Modulation of N-methyl-D-aspartate receptor-mediated increases in cytosolic calcium in cultured rat cerebellar granule cells.

The concentration of intracellular free Ca2+ ([Ca2+]i) was measured in rat cerebellar granule cells using the fluorescent indicator fura-2. Culturing the cells as monolayers on plastic squares which could be placed into cuvettes allowed measurements of [Ca2+]i to be performed on large and homogeneous populations of CNS neurons. Granule cells so cultured maintained low levels of [Ca2+]i (around 90 nM) which increased promptly upon the addition of various excitatory amino acids including N-methyl-D-aspartate (NMDA). Increases in [Ca2+]i elicited by NMDA were inhibited by Mg2+ (1 mM) and often potentiated by glycine (1 microM). The addition of TTX or strychnine (5 microM each) did not alter responses to NMDA or NMDA plus glycine. Cytosolic Ca2+ responses to NMDA/glycine were dependent on the presence of extracellular Ca2+ and were unaffected by concentrations of nifedipine or verapamil that blocked increases in [Ca2+]i elicited by K+ depolarization. Responses elicited by NMDA/glycine were inhibited competitively by 2-amino-5-phosphonovalerate or 3-((+-)-2-carboxypiperazin-4-yl)-propyl-1- phosphonic acid and non-competitively by MK-801 or Mg2+. HA-966 and 7-chlorokynurenate inhibited responses to NMDA alone and blocked competitively the potentiating effects of glycine. The results demonstrate NMDA-mediated increases in [Ca2+]i in cerebellar granule cells that arise solely from influx of extracellular Ca2+ through dihydropyridine-insensitive channels. The strict dependence of the NMDA-evoked response on extracellular Ca2+ provides little evidence for a coupling of NMDA receptors to inositol phosphate metabolism and mobilization of intracellular Ca2+. The effect of various agents on NMDA/glycine-induced increases in [Ca2+]i parallels their effects on ligand binding to or current flow through the NMDA receptor-channel complex. The measurement of cytosolic Ca2+ in this preparation of neuronal cells thus appears especially well suited for assessing, on a functional level, the regulation of NMDA receptors in the CNS.     

NMDA-induced increase in [Ca2+]i and 45Ca2+ uptake in acutely dissociated brain cells derived from adult rats.

A preparation of acutely dissociated brain cells derived from adult (3-month-old) rat has been developed under conditions preserving the metabolic integrity of the cells and the function of N-methyl-D-aspartate (NMDA) receptors. The effects of glutamate and NMDA on [Ca2+]i measured with fluo3 and 45Ca2+ uptake have been studied on preparations derived from hippocampus and cerebral cortex. Glutamate (100 microM) and N-methyl-DL-aspartate (200 microM) increased [Ca2+]i by 26-12 nM and 23-9 nM after 90 s in cerebral cortex and hippocampus, and stimulated 45Ca2+ uptake about 16-10% in the same regions. The increases in [Ca2+]i and 45Ca2+ uptake were inhibited by 40% in the presence of 1 mM MgCl2 and by 90-50% in the presence of MK-801. The results indicate (a) that a large fraction of the [Ca2+]i response to glutamate in freshly dissociated brain cells from the adult rat involves NMDA receptors, (b) when compared with results in newborn rats, there is a substantial blunting of the [Ca2+]i increase in adult age.     

Redox modulation of NMDA receptor-mediated Ca2+ flux in mammalian central neurons

NMDA receptor-mediated Ca2+ flux was studied in cultured rat retinal ganglion cells and neocortical neurons. Intracellular free calcium ([Ca2+]i was measured with fura-2 fluorescence imaging. Baseline [Ca2+]i was 59 +/- 5 nM. In low [Mg2+]o, 200 microM NMDA reversibly increased [Ca2+]i to 421 +/- 70 nM. This rise in [Ca2+]i was blocked by the NMDA antagonists APV (200 microM) or [Mg2+]o (1 mM), but only slightly inhibited by the non-NMDA antagonist CNQX (10 microM). Chemical reduction with dithiothreitol (DTT) had no effect on resting [Ca2+]i. However, DTT increased the NMDA-induced rise in [Ca2+]i approximately 1.6-fold; the oxidizing agent dithiobisnitrobenzoic acid (DTNB) reversed this effect. In patch-clamp experiments, DTT increased NMDA-activated whole-cell conductance approximately 1.7-fold in low and high [Ca2+]o. The Ca2+/Na+ permeability ratio of approximately 7 for NMDA channels remained unaltered by chemical reduction. Thus, redox modulation of the NMDA receptor/channel complex results in a dramatic alteration in current magnitude but no change in ionic permeabilities.     

The effects of excitatory amino acids on intracellular calcium in single mouse striatal neurons in vitro

Using microspectrofluorimetry and the calcium-sensitive dye fura-2, we examined the effect of excitatory amino acids on [Ca2+]i in single striatal neurons in vitro. N-methyl-D-aspartic acid (NMDA) produced rapid increases in [Ca2+]i. These were blocked by DL-2-amino-5-phosphonovaleric acid (AP5), by Mg2+, by phencyclidine, and by MK801. The block produced by Mg2+ and MK801 could be relieved by depolarizing cells with veratridine. When external Ca2+ was removed, NMDA no longer increased [Ca2+]i. Furthermore, the effects of NMDA were not blocked by concentrations of La3+ that blocked depolarization induced rises in [Ca2+]i. Substitution of Na+o by Li+ did not block the effects of NMDA. Concentrations of L-glutamate greater than or equal to 10(-6) M also increased [Ca2+]i. The effects of moderate concentrations of glutamate were blocked by AP5 but not by La3+ or by substitution of Na+ by Li+. The effects of glutamate were blocked by removal of external Ca2+ but were not blocked by concentrations of Mg2+ or MK801 that completely blocked the effects of NMDA. The glutamate analogs kainic acid (KA) and quisqualic acid also increased [Ca2+]i. The effects of KA were blocked by removal of external Ca2+ but not by La3+, Mg2+, MK801, or replacement of Na+ by Li+. Although AP5 was able to block the effects of KA partially, very high concentrations were required. These results may be explained by considering the properties of glutamate-receptor-linked ionophores. Excitatory amino acid induced increases in [Ca2+]i are consistent with the possibility that Ca2+ mediates excitatory amino acid induced neuronal degeneration.     

The interactions between plasma membrane depolarization and glutamate receptor activation in the regulation of cytoplasmic free calcium in cultured cerebellar granule cells

The complex modulation of cytoplasmic free calcium concentration ([Ca2+]c) in primary cultures of cerebellar granule cells in response to glutamate receptor agonists has been the subject of several contradictory reports. We here show that 3 components of the [Ca2+]c response can be distinguished: (1) Ca2+ entry through voltage-dependent Ca2+ channels, following KCl- or receptor-evoked depolarization, (2) Ca2+ entry through NMDA receptor channels, and (3) liberation of internal Ca2+ via a metabolotropic receptor. Depolarization with KCl induced a transient [Ca2+]c response (subject to voltage inactivation) decaying to a sustained plateau (largely inhibited by nifedipine). The NMDA response was potentiated by glycine, totally inhibited by (+)5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801), and blocked by Mg2+ in a voltage-sensitive manner. Polarized cells displayed small responses to quisqualate (QA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA). Depolarization enhanced a transient response to QA, but not to AMPA. Trans-1-amino-1,3-cyclopentanedicarboxylic acid (trans-ACPD), a selective agonist for the metabolotropic glutamate receptor, caused a transient elevation of [Ca2+]c, which was blocked by prior exposure to QA but not AMPA. The prolonged [Ca2+]c response to kainate (KA) can be resolved into 2 major components: an indirect NMDA receptor-mediated response due to released glutamate and a nifedipine-sensitive component consistent with depolarization-mediated entry via Ca2+ channels. 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX), QA at greater than 10 microM, and AMPA (but not trans-ACPD) reversed the KA response, consistent with an inactivation of the KA receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism underlying protective effect of MK-801 against NMDA-induced neuronal injury in vivo.

The effects of the N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 and the dihydropyridine calcium antagonist nimodipine on NMDA-induced phenomena were investigated using an in vivo fluorometric technique with indo-1. Indo-1, a fluorescent cytosolic free calcium ([Ca2+]i) indicator, was loaded into the cat cortex approximately 500 microns in depth by superfusion with the membrane-permeant indo-1 acetoxy-methyl ester (indo-1-AM). Changes in [Ca2+]i signals (400 and 506 nm) and reduced nicotinamide adenine dinucleotide (NADH) fluorescence (464 nm) were simultaneously measured directly from the cortex during ultraviolet excitation (340 nm). Superfusion of 100 microM NMDA over the exposed cortex induced an elevation of the [Ca2+]i signal ratio (400/506 nm), biphasic changes in NAD/NADH redox state (initial oxidation followed by progressive reduction), and characteristic changes in the EEG (abrupt depression in amplitude followed by an excitatory pattern of 18-22 Hz polyspikes or sharp waves). These changes were completely blocked by treatment with MK-801 and reduced by nimodipine. The mechanism underlying the protective effects of systemically administered MK-801 on the NMDA-induced neuronal injury was verified in vivo.     

Inhibiting neuronal migration by blocking NMDA receptors in the embryonic rat cerebral cortex: a tissue culture study

To investigate the role of the NMDA receptor on neuronal migration in the cerebral cortex, we performed a tissue culture study using embryonic rat brain. After we labeled progenitor cells in the ventricular zone of E16 cerebral cortex explants by [3H]thymidine, the explants were cultured for 48 h. Then distribution of labeled cells was evaluated autoradiographically. Blocking NMDA receptors by adding the NMDA receptor antagonist, (+)-5-methyl-10, 11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801: 1 or 10 microM) or d(-)-2-amino-5-phosphonopentanoic acid (d-AP5: 100 microM) to the culture medium, caused significantly decreased distribution of labeled cells in the outer intermediate zone (control 14.2+/-5.5%, 1 microM MK-801 5.8+/-7.2%, 10 microM MK-801 3.6+/-1.4%, and d-AP5 8.6+/-4.0%; mean+/-S.D.). This suggests that blocking NMDA receptors inhibits neuronal migration in the cerebral cortex. Furthermore, the influence of decreased intracellular Ca2+ concentration on neuronal migration was examined by adding intracellular Ca2+ chelator, 1, 2-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetra-(acetoxymethyl)-ester (BAPTA-AM: 5 or 25 microM). This also resulted in inhibited neuronal migration. Therefore, it seems that neuronal migration in the cerebral cortex is regulated by intracellular Ca2+ concentration, which the NMDA receptor may influence.     

Ion Dependence and Receptor Mediation of Glutamate Toxicity in the Immature Rat Hippocampal Slice

Glutamate (glu) is a major excitatory transmitter and a toxin in the brain. In the present study, the immature rat hippocampal slice was used to determine the morphology, topography, ionic mediation and receptor specificity of glu toxicity. Slices were exposed to glu for 30 min, and the damage was evaluated after 3 h of recovery in regular medium. The effects on glu toxicity of changes of [Ca2+], [Cl-] and [Na+] were determined. The receptor preference of glu was assessed by using the N-methyl-D-aspartate (NMDA) antagonist MK-801 and the kainate (KA)/quisqualate (QA) antagonist DNQX, alone or in combination. Further, to see whether glu produces cytotoxicity via osmolysis, the effects of hyperosmolal sucrose on glu toxicity were studied. Glu toxicity was similar to the previously described NMDA toxicity with regard to cytopathology, but differed in some aspects from that caused by KA and QA. The severity of the lesion was determined by the proximity of neurons to the incubation fluid, probably as a consequence of cellular accumulation of the amino acid. Omission of Ca2+ abolished glu toxicity in all neurons except the granule cells of the outer blade. This population was completely protected when Ca2+ was omitted and [Cl-] was reduced. Elevation of [Ca2+] markedly aggravated the lesion caused by glu. Substitution of isethionate for Cl- worsened the glu-induced damage, whilst the amino acid produced qualitatively different neuropathology when choline substituted for Na+. Apparently glu did not damage hippocampal nerve cells through an osmolytic mechanism as medium supplemented with 100 mM sucrose increased the toxicity of glu. Since the lesion produced by glu was more widespread in the presence of high [Ca2+], the effects of receptor antagonists were studied under this condition. MK-801 inhibited glu toxicity whereas DNQX had no effect. Combination of MK-801 and DNQX did not offer better protection than did MK-801 alone. The results suggest that Ca2+ is the main (but not single) determinant of glu toxicity in the immature hippocampal slice. The ionic requirements of glu neurotoxicity are identical to those of NMDA, but differ from those of KA and QA. The notion that glu is a selective NMDA agonist in the present model was confirmed by the protection of MK-801, and by the lack of an effect of DNQX. This is the first report demonstrating that the toxicity of glu is mediated by NMDA receptors in brain tissue which has developed normally. The findings indicate that specific blockade of NMDA receptors may be the most rational strategy in the prevention of glu-related neuronal death occurring in certain neurological anomalies.     

Simultaneous action of MK-801 (dizclopine) on dopamine, glutamate, aspartate and GABA release from striatum isolated nerve endings

The simultaneous effect of MK-801 on the baseline- and depolarization (20 microM veratridine or 30 mM high K+)-evoked release of endogenous dopamine, glutamate (Glu), aspartate (Asp), and GABA is investigated in the same preparation of rat striatum isolated nerve endings. MK-801, in the microM range, selectively increases the baseline and high K+ depolarization-evoked release of dopamine, without causing any effect on the baseline or on the high K+-evoked release of Glu, Asp and GABA. In addition to this selective action on dopamine release, MK-801 inhibits the veratridine depolarization-evoked release of all the neurotransmitters tested, including dopamine. In SBFI and fura-2 preloaded striatal synaptosomes, MK-801 inhibits the elevation of internal Na+ (Na(i)) and the elevation of internal Ca2+ (Ca(i)) induced by veratridine depolarization. The elevation of Ca(i) induced by high K+ depolarization is unchanged by MK-801. This study reveals two separate MK-801 actions. (1) The voltage-independent action, which increases dopamine release selectively, and might contribute to the effects of MK-801 on motor coordination. (2) The voltage-dependent action, which inhibits all the veratridine-evoked responses including the evoked release of the excitatory amino acids (which are particularly concentrated in striatum nerve endings), and might contribute to the anticonvulsant and neuroprotective effects of MK-801.     

Hypoosmotic swelling increases protein tyrosine nitration in cultured rat astrocytes

Astrocyte swelling is observed in different types of brain injury. We studied a potential contribution of swelling to protein tyrosine nitration (PTN) by using cultured rat astrocytes exposed to hypoosmotic (205 mosmol/L) medium. Hypoosmolarity (2 h) increases total PTN by about 2-fold in 2 h. The hypoosmotic PTN is significantly inhibited by the NMDA receptor antagonist MK-801, the nitric oxide synthase (NOS) inhibitor L-NMMA, the extracellular Ca2+ chelator EGTA and the calmodulin antagonist W13, suggesting the involvement of NMDA receptor activation, influx of extracellular Ca2+ and Ca2+/calmodulin-dependent NO synthesis. Further, superoxide dismutase plus catalase and uric acid strongly inhibit hypoosmotic PTN, suggesting the involvement of the toxic metabolite peroxynitrite (ONOO-) as a nitrating agent. Hypoosmotic astrocyte swelling rapidly stimulates generation of reactive oxygen intermediates; this process is prevented by MK-801 and EGTA. In addition, MK-801 inhibits the hypoosmotic elevation of [Ca2+]i. The findings support the view that astrocyte swelling as induced, for example, by toxins relevant for hepatic encephalopathy is sufficient to produce oxidative stress and PTN and thus contributes to altered astroglial and neuronal function.     

Effect of the removal of extracellular Ca2+ on the response of cytosolic concentrations of Ca2+ to ouabain in carotid body glomus cells of adult rabbits.

Effect of the removal of extracellular Ca2+ on the response of cytosolic concentrations of Ca2+ ([Ca2+]i) to ouabain, an Na+/K+ exchanger antagonist, was examined in clusters of cultured carotid body glomus cells of adult rabbits using fura-2AM and microfluorometry. Application of ouabain (10 mM) induced a sustained increase in [Ca2+]i (mean+/-S.E.M.; 38+/-5% increase, n=16) in 55% of tested cells (n=29). The ouabain-induced [Ca2+]i increase was abolished by the removal of extracellular Na+. D600 (50 microM), an L-type voltage-gated Ca2+ channel antagonist, inhibited the [Ca2+]i increase by 57+/-7% (n=4). Removal of extracellular Ca2+ eliminated the [Ca2+]i increase, but subsequent washing out of ouabain in Ca2+-free solution produced a rise in [Ca2+]i (62+/-8% increase, n=6, P<0.05), referred to as a [Ca2+]i rise after Ca2+-free/ouabain. The magnitude of the [Ca2+]i rise was larger than that of ouabain-induced [Ca2+]i increase. D600 (5 microM) inhibited the [Ca2+]i rise after Ca2+-free/ouabain by 83+/-10% (n=4). These results suggest that ouabain-induced [Ca2+]i increase was due to Ca2+ entry involving L-type Ca2+ channels which could be activated by cytosolic Na+ accumulation. Ca2+ removal might modify the [Ca2+]i response, resulting in the occurrence of a rise in [Ca2+]i after Ca2+-free/ouabain which mostly involved L-type Ca2+ channels.     

N-methyl-d-aspartate receptor-mediated processing of beta-amyloid precursor protein in rat hippocampal slices: in vitro--superfusion study

Abnormal proteolytic degradation of the beta amyloid precursor protein (beta-APP) may result in accumulation of potentially neurotoxic beta amyloid (betaA). The role of various receptors in the regulation of beta-APP processing has been suggested. This study aimed to determine how NMDA receptors and Ca2+ ions regulate proteolysis of beta-APP in rat hippocampus in vitro. Adult rat hippocampal slices were superfused with NMDA-containing media, and immunoreactivity of soluble beta-APP derivatives was detected in dialysates. Application of 100 microM and 250 microM NMDA for 20 min in Ca2+-containing medium induced dose-dependent release of aminoterminal beta-APP derivatives, and a fragment of Abeta sequence, whereas carboxy-terminal fragments of beta-APP were only slightly detected. This indicates activation of beta-APP processing, and release of its soluble cleavage products. This effect was inhibited by NMDA receptor antagonist 1 microM MK-801 and 100 microM CPP in Ca2+-free medium, thus indicating that NMDA receptors and calcium ions mediate proteolytic non-amyloidogenic degradation of the beta-APP.     

Mode of action of taurine as a neuroprotector

Previously, it has been shown that taurine exerts its protective function against glutamate-induced neuronal excitotoxicity through its action in reducing glutamate-induced elevation of intracellular free calcium, [Ca2+]i. Here, we report the mechanism underlying the effect of taurine in reducing [Ca2+]i. We found that taurine inhibited glutamate-induced calcium influx through L-, P/Q-, N-type voltage-gated calcium channels (VGCCs) and NMDA receptor calcium channel. Surprisingly, taurine had no effect on calcium influx through NMDA receptor calcium channel when cultured neurons were treated with NMDA in Mg2+-free medium. Since taurine was found to prevent glutamate-induced membrane depolarization, we propose that taurine protects neurons against glutamate excitotoxicity by preventing glutamate-induced membrane depolarization, probably through its effect in opening of chloride channels and, therefore, preventing the glutamate-induced increase in calcium influx and other downstream events.     

Presynaptic site of action underlying the ethanol-induced inhibition of norepinephrine release evoked by stimulation of N-methyl-D-aspartate (NMDA) receptors in rat cerebral cortex.

Rat brain cortex synaptosomes pre-incubated with [3H]norepinephrine were used (1) to provide evidence that part of the NMDA receptors mediating stimulation of norepinephrine (NE) release are located on the noradrenergic varicosities themselves, (2) to characterize these receptors and (3) to examine whether ethanol specifically inhibits the NMDA-evoked NE release via a presynaptic site of action. In synaptosomes superfused with Mg(2+)-free Krebs-Henseleit solution, NMDA (2-min exposure) stimulated tritium overflow in a concentration- and glycine-dependent manner. The stimulatory effect of NMDA was not altered by tetrodotoxin but was abolished by omission of Ca2+ from the superfusion fluid and was considerably reduced in the presence of 1.2 mM Mg2+. DL-(E)-2-Amino-4-methyl-5-phosphono-3-pentanoic acid (CGP 37849; a competitive NMDA receptor antagonist) produced a parallel shift of the concentration-response curve for NMDA to the right, whereas dizocilpine (MK-801; an antagonist at the phencyclidine, PCP, recognition site of the NMDA-gated ion channel) reduced the maximum effect of NMDA. Ethanol inhibited the NMDA-evoked tritium overflow in a concentration-dependent manner. In contrast, in synaptosomes superfused with Ca(2+)-free Krebs-Henseleit solution containing 15 mM K+ throughout, ethanol did not affect the tritium overflow evoked by 2 min introduction of 75 microM Ca2+ into the superfusion fluid. This Ca(2+)-evoked overflow was also not altered by tetrodotoxin and dizocilpine, but was inhibited by the inorganic Ca2+ channel antagonist Cd2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulatory effects of aluminum, calcium, lithium, magnesium, and zinc ions on [3H]MK-801 binding in human cerebral cortex

The independent and combined effects of Ca2+, Mg2+, Zn2+, Al3+ and Li+ on [3H]MK-801 binding in human cerebral cortical membranes were studied to further characterize the modulatory effects of metal ions on the N-methyl-D-aspartate (NMDA) receptor-ionophore. Glycine, in the presence of glutamate, significantly intensified the Mg2+ inhibition of [3H]MK-801 binding whereas it masked the Ca2+ enhancement and slightly diminished the Zn2+ inhibition. Both Ca2+ and Mg2+ reduced the Zn2+ inhibitory potency. Aluminum demonstrated a potent, relatively glycine-insensitive inhibition of [3H]MK-801 binding as an amorphous Al(OH)3 polymer rather than as the free ion. Cationic modulation of the NMDA receptor-ionophore appears to be regulated at multiple sites which have significant allosteric interactions.     

Excitatory amino acid regulation of intracellular Ca2+ in isolated catfish cone horizontal cells measured under voltage- and concentration-clamp conditions.

[Ca2+]i was measured using fura-2-loaded isolated catfish horizontal cells in the presence of L-glutamate and the glutamate analogs kainate (KA), quisqualate (QA), and NMDA. Caffeine was used to release Ca2+ from intracellular stores. Cell membrane potential was controlled with a voltage clamp to prevent activation of voltage-dependent Ca2+ channels in the presence of agonist. All excitatory amino acid agonists produced a rapid and sustained rise in [Ca2+]i with the order of potency being QA greater than Glu greater than KA greater than NMDA. The agonist-induced [Ca2+]i increase was blocked in reduced [Ca2+]o and by 6-cyano-7-nitroquinoxaline-2,3-dione and 2-amino-5-phosphonopentanoate, which are specific blockers for QA/KA and NMDA receptors, respectively. The metabotropic receptor agonist trans-1-amino-1,3-cyclopentanedicarboxylic acid (ACPD; 10-200 microM) had no effect on [Ca2+]i. Hill coefficients from curves fitted to concentration-response data suggested an amplification of the Ca2+ signal that was interpreted as calcium-induced calcium release (CICR) from intracellular Ca2+ stores. Caffeine (10 mM) produced a rapid transient rise in [Ca2+]i, confirming the existence of a Ca(2+)-sensitive store. Following caffeine-induced depletion of Ca2+ from intracellular stores, agonists were still able to produce increases in [Ca2+]i, confirming Ca2+ influx through the agonist-gated channel. The agonist-induced increase in [Ca2+]i was decreased following caffeine-induced depletion, confirming a process of CICR. These results are consistent with the hypothesis that excitatory amino acids can produce direct modulation of [Ca2+]i by influx through the agonist-gated channel and by CICR from intracellular stores.     

Secreted phospholipase A2 potentiates glutamate-induced calcium increase and cell death in primary neuronal cultures

Secreted phospholipases A2 (sPLA2s) modulate neuronal survival and neurotransmitter release. Here we show that sPLA2 (group III) synergistically increases glutamate-induced cell death and intracellular calcium ([Ca2+]i) in cultured primary cortical and hippocampal neurons. Whereas 1 microM glutamate elicited transient [Ca2+]i increases in all neurons that recovered 66% to baseline, 25 ng/ml sPLA2 pretreatment resulted in sustained [Ca2+]i increases, with only 5% recovery. At 250 nM glutamate, 25% of neurons failed to respond, and the average recovery time was 101 +/- 12 sec; sPLA2 increased recovery time to 158 +/- 6 sec, and only 2% of cells failed to respond. Both the noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 and the calcium-channel blocker cobalt inhibited this effect. Experiments with the glutamate uptake inhibitor L-trans-pyrollidine-2,4-dicarboxylic acid (2.5 microM) indicated that glutamate uptake sites are not a likely modulation point by sPLA2, whereas arachidonic acid (AA) potentiated calcium responses to glutamate. Thus the enhancement of glutamate-induced [Ca2+]i increases by sPLA2 may be due to modulation at NMDA receptors and/or calcium channels by AA. These results indicate that sPLA2 affects neuronal responses to both nontoxic (0.1-10 microM) and toxic (=25 microM) concentrations of glutamate, implicating this enzyme in neuronal functions in pathology.     

Influence of MK-801 on brain extracellular calcium and potassium activities in severe hypoglycemia

The purpose of the present study was to examine the effect of blockade of N-methyl-D-aspartate (NMDA) receptors on the depolarization associated with severe hypoglycemia, which is commonly preceded by one or a few transient depolarizations reminiscent of cortical spreading depression (CSD). In the cerebral cortices of rats [K+]e and [Ca2+]e were measured with ion-selective microelectrodes. NMDA blockade was achieved by injection of MK801 in doses that block CSD. In control rats, the latency from the time point when blood glucose reached minimal levels to onset of ionic shifts was 33.2 +/- 3.5 min, and [K+]e rose from 3.2 +/- 0.2 to 55 +/- 5 mM. All variables remained unchanged in rats treated with MK801. In another four rats treated with MK801, [Ca2+]e declined from 1.06 +/- 0.22 to 0.12 +/- 0.02 mM. Plasma glucose measurements indicated that the cortex depolarized at a plasma glucose concentration between 0.7 and 0.8 mM, i.e., within a narrow range, suggesting a threshold phenomenon. In conclusion, activation of NMDA receptors seems of minor importance for hypoglycemic depolarization. The ionic transients that precede the persistent hypoglycemic depolarization are probably mediated by mechanisms distinct from those of electrically induced CSD.     

The effect of age on binding of MK-801 in the cat visual cortex.

We have examined the effect of age on the binding of (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine maleate (MK-801) in the cat visual cortex. We hypothesized that this binding might change with age because: (1) MK-801 binds to a site associated with the N-methyl-D-aspartate (NMDA) receptor; (2) the NMDA receptor complex has been implicated in neural plasticity; (3) plasticity in the cat visual cortex is age dependent. We used standard receptor binding techniques to measure MK-801 binding in membrane homogenates in cats aged 7 days (d), 21 d, 43 d, 83 d, 7-8 months (mo) and over 2 years. Glutamate (100 microM), glycine (30 microM) and spermidine (20 microM) were used to enhance binding. We found that MK-801 binding is maximal at about 6 weeks of age, decreases slightly by 83 days and then decreases more dramatically in adults. Saturation analysis showed that the of binding with age resulted from variation in number of binding sites and not from variation in affinity. The ability of Mg2+ to inhibit MK-801 binding did not change with age. Dark rearing did not alter the development of MK-801 binding sites.     

Age-related changes in NMDA-induced [3H]acetylcholine release from brain slices of senescence-accelerated mouse.

From the brain slices of normal mice (ddY strain, subcloned from dd strain in National Institute of Health in Japan), N-methyl-D-aspartic acid (NMDA) at 0.01-1 mM evoked [3H]acetylcholine (ACh) release in a concentration dependent manner. [3H]ACh release evoked by 1 mM NMDA was significantly inhibited by 2-amino-5-phosphonovaleric acid (APV), phencyclidine (PCP) and 5-methyl-10,11-dihydroxy-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801). The effects of NMDA were not seen in the Ca2+ free medium and were inhibited by physiological concentration (0.83 mM) of Mg2+. NMDA seems to cause ACh release from nerve terminals through the receptor-ion channel mediated mechanism in the mouse brain. Based upon these results, we determined the activity of a high K(+)- or NMDA-evoked [3H]ACh release using prone/8 strain of senescence-accelerated mouse (SAM-P/8) (a murine model of accelerated aging and memory dysfunction) and SAM-resistance/1 strain (SAM-R/1) (normal aging mice as the control) and these release activities were compared between both strains and during aging. [3H]ACh release evoked by 30 mM KCl was significantly lower than that of age-matched SAM-R/1 at 9 and 12 months. NMDA evoked the [3H]ACh release at 2, 6, 10 and 14 months in R/1 mice. In SAM-P/8 mice the activity of NMDA-evoked release was seen at 2 months, but markedly decreased afterwards. Nonsignificant difference was observed on the uptake of [3H]choline and on the spontaneous release of [3H]ACh between SAM-P/8 and SAM-R/1 strains, and during aging.(ABSTRACT TRUNCATED AT 250 WORDS)