Imidazoline-induced neuroprotective effects result from blockade of NMDA receptor channels in neuronal cultures

Imidazolines have been shown to be neuroprotective in focal and global ischemia in the rat. However, their mechanism of action is still unclear. We have studied the neuroprotective effects of imidazolines against NMDA-induced neuronal death and hypoxic insult in cerebellar and striatal neuronal cultures. All of the imidazolines tested decreased the NMDA-mediated neurotoxicity in a non-competitive manner. Antazoline was the most effective (IC(50) of 5 microM, maximal neuroprotection reaching 90% at 100 microM). The neuroprotective effects were still present when the imidazolines were applied during the post-insult period. Antazoline, idazoxan and guanabenz also showed neuroprotective effects against hypoxia-induced neuronal death (neuroprotection reaching 95% for antazoline at 100 microM). Antazoline was still active if applied during the reoxygenation period (15% neuroprotection). To determine the mechanism of the neuroprotective effects, the possible interaction of imidazolines with NMDA receptors was studied. Imidazolines dose-dependently and non-competitively inhibited NMDA currents. As found for the neuroprotective effects, antazoline was the most effective imidazoline, with an IC(50) of 4 microM and a maximal inhibition of 90% at 100 microM. This blockade was rapid, reversible and voltage-dependent. We compared these effects to those of the classical non-competitive antagonist of NMDA channels, MK-801. In contrast to imidazolines, blockade of the NMDA current by MK-801 was voltage-independent and reversible only at positive potentials. When co-applied with MK-801, antazoline prevented the long lasting blockade of the NMDA current by MK-801. These results are consistent with the existence of overlapping binding sites for these drugs on the NMDA receptor channel. They indicate that imidazolines exert a strong neuroprotective effect against excitotoxicity and hypoxia in cerebellar and striatal primary neuronal cultures by inhibiting NMDA receptors. Since these effects were non-competitive, imidazolines appear to be interesting new drugs with therapeutic potential.     

Postnatal changes in N-methyl-D-aspartate receptor binding and stimulation by glutamate and glycine of [3H]-MK-801 binding in human temporal cortex.

1. Homogenates of human infant and adult temporal cortex were used to measure [3H]-TCP and [3H]-MK-801 binding to the N-methyl-D-aspartate (NMDA)-coupled ion channel phencyclidine site. 2. Both [3H]-TCP and [3H]-MK-801 binding increased in infant cortex by > 100% between term and 26 weeks suggesting that the numbers of NMDA receptors increase during postnatal brain development. 3. [3H]-MK-801 binding was measured under non-equilibrium conditions in temporal cortex homogenates with the addition of 100 microM of L-glutamate plus a range of concentrations (0.05 microM-100 microM) of glycine. Glutamate and glycine increased [3H]-MK-801 binding by stimulating NMDA receptors and improving [3H]-MK-801 access to ion channel binding sites; maximum stimulation in adult and infant temporal cortex was achieved with 100 microM glutamate plus 5 microM glycine; a higher concentration of glycine (50 microM) reduced [3H]-MK-801 binding to below maximum. 4. The stimulation by 100 microM glutamate plus 5 microM glycine of [3H]-MK-801 binding in infant temporal cortex was affected by postnatal age. For example, although the stimulation of [3H]-MK-801 binding in 5-6 week infant cortex (236% of basal) was similar to adult cortex (230% of basal), in samples taken from infants aged 5-6 months glycine (plus glutamate) stimulation of [3H]-MK-801 binding (392% of basal) was substantially greater than that measured in adult temporal cortex. 5. The binding of [3H]-glycine to the glycine modulatory site associated with the NMDA receptor in infant cortex also increased with postnatal age by > 100% between term and 26 weeks.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantitative autoradiography of [3H]-MK-801 binding sites in mammalian brain.

1. An in vitro receptor autoradiography procedure is described for visualizing binding sites for the excitatory amino acid antagonist radiolabelled MK-801, in rat and gerbil brain sections. 2. Ten micron sections were labelled by incubation at room temperature for 20 min in 30 nM [3H]-MK-801. This was followed by 2 rinses for 20 s in fresh buffer solution. Specifically bound ligand determined with 100 microM unlabelled MK-801 amounted to 55-60% of total. 3. Phencyclidine, (+/-)-SKF 10047, ketamine and 2-aminophosphonovaleric acid (APV) (all 100 microM) prevented the specific binding of [3H]-MK-801. L-Glutamate and N-methyl D-aspartate (NMDA) (100 microM) had no effect. However, L-glutamate prevented the inhibition by APV. 4. The highest concentrations of [3H]-MK-801 binding sites occurred in the hippocampal formation, cerebral cortex, olfactory bulb and thalamus. Very low levels were detected in the brain stem and cerebellum. 5. The distribution of [3H]-MK-801 binding sites was comparable to that of NMDA sites and phencyclidine sites (labelled with [3H]-TCP) but not with high-affinity sigma sites labelled with [3H]-3-PPP. 6. The density of [3H]-MK-801 binding sites in the gerbil hippocampus was examined 1, 2, 6 and 22 days after unilateral carotid artery occlusion for 10 min. Only at 6 and 22 days was the binding reduced (by 36% and 46% respectively) in the CA1 region whereas a significant neuronal loss was apparent at day 2. In CA2 a decrease in binding was only evident at day 22. 7. These results indicate that binding sites for [3H]-MK-801 can be detected in mammalian brain sections by receptor autoradiography. Their distribution supports an association with the NMDA receptor complex and the loss in the hippocampus after carotid artery occlusion indicates their presence on pyramidal cells is vulnerable to ischaemic insult.     

Evidence for imidazoline receptors involvement in the agmatine antidepressant-like effect in the forced swimming test

This study investigated the involvement of the imidazoline receptors in the antidepressant-like effect of agmatine in the forced swimming test. The antidepressant-like effects of agmatine (10 mg/kg, i.p.) in the forced swimming test was blocked by pretreatment of mice with efaroxan (1 mg/kg, i.p., an imidazoline I1/alpha2-adrenoceptor antagonist), idazoxan (0.06 mg/kg, i.p., an imidazoline I2/alpha2-adrenoceptor antagonist) and antazoline (5 mg/kg, i.p., a ligand with high affinity for the I2 receptor). A subeffective dose of agmatine (0.001 mg/kg, i.p.) produced a synergistic antidepressant-like effect with clonidine (0.06 mg/kg, i.p, an imidazoline I1/alpha2-adrenoceptor agonist), moxonidine (0.5 mg/kg, i.p., an imidazoline I1/alpha2-adrenoceptor agonist), antazoline (1 mg/kg, i.p.) and MK-801 (0.001 mg/kg, i.p., a non-competitive NMDA receptor antagonist), but not with efaroxan (1 mg/kg, i.p.) and idazoxan (0.06 mg/kg, i.p.). Pretreatment of mice with yohimbine (1 mg/kg, i.p., an alpha2-adrenoceptor antagonist) blocked the synergistic antidepressant-like effect of agmatine (0.001 mg/kg, i.p.) with clonidine (0.06 mg/kg, i.p). A subeffective dose of MK-801 (0.001 mg/kg, i.p.) produced a synergistic antidepressant-like effect with antazoline (5 mg/kg, i.p.), but not with efaroxan (1 mg/kg, i.p.) or idazoxan (0.06 mg/kg, i.p.). In conclusion, this study suggests that the anti-immobility effect of agmatine in the forced swimming test is dependent on its interaction with imidazoline I1 and I2 receptors.     

Effects of memantine on recombinant rat NMDA receptors expressed in HEK 293 cells.

1. The actions of the uncompetitive N-methyl-D-aspartate (NMDA) receptor antagonists, memantine (1-amino-3,5-dimethyladamantane) and (+)-MK-801 ((+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate, dizocilpine), on recombinant NMDA receptors has been studied by use of the whole-cell patch clamp technique. 2. Human embryonic kidney (HEK) 293 cells were transiently transfected with different NMDA receptor subunit combinations (NR1a/NR2A, NR1a/NR2B and NR1a/NR2D). A mutant form of the green fluorescent protein (GFP) cotransfected with the NMDA receptor subunits to enable the visualization of transfected cells. 3. Memantine (0.3-30 microM) blocked L-glutamate (100 microM)-mediated currents in a concentration-dependent manner in NR1a/NR2A, NR1a/NR2B and NR1a/NR2D transfected cells with IC50 values (at -70 mV) of 0.93 +/- 0.15 microM, 0.82 +/- 0.12 microM and 0.47 +/- 0.06 microM (mean +/- s.c. mean), respectively. 4. The memantine-induced block was strongly voltage-dependent. Alteration of the holding potential from -70 mV to +60 mV resulted in an e-fold increase in the IC50 values per 30-33 mV change in membrane potential, for all 3 subunit combinations investigated. 5. The kinetics of the actions of memantine (30 microM) were investigated for the NR1a/2A combination, in 6 cells (13-15 determinations). At -70 mV, the block and recovery from block were both best described by two exponentials with time-constants of 201 +/- 23 ms (81 +/- 2%) and 3.9 +/- 0.6 s and 597 +/- 94 ms (18 +/- 1%) and 18.6 +/- 2.4 s, respectively. The predominant effect of depolarization was to increase the weight of the faster recovery time-constant. Kinetic analysis suggests that these results are consistent with previously proposed Markov models. 6. (+)-MK-801 was studied briefly for comparative purposes. (+)-MK-801 (200 nM) preferentially blocked NMDA receptor currents (at -70 mV) in NR1a/NR2A and NR1a/NR2B (82 +/- 10% and 93 +/- 2% depressions) compared to NR1a/NR2D (38 +/- 7%) transfected cells. (+)-MK-801 appeared to be less voltage-dependent than memantine on all three receptor combinations. 7. In conclusion, memantine was a voltage-dependent antagonist of recombinant rat NMDA receptors expressed in HEK 293 cells but showed little selectivity between the subunits investigated. Its actions on these recombinant receptor combinations are similar to its actions on native NMDA receptors.     

N-methyl-D-aspartate exposure blocks glutamate toxicity in cultured cerebellar granule cells.

Exposure of cultured cerebellar granule cells to glutamate results in a concentration-dependent (EC50 = 22.7 +/- 0.4 microM) and delayed (24-72 hr) neurotoxicity, which is blocked by the specific N-methyl-D-aspartate (NMDA) receptor antagonists 2-amino-5-phosphovalerate and MK-801 but is unaffected by the non-NMDA receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione and 6,7-dinitroquinoxaline-2,3-dione. Although glutamate toxicity in these cells is mediated by the NMDA subtype of glutamate receptor, pretreatment of cerebellar granule cells with subtoxic concentrations of NMDA markedly antagonizes the neurotoxic actions of glutamate, with an IC50 of 55 +/- 4 microM. The neuroprotective effect of NMDA requires a preincubation time of approximately 120 min to be fully manifested and does not require the presence of NMDA during glutamate exposure. These data demonstrate that NMDA receptors mediate both neurotoxicity and neuroprotection in cerebellar granule cells. Among four glutamate receptor agonists tested (NMDA, quisqualate, ibotenate, and kainate), only NMDA was able to provide a robust neuroprotection against glutamate toxicity. Quisqualate was neither neurotoxic nor neuroprotective, whereas ibotenate, which was nontoxic by itself, induced a small degree of neuroprotection. In contrast, kainate, which was neurotoxic to cerebellar granule cells, also provided considerable neuroprotection against glutamate toxicity. Because preincubation of cerebellar granule cells with NMDA fails to alter NMDA receptor-mediated phosphoinositide hydrolysis or the specific binding of [3H]MK-801 to NMDA receptors, it appears that the neuroprotective effects of NMDA are not due to NMDA receptor desensitization.     

The novel anticonvulsant MK-801 binds to the activated state of the N-methyl-D-aspartate receptor in rat brain.

The influence of endogenous and exogenous acidic amino acids on the binding of [3H]-MK-801, a selective, non-competitive antagonist of N-methyl-D-aspartate (NMDA) receptors, has been investigated in rat cerebral cortex crude synaptic membranes (CSM). Removal of endogenous glutamate and aspartate from CSM by repeated washing reduced the affinity of [3H]-MK-801 for its binding site (with no change in the total number of binding sites) and increased NMDA-sensitive L-[3H]-glutamate binding. In washed CSM, competitive NMDA antagonists of the DL-alpha-amino-omega-phosphonocarboxylate series reduced [3H]-MK-801 binding and NMDA-sensitive L-[3H]-glutamate binding, the most active compounds being 2-amino-5-phosphonovalerate (AP5) and 2-amino-7-phosphono-heptanoate (AP7). Exogenous excitatory amino acid agonists enhanced the binding of [3H]-MK-801 to washed CSM by up to 700%. A selective involvement of NMDA receptors in these effects was indicated by the excellent correlation between EC50s for stimulation of [3H]-MK-801 binding and IC50s for inhibition of NMDA-sensitive L-[3H]-glutamate binding in the same membranes. The selective, competitive NMDA receptor antagonist D-AP5 blocked the L-glutamate-induced increase in [3H]-MK-801 binding in a competitive manner with a pA2 value of 6.0. These results seem to reflect a molecular interaction between two distinct components of the NMDA receptor complex: the transmitter recognition site and the site through which MK-801 exerts its antagonist effects, possibly the ion channel.     

Neuroprotective profile of lifarizine (RS-87476) in rat cerebrocortical neurones in culture.

1. The ability of the neuroprotective agent, lifarizine (RS-87476), to mitigate veratridine-, cyanide- and glutamate-induced toxicity in rat embryonic cerebrocortical neurones in primary culture has been compared with that of tetrodotoxin (TTX), nitrendipine, (+)-MK-801 and (-)-MK-801. Lactate dehydrogenase (LDH) released into the culture medium was used as the indicator of cell viability. 2. Incubation of cultures for 16 h in a medium containing veratridine (10(-4) M), sodium glutamate (10(-3) M) or sodium cyanide (10(-3) M) resulted in consistent elevations of LDH activity in the culture medium. The ability of compounds to attenuate these elevations was expressed as the concentration required to inhibit the increases in LDH release by 50% (IC50). 3. Neurotoxicity induced by veratridine was inhibited by lifarizine (IC50 = 4 x 10(-7) M), TTX (IC50 = 3 x 10(-8) M) and nitrendipine (IC50 = 3 x 10(-5) M). In contrast, (+)-MK-801 (up to 3 x 10(-5) M) was ineffective against this insult. 4. Glutamate-induced neurotoxicity was inhibited by (+)-MK-801 (IC50 = 1.4 x 10(-8) M) and to a lesser extent by (-)-MK-801 (IC50 = 1 x 10(-7) M), but was unaffected by lifarizine, TTX or nitrendipine (up to 10(-6) M). 5. (+)-MK-801 was effective against sodium cyanide-induced neurotoxicity (IC50 = 1.9 x 10(-8) M), whereas lifarizine and TTX (up to 10(-6) M) and nitrendipine (up to 3 x 10(-6) M) were without protective activity against this insult.(ABSTRACT TRUNCATED AT 250 WORDS)     

Anticonvulsant preclinical profile of CHF 3381: dopaminergic and glutamatergic mechanisms

Following intraperitoneal or oral administrations, CHF 3381 ([n-(2-indanyl)-glycinamide hydrochloride]) protected rats against maximal electroshock (MES) test seizures. As glutamatergic pathways play a pivotal role in epilepsy, to better characterize the molecular mechanisms of action of CHF 3381, the drug effects on the binding of the excitatory amino acid antagonist [3H]-MK-801 in the presence of n-methyl-D-aspartate (NMDA), spermidine, or the combination of both ligands, were studied. CHF 3381 inhibited the [3H]-MK-801 specific binding in a noncompetitive fashion in respect to NMDA and polyamines recognition sites. CHF 3381 failed to change the kinetic characteristic of glycine B receptors labeled with [3H]-glycine; in contrast, it significantly increased K(d) values when the receptors were labeled with the more specific compound [3H]-MDL 105,519. CHF 3381 antagonized dopamine (DA)-induced behavioral responses and inhibited, in a glycine-dependent manner, the NMDA-induced [3H]-DA release from rat striatal slices, but it failed to change either the kinetic characteristics of D1, D2, or D3 receptors in synaptic plasma membranes (SPM) or the [3H]-DA uptake from striatal synaptosomes. Moreover, in primary cell cultures of cortical neurons, this drug exhibited glycine-independent neuroprotective effects against glutamate-induced excitotoxicity. It is concluded that this compound could have a potential use in several disease states where a pathological high level of NMDA receptor activation is thought to occur.     

Neuroprotective lignans from the bark of Machilus thunbergii

The CH (2)Cl (2) fraction of the bark of Machilus thunbergii Sieb. et Zucc. (Lauraceae) significantly protected primary cultures of rat cortical cells exposed to the excitotoxic amino acid, L-glutamate. (-)-Isoguaiacin, meso-dihydroguaiaretic acid, machilin A, (+)-galbelgin, licarin A, (-)-sesamin, and (+)-guaiacin were isolated by bioactivity-guided fractionation from the CH (2)Cl (2) fraction and further separated using chromatographic techniques. Isoguaiacin, meso-dihydroguaiaretic acid, licarin A and (+)-guaiacin had significant neuroprotective activities against glutamate-induced neurotoxicity in primary cultures of rat cortical cells at concentrations ranging from 0.1 microM to 10.0 microM.     

Cis-2,4-methanoglutamate is a potent and selective N-methyl-D-aspartate receptor agonist

Cis- and trans-2,4-methanoglutamate were compared with L-glutamate as acidic amino acid ligands. Cis-2,4-methanoglutamate had a Ki of 0.052 microM against N-methyl-D-aspartate (NMDA)-specific L-[3H]glutamate binding compared with 0.050 microM for L-glutamate. Cis-2,4-methanoglutamate exhibited no significant affinity against [3H]kainate or [3H]alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate ([3H]AMPA) binding. Trans-2,4-methanoglutamate had no significant affinity for any of these sites. Cis-2,4-methanoglutamate increased [3H]N-1[2-thienyl]cyclohexyl-3,4-piperidine [( 3H]TCP) binding with EC50 of 0.35 +/- 0.14 microM. It produced an inward current in rat brain mRNA-injected Xenopus oocytes which was blocked by the NMDA antagonist, D-2-amino-7-phosphonoheptanoate (D-AP7). Cis-2,4-methanoglutamate (EC50 = 15.9 microM) was 100-fold more potent than L-glutamate (EC50 = 1,584 microM) in reducing the excitatory postsynaptic potential in CA1 of hippocampal slices. Cis-2,4-methanoglutamate is the most potent, selective NMDA agonist known.     

Donepezil attenuates excitotoxic damage induced by membrane depolarization of cortical neurons exposed to veratridine

Long-lasting membrane depolarization in cerebral ischemia causes neurotoxicity via increases of intracellular sodium concentration ([Na+]i) and calcium concentration ([Ca2+]i). Donepezil has been shown to exert neuroprotective effects in an oxygen-glucose deprivation model. In the present study, we examined the effect of donepezil on depolarization-induced neuronal cell injury resulting from prolonged opening of Na+ channels with veratridine in rat primary-cultured cortical neurons. Veratridine (10 microM)-induced neuronal cell damage was completely prevented by 0.1 microM tetrodotoxin. Pretreatment with donepezil (0.1-10 microM) for 1 day significantly decreased cell death in a concentration-dependent manner, and a potent NMDA receptor antagonist, dizocilpine (MK801), showed a neuroprotective effect at the concentration of 10 microM. The neuroprotective effect of donepezil was not affected by nicotinic or muscarinic acetylcholine receptor antagonists. We further characterized the neuroprotective properties of donepezil by measuring the effect on [Na+]i and [Ca2+]i in cells stimulated with veratridine. At 0.1-10 microM, donepezil significantly and concentration-dependently reduced the veratridine-induced increase of [Ca2+]i, whereas MK801 had no effect. At 10 microM, donepezil significantly decreased the veratridine-induced increase of [Na+]i. We also measured the effect on veratridine-induced release of the excitatory amino acids, glutamate and glycine. While donepezil decreased the release of glutamate and glycine, MK801 did not. In conclusion, our results indicate that donepezil has neuroprotective activity against depolarization-induced toxicity in rat cortical neurons via inhibition of the rapid influx of sodium and calcium ions, and via decrease of glutamate and glycine release, and also that this depolarization-induced toxicity is mediated by glutamate receptor activation.     

Effects of polyamines on the binding of [3H]MK-801 to the N-methyl-D-aspartate receptor: pharmacological evidence for the existence of a polyamine recognition site

A heat-stable factor of low molecular weight that increases the binding of [3H]MK-801 to rat brain membranes in the presence of maximally effective concentrations of L-glutamate and glycine was purified from bovine brain by reverse phase and ion-exchange high pressure liquid chromatography. The stimulatory activity was due to the presence of spermidine in the active fractions. Polyamines including spermine and spermidine are found in high concentrations in mammalian tissue. These compounds increase the affinity of N-methyl-D-aspartate (NMDA) receptors for [3H]MK-801 when assays are carried out in the presence of 100 microM L-glutamate and 100 microM glycine. At concentrations of 1 to 300 microM, a number of di- and triamines, including NH2(CH2)3NH2, NH2(CH2)3NH(CH2)2NH2, and NH2(CH2)3NH(CH2)3NH2, have partial or full agonist-like activity similar to that of spermidine. Other polyamines, including putrescine, cadaverine, NH2(CH2)2NH(CH2)2NH2, and CH3NH(CH2)3NHCH3, at concentrations of 1 to 100 microM, inhibited the binding of [3H]MK-801 in the presence of spermine, L-glutamate, and glycine but not in the presence of only L-glutamate and glycine. It is concluded that these compounds are selective antagonists of the effects of spermine at the NMDA receptor. These results suggest that there may be a polyamine recognition site on the NMDA receptor complex.     

Gacyclidine: a new neuroprotective agent acting at the N-methyl-D-aspartate receptor

Gacyclidine is a new phencyclidine derivative with neuroprotective properties. Tritiated gacyclidine and its enantiomers bind to NMDA receptors with binding parameters similar to those of other non-competitive NMDA receptor antagonists. The (-)enantiomer, (-)GK11, exhibits an affinity (2.5 nM) similar to that of dizocilpine (MK-801), while the (+)enantiomer, (+)GK11, has a 10 times lower affinity. When its interaction with NMDA receptors is prevented, gacyclidine binds also to "non-NMDA" binding sites which are mainly located in the molecular layer of the cerebellum on the dendritic tree of Purkinje cells. These binding sites do not appear to be related to any known neurotransmitters. In primary cortical cultures, gacyclidine and its enantiomers, at 0.1 to 5.0 microM, prevent glutamate-induced neuronal death. In rats, in vivo neurotoxicity of gacyclidine is far low than that of MK-801. No necrotic neurons were detected in animals sacrificed at 18 or 96 h after treatment with gacyclidine (1, 5, 10 or 20 mg/kg i.v.). At the highest (20 mg/kg) but not the lower doses (1-100 mg/kg) electron microscopy revealed the presence of few cytoplasmic or intramitochondrial vacuoles. In soman-treated monkeys gacyclidine enhanced neuroprotective activity of "three drugs cocktail" (atropine + diazepam + pralidoxime). Moreover, in rats, gacyclidine exerts a dose- and time-dependent neuroprotection in three models of spinal cord lesion. Beneficial effects of gacyclidine include reduction of lesion size and improvement of functional parameters after injury. In traumatic brain injury models gacyclidine improves also behavioral parameters and neuronal survival. Optimal protection is obtained when gacyclidine is administered at 0 to 30 min after injury. It is, therefore, concluded that gacyclidine exhibits neuroprotective effects similar to those of other NMDA receptor antagonists, with the advantage of being substantially less neurotoxic maybe due to its interaction with "non-NMDA" binding sites.     

In vivo labelling of the NMDA receptor channel complex by [3H]MK-801

An in vivo radioligand binding assay for the N-methyl-D-aspartate (NMDA) receptor channel complex in the mouse brain has been developed using the non-competitive NMDA receptor antagonist [3H]MK-801. In vivo binding of [3H]MK-801 was displaced by MK-801 (ED50 = 0.17 mg/kg i.p.), (-)-MK-801 (1.0 mg/kg), thienylcyclohexylpiperidine (1.8 mg/kg), etoxadrol (5.1 mg/kg) and (+)-SKF 10,047 (34.5 mg/kg). The potency of these drugs in this in vivo binding assay was highly correlated (r = 0.97) with their functional effects as antagonists of N-methyl-DL-aspartate-induced tonic convulsions.     

Effect of neurosteroids on glutamate binding sites and glutamate uptake in rat hippocampus

Effects of some neurosteroids on the binding of [3H]-glutamate, [3H]-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and [3H]-MK-801, as well as on the [3H]-glutamate uptake were examined in rat hippocampus. The following compounds were evaluated: (a) positive modulators of the GABA(A) receptor: 5alpha-pregnan-3alpha-ol-20-one (allopregnanolone), 5alpha-pregnane-3alpha,21-diol-20-one (allotetrahydrodeoxycorticosterone), 5alpha-pregnan-3alpha-ol-11,20-dione (alphaxalone) and 5alpha-androstan-3alpha-ol-17-one (androsterone); (b) compounds showing GABA(A)-antagonistic and/or N-methyl-D-aspartic acid (NMDA)-agonistic properties: dehydroepiandrosterone sulfate and pregnenolone sulfate; (c) a substance which, apart from its GABA(A)-agonistic potency, has a NMDA-antagonistic action: 5beta-pregnan-3alpha-ol-20-one. None of those neurosteroids tested at concentrations of 0.001-100 microM affected the binding of [3H]-glutamate, [3H]-AMPA and [3H]-MK-801 or the glutamate uptake. The present study suggests that the previously reported inhibitory effects of neurosteroids on excitatory amino acid-induced seizures and neurotoxicity can be linked neither to the direct interaction of these compounds with the above binding sites on glutamate receptor complexes, nor to the glutamate uptake mechanism.     

Modulation of dopamine and noradrenaline release and of intracellular Ca2+ concentration by presynaptic glutamate receptors in hippocampus.

1. We studied the release of [3H]-dopamine and [3H]-noradrenaline (NA) from hippocampal synaptosomes induced by glutamate receptors and the associated Ca2+ influx through Ca2+ channels. The release of tritiated neurotransmitters was studied by use of superfusion system and the intracellular free Ca2+ concentration ([Ca2+]i) was determined by a fluorimetric assay with Indo-1 as a probe for Ca2+. 2. Presynaptic glutamate receptor activation induced Ca(2+)-dependent release of [3H]-dopamine and [3H]-NA from rat hippocampal synaptosomes. Thus, L-glutamate induced the release of both neurotransmitters in a dose-dependent manner (EC50 = 5.62 microM), and the effect of 100 microM L-glutamate was inhibited by 83.8% in the presence of 10 microM 6-cyano-7-nitroquinoxaline-2,3-dioxine (CNQX), but was not affected by 1 microM (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine (MK-801). 3. Other glutamate receptor agonists also stimulated the Ca(2+)-dependent release of [3H]-dopamine and [3H]-NA as follows: N-methyl-D-aspartate (NMDA), at 200 microM, released 3.65 +/- 0.23% of the total 3H catecholamines, and this effect was inhibited by 81.2% in the presence of 1 microM MK-801; quisqualate (50 microM), S-alpha-amino-3-hydroxy-5-methyl-4-isoxazolopropionic acid (AMPA) (100 microM) or kainate (100 microM) released 1.57 +/- 0.26%, 1.93 +/- 0.17% and 2.09 +/- 0.22%, of the total 3H catecholamines, respectively. 4. The ionotropic glutamate receptor agonist, AMPA, induced an increase in the [Ca2+]i which was inhibited by 58.6% in the presence of 10 microM CNQX.(ABSTRACT TRUNCATED AT 250 WORDS)     

sigma receptor ligands attenuate N-methyl-D-aspartate cytotoxicity in dopaminergic neurons of mesencephalic slice cultures

We investigated the potential neuroprotective effects of several sigma receptor ligands in organotypic midbrain slice cultures as an excitotoxicity model system. When challenged with 100-microM N-methyl-D-aspartate (NMDA) for 24 h, dopaminergic neurons in midbrain slice cultures degenerated, and this was prevented by (5R, 10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,b]-cyclohepten-5, 10-imine (MK-801; 1-10 microM). Concomitant application of ifenprodil (1-10 microM) or haloperidol (1-10 microM), both of which are high-affinity sigma receptor ligands, significantly attenuated the neurotoxicity of 100 microM NMDA. The sigma(1) receptor-selective ligand (+)-N-allylnormetazocine ((+)-SKF 10047; 1-10 microM) was also effective in attenuating the toxicity of NMDA. The effect of R(-)-N-(3-phenyl-1-propyl)-1-phenyl-2-aminopropane hydrochloride ((-)-PPAP), a sigma receptor ligand with negligible affinity for the phencyclidine site of NMDA receptors, was also examined. (-)-PPAP (3-100 microM) caused a concentration-dependent reduction of NMDA cytotoxicity, with significant protection at concentrations of 30 and 100 microM. In contrast, (+)-SKF 10047 (10 microM) and (-)-PPAP (100 microM) showed no protective effects against cell death induced by the Ca(2+) ionophore ionomycin (1-3 microM). These results indicate that sigma receptor ligands attenuate the cytotoxic effects of NMDA on midbrain dopaminergic neurons, possibly via inhibition of NMDA receptor functions.     

N-methyl-D-aspartate increases cytoplasmic free calcium in mouse hippocampus.

The effect of N-methyl-D-aspartate (NMDA) and L-glutamate on the concentration of intracellular free calcium (Cai) and on uptake of the calcium was determined in microsacs and synaptosomes isolated from mouse brain. L-Glutamate and NMDA increased Cai in hippocampal microsacs but had little or no effect on Cai in microsacs, isolated from cortex or cerebellum or in synaptosomes. N-Methyl-D-aspartate also increased uptake of calcium, measured using 45Ca into hippocampal microsacs. The EC50 values for NMDA-stimulated increases in Cai and uptake of calcium in microsacs were about 30 microM. Maximum responses were observed with 100 microM NMDA. Increases in Cai stimulated by NMDA were dependent on extracellular calcium, indicating that NMDA increased Cai in microsacs by increasing conductance through an NMDA receptor-operated cation channel, rather than by releasing calcium from intracellular stores. The NMDA antagonists, 5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801), 2-amino-5-phosphonopentanoic acid (AP-5), magnesium and zinc blocked responses to NMDA. This demonstrates NMDA-mediated effects on ion flux in a cell-free preparation from brain. This preparation may be useful for study of the in vitro effects of drugs or toxins on NMDA receptors in brain.     

Effects of several cerebroprotective drugs on NMDA channel function: evaluation using Xenopus oocytes and [3H]MK-801 binding.

The effects of several cerebroprotective and nootropic drugs on the function of excitatory amino acid (EAA) receptor subtypes expressed in Xenopus oocytes after injection of rodent brain poly(A)+ mRNA were investigated. The oocyte response to N-methyl-D-aspartate (NMDA) in the presence of glycine (Gly) was inhibited dose-dependently by bifemelane, indeloxazine, vinpocetine and vincamine while no effect was observed by idebenone, Ca hopantenate, aniracetam or piracetam. Bifemelane, indeloxazine and vinpocetine suppressed the maximum response of NMDA and Gly without affecting their EC50 values. Unlike Mg2+, they did not affect the current-voltage relationship of the NMDA response below 0 mV. On the non-NMDA-type responses of the injected oocytes to kainate (KA), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and quisqualate (QA), no significant effects were observed by these drugs at 100 microM. On the binding of [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imi ne (MK-801) to brain membranes, the estimated IC50 values were 88 microM for bifemelane, 102 microM for indeloxazine, and 115 microM for vinpocetine. The dissociation rate of [3H]MK-801 was significantly slowed by Zn2+ and vinpocetine, but not affected by bifemelane or indeloxazine. The Kd value for [3H]MK-801 binding was increased by bifemelane and indeloxazine while Bmax was unchanged. These results suggest that the inhibition of NMDA channels by vinpocetine shows a similarity to the action of Zn2+ which closes the gate of the NMDA channel. In contrast, bifemelane and indeloxazine may affect the phencyclidine (PCP)-site in the open channels and inhibit NMDA function.