Bihua Feng

Extrasynaptic NR2B and NR2D subunits of NMDA receptors shape ‘superslow’ afterburst EPSC in rat hippocampus

In conditions of facilitated synaptic release, CA3/CA1 synapses generate anomalously slow NMDA receptor‐mediated EPSCs (EPSCNMDA). Such a time course has been attributed to the cooperation of synapses through glutamate spillover. Imitating a natural pattern of activity, we have applied short bursts (2–7 stimuli) of high‐frequency stimulation and observed a spike‐to‐spike slow‐down of the EPSCNMDA kinetics, which accompanied synaptic facilitation. It was found that the early component of the EPSCNMDA and the burst‐induced late component of the EPSCNMDA have distinct pharmacological properties. The competitive NMDA antagonist R‐(−)‐3‐(2‐carboxypiperazine‐4‐yl)‐propyl‐1‐phosphonic acid (D‐CPP), which has higher affinity to NR2A than to NR2B subunits and lowest affinity at NR2D subunits, significantly slowed down the decay rate of the afterburst EPSC while leaving the kinetics of the control current unaffected. In contrast, ifenprodil, a highly selective NR2B antagonist, and [±]‐cis‐1‐[phenanthren‐2yl‐carbonyl]piperazine‐2,3‐dicarboxylic acid (PPDA), a competitive antagonist that is moderately selective for NR2D subunits, more strongly inhibited the late component of the afterburst EPSCNMDA. The receptors formed by NR2B and (especially) NR2D subunits are known to have higher agonist sensitivity and much slower deactivation kinetics than NR2A‐containing receptors. Furthermore, NR2B is preferentially and NR2D is exclusively located on extrasynaptic membranes. As the density of active synapses increases, the confluence of released glutamate makes EPSC decay much longer by activating more extrasynaptic NR2B‐ and NR2D‐subunit‐containing receptors. Long‐term potentiation (LTP) induced by successive rounds of burst stimulation is accompanied by a long‐term increase in the contribution of extrasynaptic receptors in the afterburst EPSCNMDA.

Structure–activity analysis of a novel NR2C/NR2D-preferring NMDA receptor antagonist: 1-(phenanthrene-2-carbonyl) piperazine-2,3-dicarboxylic acid

(2S*,3R*)‐1‐(biphenyl‐4‐carbonyl)piperazine‐2,3‐dicarboxylic acid (PBPD) is a moderate affinity, competitive N‐methyl‐D‐aspartate (NMDA) receptor antagonist with an atypical pattern of selectivity among NMDA receptor 2 subunit (NR2) subunits. We now describe the activity of several derivatives of PBPD tested at both rat brain NMDA receptors using L‐[3H]‐glutamate binding assays and at recombinant receptors expressed in Xenopus oocytes. Substituting various branched ring structures for the biphenyl group of PBPD reduced NMDA receptor activity. However, substituting linearly arranged ring structures – fluorenone or phenanthrene groups – retained or enhanced activity. Relative to PBPD, the phenanthrene derivative (2S*,3R*)‐1‐(phenanthrene‐2‐carbonyl)piperazine‐2,3‐dicarboxylic acid (PPDA) displayed a 30‐ to 78‐fold increase in affinity for native NMDA receptors. At recombinant receptors, PPDA displayed a 16‐fold (NR2B) to 94‐fold (NR2C) increase in affinity over PBPD. Replacement of the biphenyl group of PBPD with a 9‐oxofluorene ring system resulted in small changes in receptor affinity and subtype selectivity. 2′‐Bromo substitution on the biphenyl group of PBPD reduced antagonist affinity 3‐ to 5‐fold at NR2A‐, NR2B‐ and NR2D‐containing receptors, but had little effect on NR2C‐containing receptors. In contrast, 4′‐fluoro substitution of the biphenyl ring of PBPD selectively increased NR2A affinity. The aromatic rings of PBPD and PPDA increase antagonist affinity and appear to interact with a region of the NMDA receptor displaying subunit heterogeneity. PPDA is the most potent and selective NR2C/NR2D‐preferring antagonist yet reported and thus may be useful in defining NR2C/NR2D function and developing related antagonists with improved NMDA receptor subtype selectivity.

Interaction with σ1 Protein, but Not N-Methyl-d-aspartate Receptor, Is Involved in the Pharmacological Activity of Donepezil

In the present study, we examined the interaction of (±)-2,3-dihydro-5,6-dimethoxy-2-[[1-(phenylmethyl)-4-piperidinyl]-methyl]-1H-inden-1-one hydrochloride (donepezil), a potent cholinesterase inhibitor, with two additional therapeutically relevant targets, N-methyl-d-aspartate (NMDA) and σ1 receptors. Donepezil blocked the responses of recombinant NMDA receptors expressed in Xenopus oocytes. The blockade was voltage-dependent, suggesting a channel blocker mechanism of action, and was not competitive at either the l-glutamate or glycine binding sites. The low potency of donepezil (IC50 = 0.7–3 mM) suggests that NMDA receptor blockade does not contribute to the therapeutic actions of donepezil. Of potential therapeutic relevance, donepezil binds to the σ1 receptor with high affinity (Ki = 14.6 nM) in an in vitro preparation (Neurosci Lett 260:5–8, 1999). Thus, we sought to determine whether an interaction with the σ1 receptor may occur in vivo under physiologically relevant conditions by evaluating the σ1 receptor dependence effects of donepezil in behavioral tasks. Donepezil showed antidepressant-like activity in the mouse-forced swimming test as did the σ1 receptor agonist igmesine. This effect was not displayed by the other cholinesterase inhibitors, rivastigmine and tacrine. The donepezil and igmesine effects were blocked by preadministration of the σ1 receptor antagonist N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(dimethylamino) ethylamine (BD1047) and an in vivo antisense probe treatment. The memory-enhancing effect of donepezil was also investigated. All cholinesterase inhibitors attenuated dizocilpine-induced learning impairments. However, only the donepezil and igmesine effects were blocked by BD1047 or the antisense treatment. Therefore, donepezil behaved as an effective σ1 receptor agonist on these behavioral responses, and an interaction of the drug with the σ1 receptor must be considered in its pharmacological actions.

Interaction with 1 Protein, but not NMDA Receptor, is Involved in the Pharmacological Activity of Donepezil

In the present study, we examined the interaction of (±)-2,3-dihydro-5,6-dimethoxy-2-[[1-(phenylmethyl)-4-piperidinyl]-methyl]-1H-inden-1-one hydrochloride (donepezil), a potent cholinesterase inhibitor, with two additional therapeutically relevant targets, N-methyl-d-aspartate (NMDA) and σ1 receptors. Donepezil blocked the responses of recombinant NMDA receptors expressed in Xenopus oocytes. The blockade was voltage-dependent, suggesting a channel blocker mechanism of action, and was not competitive at either the l-glutamate or glycine binding sites. The low potency of donepezil (IC50 = 0.7–3 mM) suggests that NMDA receptor blockade does not contribute to the therapeutic actions of donepezil. Of potential therapeutic relevance, donepezil binds to the σ1 receptor with high affinity (Ki = 14.6 nM) in an in vitro preparation (Neurosci Lett 260:5–8, 1999). Thus, we sought to determine whether an interaction with the σ1 receptor may occur in vivo under physiologically relevant conditions by evaluating the σ1 receptor dependence effects of donepezil in behavioral tasks. Donepezil showed antidepressant-like activity in the mouse-forced swimming test as did the σ1 receptor agonist igmesine. This effect was not displayed by the other cholinesterase inhibitors, rivastigmine and tacrine. The donepezil and igmesine effects were blocked by preadministration of the σ1 receptor antagonist N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(dimethylamino) ethylamine (BD1047) and an in vivo antisense probe treatment. The memory-enhancing effect of donepezil was also investigated. All cholinesterase inhibitors attenuated dizocilpine-induced learning impairments. However, only the donepezil and igmesine effects were blocked by BD1047 or the antisense treatment. Therefore, donepezil behaved as an effective σ1 receptor agonist on these behavioral responses, and an interaction of the drug with the σ1 receptor must be considered in its pharmacological actions.

The effect of competitive antagonist chain length on NMDA receptor subunit selectivity.

The widely-used N-methyl-D-aspartate (NMDA) receptor antagonists (R)-4-(3-phosphonopropyl) piperazine-2-carboxylic acid ((R)-CPP) and (R)-2-amino-7-phosphonoheptanoate ((R)-AP7) are frequently used as general NMDA receptor antagonists and assumed not to display significant selectivity among NMDA receptor NR2 subunits. However, electrophysiological studies have suggested that certain longer chain N-methyl-D-aspartate (NMDA) receptor competitive antagonists, such as (R)-CPP are ineffective at subpopulations of NMDA receptors in the red nucleus, superior colliculus, and hippocampus. Using recombinant receptors expressed in Xenopus oocytes, we have examined the effect of antagonist chain length on NR2 subunit selectivity. All antagonists displayed the potency order (high to low affinity) of NR2A > NR2B > NR2C > NR2D, however the longer chain antagonists (having 7 instead of 5 bond lengths between acidic groups) displayed much greater subunit selectivity than their short-chain homologues. For example (R)-CPP displayed a 50-fold difference in affinity between NR2A-containing and NR2D-containing NMDA receptors, while the shorter chain homologue 4-(phosphonomethyl) piperazine-2-carboxylic acid (PMPA) displayed only a 5-fold variation in affinity. These results can account for the earlier physiological findings and suggest that longer chain antagonists such as (R)-CPP and (R)-AP7 should not be used as general NMDA receptor antagonists.

N -Methyl-d-aspartate (NMDA) Receptor NR2 Subunit Selectivity of a Series of Novel Piperazine-2,3-dicarboxylate Derivatives: Preferential Blockade of Extrasynaptic NMDA Receptors in the Rat Hippocampal CA3-CA1 Synapse

N-Methyl-d-aspartate (NMDA) receptor antagonists that are highly selective for specific NMDA receptor 2 (NR2) subunits have several potential therapeutic applications; however, to date, only NR2B-selective antagonists have been described. Whereas most glutamate binding site antagonists display a common pattern of NR2 selectivity, NR2A > NR2B > NR2C > NR2D (high to low affinity), (2S*,3R*)-1-(phenanthrene-2-carbonyl)piperazine-2,3-dicarboxylic acid (PPDA) has a low selectivity for NR2C- and NR2D-containing NMDA receptors. A series of PPDA derivatives were synthesized and then tested at recombinant NMDA receptors expressed in Xenopus laevis oocytes. In addition, the optical isomers of PPDA were resolved; the (−) isomer displayed a 50- to 80-fold greater potency than the (+) isomer. Replacement of the phenanthrene moiety of PPDA with naphthalene or anthracene did not improve selectivity. However, phenylazobenzoyl (UBP125) or phenylethynylbenzoyl (UBP128) substitution significantly improved selectivity for NR2B-, NR2C-, and NR2D-containing receptors over NR2A-containing NMDA receptors. Phenanthrene attachment at the 3 position [(2R*,3S*)-1-(phenanthrene-3-carbonyl)piperazine-2,3-dicarboxylic acid (UBP141); (2R*,3S*)-1-(9-bromophenanthrene-3-carbonyl)piperazine-2,3-dicarboxylic acid (UBP145); (2R*,3S*)-1-(9-chlorophenanthrene-3-carbonyl)piperazine-2,3-dicarboxylic acid (UBP160); and (2R*,3S*)-1-(9-iodophenanthrene-3-carbonyl)piperazine-2,3-dicarboxylic acid (UBP161)] displayed improved NR2D selectivity. UBP141 and its 9-brominated homolog (UBP145) both display a 7- to 10- fold selectivity for NR2D-containing receptors over NR2B- or NR2A-containing receptors. Schild analysis indicates that these two compounds are competitive glutamate binding site antagonists. Consistent with a physiological role for NR2D-containing receptors in the hippocampus, UBP141 (5 μM) displayed greater selectivity than PPDA for inhibiting the slow-decaying component of the NMDA receptor-mediated CA3-CA1 synaptic response in rat hippocampal slices. UBP125, UBP128, UBP141, and UBP145 may be useful tools for determining the function of NMDA receptor subtypes.