The interactions of putative neuroprotectant compounds with NMDA ion channels
Creators: name: Sawyer, Dale C.
Publisher: name: University of British Columbia
Year: 2009

description: 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 &lt; 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.