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Conantokins and variants derived from cone snail venom inhibit naloxone-induced withdrawal jumping in morphine-dependent mice.

The biochemical processes underlying opiate addiction are complex, but n-methyl-d-aspartate receptor (NMDAR) dysfunction appears to be one contributing factor. NMDAR antagonists, including MK-801 and memantine, have previously been shown to assuage symptoms stemming from opiate withdrawal. The conantokins are a small family of naturally occurring peptide toxins known to specifically antagonize the NMDAR. In the present study, the effects of wild-type and variant conantokins on the suppression of naloxone-induced jumping in morphine-dependent mice were evaluated. Our results demonstrate that NR2B-selective conantokins, viz., con-G, con-G[S(16)Y] and con-G[gamma(7)K], are potent inhibitors of naloxone-induced jumping at low doses (2-15 nmol/kg) compared with con-T, con-R[1-17], and small-molecule antagonists of the NMDAR, including the NR2B-selective agent, ifenprodil. We conclude that the NR2B-selective conantokins may find utility as neuropharmacological tools for probing NMDAR-related mechanisms of opiate dependence.

Animals↗

Effects of distinct classes of N-methyl-D-aspartate receptor antagonists on seizures, axonal sprouting and neuronal loss in vitro: suppression by NR2B-selective antagonists.

Chronic treatment with high-affinity, competitive N-methyl-D-aspartate receptor (NMDAR) antagonists can promote axonal sprouting, induce neuronal loss and exacerbate seizures associated with temporal lobe epilepsy. Whether moderate-affinity uncompetitive and NR2B subunit-selective NMDAR antagonists elicit similar responses remains largely unexplored. We directly compared the effects of distinct classes of NMDAR antagonists on electrographic seizures, axonal sprouting and neuronal survival using electrophysiological recordings and histology in hippocampal slice cultures treated chronically with vehicle, D-APV (high-affinity competitive), Ro 25-6981 or ifenprodil (NR2B-selective), or memantine (moderate-affinity uncompetitive). Granule cell layer field potential recordings revealed multiple spontaneous electrographic seizures in vehicle-treated cultures following GABA(A) receptor blockade. Compared to vehicle, seizures were dramatically reduced in cultures treated with NR2B selective antagonists and slightly increased in cultures treated with moderate-affinity uncompetitive or high-affinity competitive antagonists. In general, compared to vehicle, cultures treated with NR2B selective antagonists exhibited less sprouting of granule cell mossy fiber axons (MFS) and more granule cell layer neurons. Cultures treated with high-affinity competitive or moderate-affinity uncompetitive NMDAR antagonists showed increased MFS and fewer granule cell layer neurons. These data reveal differential effects of distinct classes of NMDAR antagonists on seizure expression, axonal sprouting and neuronal survival and suggest an association between these responses.

Animals↗

Peripherally acting NMDA receptor/glycineB site receptor antagonists inhibit morphine tolerance.

The present study focused on the role of peripheral ionotropic N-methyl-D-aspartate (NMDA) receptors in the development of tolerance to morphine-induced antinociception. An initial experiment revealed that NMDA channel blocker memantine, and NMDA receptor/glycine(B) site antagonist MRZ 2/576 inhibited maximal electroshock-induced convulsions (MES) in female NMR mice with respective potency of 5.93 and 20.8 mg/kg, while other NMDA receptor/glycine(B) site antagonists MRZ 2/596 and MDL 105,519 were ineffective, supporting lack of CNS activity of the latter two agents. This observation was also supported by blood-brain barrier experiments in vitro. In male Swiss mice, morphine (10 mg/kg) given for 6 days twice a day (b.i.d.) produced tolerance to its antinociceptive effects in the tail-flick test. The NMDA receptor/glycine(B) site antagonists, MRZ 2/576 at 0.03, 0.1, 0.3 mg/kg and MRZ 2/596 at 0.1, 0.3, 3 and 10 mg/kg attenuated the development of morphine tolerance. Similarly, in male C57/Bl mice, morphine (10 mg/kg) given for 6 days b.i.d. produced tolerance to its antinociceptive effects in the tail-flick test. Like in Swiss mice, in C57/Bl mice morphine tolerance was attenuated by both MRZ 2/576 and MRZ 2/596. Another NMDA receptor/glycine(B) site receptor antagonist, MDL 105,519 (that very weakly penetrates to the central nervous system) also inhibited morphine tolerance at the dose of 1 but not 0.1 mg/kg. Moreover, both naloxone hydrochloride (5 and 50 mg/kg) and centrally inactive naloxone methiodide (50mg/kg) inhibited morphine tolerance suggesting the involvement of peripheral opioid receptors in this phenomenon. The present data suggest that blockade of NMDA receptor/glycine(B) sites in the periphery may attenuate tolerance to the antinociceptive effects of morphine.

Animals↗

Selective cortical VGLUT1 increase as a marker for antidepressant activity.

The two recently characterized vesicular glutamate transporters (VGLUT) presynaptically mark and differentiate two distinct excitatory neuronal populations and thus define a cortical and a subcortical glutamatergic system (VGLUT1 and VGLUT2 positive, respectively). These two systems might be differentially implicated in brain neuropathology. Still, little is known on the modalities of VGLUT1 and VGLUT2 regulations in response to pharmacological or physiological stimuli. Given the importance of cortical neuronal activity in psychosis we investigated VGLUT1 mRNA and protein expression in response to chronic treatment with commonly prescribed psychotropic medications. We show that agents with antidepressant activity, namely the antidepressants fluoxetine and desipramine, the atypical antipsychotic clozapine, and the mood stabilizer lithium increased VGLUT1 mRNA expression in neurons of the cerebral cortex and the hippocampus and in concert enhanced VGLUT1 protein expression in their projection fields. In contrast the typical antipsychotic haloperidol, the cognitive enhancers memantine and tacrine, and the anxiolytic diazepam were without effect. We suggest that VGLUT1 could be a useful marker for antidepressant activity. Furthermore, adaptive changes in VGLUT1 positive neurons could constitute a common functional endpoint for structurally unrelated antidepressants, representing promising antidepressant targets in tracking specificity, mechanism, and onset at action.

Animals↗

Analgesic effects of mGlu1 and mGlu5 receptor antagonists in the rat formalin test.

mGlu1 and mGlu5 receptors have been implicated in pain associated with inflammation. In the present study, the formalin test was used to measure sustained pain with components of tissue injury. The aims of the present study were to assess: (i) the role of mGlu1 and mGlu5 receptors in inflammatory pain using selective antagonist EMQMCM, 1.25-5 mg/kg, as the mGlu1 receptor antagonist, and MPEP or MTEP, 2.5-10 mg/kg, as mGlu5 receptor antagonist; (ii) the possible interaction between mGlu1 and mGlu5 receptor antagonists and morphine; and (iii) whether tolerance develops to the analgesic effects of these antagonists after prolonged treatment. EMQMCM, MTEP and MPEP significantly reduced the manifestation of both phases of formalin response. However, all these mGlu receptor antagonists did not affect the withdrawal latencies in a model of acute pain (Hargreaves test), which has a different underlying mechanism. In the present study, the suppressive effect on formalin-induced pain behaviour was much stronger when mGlu1 and mGlu5 receptor antagonists were co-injected compared to administration of a single antagonist, but this effect was not seen when mGlu receptor antagonist was co-administered with morphine. This is in contrast to the pronounced inhibitory effects after co-treatment with morphine and the uncompetitive NMDA receptor antagonist memantine. The present study also provides the first direct in vivo evidence that prolonged administration of MTEP (5 mg/kg) over 7 days leads to the development of tolerance to its antinociceptive effects. Such tolerance was not observed when EMQMCM (5 mg/kg) was administered in the same manner. In conclusion, these results provide additional arguments for the role of group I mGlu receptors in pain with inflammatory conditions.

Analgesics↗

Response properties of antral mechanosensitive afferent fibers and effects of ionotropic glutamate receptor antagonists.

The ionotropic glutamate receptors N-methyl-d-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are present peripherally in the primary sensory afferent neurons innervating the viscera. Multiple studies have reported roles of glutamate receptors in gastric functions. However, no study has previously shown the direct influence of ionotropic glutamate receptor antagonist on vagal sensory neurons. The objective of this study was to investigate the effects of NMDA and AMPA receptor antagonists on mechanotransduction properties of vagal afferent fibers innervating the rat stomach. Action potentials were recorded from the hyponodal vagus nerve innervating the antrum of the Long-Evans rats. For antral distension (AD), a small latex balloon was inserted into the stomach and positioned in the antrum. The antral contractions were recorded with solid-state probe inserted into the water-filled balloon. Antral units were identified to isovolumic (0.2-1 ml) or isobaric AD (5-60 mm Hg). NMDA and AMPA receptor antagonists were injected in a cumulative fashion (1-100 micromol/kg, i.v.). After the conclusion of experiment, the abdomen was opened and receptive field was mapped by probing the serosa of the stomach. Thirty-two fibers were identified to AD. The receptive fields of 26 fibers were located in the posterior part of the antrum. All fibers exhibited spontaneous firing (mean: 7.00+/-0.97 impulses/s). Twenty fibers exhibited a rhythmic firing that was in phase with antral contractions, whereas 12 fibers exhibited non-rhythmic spontaneous firing unrelated to spontaneous antral contraction. Both groups of fibers exhibited a linear increase in responses to graded isovolumic or isobaric distensions. NMDA (memantine HCl and dizocilpine (MK-801)) and AMPA/kainate (6-cyano-7-nitroquinoxaline 2,3-dione; CNQX) receptor antagonists dose-dependently attenuated the mechanotransduction properties of these fibers to AD. However, competitive NMDA antagonist dl-2-amino-5 phosphopentanoic acid (AP-5) had no effect. The study documents that glutamate receptor antagonists can attenuate responses of gastric vagal sensory afferent fibers innervating the distal stomach, offering insight to potential pharmacological agents in the treatment of gastric disorders.

Action Potentials↗

Complementary therapy for the treatment of glaucoma: a perspective.

Although neuroprotective strategies and pharmaceutical agents have been initiated in the treatment of numerous disorders of the central and peripheral nervous systems, including trauma, epilepsy, stroke, Huntington disease, amyotrophic lateral sclerosis,and AIDS dementia, none have yet been applied to the treatment of glaucoma. A prospective, placebo-controlled, multi-institutional trial of memantine is underway. One would not expect the treatment modalities that form the bases of nonpharmaceutical, traditional medical systems to be used to lower IOP. Glaucoma was unknown when these medicinal treatments were developed over the centuries. Their primary use is in improving the cardiovascular and immune systems and in what is now called neuro-protection. Rather than single compounds that target a specific receptor and have demarcated side effects in other systems, plant products are a blend of many compounds and, according to those most versed in them, they achieve a balanced therapy, helping in specific symptomatic complexes while reducing side effects through ameliorating effects in other areas. It is not insignificant that, now that the rain forests are rapidly dwindling, together with their inhabitants and the knowledge of medicinal plants (especially in South America), the pharmaceutical companies are spending large amounts of money in a sudden, almost frantic attempt to gather the knowledge about rainforest plants before all has been completely lost. Proof of effects clinically in a chronic disease such as glaucoma remains largely lacking, and controlled trials are unlikely to be initiated, except perhaps through the National Institutes of Health, because these compounds have been in the public domain for many years. Perhaps those as yet unknown or un-recorded are patentable and perhaps these include drugs known only to small surviving communities of hunter-gatherers, which explains the pharmaceutical interest in these areas. When more accurate and rapid means of assessment of progression of glaucomatous damage than perimetry and optic nerve head photography are eventually developed and trials can be reduced in time, number of subjects, or even the use of nonhuman subjects for the bulk of studies, studies could be done for verification of effect of various compounds and also comparative studies. At the present time, GBE is the best documented of all the complementary medicinal agents and seems to have the greatest potential value. Ginkgo biloba extract has numerous properties that theoretically should be beneficial in treating non-IOP-dependent mechanisms in glaucoma. Its multi-ple beneficial actions, including increased ocular blood flow, antioxidant activity, platelet activating factor inhibitory activity, nitric oxide inhibition, and neuroprotective activity, combine to suggest that GBE could prove to be of major therapeutic value in the treatment of glaucoma.

Complementary Therapies↗

Effects of NMDA receptor antagonists on acute mu-opioid analgesia in the rat.

Mixed research findings have led to a debate regarding the effect of N-methyl-D-aspartate (NMDA) receptor antagonists on opiate analgesia. NMDA antagonists have been found in various studies to enhance, to inhibit, or to have no effect on opiate analgesia. The present research used a single protocol to explore the effects of six NMDA receptor antagonists on acute morphine (3.0 mg/kg s.c.) and fentanyl (0.05 mg/kg s.c.) analgesia in adult male Sprague-Dawley rats. NMDA receptor antagonists were selected based on their abilities to block various sites on the NMDA receptor complex, including the noncompetitive antagonists MK-801 (0.1 and 0.3 mg/kg i.p.), dextromethorphan (10.0 and 30.0 mg/kg i.p.), and memantine (3.0 and 10.0 mg/kg i.p.), a glycine site antagonist, (+)-HA-966 (10.0 and 30.0 mg/kg i.p.), a competitive antagonist, LY235959 (1.0 and 3.0 mg/kg i.p.), and a polyamine site antagonist, ifenprodil (1.0 and 3.0 mg/kg i.p.). Analgesia was assessed using the tail-flick test. A single dose of each opiate was used. The low doses of the antagonists, which are known to produce significant neural and behavioral actions at NMDA receptors, had no effect on morphine or fentanyl analgesia. At the higher doses, morphine analgesia was significantly enhanced by LY235959 (3.0 mg/kg), and fentanyl analgesia was significantly enhanced by LY235959 (3.0 mg/kg), dextromethorphan (30.0 mg/kg), and (+)-HA-966 (30.0 mg/kg). Enhancement of analgesia occurred without any apparent adverse side effects. None of the NMDA antagonists affected tail-flick responses on their own, except the higher dose of LY235959 (3.0 mg/kg), which produced a mild analgesic effect. Because no consistent effects were observed, the data suggest that NMDA receptors are not involved in acute mu-opioid analgesia. The mechanisms underlying the enhancement of opiate analgesia by selected NMDA antagonists, such as LY235959, dextromethorphan, and (+)-HA-966, remain to be determined.

Analgesics, Opioid↗

Role of NMDA, nicotinic, and GABA receptors in the steady-state visual-evoked potential in rats.

Agonists and antagonists at the NMDA, GABA, and nicotinic acetylcholine receptors were administered to adult male rats to evaluate the contribution of these pathways to the visual-evoked potential (VEP). Rats were presented with an onset/offset pattern at a temporal frequency (4.55 Hz) resulting in a steady-state VEP. Averaged VEPs were Fourier transformed and VEP amplitudes were calculated at 1x stimulus frequency (F1) and 2x stimulus frequency (F2). About 30 min after administration, NMDA (10 mg/kg, i.p.; n = 9) increased F1 amplitude by 350% and decreased F2 amplitude by 48%. Memantine (4.5 mg/kg, i.p.; n = 10) increased F1 amplitude by 50%, 10 min post-injection. Similarly, nicotine (0.1 mg/kg, s.c.; n = 9) increased F1 amplitude by 55%, 20 min after drug administration. Muscimol (1 mg/kg, i.p.; n = 10) increased F1 amplitude significantly from 20 to 45 min post-injection. Mecamylamine (6 mg/kg, i.p.; n = 10) decreased F2 amplitude by 70% during the 60-min testing session. Bicuculline (0-0.5 mg/kg, i.p.; n = 8-10 rats/dose) did not significantly alter either F1 or F2 amplitudes. Results indicate important roles for glutamate and nicotinic acetylcholine receptors in both F1 and F2, while GABA receptors contribute to F1.

Animals↗

Simple pharmacological test battery to assess efficacy and side effect profile of centrally acting muscle relaxant drugs.

INTRODUCTION: Centrally muscle relaxants (CMRs) are used mainly for treating muscle spasticities of neurological origin, and painful muscle spasms due to rheumatologic conditions. Their use is frequently associated with dose-limiting adverse effects. New drugs with improved side-effect characteristics are badly needed. However, there is no general agreement in the pharmacological literature on what methods are adequate to assess CMR effect and side effects in behaving rodents, which may hinder the development of new drugs. Here we report on the establishment of a simple pharmacological test battery, which was used to compare efficacies and side effect profiles of 11 compounds with central muscle relaxant action, in mice (intraperitoneal application). METHODS: For measuring muscle relaxant activity, (1) a new tremor model (GYKI 20039-induced tremor) and (2) the morphine-induced Straub-tail assay were used. The former, newly developed method has advantages over harmaline- or LON-954-induced tremor. For detecting side effect liability (ataxia, sedation, impairment of voluntary motor functions), (1) the rota-rod test, (2) measurement of spontaneous motility, (3) the weight-lifting test and (4) the thiopental sleep test were used. RESULTS: Among the 11 muscle relaxant compounds tested (tolperisone, eperisone, silperisone, diazepam, baclofen, tizanidine, afloqualon, mephenesin, zoxazolamine, memantine and carisoprodol), the calculated safety ratios (i.e. ID50 for side effect/ID50 for muscle relaxant effect) varied in a wide range. Silperisone seems to have the most advantageous profile (safety ratios range between 1.7 and 3.3 in the different pairs of assays) compared to the other tested drugs with lower (one or more ratios below 1.5, and often far below 1) and more varying ratios. DISCUSSION: Therapeutic indices calculated from the results of these in vivo experiments for the clinically used muscle relaxants are in agreement with their adverse effect profiles in humans. Thus the present test battery seems to be suitable for predicting the possible clinical utility of newly synthesized compounds.

Animals↗

Opposite effects of glutamate antagonists and antiparkinsonian drugs on the activities of DOPA decarboxylase and 5-HTP decarboxylase in the rat brain.

This study measured the activities of L-DOPA and 5-HTP decarboxylase (DDC and 5-HTPDC) in the substantia nigra and corpus striatum of reserpine-treated rats. Acute injection of the NMDA receptor antagonists CGP 40116 (5 mg/kg) and HA 966 (5 mg/kg), and to a lesser extent eliprodil (10 mg/kg), greatly elevated DDC in both structures, whilst having no effect on (nigra) or inhibiting (striatum) 5-HTPDC. L-DOPA (25 mg/kg) on its own inhibited both enzymes in either brain region. The weak NMDA receptor-channel blockers (and antiparkinsonian drugs) budipine (10 mg/kg), memantine (40 mg/kg) and amantadine (40 mg/kg) strongly increased DDC, whilst not affecting or decreasing 5-HTPDC activity in nigra and striatum. The L-DOPA-induced suppression of DDC was mostly reversed by all three antiparkinsonian drugs, whilst L-DOPA-induced inhibition of 5-HTPDC was only reversed by CGP 40116 (striatum only). It is concluded that glutamate exerts a differential physiological influence on the biosynthesis of dopamine and 5-HT in the brain, by tonically suppressing DDC and tonically stimulating 5-HTPDC. The L-DOPA-induced reduction in DDC may help to explain the eventual loss of efficacy of L-DOPA therapy in parkinsonian patients. It is suggested, however, that it may be possible to extend the lifetime of L-DOPA therapy with drugs which potentiate the activity of DDC, such as budipine and the 1-aminoadamantanes.

Animals↗

Uncompetitive NMDA receptor antagonists potentiate morphine antinociception recorded from the tail but not from the hind paw in rats.

We investigated the effects of pretreatment with low-affinity, uncompetitive NMDA receptor antagonists on morphine-induced antinociception in rats using the same intensity of thermal stimulus applied to the tail and the paws. Similar baseline responses to thermal stimuli of the same intensity were recorded from tails and hind paws. However, morphine produced equal antinociception from the tail and hind paw when used at doses of 2.5 and 6 mg/kg, respectively. These doses were used in further experiments. Thirty minutes before morphine, rats were administered the NMDA receptor antagonists dextromethorphan (2.5--30 mg/kg), memantine (2.5--15 mg/kg) and MRZ 2/579 (1-amino-1,3,3,5,5-pentamethyl-cyclohexane HCl) (1.25--10 mg/kg). All three compounds significantly and dose-dependently potentiated morphine-induced antinociception recorded from the tail. However, none of these NMDA receptor antagonists affected morphine antinociception recorded from the paw. These findings suggest that low-affinity NMDA receptor antagonists modulate differently morphine antinociceptive activity recorded from the tail and hind paws.

Analgesics↗

Screening of novel pentacyclo-undecylamines for neuroprotective activity.

A novel series of pentacyclo-undecylamines with 8-benzylamino-8,11-oxapentacyclo[5.4.0.0(2,6).0(3,10).0(5,9)]undecane (NGP1-01) as the lead compound was synthesised and screened for neuroprotective activity in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) parkinsonian mouse model. We hypothesise that these compounds may attenuate excitotoxic neuronal cell death mediated through the NMDA receptor (similar to memantine), and through calcium channel block. The pentacyclo-undecylamines (300 mg/kg) were administered to C57BL/6 mice 30 min before intraperitoneal (i.p.) MPTP administration (35 mg/kg). Striatal dopamine, 3,4-hydroxyphenylacetic acid (DOPAC), and homovanillic acid levels were analysed 10 days later by means of HPLC with electrochemical detection. Increased levels of DOPAC and homovanillic acid were observed when some of the test compounds were administered together with MPTP (compared to animals receiving only MPTP). One compound in the series, 8-phenylethylamino-8,11-oxapentacyclo[5.4.0.0(2,6).0(3,10).0(5,9)]undecane, attenuated MPTP-induced striatal dopamine depletion when compared to animals treated with MPTP only (p<0.05).

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗

(2-Naphthoxy)acetyl chloride, a simple fluorescent reagent.

In continuing the search for fluorescent reagents for analytical derivatization in chromatography, we found a simple chemical, (2-naphthoxy)acetyl chloride, with potential fluorophore/chromophore characteristics for the highly sensitive detection of analytes with an amino function. The reagent has an auxochrome (a substituted alkoxy moiety) attached to the fluorophoric/chromophoric naphthalene system, resulting in favorable spectrophotometric properties. The reagent can be easily prepared from (2-naphthoxy)acetic acid and has been used in organic synthesis; it is initially introduced as a fluorescent reagent to derivatise amantadine and memantine (amino pharmaceuticals) as model analytes. The resulting naphthoxy derivatives of the drugs can be analyzed at sub-microM levels by HPLC with fluorimetric detection (excitation wavelength 227 nm, emission wavelength 348 nm). Application of the reagent to the fluorimetric derivatization of important biological amines for sensitive detection can be expected.

Calibration↗

Treatment with donepezil in Alzheimer patients with and without cerebrovascular disease.

Donepezil, a selective acetylcholinesterase inhibitor, is approved for the symptomatic treatment of mild to moderate Alzheimer's disease (AD). In a post-marketing surveillance (PMS) study in Germany, patients under routine treatment conditions were observed while treatment was switched from other antidementia drugs (i.e., nootropics) to donepezil. A total of 913 patients were enrolled (60.1% female, mean+/-S.D. age 73.4+/-8.6 years, mean Mini-Mental Status Examination [MMSE] 18.0+/-5.3), and were treated with donepezil (5 or 10 mg/day according to recommended dosing). 709/913 (77.1%) of patients had been pretreated with other antidementive drugs (piracetam, memantine, ginkgo, and others). In 29.6% of patients, investigators documented concomitant cerebrovascular disease (CVD+) according to their clinical judgment. Observation period was 3 months for the individual patient. Efficacy parameters were changes in MMSE, global clinical (investigators) judgment of efficacy, and a clinical judgment about the patients' quality of life (QoL). Adverse events were also analyzed. The objective of the present investigation was to compare-in a "real-life" setting-the differential efficacy and tolerability of donepezil in AD patients with and without concomitant cerebrovascular disease. After 3 months, patients had improved by a mean MMSE change from baseline of 2.2 points (CVD+: 2.4 pts, CVD-: 2.1 pts). QoL was judged "improved" in 70.0% of patients (CVD+: 72.5%, CVD-: 69.6%). Adverse events were reported in 85/913 (9.3%) of patients (CVD+: 11.2%, CVD-: 7.9%). Reported adverse events were substantially less than reported previously in controlled clinical trials. This suggests that donepezil therapy is effective and well tolerated in AD patients, both with and without concomitant cerebrovascular disease.

Adult↗

Increased expression of neuronal Src and tyrosine phosphorylation of NMDA receptors in rat brain after systemic treatment with MK-801.

We have observed that systemic treatment with the uncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 increases Src expression and NMDA receptor phosphorylation in rat brain. A partial cDNA encoding rat neuronal Src was isolated and its sequence was used to design specific oligonucleotide probes. Systemically administered MK-801 (5 mg/kg for 4 h) increased by 28+/-4% mRNA expression of neuronal Src in the superficial layers of the parietal cortex. This effect was observed at doses as low as 0.2 mg/kg. A similar, although more modest, induction was observed 6 h after phencyclidine (15 mg/kg) administration, but not after high doses of memantine and ketamine. The MK-801-induced effect was not blocked by pretreatment with clozapine. Consistent with the increase in mRNA levels, cortical Src protein was increased to 186 +/- 24% of control 24 h after MK-801 treatment. Total cellular Src activity was also increased in parietal cortex homogenates 4 h after MK-801 (5 mg/kg). Moreover, MK-801 treatment (0.5 mg/kg and 5 mg/kg for 4 h) increased tyrosine phosphorylation, but not protein levels, of the NMDA receptor subunit NR2A. These results provide evidence for a contribution of Src and tyrosine phosphorylation of NMDA receptors in the pharmacological actions of uncompetitive NMDA receptor antagonists.

Amino Acid Sequence↗

Prevention of cocaine-induced convulsions and lethality in mice: effectiveness of targeting different sites on the NMDA receptor complex.

N-methyl-D-aspartate (NMDA) receptors appear to be involved in the behavioral toxic effects of cocaine. Therefore, different classes of NMDA receptor antagonists were compared for their ability to attenuate cocaine-induced convulsions and lethality in male, Swiss Webster mice. The mice were pre-treated (i.p.) with vehicle or an antagonist from one of the following classes: NMDA/glycine site antagonist (7-chlorokynurenic acid, ACEA-1021, ACEA-1031, ACEA-1328, DCQX, R(+)-HA-966), competitive antagonist (CPP, D-AP7), channel blocker (MK-801, memantine), or allosteric modulator (ifenprodil, CP-101,606, Co 101022, haloperidol). After a 15 min pre-treatment period, the mice were administered a convulsive (60 mg/kg, i.p.) or lethal (125 mg/kg, i.p.) dose of cocaine, equivalent to the calculated ED/LD97 values. Pre-treatment with competitive or NMDA/glycine site antagonists dose-dependently attenuated cocaine-induced convulsions and lethality (P<0.05). Pre-treatment with channel blockers or allosteric modulators of the NMDA receptor protected against cocaine-induced convulsions (P<0.05), but were ineffective or less effective than the competitive and glycine site antagonists in preventing death. The glutamate release inhibitor riluzole failed to prevent both the convulsions and lethality induced by cocaine. Significantly, post-treatment with NMDA/glycine site antagonists (ACEA-1021, ACEA-1031, ACEA-1328) after a cocaine overdose prevented death in a significant number of animals. The data suggest that NMDA receptors are involved in the pathophysiology of a cocaine overdose.

Animals↗

Novel approaches in diagnosis and therapy of Creutzfeldt-Jakob disease.

The scrapie prion protein, PrP(Sc), as well as its peptide fragment, PrP106-126, are toxic on neuronal cells, resulting in cell death by an apoptotic, rather than necrotic mechanism. The apoptotic process of neuronal cells induced by prion protein supports diagnosis and offers potential targets for therapeutic intervention of the prion diseases. Among the cerebrospinal fluid (CSF) proteins, which may serve as markers of neuronal cell death associated with prion diseases, the 14-3-3 protein(s) turned out to be the most promising one. A new sensitive assay allows the detection of even small changes in the normally low levels of these proteins. In vitro, the toxic effects displayed by PrP(Sc) and its peptide fragment can be blocked by antagonists of N-methyl-D-aspartate (NMDA) receptor channels, like Memantine. Also Flupirtine, a non-opiod analgesic drug, which is already in clinical use, was found to display in vitro a strong cytoprotective effect on neurons treated with PrP(Sc) or PrP106-126. This drug acts like a NMDA receptor antagonists, but does not bind to the receptor. Clinical trials on prion diseases with Flupirtine are in progress. Flupirtine was found to enhance the intracellular levels of the antiapoptotic protein Bcl-2 and the antioxidative agent glutathione (GSH). Due to its favourable pharmacokinetic profile, Flupirtine is considered to be a promising drug to prevent neuronal death in Creutzfeldt-Jakob disease (CJD) and other neurodegenerative disorders occurring with age, e.g. Alzheimer's disease.

Animals↗