Non-competitive Channel Blocker Research Articles

Effect of the General Anaesthetic Ketamine on Electrical and Ca2+ Signal Propagation in Arabidopsis thaliana

The systemic electrical signal propagation in plants (i.e., from leaf to leaf) is dependent on GLUTAMATE RECEPTOR-LIKE proteins (GLRs). The GLR receptors are the homologous proteins to the animal ionotropic glutamate receptors (iGluRs) which are ligand-gated non-selective cation channels that mediate neurotransmission in the animal’s nervous system. In this study, we investigated the effect of the general anaesthetic ketamine, a well-known non-competitive channel blocker of human iGluRs, on systemic electrical signal propagation in Arabidopsis thaliana. We monitored the electrical signal propagation, intracellular calcium level [Ca2+]cyt and expression of jasmonate (JA)-responsive genes in response to heat wounding. Although ketamine affected the shape and the parameters of the electrical signals (amplitude and half-time, t1/2) mainly in systemic leaves, it was not able to block a systemic response. Increased [Ca2+]cyt and the expression of jasmonate-responsive genes were detected in local as well as in systemic leaves in response to heat wounding in ketamine-treated plants. This is in contrast with the effect of the volatile general anaesthetic diethyl ether which completely blocked the systemic response. This low potency of ketamine in plants is probably caused by the fact that the critical amino acid residues needed for ketamine binding in human iGluRs are not conserved in plants’ GLRs.

Evaluation of the effect of the neurotoxin MPTP and the antiparkinsonian drug hemantane on the DNA integrity in the brain structures of C57BL/6 mice

Hemantane (N-adamant-2-yl-hexamethylenimine hydrochloride) is an antiparkinsonian drug with a multicomponent mechanism of action, including a modulating effect on the activity of dopamine and serotonergic mediator systems, a selective inhibitory effect on MAO-B, properties of a low-affinity non-competitive channel blocker of glutamate NMDA receptors, has moderate antiradical and anti-inflammatory activity. The aim of this study was to assess the effect of the neurotoxin MPTP, which is used for modeling of parkinsonian syndrome, and the hemantane on the DNA integrity in the striatum and frontal cortex of the brain of C57BL/6 mice by the DNA comet assay – gel electrophoresis of DNA single cells. Results. In the first experiment, hemantane was administered once a day for 5 days before the MPTP (20 mg/kg, i.p.), then together with MPTP once a day for 5 days. In the second experiment hemantane 10 mg/kg was injected preliminarily for 4 days and 40 minutes before MPTP 30 mg/kg. The obtained results confirm the absence of an effect on DNA of hemantane at a therapeutic dose of 10 mg / kg. In the experimental schemes used, we did not reveal the expected increase in the level of DNA damage under the influence of MPTP, and, accordingly, we were not able to assess the protective effect of hemantane.

Structural basis of ketamine action on human NMDA receptors.

Ketamine is a non-competitive channel blocker of N-methyl-D-aspartate (NMDA) receptors1. A single sub-anaesthetic dose of ketamine produces rapid (within hours) and long-lasting antidepressant effects in patients who are resistant to other antidepressants2,3. Ketamine is a racemic mixture of S- and R-ketamine enantiomers, with S-ketamine isomer being the more active antidepressant4. Here we describe the cryo-electron microscope structures of human GluN1-GluN2A and GluN1-GluN2B NMDA receptors in complex with S-ketamine, glycine and glutamate. Both electron density maps uncovered the binding pocket for S-ketamine in the central vestibule between the channel gate and selectivity filter. Molecular dynamics simulation showed that S-ketamine moves between two distinct locations within the binding pocket. Two amino acids-leucine 642 on GluN2A (homologous to leucine 643 on GluN2B) and asparagine 616 on GluN1-were identified as key residues that form hydrophobic and hydrogen-bond interactions with ketamine, and mutations at these residues reduced the potency of ketamine in blocking NMDA receptor channel activity. These findings show structurally how ketamine binds to and acts on human NMDA receptors, and pave the way for the future development of ketamine-based antidepressants.

NMDA Receptor and L-Type Calcium Channel Modulate Prion Formation.

Transmissible neurodegenerative prion diseases are characterized by the conversion of the cellular prion protein (PrPC) to misfolded isoforms denoted as prions or PrPSc. Although the conversion can occur in the test tube containing recombinant prion protein or cell lysates, efficient prion formation depends on the integrity of intact cell functions. Since neurons are main targets for prion replication, we asked whether their most specialized function, i.e. synaptic plasticity, could be a factor by which PrPSc formation can be modulated.Immortalized gonadotropin-releasing hormone cells infected with the Rocky Mountain Laboratory prion strain were treated with L-type calcium channels (LTCCs) and NMDA receptors (NMDARs) stimulators or inhibitors. Western blotting was used to monitor the effects on PrPSc formation in relation to ERK signalling.Infected cells showed enhanced levels of phosphorylated ERK (pERK) compared with uninfected cells. Exposure of infected cells to the LTCC agonist Bay K8644 enhanced pERK and PrPSc levels. Although treatment with an LTCC blocker (nimodipine) or an NMDAR competitive antagonist (D-AP5) had no effects, their combination reduced both pERK and PrPSc levels. Treatment with the non-competitive NMDAR channel blocker MK-801 markedly reduced pERK and PrPSc levels.Our study shows that changes in LTCCs and NMDARs activities can modulate PrPSc formation through ERK signalling. During synaptic plasticity, while ERK signalling promotes long-term potentiation accompanied by expansion of post-synaptic lipid rafts, other NMDA receptor-depending signalling pathways, p38-JNK, have opposing effects. Our findings indicate that contrasting intracellular signals of synaptic plasticity can influence time-dependent prion conversion.

Structure-Dependent Activity of Natural GABA(A) Receptor Modulators.

GABA(A) receptors are ligand-gated ion channels consisting of five subunits from eight subfamilies, each assembled in four hydrophobic transmembrane domains. This pentameric structure not only allows different receptor binding sites, but also various types of ligands, such as orthosteric agonists and antagonists, positive and negative allosteric modulators, as well as second-order modulators and non-competitive channel blockers. A fact, that is also displayed by the variety of chemical structures found for both, synthetic as well as nature-derived GABA(A)-receptor modulators. This review covers the literature for natural GABA(A)-receptor modulators until the end of 2017 and discusses their structure-activity relationship.

Probing the structure of the uncoupled nicotinic acetylcholine receptor

In the absence of activating anionic lipids and cholesterol, the nicotinic acetylcholine receptor (nAChR) from Torpedo adopts an uncoupled conformation that does not usually gate open in response to agonist. The uncoupled conformation binds both agonists and non-competitive channel blockers with a lower affinity than the desensitized state, consistent with both the extracellular agonist-binding and transmembrane channel-gating domains individually adopting resting-state like conformations. To test this hypothesis, we characterized the binding of the agonist, acetylcholine, and two fluorescent channel blockers, ethidium and crystal violet, to resting, desensitized and uncoupled nAChRs in reconstituted membranes. The measured Kd for acetylcholine binding to the uncoupled nAChR is similar to that for the resting state, confirming that the agonist binding site adopts a resting-state like conformation. Although both ethidium and crystal violet bind to the resting and desensitized channel pores with distinct affinities, no binding of either probe was detected to the uncoupled nAChR. Our data suggest that the transmembrane domain of the uncoupled nAChR adopts a conformation distinct from that of the resting and desensitized states. The lack of binding is consistent with a more constricted channel pore, possibly along the lines of what is observed in crystal structures of the prokaryotic homolog, ELIC.

Pax2-Islet1 Transgenic Mice Are Hyperactive and Have Altered Cerebellar Foliation

The programming of cell fate by transcription factors requires precise regulation of their time and level of expression. The LIM-homeodomain transcription factor Islet1 (Isl1) is involved in cell-fate specification of motor neurons, and it may play a similar role in the inner ear. In order to study its role in the regulation of vestibulo-motor development, we investigated a transgenic mouse expressing Isl1 under the Pax2 promoter control (Tg+/−). The transgenic mice show altered level, time, and place of expression of Isl1 but are viable. However, Tg+/− mice exhibit hyperactivity, including circling behavior, and progressive age-related decline in hearing, which has been reported previously. Here, we describe the molecular and morphological changes in the cerebellum and vestibular system that may cause the hyperactivity of Tg+/− mice. The transgene altered the formation of folia in the cerebellum, the distribution of calretinin labeled unipolar brush cells, and reduced the size of the cerebellum, inferior colliculus, and saccule. Age-related progressive reduction of calbindin expression was detected in Purkinje cells in the transgenic cerebella. The hyperactivity of Tg+/− mice is reduced upon the administration of picrotoxin, a non-competitive channel blocker for the γ-aminobutyric acid (GABA) receptor chloride channels. This suggests that the overexpression of Isl1 significantly affects the functions of GABAergic neurons. We demonstrate that the overexpression of Isl1 affects the development and function of the cerebello-vestibular system, resulting in hyperactivity.

Anxiolytic- and antidepressant-like effects of the methadone metabolite 2-ethyl-5-methyl-3,3-diphenyl-1-pyrroline (EMDP)

The enhancement of GABAergic and monoaminergic neurotransmission has been the mainstay of pharmacotherapy and the focus of drug-discovery for anxiety and depressive disorders for several decades. However, the significant limitations of drugs used for these disorders underscores the need for novel therapeutic targets. Neuronal nicotinic acetylcholine receptors (nAChRs) may represent one such target. For example, mecamylamine, a non-competitive antagonist of nAChRs, displays positive effects in preclinical tests for anxiolytic and antidepressant activity in rodents. In addition, nicotine elicits similar effects in rodent models, possibly by receptor desensitization. Previous studies (Xiao et al., 2001) have identified two metabolites of methadone, EMDP (2-ethyl-5-methyl-3,3-diphenyl-1-pyrroline) and EDDP (2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine), which are considered to be inactive at opiate receptors, as relatively potent noncompetitive channel blockers of rat α3β4 nAChRs. Here, we show that these compounds are likewise highly effective blockers of human α3β4 and α4β2 nAChRs. Moreover, we show that they display relatively low affinity for opiate binding sites labeled by [3H]-naloxone. We then evaluated these compounds in rats and mice in preclinical behavioral models predictive of potential anxiolytic and antidepressant efficacy. We found that EMDP, but not EDDP, displayed robust effects predictive of anxiolytic and antidepressant efficacy without significant effects on locomotor activity. Moreover, EMDP at behaviorally active doses, unlike mecamylamine, did not produce eyelid ptosis, suggesting it may produce fewer autonomic side effects than mecamylamine. Thus, the methadone metabolite EMDP may represent a novel therapeutic avenue for the treatment of some affective disorders.

Allosteric ligands and their binding sites define γ-aminobutyric acid (GABA) type A receptor subtypes.

GABAA receptors (GABA(A)Rs) mediate rapid inhibitory transmission in the brain. GABA(A)Rs are ligand-gated chloride ion channel proteins and exist in about a dozen or more heteropentameric subtypes exhibiting variable age and brain regional localization and thus participation in differing brain functions and diseases. GABA(A)Rs are also subject to modulation by several chemotypes of allosteric ligands that help define structure and function, including subtype definition. The channel blocker picrotoxin identified a noncompetitive channel blocker site in GABA(A)Rs. This ligand site is located in the transmembrane channel pore, whereas the GABA agonist site is in the extracellular domain at subunit interfaces, a site useful for low energy coupled conformational changes of the functional channel domain. Two classes of pharmacologically important allosteric modulatory ligand binding sites reside in the extracellular domain at modified agonist sites at other subunit interfaces: the benzodiazepine site and the high-affinity, relevant to intoxication, ethanol site. The benzodiazepine site is specific for certain GABA(A)R subtypes, mainly synaptic, while the ethanol site is found at a modified benzodiazepine site on different, extrasynaptic, subtypes. In the transmembrane domain are allosteric modulatory ligand sites for diverse chemotypes of general anesthetics: the volatile and intravenous agents, barbiturates, etomidate, propofol, long-chain alcohols, and neurosteroids. The last are endogenous positive allosteric modulators. X-ray crystal structures of prokaryotic and invertebrate pentameric ligand-gated ion channels, and the mammalian GABA(A)R protein, allow homology modeling of GABA(A)R subtypes with the various ligand sites located to suggest the structure and function of these proteins and their pharmacological modulation.

Analysis of γ-aminobutyric acid (GABA) type A receptor subtypes using isosteric and allosteric ligands.

The GABAA receptors (GABAARs) play an important role in inhibitory transmission in the brain. The GABAARs could be identified using a medicinal chemistry approach to characterize with a series of chemical structural analogues, some identified in nature, some synthesized, to control the structural conformational rigidity/flexibility so as to define the 'receptor-specific' GABA agonist ligand structure. In addition to the isosteric site ligands, these ligand-gated chloride ion channel proteins exhibited modulation by several chemotypes of allosteric ligands, that help define structure and function. The channel blocker picrotoxin identified a noncompetitive channel blocker site in GABAARs. This ligand site is located in the transmembrane channel pore, whereas the GABA agonist site is in the extracellular domain at subunit interfaces, a site useful for low energy coupled conformational changes of the functional channel domain. Also in the trans-membrane domain are allosteric modulatory ligand sites, mostly positive, for diverse chemotypes with general anesthetic efficacy, namely, the volatile and intravenous agents: barbiturates, etomidate, propofol, long-chain alcohols, and neurosteroids. The last are apparent endogenous positive allosteric modulators of GABAARs. These binding sites depend on the GABAAR heteropentameric subunit composition, i.e., subtypes. Two classes of pharmacologically very important allosteric modulatory ligand binding site reside in the extracellular domain at modified agonist sites at other subunit interfaces: the benzodiazepine site, and the low-dose ethanol site. The benzodiazepine site is specific for certain subunit combination subtypes, mainly synaptically localized. In contrast, the low-dose (high affinity) ethanol site(s) is found at a modified benzodiazepine site on different, extrasynaptic, subtypes.

Identifying the Binding Site of Novel Methyllycaconitine (MLA) Analogs at α4β2 Nicotinic Acetylcholine Receptors

Neuronal nicotinic acetylcholine receptors (nAChR) are ligand gated ion channels that mediate fast synaptic transmission. Methyllycaconitine (MLA) is a selective and potent antagonist of the α7 nAChR, and its anthranilate ester side-chain is important for its activity. Here we report the influence of structure on nAChR inhibition for a series of novel MLA analogs, incorporating either an alcohol or anthranilate ester side-chain to an azabicyclic or azatricyclic core against rat α7, α4β2, and α3β4 nAChRs expressed in Xenopus oocytes. The analogs inhibited ACh (EC(50)) within an IC(50) range of 2.3-26.6 μM. Most displayed noncompetitive antagonism, but the anthranilate ester analogs exerted competitive behavior at the α7 nAChR. At α4β2 nAChRs, inhibition by the azabicyclic alcohol was voltage-dependent suggesting channel block. The channel-lining residues of α4 subunits were mutated to cysteine and the effect of azabicyclic alcohol was evaluated by competition with methanethiosulfonate ethylammonium (MTSEA) and a thiol-reactive probe in the open, closed, and desensitized states of α4β2 nAChRs. The azabicyclic alcohol was found to compete with MTSEA between residues 6' and 13' in a state-dependent manner, but the reactive probe only bonded with 13' in the open state. The data suggest that the 13' position is the dominant binding site. Ligand docking of the azabicyclic alcohol into a (α4)(3)(β2)(2) homology model of the closed channel showed that the ligand can be accommodated at this location. Thus our data reveal distinct pharmacological differences between different nAChR subtypes and also identify a specific binding site for a noncompetitive channel blocker.

Regulation and Function of Aquaporins in Human Urothelium

PURPOSE We have previously provided molecular evidence that aquaporins (AQP) are expressed by human urothelium, suggesting a potential role in water and solute transport (Rubenwolf et al, 2008). The aim of this work was to investigate factors that regulate AQP expression in human urothelium and to develop functional assays to examine the potential role in urothelial physiology. MATERIAL AND METHODS Normal Human Urothelial (NHU) cell lines were established and the effect on AQP protein expression of exposing NHU cell cultures to osmotic stress was examined by immunochemistry. The barrier properties of differentiated urothelial constructs were assessed by measuring transepithelial electrical resistance (TER) and permeability coefficients for 3H water and 14C urea. HgCl2 was used as a non-competitive AQP channel blocker. RESULTS AQP 3 protein expression was shown to be up-regulated by up to 13-fold in response to changing the culture medium osmolality from 295 to 500 mosm/kg. Differentiated urothelium revealed a significant barrier function (average TER: 3680 Ω.cm2); average diffusive water and urea permeability coefficients (Pd) were 10.4x10-5 and 3.7x10-5 cm/sec, respectively. Mean osmotic permeability coefficients (Posm) were 1.8-fold higher than corresponding diffusive permeability coefficients. A significant, dose-dependent (up to 4-fold) decrease of both water and urea flux across urothelium was observed upon application of HgCl2. CONCLUSIONS Our results demonstrate for the first time that expression of AQP 3 is regulated by osmolality, which suggests that urothelium is capable of reabsorbing water in response to the body's hydration status. The low, but finite, permeability of cultured urothelium to water and urea, which was reduced upon AQP inhibition, is also highly supportive of a previously unrecognised role for AQPs in body fluid regulation with potential implications on urinary tract physiology and urinary continence.

A new NMDA-receptor blocker provides potential neuroprotection

Glutamate neurotoxicity is implicated in the pathogenesis of neurodegenerative diseases such as Huntington's, Alzheimer's and Parkinson's diseases, and also plays a role in brain damage caused by head trauma, stroke and brain-tumour progression.NMDA-receptor channels are very permeable to Ca2+. Glutamate-mediated Ca2+ influx into neurons sets up a glutamate–nitric oxide–cGMP pathway, which eventually results in cell death by necrosis or apoptosis. Therefore, drug development aimed at neuroprotection has targeted the NMDA receptor. Unfortunately, at present, NMDA-receptor antagonists have serious cognitive side effects that interfere with their therapeutic benefit. These antagonists have strong affinity, and bind not only to pathologically active receptors, but also to physiologically active receptor sites, thereby interfering with the normal processes of memory and learning. Non-competitive channel blockers bind preferentially to pathologically active receptors. A potential neuroprotectant non-competitive NMDA-channel blocker would need the attributes of low molecular weight, low receptor affinity and fast on–off kinetics.Planells-Cases et al. screened a restricted oligo-N-substituted glycine-based combinatorial library to find novel antagonists of the NMDA receptor [1xA novel n-methyl-d-aspartate receptor open channel blocker with in vivo neuroprotectant activity. Planells-Cases, R. et al. J. Pharmacol. Exp. Ther. 2002; 302: 163–173Crossref | PubMed | Scopus (46)See all References[1]. The rationale for this was the finding that arginine-rich hexapeptides blocked NMDA receptors with high efficacy and exhibited neuroprotectant activity in vitro. However, in vivo, the compounds were toxic at theraputic doses. The library identified trimers of N-alkylglycine with a 3,3-diphenylpropyl amino moeity that blocked NMDA-receptor channels. Structure–activity relationships demonstrated that one compound, N20C, selectively inhibited NMDA receptors with micromolar affinity, and had fast on–off kinetics and strong voltage dependence. It did not compete for binding with glycine or glutamate, but prevented high affinity MK801 binding. The authors concluded, therefore, that N20C is a non-competitive blocker that binds to the same non-competitive site as MK801, deep in the NMDA-receptor channel pore, thereby interfering with Ca2+ permeability. In vitro and in vivo experiments also demonstrated that N20C has a neuroprotectant effect on glutamate-induced neuronal cell death.The extent of neuroprotection provided by N20C was shown to be higher than that of memantine, and a better therapeutic profile was also suggested. The compound did not exhibit toxicity at high concentrations, and animals treated with N-alkylglycine did not display any conspicuous behavioural or motor deficits. Although the authors caution that a therapeutic index needs to be determined for this new open-channel blocker, this study represents an exciting step in the discovery of a neuroprotectant drug that has the potential to treat many neurodegenerative diseases.

NMDA receptor regulation of cell death in the rat olfactory bulb.

Cell death is widespread in the developing nervous system and is under complex regulation by numerous intra- and intercellular mechanisms. Blockade of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor has been shown to promote cell death in the developing brain (Ikonomidou et al., 1999), suggesting that afferent functional activation is an important regulator of cell survival. The olfactory bulb, the first central relay for olfactory information from the nose, is well suited for examining the role of afferent activity in neuronal development. Functional deprivation is easily performed by surgical blockade of airflow to one side of the nasal passage, which results in dramatic alterations in postnatal development of the bulb (Brunjes, 1994), including enhanced neuronal loss (Frazier and Brunjes, 1988; Najbauer and Leon, 1995). The present report examined the specific role of NMDA receptor activation in regulating cell survival within the rat bulb. Pharmacological blockade of receptors with the noncompetitive channel blocker MK-801 (3 x 0.5 mg/kg i.p.) resulted in profound increases in cell death within 24 h. Furthermore, in contrast to other regions, where the effects of receptor blockade were confined to the first 2 postnatal weeks (Ikonomidou et al., 1999), enhancement of cell death was seen in the deeper granule cell-containing regions of the bulb with injections as late as postnatal day 28. In addition, the effects of MK-801 were much more dramatic than those seen after unilateral naris closure, suggesting that NMDA receptor activation may mediate additional survival pathways in the bulb beyond that provided by first nerve input.

A Conformational Intermediate between the Resting and Desensitized States of the Nicotinic Acetylcholine Receptor

The structural changes induced in the nicotinic acetylcholine receptor by two noncompetitive channel blockers, proadifen and phencyclidine, have been studied by infrared difference spectroscopy and using the conformationally sensitive photoreactive noncompetitive antagonist 3-(trifluoromethyl)-3-m-([(125)I]iodophenyl)diazirine. Simultaneous binding of proadifen to both the ion channel pore and neurotransmitter sites leads to the loss of positive markers near 1663, 1655, 1547, 1430, and 1059 cm(-)(1) in carbamylcholine difference spectra, suggesting the stabilization of a desensitized conformation. In contrast, only the positive markers near 1663 and 1059 cm(-)(1) are maximally affected by the binding of either blocker to the ion channel pore suggesting that the conformationally sensitive residues vibrating at these two frequencies are stabilized in a desensitized-like conformation, whereas those vibrating near 1655 and 1430 cm(-)(1) remain in a resting-like state. The vibrations at 1547 cm(-)(1) are coupled to those at both 1663 and 1655 cm(-)(1) and thus exhibit an intermediate pattern of band intensity change. The formation of a structural intermediate between the resting and desensitized states in the presence of phencyclidine is further supported by the pattern of 3-(trifluoromethyl)-3-m-([(125)I]iodophenyl)diazirine photoincorporation. In the presence of phencyclidine, the subunit labeling pattern is distinct from that observed in either the resting or desensitized conformations; specifically, there is a concentration-dependent increase in the extent of photoincorporation into the delta-subunit. Our data show that domains of the nicotinic acetylcholine receptor interconvert between the resting and desensitized states independently of each other and suggest a revised model of channel blocker action that involves both low and high affinity agonist binding conformational intermediates.

Antiparkinsonian and antidyskinetic activity of drugs targeting central glutamatergic mechanisms.

Motor dysfunction produced by the chronic non-physiological stimulation of dopaminergic receptors on striatal medium spiny neurons is associated with alterations in the sensitivity of glutamatergic receptors, including those of the N-methyl-D-aspartate (NMDA) subtype. Functional characteristics of these ionotropic receptors are regulated by their phosphorylation state. Lesioning the nigrostriatal dopamine system of rats induces parkinsonian signs and increases the phosphorylation of striatal NMDA receptor subunits on serine and tyrosine residues. The intrastriatal administration of certain inhibitors of the kinases capable of phosphorylating NMDA receptors produces a dopaminomimetic motor response in these animals. Treating parkinsonian rats twice daily with levodopa induces many of the characteristic features of the human motor complication syndrome and further increases the serine and tyrosine phosphorylation of specific NMDA receptor subunits. Again, the intrastriatal administration of selective inhibitors of certain serine and tyrosine kinases alleviates the motor complications. NMDA receptor antagonists, including some non-competitive channel blockers, act both palliatively and prophylactically in rodent and primate models to reverse these levodopa-induced response alterations. Similarly, in clinical studies dextrorphan, dextromethorphan, and amantadine have been found to be efficacious against motor complications. Recent observations in animal models further indicate that certain amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA) antagonists alleviate, while others exacerbate, these complications. Thus, it appears that the denervation or intermittent stimulation of striatal dopaminergic receptors differentially activates signal transduction pathways in medium spiny neurons. These in turn modify the phosphorylation state of ionotropic glutamate receptors and consequently their sensitivity to cortical input. These striatal changes contribute to symptom production in Parkinson's disease, and their prevention or reversal could prove useful in the treatment of this disorder.

Dextromethorphan ameliorates effects of neonatal hypoxia on brain morphology and seizure threshold in rats

Hypoxic injury to the brain is mediated in part by NMDA receptors. Therefore, NMDA receptor blockade with dextromethorphan (DM), a non-competitive channel blocker, was hypothesized to ameliorate injury even when given after the hypoxic insult. Rats were exposed to 8% oxygen for 3 h on postnatal day 7. Within 20 min of exposure, animals received 30 mg/kg i.p. DM or normal saline. Littermates maintained in room air for 3 h also received DM or saline. At 14 days of age, 7 days after exposure, cortical thickness and hippocampal area were measured. At 70–90 days of age, approximately two months after exposure, in a separate group of rats, seizure threshold using pentylenetetrazol (PTZ) and passive avoidance learning and retention were determined. There were no gross changes in cellular morphology and no evidence for cellular necrosis in any of the exposure groups. However, cortical thickness was decreased in animals exposed to hypoxia. DM administration prevented this decrease. Hippocampal area was unaffected. Seizure susceptibility in adulthood was increased in animals exposed to hypoxia in the neonatal period. DM prevented the decrease in seizure threshold. There was no difference in passive avoidance learning or retention as a function of neonatal exposure condition. Mild to moderate hypoxia, previously thought not to produce any histologic changes, causes significant short-term loss of cortical thickness and long-term decrease in seizure threshold. DM appears to ameliorate these effects even when given after the hypoxic insult. These results implicate the glutamate receptor system in the pathophysiology of hypoxia damage and suggest that treatment with a glutamate receptor blocker when neonatal asphyxia is suspected would help ameliorate the consequences of such an insult. © Elsevier Science B.V. All rights reserved.

Coadministered pentobarbital anesthesia postpones but does not block the motor and sleep EEG responses to MK-801.

In previous studies with Sprague-Dawley rats, we demonstrated that NMDA channel blockade during waking massively stimulates the delta (1-4 Hz) EEG of non-rapid eye movement (NREM) sleep. However, non-competitive channel blockers also produce neurotoxicity that is manifested by posterior cingulate vacuolization and heat shock protein production. These neurotoxic effects can be blocked by coadministering gabaergic drugs, including barbiturates and benzodiazepines, with the MK-801. To determine whether delta stimulation by MK-801 would be similarly blocked, we administered an anesthetic dose (40 mg/kg) of pentobarbital followed immediately by 0.3 mg/kg of MK-801. Neither the MK-801 motor syndrome nor the NREM delta stimulation was blocked. When the rats recovered from nearly two hours of barbiturate anesthesia, they behaved as though they had just received the MK-801 injection, exhibiting the typical motor syndrome, spikes in the waking EEG and strong stimulation of NREM delta EEG. These findings support our previous evidence that NREM delta stimulation by NMDA channel blockade does not depend on toxic brain changes. They also raise interesting questions regarding the fate of MK-801 during pentobarbital anesthesia. We propose that the drug is not metabolized during the period of anesthesia because it is sequestered within the NMDA cation channel. However, neurons do not respond to the channel block because they have been rendered inert by the anesthesia. When the neurons emerge from anesthesia, the cascade of MK-801 events unfolds. This and other possible explanations can be tested experimentally. Establishing the fate of MK-801 during barbiturate anesthesia could shed new light on the cellular processing of non-competitive NMDA channel blockers.

N‐methyl‐d‐aspartate receptor antagonists potentiate morphine's antinociceptive effect in the rat

The interaction between morphine and three antagonists of the N-methyl-D-aspartate (NMDA) receptor. MK-801 (non-competitive channel blocker), dextromethorphan (clinically available non-competitive antagonist) and CGS19755 (competitive receptor antagonist), was examined in rats with the hot plate test. The NMDA antagonists were administered intraperitoneally and none of them caused antinociception at doses that did not produce motor deficits (0.1 mg kg-1 MK-801, 30 mg kg-1 dextromethorphan and 5 mg kg-1 CGS19755). However, pretreatment with the NMDA antagonists at these doses 30 min prior to subcutaneous injection of 5 mg kg-1 morphine significantly potentiated the antinociceptive effect of morphine, with strongest effect observed with dextromethorphan. It is suggested that blockade of NMDA receptors enhances the antinociceptive effect of morphine and NMDA antagonists may improve the analgesic efficacy of morphine in the clinic.

Effect of NMDA receptor antagonists on prostaglandin E 2-induced hyperalgesia in conscious mice

Intrathecal (i.t.) injection of prostaglandin E 2 (PGE 2) to conscious mice produced a hyperalgesic action over a wide range of dosages with two apparent peaks at 100 pg and 10 ng per mouse, which may be mediated through EP 3 and EP 2 subtypes of the PGE receptor. In the present study, the effects of NMDA receptor antagonists on hyperalgesia induced by PGE 2 were evaluated by the hot plate test at 30 min after i.t. injection. Hyperalgesia induced by a higher dose of PGE 2 (10 ng/mouse) was relieved by d-AP5 (a competitive antagonist), 7-Cl-KynA (a glycine site antagonist), and ketamine and MK801 (non-competitive channel blockers). Intrathecal injection of butaprost (10 ng/mouse), an EP 2 agonist, induced hyperalgesia, and this hyperalgesia was blocked by d-AP5, 7-Cl-KynA, ketamine, and MK801, similar to that induced by 10 ng of PGE 2. On the other hand, hyperalgesia induced by a lower dose of PGE 2 (100 pg/mouse) was blocked by d-AP5 and 7-Cl-KynA, but not by ketamine and MK801. Intrathecal injection of sulprostone (100 pg/mouse), an EP 1 and EP 3 agonist, induced hyperalgesia, and this hyperalgesia was blocked by d-AP5 and 7-Cl-KynA, but not by ketamine and MK801, similar to that induced by 100 pg of PGE 2. These results first demonstrate that the NMDA receptor is involved in the PGE 2-induced hyperalgesia and suggest that the hyperalgesic action by lower and higher doses of PGE 2 may be mediated through EP 3 and EP 2 subtypes, respectively.