| Summary: | The NMDA (N-methyl-D-aspartate) subtype of the receptors for the excitatory
amino acid L-glutamate has been implicated as a mediator of anoxic neuronal death
following periods of cerebrovascular ischaemia. Laboratory observations have
implicated it in neuronal death occurring in other situations, including hypoglycemia
and neurodegenerative disorders. Inhibiting the NMDA receptor could, therefore,
represent a useful therapeutic intervention where CNS damage is a potential outcome
resulting from conditions such as those given above. However, since NMDA receptors
are involved in a number of vital CNS functions, such as synaptic transmission, simple
all-or-none antagonism would present an unacceptable risk of toxicity and side effects.
Determination of specific characteristics of a given agent's interaction with the NMDA
receptor-ion channel complex would aid considerably in assessing that agent's clinical
utility as a safe and efficacious neuroprotectant.
The ion channel associated with the NMDA receptor is the target of a number of
uncompetitive NMDA antagonists. Four such agents have been chosen for study: (-)-
and (+)-β-cyclazocine are members of the benzomorphan class of compounds, several
others of which are known to act as NMDA antagonists; dextromethorphan (DM) is an
antitussive morphinan closely related in chemical structure to the benzomorphans; and
L-687,384, which is, like a number of other uncompetitive NMDA antagonists, a ligand
for sigma receptors. These agents were tested for their actions against responses to
NMDA. As an estimation of the potential for producing side effects, (-)-β-cyclazocine
and DM were also studied for their effects on responses to the non-NMDA glutamate
receptor agonists kainate and ⍺-amino-2,3-dihydro-5-methyl-3-oxo-4-isoxazole propanoic acid (AMPA), as well as for their actions against voltage-gated Ca²⁺, Na⁺,
and K⁺ currents. Finally, (-)-β-cyclazocine was tested in a cell viability assay to confirm
its efficacy as a neuroprotective agent.
Unitary currents through single NMDA-activated ion channels were studied using
outside-out patches isolated from cultured rat hippocampal or cortical neurons.
Currents recorded in the presence of NMDA were compared to those recorded
following the addition of one of the agents. None of the compounds studied altered the
amplitudes of the unitary currents activated by NMDA and hence did not affect the
channel conductance. The compound (-)-β-cyclazocine concentration-dependently
reduced the open state probability (P₀) of NMDA ion channels with IC₅₀ values of 84 nM
and 680 nM in hippocampal and cortical neurons, respectively. The reduction in P0 by
(-)-β-cyclazocine was attributable to decreases in mean channel .open time and mean
channel opening frequency. In hippocampal neurons, (+)-β-cyclazocine was 170x less
potent in reducing P₀ than the (-)-enantiomer (IC₅₀ = 14 pM for (+)-β-cyclazocine). DM
also reduced P0 , with IC₅₀ values of 4.4 μM and 3.8 βM in hippocampal and cortical
neurons, respectively. Again, decreases in mean open time and mean frequency were
associated with the reduction in P0 by DM. L-687,384 decreased the mean open time
of NMDA-activated unitary currents, but did not diminish their frequency.
Consequently, the action of L-687-384 against P0 was relatively weak (IC₅₀ = 61 uM).
The reductions in mean open time and mean frequency by (-)-β-cyclazocine and
DM, without effects on channel conductance, were consistent with an open-channel
block model for inhibition of NMDA activity. Analysis of the results assuming an openchannel
blockade model gave estimations of the on-rate constants (k₂) and off-rate constants (k.2) for the interactions of the compounds with NMDA ion channels in
hippocampal and cortical neurons. The k₂ values in both neuronal types were similar
for (-)-β-cyclazocine, DM, and L-687,384, all being near 10⁷ M⁻¹V⁻¹. The closeness of
the k₂ values suggests that the on-rate for a given open channel blocker is not a
determinant of its potency as an NMDA activity inhibitor. The frequency reductions by
(-)-β-cyclazocine and DM allowed for estimations of k₂, an apparent unblocking, or
"off, rate constant, from the single-channel data; this was 2.5 - 5.0 s⁻¹ and 2.5 s⁻¹ for (-)-
β-cyclazocine in hippocampal and cortical neurons, respectively, and 10 - 13 s⁻¹ for DM
in hippocampal neurons.
Optical recording of intracellular free calcium [Ca²⁺]j responses using
hippocampal or cortical neurons loaded with the Ca²⁺-sensitive fluorescent dye fura-2
allowed further elucidation of the actions of (-)-β-cyclazocine and .DM on responses to
NMDA. Both agents depressed NMDA responses with IC₅₀ values of 270 nM and 220
nM for (-)-β-cyclazocine in hippocampal and cortical neurons, respectively, and 4.1 μM
and 5.4 μM for DM in hippocampal and cortical neurons, respectively. The action of (-)-
β-cyclazocine was use-dependent, a property consistent with open-channel block. No
effect on NMDA-evoked [Ca²⁺]i responses was observed with (+)-p-cyclazocine in either
neuronal type.
In either hippocampal or cortical neurons loaded with fura-2, (-)-β-cyclazocine at
5 μM was found to have no action against [Ca²⁺]i responses to the non-NMDA agonists
kainate and AMPA. Responses evoked by exposure of the neurons to high [K⁺]-
containing medium were also unaffected, indicating that this agent did not interact with
voltage-activated Ca²⁺ channels. DM, at a concentration of 50 μM, reduced high [K⁺]-evoked responses in both neuronal types, showing this agent acted to block neuronal
voltage-activated Ca²⁺ channels.
Cell viability assays showed enantioselective neuroprotection with (-)- and ( + ) -β-cyclazocine. Hippocampal or cortical neurons were exposed to 1 mM NMDA for 24 hr
and the number of surviving neurons following this treatment were compared to the
number of neurons before the toxic NMDA exposure. When present during the NMDA
insult, (-)-β-cyclazocine protected both hippocampal and cortical neurons, with 50%
neuroprotection being achieved near 1 μM. The (+)-enantiomer was weaker as a
neuroprotectant, with < 50 % protection at a concentration of 10 μM.
The specificities of (-)-p-cyclazocine and DM were further assessed by studying
voltage-activated Na⁺ and K⁺ currents in DRG neurons and cardiac myocytes. There
was no detectable effect of either (-)-p-cyclazocine (5 μM) or DM (50 μM) to alter Na⁺ or
K⁺ currents in neurons; (-)-β-cyclazocine (5 μM) had no effect on Na⁺ or K⁺ currents in
cardiac myocytes. However, DM (50 μM) inhibited both Na⁺ and K⁺ currents in cardiac
myocytes, with use-dependent Na⁺ current reduction suggesting DM block of open Na⁺
channels. The results indicate that (-)-β-cyclazocine is a highly potent and selective
blocker of NMDA ion channels, with a concomitant neuroprotective capacity. This
compound is hence suggested as a possible therapeutic agent for conditions requiring
CNS neuroprotection. While DM is an uncompetitive NMDA antagonist, its relative
non-selectivity, as demonstrated by actions against neuronal Ca²⁺ channels and
cardiac Na⁺ and K⁺ channels, warrants caution with regard to its use at doses above
those required for its antitussive effect and hence may limit its clinical application as a neuroprotectant. === Medicine, Faculty of === Anesthesiology, Pharmacology and Therapeutics, Department of === Graduate
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