. Only one neuron displaying type II current in the course of
these experiments was treated with this concentration of the drug,
and the frequency of occurrence of type III current was <1%.
Amantadine (500 µM) decreased the peak amplitude of the type IA
current to 20 ± 3% of control (n = 6 neurons),
that of the type II current to 50% of control (n = 1)
and that of the type III current to 15% of control (n = 1) (fig. 9). Normal responses of each type were recovered 2 to 5 min
after exposure of the neurons to amantadine-free external solution
(fig. 9). Thus, amantadine reversibly inhibited each of the three types
of nicotinic current, and the greatest effects on the peak amplitude
were on type IA and type III currents. Amantadine also markedly
shortened the early decay phase of the type II and type III currents.
By fitting these currents with single-exponential decay functions with
offsets, it was found that the decay-time constant of the type II
current was decreased 4-fold, from 263 msec in the control condition to
65 msec in the presence of the drug, and the decay-time constant of the
type III current decreased 2.5-fold, from 260 msec in the control
condition to 107 msec in the presence of amantadine.
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Fig. 9.
Inhibitory effects of amantadine, applied
via U-tube, on three types of neuronal nicotinic
currents. Upper, the three traces were obtained by application to a
single neuron, via a U-tube, of a 0.5-sec pulse of ACh
(1 mM), followed 1 min later by a pulse of ACh plus amantadine (500 µM) and 1 min thereafter by a pulse of ACh (1 mM) alone. The response
to ACh (1 mM) was recovered at 2 min (not shown). Center, type II
current was obtained in another neuron using a 1-sec pulse of ACh
without amantadine and 2 min later a pulse of ACh with amantadine. The
current was recovered at 2 min after the application of amantadine.
Lower, type III current was obtained from another neuron. The 0.5-sec
pulse of ACh plus amantadine was applied to the neuron 10 min after the control, and the response to ACh was recovered 5 min after the application of amantadine. All traces were obtained at 60-mV holding
potential.
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Sensitivity of other ligand-gated ion channels to blockade by
amantadine.
The effects of amantadine (10 and 100 µM) on the
currents evoked by activation of glutamate, GABA and glycine receptors
were evaluated at 60 mV. Amantadine (100 µM) inhibited the
current induced by NMDA (50 µM) plus glycine (10 µM). A 2-sec pulse
of NMDA (50 µM) plus glycine (10 µM) was applied to the hippocampal neuron as control. After 1 min, a pulse of NMDA, glycine and amantadine (100 µM) was delivered to the same cell through the U-tube.
Amantadine, applied via U-tube alone, decreased the peak
amplitude to 62% (n = 14) of control and shortened the
decay phase of this current. After changing to a bath solution
containing amantadine (10 or 100 µM) for 10 min, a pulse of NMDA,
glycine and amantadine (100 µM) was applied. At 10 µM amantadine
the current was not affected significantly, but at 100 µM the current
amplitude was decreased to 63% (n = 5) of control.
Complete recovery was observed 10 min after changing back to the bath
solution without amantadine. There was no significant difference
between the effects of U-tube application and bath-plus-U-tube
application of amantadine on NMDA-evoked currents (fig.
10). This suggests that, for the NMDA receptor, equilibration of amantadine with the closed state causes no change in
the apparent potency of amantadine.
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Fig. 10.
Relative sensitivity of ligand-gated ion channels
to amantadine. At least five independent experiments were performed
with each agonist, and membrane potentials were held at 60 mV. Each bar represents the mean ± S.E of the amplitude in the presence of
amantadine as the percentage of control amplitude. The data for
ACh-evoked currents are summarized here. The agonists kainate, GABA,
glycine and NMDA plus glycine were applied as 2-sec pulses to different
neurons; 10 min after changing to the bath solution containing
amantadine (10 µM), a 2-sec pulse of the same agonist plus amantadine
(10 µM) was applied. Amantadine (10 µM), in the bath and in the
U-tube, had a significant effect on the type IA current induced by ACh.
For the type IA currents, the effects of amantadine (10-100 µM)
applied via a U-tube alone were less than that of
amantadine (100 µM) applied via bath and U-tube. For
the current evoked by NMDA plus glycine, the effect of amantadine (100 µM) applied via a U-tube alone was the same as that of
amantadine (100 µM) applied via bath and U-tube.
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Kainate (50 µM), GABA (50 µM) and glycine (100 µM) were applied
in 2-sec pulses to five hippocampal neurons each. Beginning 10 min
after changing to the bath solution including amantadine (10 µM), a
2-sec pulse of the same agonist plus amantadine (10 µM) was applied
to the neuron. Amantadine (10 µM) did not significantly affect the
currents evoked by kainate, GABA or glycine (fig. 10).
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Discussion |
Our study revealed that amantadine is a potent inhibitor of nAChR
function in hippocampal neurons. Exposure of the neurons to amantadine
for several minutes inhibited type IA currents, which are mediated by
7-bearing nAChRs. The effect of amantadine on the 7 nAChRs was
noncompetitive with respect to the agonist, and the IC50
(6.5 µM) (fig. 1) for inhibition of the type IA current was very
close to the concentrations of amantadine found in the striatal
dialysate of rats whose behavior was altered by treatment with
amantadine and to the concentrations found in serum and cerebrospinal fluid of patients who had received amantadine therapy for Parkinson's disease (Kornhuber et al., 1995). Thus, inhibition of
neuronal nAChRs can be expected to occur during therapy for
Parkinson's disease. Using a different experimental protocol,
amantadine was coapplied with ACh for only a few seconds, the drug was
less potent in reducing the amplitude of the type IA, 7
nAChR-mediated current (IC50 = 130 µM). Short
application also had weak effects on the peak amplitudes of type II,
42 nAChR-mediated current and type III, 34 nAChR-mediated
current, although this treatment accelerated the initial decay and
reduced the amplitude of the later, slowly decaying component of the
-BGT-insensitive type II and type III currents.
Mechanism of type IA nicotinic current blockade by amantadine.
The double-reciprocal plots of the amplitudes of type IA currents
obtained from cultured hippocampal neurons stimulated by ACh in the
absence and presence of amantadine showed that the maximal response to
ACh was decreased but the affinity of the nAChR for ACh was not
affected by amantadine. This result indicates that amantadine does not
compete with ACh for binding to the nAChR that mediates the type IA
current. The mechanism of action of amantadine on this neuronal nAChR,
composed of 7 subunits (Lindstrom, 1995; Palma et al.,
1996), is apparently similar to that on the muscle-type nAChR, where
amantadine (100 µM) did not affect the binding of
[3H]ACh or 125I--BGT to the muscle-type
nAChR of Torpedo electric organs (Tsai et al.,
1978).
The reduction of the peak amplitude of type IA current by amantadine
was voltage dependent, and the drug also shortened the decay phase of
the type IA current. One way to determine whether an open-channel
blocking action of amantadine contributes to the inhibition of the type
IA current, in the manner that it contributes to the inhibition of EPCs
(Albuquerque et al., 1978; Tsai et al., 1978;
Dumbill and Albuquerque, 1987), would be to determine whether amantadine shortens the open time of the 7 nAChR channel. This approach, however, would be very difficult because the 73-pS channels, the most common conductance state subserving type IA current, have a
very brief open time and a fast rate of desensitization under control
conditions (Castro and Albuquerque, 1993) and other subconductance
states of the 7 nAChR, whose open times are longer, occur
infrequently (Pereira et al., 1993; Castro and Albuquerque, 1993). The physiological process accounting for the decay of the type
IA current, i.e., desensitization, is determined by the rate of receptor inactivation (Castro and Albuquerque, 1993) and is voltage
independent under the control condition (fig. 5) (Alkondon and
Albuquerque, 1993). Amantadine did not shorten the initial rate of
decay of the type IA current, irrespective of voltage, but the drug
eliminated the later slow-decay phase of the current (fig. 5). Thus,
this effect of amantadine on the type IA current is apparently caused
not by open-channel blockade but by an increase in the extent of
desensitization, suggesting that amantadine decreases the rate of nAChR
reactivation. Amantadine applied through the U-tube alone was
relatively weak at inhibiting the peak amplitude of the type IA current
(IC50 = 130 µM). An explanation for this finding is that
amantadine also has a rapid action as an open-channel blocker of the
nAChR, as well as of the NMDA receptor (fig. 10). Different
pharmacodynamics and pharmacokinetics of the actions of amantadine at
two different sites on the nAChR for type IA current can explain the
findings that low micromolar concentrations of the drug act slowly and
noncompetitively, with respect to ACh, on the closed-channel
conformation and that the drug rapidly blocks open channels at high
micromolar concentrations.
The possibility that the more potent, slower, noncompetitive action of
amantadine on the nAChR may occur at the allosteric site for agonists
should be considered. Amantadine activates the muscle-type nAChR
(Dumbill and Albuquerque, 1987), and such activation was not blocked by
-BGT (E. F. R. Pereira and E. X. Albuquerque, unpublished
observations), indicating that amantadine activates the nAChR by an
action on the second agonist site (Shaw et al., 1985;
Pereira et al., 1993; Schrattenholz et al.,
1993). In the absence of endogenous agonist, such a weak allosteric
agonist action by amantadine could cooperate with a slowed rate of
reactivation of receptor in the presence of amantadine (suggested
above) to favor the desensitized state of the nAChR, accounting for the more potent depression of peak amplitude elicited by ACh when amantadine is applied to the neuron for a longer time, i.e.,
in the bath.
Comparison of the effects of amantadine on neuronal and muscle-type
nAChRs.
Although amantadine could be applied to relatively fewer
neurons showing the type II and type III currents, compared with many
neurons showing type IA currents, it was clear that the drug inhibited
the three neuronal types of nicotinic currents with different
potencies. The inhibitory potency of amantadine when drug treatment was
limited to coapplication with the agonist, i.e., via U-tube
only, appeared to be greater against the peak amplitude of the type IA
and type III currents than against the peak amplitude of the type II
current. On the other hand, amantadine shortened the decay phase of
type II and III currents substantially, but shortening of the decay
phase of the already rapidly desensitizing type IA current evoked by
ACh (1 mM) was less apparent. The kinetic changes in the type II and
type III currents suggest that amantadine may act on these nAChRs as an
open-channel blocker.
Amantadine also decreases the response of muscle-type nAChRs in frog
sartorius, rat soleus and rat diaphragm muscles (Tsai et
al., 1978). The peak amplitude of the EPC of frog sartorius muscle
at 90 mV is reduced in a concentration-dependent manner by
amantadine, with an IC50 of 64 µM (Warnick et
al., 1982). Furthermore, the binding of
[3H]perhydrohistrionicotoxin to Torpedo
electric organ membranes is inhibited by amantadine
(Ki = 60 µM) (Tsai et al., 1978).
Thus, amantadine is 10-fold less potent against EPCs of
2
nAChRs in muscle than against the type IA
current of 7 nAChRs in the hippocampal neurons (IC50 = 6.5 µM). The mechanisms of actions of amantadine on these
-BGT-sensitive receptors also differ. The effects of amantadine on
EPCs, i.e., nonlinearity in the peak amplitude-voltage
relationship and reversal of the voltage dependence of decay rate with
prolongation at depolarized potentials (Warnick et al.,
1982), suggest that ion-channel blockade of muscle-type nAChRs occurs
at hyperpolarized potentials. The greater potency of amantadine against
the type IA nicotinic current than against the EPC appears to arise
from the strong action on a closed-channel state of the 7 nAChR at
hyperpolarized potentials. To a lesser extent, a weaker action on the
open state of the neuronal nAChRs may explain inhibition of type IA
nicotinic currents at depolarized potentials, and perhaps also the type
II and type III currents.
In addition to the inhibitory effects of amantadine thus far discussed,
amantadine has been shown to augment the response mediated by nAChR
under a different condition. In the rat phrenic nerve-diaphragm
preparation, muscle twitch was potentiated for up to 45 min after the
start of application 150 µM amantadine, and only later was the
response inhibited (Tsai et al., 1978). The possibility that
amantadine may be able to potentiate nicotinic responses in brain
tissue is currently being studied.
Effects of amantadine on other ligand-gated ion channels in
hippocampal neurons.
In comparison with its effects on nAChRs in
the cultured hippocampal neurons, amantadine at 100 µM was a weak
inhibitor (60% of control current) of the NMDA-induced currents, and
amantadine at 10 µM was ineffective against NMDA-, kainate-, GABA- or
glycine-induced currents. Thus, the lower concentration of amantadine
selectively inhibited the nicotinic currents, leaving other
ligand-gated channels of hippocampal neurons relatively unaffected.
Therapeutic mechanisms of amantadine.
The pathology of
Parkinson's disease has often been attributed to an imbalance of
dopaminergic and cholinergic transmission. Recent studies of the
caudate nucleus in vitro have shown mechanisms by which
amantadine may increase the dopamine/ACh ratio in treatment of
Parkinson's disease. Amantadine above 50 µM selectively increases the release of dopamine by a direct action on storage granules, without
stimulating ACh release or changing electrically evoked dopamine or ACh
release (Jackisch et al., 1992). In slices of caudate
nucleus, lower concentrations of amantadine inhibited by a
noncompetitive mechanism the selective release of ACh evoked by NMDA
(IC50 = 30 µM amantadine) (Stoof et al., 1992)
or L-glutamate (IC50 1 µM amantadine)
(Lupp et al., 1992). Furthermore, amantadine at 50 µM and
its analog memantine at 6 µM, both levels above their respective
therapeutic concentrations, protected neurons in vitro against NMDA-receptor mediated toxicity. Memantine appeared to be a
potent uncompetitive antagonist of NMDA receptors in the retinal
ganglion (Chen et al., 1992), suggesting that this
pharmacokinetic property may account for the protection offered by the
drug against glutamate toxicity (Chen et al., 1992). The
role of nicotinic transmission in nigrostriatal function is now being
explored. Nicotine increases the release of dopamine in striatal tissue (Rapier et al., 1990) and enhances the excitability of
nigrostriatal axons in association with glutamate receptor function
(Garcia-Munoz et al., 1996). Therefore, nicotinic receptors
in the nigrostriatal pathway have the potential to affect the symptoms
of Parkinson's disease, and the effectiveness of amantadine as a
noncompetitive inhibitor of neuronal nicotinic receptors shown in our
study should be considered in evaluation of the therapeutic actions of
amantadine.
In hippocampal neurons, amantadine at therapeutic concentrations was a
potent antagonist of type IA current, which is most likely mediated by
nAChRs composed of 7 subunits (Alkondon and Albuquerque, 1995). The
biological role of the 7 nAChRs that mediate the rapidly decaying
type IA currents in the hippocampus (Clarke et al., 1985;
Séguéla et al., 1993) has been investigated in
neurons grown in culture (Alkondon and Albuquerque, 1993), dissociated
acutely (Ishihara et al., 1995; Barbosa et al.,
1996) and visualized in tissue slices (Gray et al., 1996;
Alkondon et al., 1996b). These electrophysiological studies
have located 7 nAChRs in presynaptic and postsynaptic sites on
hippocampal and olfactory bulb neurons. Whereas the specific role of
postsynaptic nAChRs is still poorly understood, presynaptic nAChRs can
modulate the release of GABA and glutamate from mammalian hippocampal
neurons (in slices, culture and acutely dissociated tissue) and from
olfactory bulb neurons (Alkondon et al., 1996a,b; Gray
et al., 1996; Albuquerque et al., 1997). Because
of the high Ca++ permeability of 7 nAChRs (Castro and
Albuquerque, 1995), amantadine would reduce Ca++ entry into
neurons during excess cholinergic transmission, and the previously
unrecognized antinicotinic effects of amantadine in the brain may have
protection effects in neurodegenerative disorders or cholinergic
toxicity. In contrast, the NMDA receptor activity in the hippocampus,
as well as the superior colliculus, was relatively unaffected by
therapeutic concentrations of amantadine (IC50 > 100 µM)
(present study; Parsons et al., 1995), a difference from the
effective antagonism of NMDA receptor activity in the caudate nucleus
(Stoof et al., 1992) that may be due to the specific subtypes of NMDA receptors in these tissues. Although other
pharmacological probes, such as MLA, -BGT or -conotoxin-ImI
(Pereira et al., 1996), have been identified as highly
selective nAChR antagonists in the hippocampus, amantadine combines
relative selectivity against nAChRs with clinical usefulness and proven
safety.
The authors are grateful to Barbara Marrow and Mabel Zelle for
expert technical assistance.
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Abstract
Introduction
![<IT>I=I</IT><SUB>max</SUB><FENCE><IT>1−</IT><FR><NU>[amantadine]<SUP><IT>n</IT><SUB><IT>H</IT></SUB></SUP></NU><DE>[amantadine]<SUP><IT>n</IT><SUB><IT>H</IT></SUB></SUP><IT>+</IT>IC<SUP><IT>n</IT><SUB><IT>H</IT></SUB></SUP><SUB><IT>50</IT></SUB></DE></FR></FENCE>](/content/vol281/issue2/fulltext/834/img001.gif)
