Institute for Biological Sciences, National Research Council of
Canada, Ottawa, Ontario, Canada,
 |
Introduction |
There
are several sites on the NMDA receptor
complex at which antagonists can interact (for a review, see Harris,
1995
). Interaction at different binding sites, and by different
mechanisms of action, might account for the diverse clinical properties
of NMDA receptor antagonists and present potential targets for the
development of new pharmaceutical agents. Uncompetitive NMDA receptor
antagonists acting at the phencyclidine site produce a block only after
the channel enters its open state after activation (Huettner and Bean, 1988), although this use-dependent blockade does not necessarily imply
occlusion of the open pore or preferential binding to the open state
(Orser et al., 1997). Use-dependent antagonism could be advantageous in
treating disease states where excessive activation of NMDA receptors
occurs, such as cerebral ischemia or epilepsy, because it allows for
preferential block of excessive activation over normal synaptic
activity. Consistent with this idea, both MK-801 and phencyclidine,
which bind with high affinity to a site within the channel pore,
exhibit neuroprotective effectiveness in various models of focal and
global ischemia (Kemp et al., 1987; Olney et al., 1989). However, their
therapeutic potential is negated by their serious neurobehavioral side
effects. Low-affinity, use-dependent NMDA receptor antagonists may have
reduced toxicities because they reach a steady-state block more rapidly
due to their rapid on-off kinetics (Rogawski, 1993), thus preventing
significant calcium entry before equilibrium is reached without
producing a supramaximal blockade. It has been suggested that the
low-affinity, use-dependent NMDA antagonists, memantine (Muller et al.,
1995; Parsons et al., 1995), amantadine (Parsons et al., 1995), and ADCI (Rogawski et al., 1991, 1995), lack serious side effects due to
their relatively rapid kinetics (Chen et al., 1992; Parsons et al.,
1993, 1995). However, a comparison of a series of uncompetitive NMDA
receptor antagonists for efficacy as antiepileptic drugs, using
electroshock in mice, demonstrated that some less potent antagonists
actually had worse therapeutic indices (Parsons et al., 1995).
Furthermore, although ADCI and ketamine are both low-affinity, use-dependent NMDA receptor antagonists, ketamine induces
psychotomimetic effects (Ginski and Witkin, 1994; Krystal et al., 1994)
despite substantially faster on-off kinetics (Parsons et al., 1995,
1996).
Recently, another low-affinity, use-dependent NMDA receptor antagonist,
AR-R15896AR, was developed. It blocks NMDA-induced toxicity in primary
cultures of cortical neurons and, at neuroprotective concentrations,
rapidly decreases the NMDA-induced calcium influx and subsequent rise
in intracellular calcium (Black et al., 1995). Electrophysiological
studies have shown it to be a use- and voltage-dependent blocker with
rapid kinetics (Mealing et al., 1997). AR-R15896AR also exhibits
neuroprotection in rodent models of global and focal ischemia and has a
favorable pharmacokinetic profile (Cregan et al., 1997; Palmer et al.,
1997). It is presently in phase IIa clinical trials with stoke patients.
The use-dependent antagonists MK-801, ADCI, memantine, amantadine, and
AR-R15896AR all exhibit some degree of trapping (Jones and Rogawski,
1992; Blanpied et al., 1997; Chen and Lipton, 1997; Mealing et al.,
1997). Trapping channel blockers permit agonist dissociation and
channel closure while the antagonist is bound to its site in the
channel, whereas sequential blockers prevent the channel from closing
while blocked. Blanpied et al. (1997) suggested that some drugs may
show a combination of these effects. With MK-801 and ADCI, trapping
appears to be complete, but in the case of memantine, about one sixth
of the blocked channels release, rather than trap, the blocker
(Blanpied et al., 1997). This partial trapping could not be attributed
to different mechanisms of action of memantine on a heterogeneous
population of NMDA receptor subtypes. In pathophysiological situations
where there is repetitive NMDA receptor stimulation, trapping could
potentially result in an undesirable accumulation of antagonist,
producing a supramaximal blockade. The extent of this accumulation of
block would be interdependent on the degree of trapping, on-off
kinetics, and the frequency of repetitive stimulation. Here, we
investigated the trapping characteristics of three low-affinity,
use-dependent NMDA receptor antagonists: AR-R15896AR, ketamine, and
memantine. These antagonists have similar intermediate blocking
kinetics and strong voltage dependence, yet ketamine and memantine are
examples of compounds that exhibit a small and large therapeutic index,
respectively. The purpose of this study was to determine whether
differences in the degree of trapping exist among use-dependent NMDA
antagonists with similar kinetics and, if so, whether they correlate
with differences in therapeutic potential.
| |
Materials and Methods |
Chemicals and Reagents.
PBS, HEPES, MEM,
poly-L-lysine, tetrodotoxin, 9-aminoacridine, and
strychnine hydrochloride were purchased from Sigma Chemical (St. Louis,
MO). Heat-inactivated fetal bovine serum was purchased from GIBCO
Laboratories (Grand Island, NY), and heat-inactivated horse serum was
from Hyclone Laboratories (Logan, UT). NMDA, AP-5, and ketamine were
purchased from Research Biochemicals International (Natick, MA).
Memantine was purchased from Tocris Cookson (St. Louis, MO). EGTA was
purchased from Fluka Biochemika (Ronkonkoma, NY). AR-R15896AR was
provided by Astra Arcus USA (Rochester, NY).
Cell Culture.
Rat cortical neurons isolated from E18 fetuses
were grown in primary culture as described previously (Black et al.,
1995). Briefly, timed-pregnant Sprague-Dawley rats were purchased from Charles River Canada (St. Constant, Quebec, Canada). After the mother
was killed through cervical dislocation while under halothane anesthesia, the fetuses were removed from the uterus on day E18; their
brains were removed and placed in ice-cold PBS; and the cortices were
isolated. The cortical neurons were dispersed by trituration with a
10-ml pipette, and the cells were centrifuged at 250g
for 5 min at 4°C. The cells were gently resuspended in plating
medium, and viable cells, as determined by trypan blue exclusion, were
counted. The cells then were plated at 105
cells/cm2 on poly-L-lysine-coated 35-mm
culture dishes (Nunc; Roskilde, Denmark) in 2 ml of plating medium at
37°C in an atmosphere of 5% CO2/95% air. Plating medium
consisted of 80% MEM, with 20 mM glucose, 10% heat-inactivated fetal
bovine serum, and 10% heat-inactivated horse serum containing 2 mM
L-glutamine. Because the cultures contained both neurons
and glial cells, they were treated with 15 mg/ml
5-fluoro-2'-deoxyuridine and 35 mg/ml on day 5 to minimize glial cell
proliferation. On day 6, half of the medium was removed and replaced
with growth medium consisting of 90% MEM and 10% horse serum.
Experiments were performed on cultures after 14 to 19 days in vitro.
Whole-Cell Recording.
Cortical neurons grown on 35-mm
culture dishes were mounted on the stage of an inverted Zeiss
microscope equipped with Hoffman Modulation optics in a perfusion
system flowing continuously at 1 ml/min at 22°C. The bathing
solution contained (in mM): 140 NaCl, 5 KCl, 1 CaCl2, 10 HEPES, and 3 glucose, pH 7.4. Bathing solutions also contained 1 µM
tetrodotoxin to block Na+ currents, 10 µM glycine to
saturate the glycine site on NMDA receptors, and 1 µM strychnine to
block glycine-activated Cl
currents.
Patch pipettes (2-4 M
resistance) were constructed from 1.5-mm
outer diameter/1.0-mm inner diameter Pyrex 7740 glass (Corning, Big Flats, MN) using a Brown-Flaming P80 micropipette puller
(Sutter Instruments, San Francisco, CA). The pipette solution contained (in mM) 140 CsCl, 1.1 EGTA, 10 HEPES, and 2 Mg-ATP, pH 7.2.
Whole-cell currents were acquired using an Axopatch 1-D amplifier
equipped with a CV-4 headstage with a 1 G feedback resistor (Axon
Instruments, Foster City, CA). Voltage command and current acquisition
were accomplished using a personal computer equipped with a Digidata
1200 interface and pClamp 6.0 software (Axon). Data were filtered at 1 kHz and sampled at 2 kHz.
Rapid agonist or agonist-antagonist application was accomplished using
a modified DAD-12 perfusion system (ALA Scientific Instruments,
Westbury, NY). The system consisted of a custom-made manifold of eight
100-µm-diameter quartz tubes that converged into a common 100-µm
tip with minimal dead volume. The tubes were fed from pressurized
reservoirs equipped with miniature switching valves controlled by a
computer, such that solution flowed from only a single reservoir at one
time. The tip of the manifold was placed <100 µm from the
patch-clamped cell under study. Solutions were degassed before use, and
reservoirs were pressurized at 200 to 400 mm Hg. Switching between
solutions took 12 ± 1 ms (n = 12), as determined by junction
potential measurements using a 10% Cl solution
in one reservoir and normal Cl in the others.
However, whole-cell current responses to agonist were slower, such that
maximum agonist-induced current developed at 200 ± 25 ms. All
drugs were prepared fresh daily.
Data Analysis.
The block of NMDA-evoked currents was
calculated according to the formula:
![B=[(I−I<SUB><UP>B</UP></SUB>)/I]×100](/content/vol288/issue1/fulltext/204/img001.gif)
|
(1)
|
where I was determined by curve fitting the decay of
the NMDA-evoked current during the NMDA application and extrapolating the fit to the end of the NMDA/antagonist coapplication, and
IB was the current measured at the end of
NMDA/blocker coapplication. Current decays were fit to first-order
exponential curves using a Chebyshev fit method and pClamp software.
The residual block of NMDA-evoked currents was calculated according to
the formula:
![B<SUB><UP>R</UP></SUB>=[(I<SUB><UP>1st</UP></SUB>−I<SUB><UP>2nd</UP></SUB>)/I<SUB><UP>1st</UP></SUB>]×100](/content/vol288/issue1/fulltext/204/img002.gif)
|
(2)
|
where I1st was the maximal current
measured 200 ± 25 ms after onset of the first NMDA exposure, and
I2nd was the maximal current measured
200 ± 25 ms after onset of the delayed second NMDA exposure after
washout of blocker from the bath. The delay between onset of agonist
application and current measurement was to allow development of maximal
I1st and to avoid possible corruption of
data measurement by artifacts.
The block trapped, or the amount of block remaining at the beginning of
the second NMDA application as a percentage of the initial block
produced at the end of the previous NMDA/antagonist coapplication, was
calculated according to the formula:
|
(3)
|
where B and BR are
defined in eqs. 1 and 2.
Data are expressed as mean ± S.E.M., where n is the number of
cells. Statistical comparisons were made using an unpaired Student's t test. Statistical significance was inferred at
p < .05.
| |
Results |
Onset and Relief of Antagonism.
The kinetic properties of
AR-R15896AR, ketamine, and memantine have been previously reported and,
as can be seen from Table 1, are quite
similar. However, there is considerable variability among some of this
data, possibly due to differences in cell preparations and experimental
conditions. For example, the k1 reported for ketamine varies from 0.075 to 0.21 s1. Therefore,
before designing an agonist/antagonist exposure protocol to study
trapping, we examined the development and relief of block by these NMDA
antagonists under comparable experimental conditions.
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TABLE 1
Kinetic properties of NMDA receptor antagonists
k1 and k1 were determined from
macroscopic current measurements.
|
|
Neurons were voltage-clamped at 
60 mV and 5-s applications of 10 µM
NMDA were repeated at 30-s intervals until currents were stable. NMDA
was then applied for 2 to 3 s followed by a 30-s coapplication of
NMDA/antagonist and a second extended NMDA application as shown in Fig.
1. The fractional block (B) of
NMDA-evoked currents was calculated using eq. 1, and the curve fit of
the decay of the initial NMDA-induced current extrapolated to the end
of the trace is shown as a dashed line. The validity of using this
method to estimate block has been demonstrated previously (Mealing et al., 1997). AR-R15896AR (50 µM) produced an 81 ± 1% (n = 4) block, as measured at the end of the 30-s coapplication, whereas 10 µM memantine and 10 µM ketamine produced 83 ± 4% (n = 5) and 87 ± 1% (n = 4) blocks, respectively. The
development and relief from block after addition or removal of
antagonist were fit to first-order exponential curves, which are shown
as solid lines superimposed on the data traces in Fig. 1. We did not
attempt to fit the initial (200 ms), development, or relief of block,
because an artifact of perfusion switching was frequently present
during this time, possibly masking a rapid second exponential process.
These transient relaxations in the current, which can be observed at
application or removal of agonist, were due to increased compliance in
the perfusion system as a result of outgassing in the perfusion line. Block and relief from block by AR-R15896AR (Fig. 1, top trace) had a
on of 2.4 s and a
off of 2.8 s, whereas 10 µM memantine (Fig. 1, middle trace) had a on of 3.5 s
and a off of 9.8 s, and 10 µM ketamine
(Fig. 1, bottom trace) had a on of 5.2 s
and a off of 10.5 s. Although the onset
and relief of block were fastest for AR-R15896AR and slowest for
ketamine, all three compounds produced a similar stable block after
30 s.
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Fig. 1.
Onset and relief of block of NMDA-induced whole-cell
currents. NMDA (10 µM) was applied for 2 to 3 s followed by a
30-s coapplication of NMDA/antagonist and a second extended NMDA
application. Neurons were voltage-clamped at 60 mV. Top trace, 50 µM AR-R15986AR. Middle trace, 10 µM memantine. Bottom trace, 10 µM ketamine. Dashed lines indicate the curve fit of the decay of the
initial NMDA-induced current and the extrapolation of this fit to the
end of the trace. Dotted lines indicate the resting current level.
Solid lines superimposed on the data indicate the first-order
exponential curve fits for onset and relief from block. Light shaded
bar indicates NMDA application. Dark shaded bar indicates antagonist
application. Scale bar, 100 pA, 10 s.
|
|
Degree of Trapping.
To study trapping, we used a low agonist
concentration (10 µM NMDA) and sufficiently high concentrations of
each antagonist to produce a near-maximal block, thus optimizing the
opportunity for trapping by minimizing the proportion of blocked
receptors that were liganded at steady state (Blanpied et al., 1997). A 3-s NMDA application immediately followed by an NMDA/antagonist coapplication 2 to 60 s in duration was followed 120 s later
by a second 20-s NMDA application. During the washout period, 80 µM
AP-5 was included in the perfusate to prevent possible "occult" channel opening due to NMDA contamination but was removed 1 s before NMDA application to permit its dissociation from the
ligand-binding site. The difference in degree of trapping by
AR-R15896AR 120 s after 2-, 10-, and 60-s coapplications with NMDA
is shown in Fig. 2. Transient artifacts
similar to those in Fig. 1 were observed on coapplication of
NMDA/antagonist that were temporally distant from the regions in the
recordings important to determination of IB,
I1st, or I2nd. We
rejected all data that contained such perfusion artifacts during
application of NMDA, where I1st, or
I2nd were measured. Trapping by 50 µM AR-R15896AR, 10 µM ketamine, and 10 µM memantine 120 s after a 30-s coapplication are shown in Fig. 3.
The residual block that remained trapped 120 s after washout was
calculated according to eq. 2 by comparing the maximal current during
the first 200 ± 25 ms of the first NMDA exposure with that of the
second NMDA exposure. The fractional block trapped was then determined
according to eq. 3. The increase in degree of trapping as a result of
extending the duration of agonist/antagonist coapplication is plotted
in Fig. 4A. The development of trapping
with all three NMDA receptor antagonists could be fit to single
exponential curves with s for AR-R15896AR, ketamine, and memantine
of 3.0, 6.0, and 3.5 s, respectively. In all cases, there was no
significant (p > .05) increase in trapping by
extending the agonist/antagonist coapplication duration beyond 30 s. The blocks caused by a 30-s coapplication of 50 µM AR-R15896AR, 10 µM ketamine, or 10 µM memantine were not significantly different,
being 82 ± 1% (n = 5), 80 ± 2% (n = 5), and
81 ± 2% (n = 7), respectively. However, after 120 s of
washout, there were significant differences in the degree of trapping
between each of the antagonists, as 54 ± 3% of the AR-R15896AR block, 86 ± 1% of the ketamine block, and 71 ± 4% of the
memantine block remained trapped (Fig. 4B). Trapping was also examined
using higher antagonist concentrations. Significantly
(p < .05) higher initial blocks were produced by
100 µM AR-R15896AR, 20 µM ketamine, and 20 µM memantine, being
88 ± 2% (n = 3), 96 ± 1% (n = 3), and 92 ± 1% (n = 5), respectively. However, with all three compounds, the percentage of block trapped was not significantly different from
that measured at the lower antagonist concentrations, being 57 ± 5%, 87 ± 5%, and 76 ± 3%, respectively. The 76 ± 3% trapping caused by a 30-s coapplication of 20 µM memantine agrees
well with the 19 ± 2% release from block reported by Blanpied et
al. (1997) using a 60-s coapplication and a holding potential of 67 mV. Trapping was not investigated at lower antagonist concentrations because the magnitude of change in current amplitude was too small to
resolve.
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Fig. 2.
Trapping by 50 µM AR-R15896AR after 2-, 10-, and
60-s coapplications with 10 µM NMDA. A 3-s NMDA application
immediately followed by an NMDA/AR-R15896AR coapplication 2 s (top
left trace), 10 s (middle left trace), or 60 s in duration
(bottom left trace) was followed 120 s later by a second NMDA
application 20 s in duration (middle traces). The left and middle
traces are superimposed to show the difference in initial current
amplitude between the first and second NMDA exposures (indicated by
arrows) after 2-s (top right trace), 10-s (middle right trace), or 60-s
(bottom right trace) exposure to AR-R15896AR. During the washout
period, 80 µM AP-5 was included in the perfusate but was removed
1 s before NMDA application. Light shaded bar indicates NMDA
application. Dark shaded bar indicates antagonist application. Dashed
lines indicate curve fit of current decay during the initial NMDA
application extrapolated to the end of the NMDA/antagonist
coapplication. Scale bar, 100 pA, 10 s.
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Fig. 3.
Degree of trapping exhibited by several NMDA
antagonists 120 s after an extended agonist/antagonist
coapplication. The first NMDA/antagonist coapplication is
superimposed over a second NMDA application 120 s later to show
the difference in initial current amplitude (indicated by arrows).
During the washout period, 80 µM AP-5 was included in the perfusate
but was removed 1 s before NMDA application. Top left trace, 20-s
application of 50 µM 9-AA. Top right trace, 30-s application of 50 µM AR-R15896AR. Bottom left trace, 30-s application of 10 µM
memantine. Bottom right trace, 30-s application of 10 µM ketamine.
Dashed lines indicate curve fit of current decay during the initial
NMDA application extrapolated to the end of the NMDA/antagonist
coapplication. Scale bar, 50 pA, 5 s.
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Fig. 4.
A, The increase in degree of trapping as a result of
extending agonist/antagonist coapplication duration. With all three
NMDA antagonists, the development of trapping was fit to single
exponential curves. The s for AR-R15896AR ( ), ketamine ( ), and
memantine ( ) were 3.0, 6.0, and 3.5 s, respectively. In all
cases, there was no significant (p > .05) increase
in trapping by extending the agonist/antagonist coapplication duration
beyond 30 s. Data points are the mean ± S.E.M. of
observations from three to nine cells. B, The block caused by a 30-s
coapplication of 50 µM AR-R15896AR, 10 µM ketamine, or 10 µM
memantine was not significantly different, being 82 ± 1% (n = 5), 80 ± 2% (n = 5), and 81 ± 2% (n = 7),
respectively. However, after 120 s of washout, there were
significant differences in the degree of trapping between each of the
antagonists, as only 54 ± 3% of the AR-R15896AR block, 86 ± 1% of the ketamine block, and 71 ± 4% of the memantine block
remained trapped. Significantly (p < .05) higher
initial blocks were produced by 100 µM AR-R15896AR, 20 µM ketamine,
and 20 µM memantine, being 88 ± 2% (n = 3), 96 ± 1% (n = 3), and 92 ± 1% (n = 5), respectively.
However, the percentage of block trapped was not significantly
different from that measured at the lower antagonist concentrations,
being 57 ± 5%, 87 ± 5%, and 76 ± 3%,
respectively.
|
|
To ensure that we could also demonstrate the absence of trapping using
our protocol, we tested the sequential NMDA receptor antagonist 9-AA
(Fig. 3). In contrast to AR-R15896AR, ketamine, or memantine, there was
a complete lack of trapping and large inward tail currents were
observed immediately after cessation of agonist/antagonist
coapplication. These observations are consistent with a sequential
mechanism of block because before dissociation of agonist, channels
that are in the blocked state must first return to a closed state via
an open state (Benveniste and Mayer, 1995).
| |
Discussion |
Trapping channel blockers permit agonist dissociation and channel
closure when the antagonist is bound to its site in the channel,
whereas sequential blockers prevent the channel from closing while
blocked. Sequential and trapping channel blockers differ in the
dependence of their block on agonist concentration. Although the
mechanisms underlying trapping are not well understood, conformational
changes in gating could sterically prevent movement of a trapping
channel blocker out of the channel or, on the other hand, the binding
of a sequential blocker might prevent movement of a channel's gate.
In this comparative study, we examined NMDA receptor antagonist
trapping. Three low-affinity, use- and voltage-dependent antagonists were chosen whose kinetics were sufficiently similar that, at the
concentrations used, they produced an equivalent, near-maximal, steady-state block within 30 s, which could be completely relieved within 50 s of exposure to NMDA. We report that despite the
similarities of the initial block produced by these compounds, ketamine
exhibited significantly more trapping than memantine or AR-R15896AR and that memantine exhibited significantly more trapping than AR-R15896AR.
The 76 ± 3% trapping observed using 20 µM memantine agrees
with the 19 ± 2% release from block reported by Blanpied et al. (1997), although we found that a 30-s, rather than a 60-s,
agonist/antagonist coapplication duration was sufficient to produce a
steady-state block. We also demonstrated an absence of trapping using
the sequential NMDA-receptor antagonist 9-AA. The large inward tail
currents observed immediately after cessation of NMDA/9-AA
coapplication are also consistent with a sequential mechanism of block
(Benveniste and Mayer, 1995). Together, the confirmation of comparable
trapping values to those published for memantine and the absence of
trapping by 9-AA verify our ability to measure trapping over a broad
spectrum. We did not measure currents (I1st
and I2nd) until 200 ± 25 ms after
agonist application due to the technical limitations mentioned previously. During the delay before the measurement of
I2nd, some degree of untrapping probably
occurred, which may have affected the accuracy of our trapping
measurements. However, the off rates for all three antagonists are slow
by comparison with the brief delay in current measurement. There was
very little relief from block within 200 ms for any of the antagonists,
as is shown in Fig. 1, so the error in determining trapping of block
due to unblock during this short time delay in current measurement was negligible.
All three NMDA receptor antagonists show use- and voltage-dependent
antagonism, suggesting activity at a site within the channel pore.
Trapping was not complete with any of these antagonists. This is not
consistent with linear models of trapping block (Lingle, 1983; Blanpied
et al., 1997) but can occur in cyclized models of trapping block
(Gurney and Rang, 1984; Benveniste and Mayer, 1995). Consider the
following model, adapted from Gurney and Rang (1984):
where Ag is agonist, B is blocker, C is closed channel, O is open
channel, OB is open-blocked channel, CB is closed-blocked channel,
l± and k± are the rate constants between
transition states, and 
B is rate of agonist
unbinding from an open-blocked channel.
For a sequential block to occur, k > 0, B = 0, and l = 0. For a trapping
block, k > 0, B > 0, and l = 0. For a nonsequential block, k > 0, B > 0, and l > 0. In this model, it was assumed that the rate constant l
for the escape from the closed channel is very small. However, if this
rate constant is much larger than zero, then closed-channel egress
occurs. It has also been suggested that some blockers may exhibit a
combination of trapping and sequential block (Blanpied et al., 1997).
This could account for the differences in trapping observed in the
present study. However, the appearance of tail currents with 9-AA, but
not with AR-R15896AR, memantine, or ketamine, does not favor a
sequential mechanism of block by these latter compounds. In this
regard, the cyclic models imply that channel closure and occlusion
of blocker are separable events. It then is possible to envision that a
blocker may retard or escape the conformational relaxation that could
result in trapping while still allowing channel closure to occur. As
pointed out by Benveniste and Mayer (1995), it may be that trapping and
sequential channel block are opposite ends of a spectrum rather than
two fundamentally different mechanisms. The lipophilic nature of many
open-channel blockers can result in the blocker leaking from the
antagonist site when the channel is closed, resulting in variable
degrees of trapping (Chen and Lipton, 1997). However, the rank order of degree of trapping, ketamine > memantine > AR-R15896AR,
does not correlate with the compound lipophilicity [c-logP values for
ketamine, memantine, and AR-R15896AR of 2.15, 3.18, and 1.63, respectively (R. Murray, personal communication)], suggesting that
variation in diffusion is an inadequate explanation for differences in trapping.
Activity at other binding sites on the NMDA receptor also varies with
antagonist. For example, memantine antagonism is not affected by
glycine, suggesting that there is no interaction with the
strychnine-insensitive glycine modulatory site (Parsons et al., 1993),
whereas AR-R15896AR does show a potentially competitive interaction at
the glycine modulatory site (Mealing et al., 1997). Ketamine inhibits
the NMDA receptor by an open-channel block and by closed-channel block
from the membrane phase (Orser et al., 1997). Therefore, the
differences in trapping that we observed may not be entirely due to
differences in antagonist action within the channel pore; there may
also be a minor contribution from block at other sites on the NMDA
receptor complex. In the case of AR-R15896AR, which shows the least
trapping, at 60 mV, the contribution to the block due to antagonism
at the glycine site is very small (Mealing et al., 1997).
Among the three low-affinity, use-dependent NMDA-receptor antagonists
tested in this study, a correlation can be drawn between degree of
trapping and therapeutic safety margin. Ketamine, which has been
reported to induce psychotomimetic effects (Ginski and Witkin, 1994;
Krystal et al., 1994), showed 86 ± 1% trapping, whereas
memantine and AR-R15896AR, which have improved therapeutic safety
profiles (Parsons et al., 1995; Palmer et al., 1996, 1997), trapped
71 ± 4% and 54 ± 3% of their initial block, respectively. This significant reduction in trapping, from ketamine to memantine to
AR-R15896AR, is the same order as that for reduction of severity of
side effects as measured with the Inverted Screen Test or Delayed Matching to Sample Test, which provide an indication of neuromuscular or memory impairment, respectively. For example, therapeutic ratios (ED50 for neuromuscular impairment divided by the
ED50 for protection against maximal electroshock)
for ketamine, memantine, and AR-R15896AR of 2.28, 3.28, and 8.55, respectively, have been observed (C. Crammer, personal communication).
Therefore, use-dependent NMDA receptor antagonists that exhibit less
trapping may provide the safest compounds for therapeutic use in
disease states where repetitive NMDA receptor activation could
potentially lead to an undesirable supramaximal accumulation of block.
It remains to be demonstrated, however, that the differences in
trapping observed in this study result in differences in the
accumulation of block during a repetitive NMDA receptor stimulation
protocol that mimics a pathophysiological situation.
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Abstract
Introduction
-aminoethyl
ether)-N,N,N',N'-tetraacetic
acid;
HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid;
MEM, Eagle's minimal essential medium;
MK-801, dizocilpine;
PBS, Dulbecco's phosphate-buffered saline.
