In silico target fishing for the potential targets and molecular mechanisms of baicalein as an antiparkinsonian agent: discovery of the protective effects on NMDA receptor-mediated neurotoxicity.

The flavonoid baicalein has been proven effective in animal models of parkinson's disease; however, the potential biological targets and molecular mechanisms underlying the antiparkinsonian action of baicalein have not been fully clarified. In the present study, the potential targets of baicalein were predicted by in silico target fishing approaches including database mining, molecular docking, structure-based pharmacophore searching, and chemical similarity searching. A consensus scoring formula has been developed and validated to objectively rank the targets. The top two ranked targets catechol-O-methyltransferase (COMT) and monoamine oxidase B (MAO-B) have been proposed as targets of baicalein by literatures. The third-ranked one (N-methyl-d-aspartic acid receptor, NMDAR) with relatively low consensus score was further experimentally tested. Although our results suggested that baicalein significantly attenuated NMDA-induced neurotoxicity including cell death, intracellular nitric oxide (NO) and reactive oxygen species (ROS) generation, extracellular NO reduction in human SH-SY5Y neuroblastoma cells, baicalein exhibited no inhibitory effect on [(3) H]MK-801 binding study, indicating that NMDAR might not be the target of baicalein. In conclusion, the results indicate that in silico target fishing is an effective method for drug target discovery, and the protective role of baicalein against NMDA-induced neurotoxicity supports our previous research that baicalein possesses antiparkinsonian activity.

Glutamate and NMDA receptors activation leads to cerebellar dysfunction and impaired motor coordination in unilateral 6-hydroxydopamine lesioned Parkinson's rat: functional recovery with bone marrow cells, serotonin and GABA.

Parkinson's disease (PD) is a chronic progressive neurodegenerative movement disorder characterised by a profound and selective loss of nigrostriatal dopaminergic neurons. In Parkinson's disease, degeneration of dopaminergic neurons involves motor structures including basal ganglia and cerebellum. Glutamate-mediated degeneration of the cerebellum contributes to motor dysfunction in Parkinson's disease. Targeting neurotransmitter system beyond the dopamine system is of important, both for the motor and for the nonmotor problems of Parkinson's disease. The aim of this study is to assess the glutamate and NMDA receptor functional regulation and motor performance of 6-hydroxydopamine-induced Parkinson's rat and the effects of serotonin (5-HT), gamma aminobutyric acid (GABA) and bone marrow cells supplementation infused intranigrally to substantia nigra individually and in combination. Scatchard analysis of total glutamate and NMDA receptor binding parameters showed a significant increase in B (max) (P < 0.001) in the cerebellum of 6-hydroxydopamine infused rat compared to control. Real-Time PCR amplification of NMDA2B, mGluR5, and bax were significantly (P < 0.001) upregulated in cerebellum of 6-hydroxydopamine infused rats compared to control. Activation of the glutamate and NMDA receptors gave rise to an increased cAMP and IP3 content in the cerebellum. Gene expression studies of GLAST and CREB showed a significant (P < 0.001) down regulation in 6-OHDA infused rats compared to control. Behavioural studies were carried out to confirm the biochemical and molecular studies. Serotonin and GABA along with bone marrow cells in combination showed reversal of glutamate receptors and motor abnormality shown in the Parkinson's rat model. The therapeutic significance in Parkinson's disease is of prominence.

Stereoselective synthesis and preliminary evaluation of new D-3-heteroarylcarbonylalanines as ligands of the NMDA receptor.

New N-heteroarylcarbonylalanines of the D-series were stereoselectively prepared from enoates derived from D-mannitol. These compounds were active in binding and functional assays of the NMDA sub-type of glutamate receptors. A pyridine derivative inhibited MK801 binding, protected neurons from excitotoxic damage and blocked NMDA-induced currents in neurons. A thiophene derivative positively modulated the NMDA receptor, possibly through the allosteric glycine site.

Proteolysis of the N-methyl-d-aspartate receptor by calpain in situ.

N-Methyl-D-aspartate (NMDA) receptors are calcium-permeable glutamate receptors that play putative roles in learning, memory, and excitotoxicity. NMDA receptor-mediated calcium entry can activate the calcium-dependent protease calpain, leading to substrate degradation. The major NMDA receptor 2 (NR2) subunits of the receptor are in vitro substrates for calpain at selected sites in the C-terminal region. In the present study, we assessed the ability of calpain-mediated proteolysis to modulate the NR1a/2A subtype in a heterologous expression system. Human embryonic kidney (HEK293t) cells, which endogenously express calpain, were cotransfected with NR1a/2A in addition to the calpain inhibitor calpastatin or empty vector as control. Receptor activation by glutamate and glycine as co-agonists led to calpain activation as measured by succinyl-L-leucyl-L-leucyl-L-valyl-L-tyrosyl-aminomethyl coumarin (Suc-LLVY-AMC). Calpain activation also resulted in the degradation of NR2A and decreased binding of (125)I-MK-801 ((125)I-dizocilpine) to NR1a/2A receptors. No stable N-terminal fragment of the NMDA receptor was formed after calpain activation, suggesting calpain regulation of NMDA receptor levels in ways distinct from that previously observed with in vitro cleavage. NR2 subunit constructs lacking the final 420 amino acids were not degraded by calpain. Agonist-stimulated NR1a/2A-transfected cells also had decreased calcium uptake and produced lower changes in agonist-stimulated intracellular calcium compared with cells cotransfected with calpastatin. Calpastatin had no effect on either calcium uptake or intracellular calcium levels when the NR2A subunit lacked the final 420 amino acids. These studies demonstrate that NR2A is a substrate for calpain in situ and that this proteolytic event can modulate NMDA receptor levels.

Lower sensitivity to stress and altered monoaminergic neuronal function in mice lacking the NMDA receptor epsilon 4 subunit.

NMDA receptors, an ionotropic subtype of glutamate receptors (GluRs), play an important role in excitatory neurotransmission, synaptic plasticity, and brain development. They are composed of the GluRzeta subunit (NR1) combined with any one of four GluRepsilon subunits (GluRepsilon1-GluRepsilon4; NR2A-NR2D). Although the GluRzeta subunit exists in the majority of the CNS throughout all stages of development, the GluRepsilon subunits are expressed in distinct temporal and spatial patterns. In the present study, we investigated neuronal functions in mice lacking the embryonic GluRepsilon4 subunit. GluRepsilon4 mutant mice exhibited reductions of [(3)H]MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d] cyclohepten-5,10-imine maleate] binding and (45)Ca(2+) uptake through the NMDA receptors. The expression of GluRzeta subunit protein, but not GluRepsilon1 and GluRepsilon2 subunit proteins, was reduced in the frontal cortex and striatum of the mutant mice. A postmortem examination in GluRepsilon4 mutant mice revealed that tissue contents of norepinephrine, dopamine, serotonin, and their metabolites were reduced in the hippocampus and that dopamine, as well as serotonin, metabolism was upregulated in the frontal cortex, striatum, hippocampus, and thalamus. To clarify the phenotypical influences of the alteration in neuronal functions, performances in various behavioral tests were examined. GluRepsilon4 mutant mice showed reduced spontaneous locomotor activity in a novel environment and less sensitivity to stress induced by the elevated plus-maze, light-dark box, and forced swimming tests. These findings suggest that GluRepsilon4 mutant mice have dysfunctional NMDA receptors and altered emotional behavior probably caused by changes in monoaminergic neuronal activities in adulthood.

NMDA recepter-mediated neuroprotection by essential oils from the rhizomes of Acorus gramineus.

Acori graminei Rhizoma (AGR) is shown to exhibit a number of pharmacological actions including sedation and anticonvulsive action. To further characterize its actions in the CNS, the present study evaluated the effects of essential oils (EO) from AGR on the excitotoxic neuronal cell death induced in primary rat cortical cell cultures. EO inhibited the glutamate-induced excitotoxicity in a concentration-dependent manner, with the IC50 of 0.241 mg/ml. EO exerted more potent neuroprotection against the toxicity induced by NMDA (IC50 = 0.139 mg/ml). In contrast, the AMPA-induced toxicity was not inhibited by EO. Receptor-ligand binding studies were performed to investigate the neuroprotective action mechanism. EO dramatically inhibited the specific bindings of a use-dependent NMDA receptorion channel blocker [3H]MK-801, indicating an NMDA receptor antagonist-like action. However, the bindings of [3H]MDL 105,519, a ligand selective for the glycine binding site of NMDA receptor, were not considerably inhibited. These results demonstrated that EO extracted from AGR exhibited neuroprotective effects on cultured cortical neurons through the blockade of NMDA receptor activity, and that the glycine binding site appeared not to be the major site of action.

Hyperfunction of dopaminergic and serotonergic neuronal systems in mice lacking the NMDA receptor epsilon1 subunit.

NMDA receptors, an ionotropic subtype of glutamate receptors (GluRs) forming high Ca(2+)-permeable cation channels, are composed by assembly of the GluRzeta subunit (NR1) with any one of four GluRepsilon subunits (GluRepsilon1-4; NR2A-D). In the present study, we investigated neuronal functions in mice lacking the GluRepsilon1 subunit. GluRepsilon1 mutant mice exhibited a malfunction of NMDA receptors, as evidenced by alterations of [(3)H]MK-801 binding as well as (45)Ca(2+) uptake through the NMDA receptors. A postmortem brain analysis revealed that both dopamine and serotonin metabolism were increased in the frontal cortex and striatum of GluRepsilon1 mutant mice. The NMDA-stimulated [(3)H]dopamine release from the striatum was increased, whereas [(3)H]GABA release was markedly diminished in GluRepsilon1 mutant mice. When (+)bicuculline, a GABA(A) receptor antagonist, was added to the superfusion buffer, NMDA-stimulated [(3)H]dopamine release was significantly increased in wild-type, but not in the mutant mice. GluRepsilon1 mutant mice exhibited an increased spontaneous locomotor activity in a novel environment and an impairment of latent learning in a water-finding task. Hyperlocomotion in GluRepsilon1 mutant mice was attenuated by treatment with haloperidol and risperidone, both of which are clinically used antipsychotic drugs, at doses that had no effect in wild-type mice. These findings provide evidence that NMDA receptors are involved in the regulation of behavior through the modulation of dopaminergic and serotonergic neuronal systems. In addition, our findings suggest that GluRepsilon1 mutant mice are useful as an animal model of psychosis that is associated with NMDA receptor malfunction and hyperfunction of dopaminergic and serotonergic neuronal systems.

Inhibition of excitotoxic neuronal death by methanol extract of Acori graminei rhizoma in cultured rat cortical neurons.

Acori graminei rhizoma (AGR) are reported to exhibit a number of pharmacological actions in the central nervous system. The effects of the methanol extract of AGR on excitotoxic neuronal death were evaluated in the present study using cultured rat cortical neurons. Based on the phase-contrast microscopic examinations of cultures and lactate dehydrogenase activities measured in the culture media, the glutamate-induced excitotoxicity was significantly inhibited by the extract. The inhibitory action of the extract was more potent and selective for the N-methyl-D-aspartate (NMDA) receptor-mediated toxicity. The AGR extract competed with [3H]MDL 105,519 for the specific binding to the glycine site of the NMDA receptor with the IC(50) value of 164.7 microg/ml. Modulation of the NMDA receptor activity by the extract was determined using [3H]MK-801 binding studies. The reduction of the binding in the presence of the extract indicated the receptor inactivation by AGR. These results demonstrated that the methanol extract of AGR exhibited protective action against excitotoxic neuronal death, and that the neuroprotective action was primarily due to the blockade of NMDA receptor function by the interaction with the glycine binding site of the receptor.

Cross-tolerance in drug response and differential changes in central nicotinic and N-methyl-D-aspartate receptor binding following chronic treatment with either (+)- or (-)-nicotine.

The present study compared the effects of chronic treatment (21 days) with (+)-nicotine and (-)-nicotine on exploratory behaviours and body temperature in rats. Chronic treatment with either (+)- or (-)-nicotine induced tolerance in exploratory behaviours. A cross-tolerance between the two stereoisomers was also observed. Body temperature was differentially affected by chronic (+)- and (-)-nicotine, but cross-tolerance was still observed. In addition, nicotinic acetylcholine receptor (nAChR) and N-methyl-D-aspartate (NMDA) receptor binding in rats treated with (+)- and (-)-nicotine were measured by receptor autoradiography. Chronic treatment with (+)- and (-)-nicotine significantly increased nAChR binding in different brain regions. Furthermore, significant differences in the induction of nAChR binding by chronic (+)- and (-)-nicotine were detected in several brain regions. Chronic treatment with (+)-nicotine significantly increased NMDA receptor binding in the CA1 region of the hippocampus. However, (-)-nicotine had no effect on NMDA receptor binding in any of the regions analysed. These results demonstrate differences between chronic treatment with (+)- and (-)-nicotine in physiological responses such as body temperature and biochemical changes such as changes in nAChRs and NMDA receptor binding, although a cross-tolerance between the two enantiomers in behaviour and body temperature is also observed.

Modulation by both diphenyliodonium and diphenyleneiodonium of [3H]MK-801 binding to rat brain synaptic membranes.

Binding of [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imi ne (MK-801) was significantly inhibited by the addition of several different compounds related to generation of nitric oxide (NO) at 100 microM in rat brain synaptic membranes. These included sodium nitroprusside, diphenyliodonium (DI), diphenyleneiodonium (DPI) and methylene blue. However, neither S-nitroso-N-acetylpenicillamine nor S-nitroso-L-glutathione inhibited binding at 100 microM. Both DI and DPI inhibited binding in a concentration-dependent manner at a concentration range of over 1 microM, while further addition of spermidine (SPD) significantly attenuated the potency of DPI to inhibit binding without affecting that of DI. In contrast, SPD induced significant potentiation of the ability of unlabelled MK-801 to displace [3H]MK-801 binding in a fashion sensitive to antagonism by the novel polyamine antagonist bis-(3-aminopropyl)nonanediamine. This novel polyamine antagonist also prevented the reversing effect of SPD on inhibition by DPI of [3H]MK-801 binding. Moreover, DPI competitively exacerbated the ability of SPD to potentiate [3H]MK-801 binding in the presence of both L-glutamic acid and glycine at maximally effective concentrations. On the other hand, SPD was effective in reversing the inhibition by DPI in cerebellar, but not hippocampal, synaptic membranes. These results suggest that both DI and DPI may modulate synaptic responses mediated by the N-methyl-D-aspartate receptor through inhibition of opening processes of the ion channel in a manner irrespective of generation of NO radicals in particular situations. Possible involvement of the polyamine domain in the inhibition by DPI is also suggested.

Harmaline competitively inhibits [3H]MK-801 binding to the NMDA receptor in rabbit brain.

Harmaline, a beta-carboline derivative, is known to produce tremor through a direct activation of cells in the inferior olive. However, the receptor(s) through which harmaline acts remains unknown. It was recently reported that the tremorogenic actions of harmaline could be blocked by the noncompetitive NMDA channel blocker, MK-801. This study examined whether the blockade of harmaline's action, in the rabbit, by MK-801 was due to a pharmacological antagonism at the MK-801 binding site. This was accomplished by measurement of [3H]MK-801 binding in membrane fractions derived from tissue containing the inferior olivary nucleus and from cerebral cortex. Harmaline completely displaced saturable [3H]MK-801 binding in both the inferior olive and cortex with apparent IC50 values of 60 and 170 microM, respectively. These IC50 values are consistent with the high doses of harmaline required to produce tremor, e.g., 10-30 mg/kg. Non-linear curve fitting analysis of [3H]MK-801 saturation experiments indicated that [3H]MK-801 bound to a single site and that harmaline's displacement of [3H]MK-801 binding to the NMDA receptor was competitive as indicated by a shift in Kd but not in Bmax. In addition, a Schild plot gave a slope that was not significantly different from 1 indicating that harmaline was producing a displacement of [3H]MK-801 from its binding site within the NMDA cation channel and not through an action at the glutamate or other allosteric sites on the NMDA receptor. These findings provide in vitro evidence that the competitive blockade of harmaline-induced tremor by MK-801 occurs within the calcium channel coupled to the NMDA receptor. Our hypothesis is that harmaline produces tremor by acting as an inverse agonist at the MK-801 binding site and thus opening the cation channel.

Anticonvulsant effect of reduced NMDA receptor expression in audiogenic DBA/2 mice.

Pretreatment of DBA/2 mice (n = 14-15 per group) with an 18-mer antisense probe to the NMDA-receptor submit NR1 (2 x 1 micrograms, or 2 x 83 pmol, NR1 antisense probe intracerebroventricularly, -29 and -7 h before testing for seizure response) resulted in almost complete suppression of sound-induced clonic seizures. A saline-treated group gave a 100% seizures response, while the group treated with NR1 antisense probe gave a 7% seizure response to the sound stimulus. The group treated with NR1 nonsense-probe showed no anticonvulsant protection (93% seizure response). The anticonvulsant protection observed following NR1 antisense administration was of relatively short duration, with seizure response gradually returning to control levels 12 to 24 h following the termination of antisense administration. When NR1 receptor levels were assessed by receptor autoradiography ([3H]-MK 801 and -CGP 39653 binding) in the same groups of mice, significant (20%) reductions in NR1 levels were observed in the retrosplenial cortex and the overall cortex. The seizure-induced expression of c-fos and NGFI-A in thalamus, hypothalamus, inferior colliculus and medical geniculate seen in vehicle- and NR1 nonsense-treated mice was completely blocked by NR1 antisense pretreatment.

Decrease of (+)-3-[125I]MK-801 binding to NMDA brain receptors revealed at puberty in rats treated neonatally with monosodium glutamate.

Obesity, altered pattern of gonadal hormone secretion, advanced vaginal opening, irregular cycling, altered sexual behavior and infertility are the effects of the neonatal administration of monosodium glutamate (MSG) to rodents. These are the consequences of lesions located mainly in the hypothalamic region. It is believed that the receptors to N-methyl-D-aspartic acid (NMDA) actively participate in the onset and development of such lesions, on the other hand, they may be altered by neuronal dysfunction as well, seriously compromising the glutamatergic pathways that are involved in the neuroendocrine regulation. To clarify the scope of the lesion induced by MSG and its probable effects on the NMDA receptors, we measured them with a very sensitive ligand for autoradiography, (+)-3-[125I]MK-801. Coronal cuts at the level of the arcuate-median eminence of brains from 4-, 8- and 40-day-old rats treated neonatally with MSG (4 mg/g) or saline (controls) were examined. In the normal hypothalamus, NMDA receptor labelling was higher in the young animals than in the 40-day-old animals, and this was observed in both control and treated rats. NMDA receptor labelling of rats at puberty was very low, and no apparent differences were observed between groups. In contrast, in areas where an increase in NMDA binding sites normally occurs with development, a significant impairment of the normal augmentation of MK-801 binding was revealed. In the hippocampal layers, stratum radiatum and stratum oriens and in the cerebral cortex of 40-day-old rats treated with MSG a lower amount of binding was observed, of about 50% fewer sites compared to the untreated controls at the level of CA3 and in the outer layer of the parietal cortex. These results suggest that at an early stage of the MSG lesion the NMDA receptors located in the hypothalamus and other brain areas are apparently expressed normally, but at puberty the effects of the lesion are revealed in the hippocampus and cerebral cortex by a decrease in the density of binding. Thus, the abnormal neuroendocrine and behavioral responses displayed by the MSG-treated rats may be contributed partially by the alteration of the NMDA receptors in these areas.

N-methyl-D-aspartate R1 messenger RNA and [125I]MK-801 binding decrease in rat spinal cord after unilateral hind paw inflammation.

Recent evidence suggests that N-methyl-D-aspartate receptors play an important role in the etiology and maintenance of chronic nociception. Previous studies have demonstrated that tissue injury or stimulation of nociceptive afferent projections results in the expansion of receptive fields, hyperalgesia and C-fiber-induced wind-up, events that can be inhibited by N-methyl-D-aspartate antagonists. This study examines the effect of unilateral hind paw inflammation on N-methyl-D-aspartate R1 messenger RNA and [125I]dizocilpine maleate binding in the L4-L5 segments of the lumbar spinal cord of rats. Spinal cords were examined at 7.5 h, three, seven and 20 days after injection of the left hind paw with 120 microliters of complete Freund's adjuvant. N-methyl-D-aspartate R1 messenger RNA, as measured with in situ hybridization, was observed to decrease bilaterally in laminae I, II and X of the lumbar spinal cord. This decrease was evident in laminae I and II at 7.5 h and three days after hind paw injection. In lamina X, a postinjection decrease in hybridization signal was observed at 7.5 h and seven days. A bilateral decrease in [125I]dizocilpine maleate binding was observed in laminae I and II at three, seven and 20 days after paw injection. This observed decrease in binding at the N-methyl-D-aspartate receptor suggests a compensatory mechanism by which N-methyl-D-aspartate-mediated nociceptive events may be modulated.

Preferential induction by stress of the N-methyl-D-aspartate recognition domain in discrete structures of rat brain.

Immobilization stress in water for 3 h was effective in inducing significant potentiation of [3H](+)-5-methyl-10,11-dihydro-5H- dibenzo [a,d] cyclohepten-5,10-imine ([3H]MK-801) binding 5 days after the stressful manipulation in rat hypothalamus and cerebellum when determined before equilibrium in the absence of any added agonists, in addition to resulting in marked reduction of rearing behaviors of animals. However, the stressful manipulation failed to modulate the [3H]MK-801 binding in other central regions examined, and binding of either [3H]DL-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid or [3H]kainic acid was not significantly affected in all brain structures studied 5 days after the stress application. In contrast, the stressful procedures potentiated binding of both L-[3H]glutamic ([3H]Glu) and [3H]DL-(E)-2-amino-4-propyl-5-phosphono-3-pentenoic ([3H]CGP-39653) acids in the hypothalamus and cerebellum 5 days later, without affecting binding of [3H]-glycine and 5,7-dichloro[3H]kynurenic acid. The systemic administration of corticosterone mimicked the stress manipulation at doses of 5-50 mg/kg in terms of inducing significant enhancement of binding of both [3H]Glu and [3H]CGP-39653 in the hypothalamus and cerebellum when determined 5 days after the single administration. The translation inhibitor cycloheximide was effective in preventing the stress-induced potentiation of [3H]Glu binding in the cerebellum, without altering that in the hypothalamus. Furthermore, the stressful handling significantly increased the densities of [3H]Glu binding sites in the hypothalamus and cerebellum, with the affinities being unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)

Regional profile of developmental changes in the sensitivity of the N-methyl-D-aspartate receptor to polyamines.

The NMDA receptor exhibits increased sensitivity to stimulation during early development compared with the adult. In this study, we examined modulation of the NMDA receptor by polyamines during development to see if it correlates with differences in the functional responsiveness of the NMDA receptor. [3H]MK-801 binding was measured in discrete brain regions in the presence and absence of polyamines in 3-, 7-, 15-, 25-, and 60-day-old Sprague-Dawley rats. [3H]MK-801 binding increased between postnatal days 3 and 15, with adult levels of binding being reached between days 15 and 25. Spermidine (75 microM) caused maximal stimulation of [3H]MK-801 binding during early development, ranging from 250% in the thalamus to 450% in the caudate putamen at postnatal day 3. This effect gradually declined to levels seen in the adult by postnatal days 15-25. During all developmental stages, the stimulation seen was greater in the caudate putamen compared with the hippocampus. Diethylenetriamine (1 mM) exhibited similar developmental and regional heterogeneity in its effects on [3H]MK-801 binding, producing substantial stimulation of binding in the neonate, but not in the adult. The EC50 and Emax values for the stimulatory effect of spermidine were significantly higher at day 7 compared with the adult. Unlike spermidine and diethylenetriamine, there was no regional variation in the effects of the putative "polyamine site" inverse agonist 1,10-diaminodecane at any age and only a slightly attenuated inhibition at postnatal day 3 compared with the adult.(ABSTRACT TRUNCATED AT 250 WORDS)

Further studies of the role of hyperthermia in methamphetamine neurotoxicity.

The depletion of striatal dopamine (DA) that can occur after methamphetamine (METH) administration has been linked to METH-induced hyperthermia. The relationship between METH-induced hyperthermia, neurotoxicity (striatal DA depletions) and compounds that protect against METH neurotoxicity was further investigated in this study. Typically, rats exposed to METH die when their body temperatures exceed 41.3 degrees C but such hyperthermic rats can be saved by hypothermic intervention. Subsequently, rats saved by hypothermic intervention have greater depletion of striatal DA at an earlier time of onset (18 hr or less post-METH) than do METH-exposed rats that do not attain such high temperatures. Striatal damage was present 3 days post-METH in these hyperthermic rats, as assessed by silver degeneration of terminals and increases in the astrocytes that express glial fibrillary acidic protein immunoreactivity. By contrast, alterations in the number of [3H]dizoclipine (MK-801) binding sites in cortical or striatal membranes at 1, 3 or 14 days post-METH were not detected. The experiments showed that mean and maximal body temperature correlated well with striatal DA concentrations 3 days post-METH (r = -0.77, n = 58), which suggests a role for hyperthermia in METH neurotoxicity. However, hyperthermia (alone or with haloperidol present) induced by high ambient temperatures did not deplete striatal DA in the absence of METH. Haloperidol, diazepam and MK-801 all reduced METH-induced striatal DA depletion to a degree predicted by their inhibition of hyperthermia and increased ambient temperature abolished their neuroprotection. Although an interleukin-1 receptor antagonist reduced maximal body temperature enough to lower the lethality rate, it did not reduce the temperature sufficiently to block METH neurotoxicity. It was concluded that short- and long-term decreases in striatal DA levels depend on the degree of hyperthermia produced during METH exposure but cannot be produced by hyperthermia alone. In addition, several agents that block DA depletions do so by inhibiting METH-induced hyperthermia. Finally, the results suggested a role for interleukin-1 in the extreme hyperthermia and lethality produced by METH.

Preservation of redox, polyamine, and glycine modulatory domains of the N-methyl-D-aspartate receptor in Alzheimer's disease.

This study used [3H]dizocilpine ([3H]MK-801) binding to the N-methyl-D-aspartate (NMDA) receptor to examine redox, polyamine, and glycine modulatory sites in membranes derived from the superior frontal and the superior temporal cortex of patients with Alzheimer's disease. In control subjects the competitive polyamine site antagonist arcaine inhibited [3H]dizocilpine binding in a dose-dependent fashion and this curve was shifted to the right by the addition of 50 microM spermidine. Arcaine inhibition of binding was more potent in the temporal cortex than in the frontal cortex, in both the absence and presence of 50 microM spermidine. In Alzheimer's disease, arcaine inhibition of [3H]dizocilpine binding (in both the absence and the presence of spermidine) was not different from control in either of the two brain areas examined. The sulfhydryl redox site of the NMDA receptor was assessed using the oxidizing agent 5,5'-dithio-bis(2-nitrobenzoic acid), which inhibited binding in a dose-dependent fashion. This inhibition was similar in patients with Alzheimer's disease and control subjects. Glycine-stimulated [3H]dizocilpine binding was also unaffected in patients with Alzheimer's disease. However, in the temporal cortex there was a significant age-associated decline in [3H]dizocilpine binding in the presence of 100 microM glutamate (Rs = -0.71) and 100 microM glutamate plus 30 microM glycine (Rs = -0.90). There was also an age-related increase in arcaine IC50 (which reflects an age-related decrease in arcaine affinity) in the frontal cortex, determined both in the absence (Rs = 0.83) and the presence (Rs = 0.79) of spermidine. These data indicate that the NMDA receptor and its modulatory redox, polyamine, and glycine subsites are intact in patients with Alzheimer's disease and that the modulatory activity of polyamine and glycine sites decline with aging.

Similar effects of glycine, zinc and an oxidizing agent on [3H]dizocilpine binding to the N-methyl-D-aspartate receptor in neocortical tissue from suicide victims and controls.

This study used [3H]dizocilpine (MK-801) binding to the N-methyl-D-aspartate (NMDA) receptor to examine glycine, redox and zinc modulatory sites in membranes derived from the frontal and parietal cortex of control subjects (n = 8) and suicide victims (n = 6). [3H]dizocilpine binding in the presence of glutamate and glutamate plus glycine was similar in control and suicide subjects. The sulphydryl redox site was assessed using the oxidizing agent 5,5'-dithio-bis (2-nitrobenzoic acid), which inhibited binding in a dose-dependent fashion. Both redox and zinc sites were unaffected in the frontal and parietal cortex of suicide victims. These data indicate that the NMDA receptor and its glycine, redox and zinc subsites are preserved in the neocortex of suicide victims.

Regional heterogeneity of polyamine effects on the N-methyl-D-aspartate receptor in rat brain.

Polyamines have pronounced effects on N-methyl-D-aspartate (NMDA) receptors in vitro and may be important modulators of NMDA receptor activity in vivo. There is considerable regional heterogeneity in the effects of polyamines on [3H]MK-801 binding in rat brain sections. For example, spermidine enhances the binding of [3H]MK-801 to a much greater extent in the striatum than in the cortex. To further explore the basis for this regional heterogeneity, the effects of polyamines on [3H]MK-801 binding were measured in well-washed membranes prepared from frontal cortex and striatum. There was no difference in the concentration-response relationship for spermidine or the KD for [3H]MK-801 in the presence of 75 microM spermidine, suggesting that the regional difference seen in tissue sections is due to an endogenous factor that is either removed or inactivated during the preparation of membranes. Comparison of spermidine concentration-response curves in washed and unwashed tissue sections revealed that washing selectively enhanced the Emax value in the ventromedial caudate putamen without changing the EC50. This is consistent with the possibility that a noncompetitive polyamine antagonist is being removed from this region during washing. There was no regional variability in the effects of the putative inverse agonist 1,10-diaminodecane, consistent with recent suggestions that this polyamine inhibits the NMDA receptor at a site distinct from the one at which polyamines act to enhance NMDA receptor function. Agents that modulate the redox state of the NMDA receptor did not eliminate the regional heterogeneity of polyamine effects. Furthermore, the stimulatory effect of glycine in these regions did not correlate with that of spermidine. These results suggest the existence of one or more endogenous factors that noncompetitively influence the effects of polyamines in a region-specific manner.