Inhibition by dizocilpine (MK-801) of striatal dopamine release induced by MPTP and MPP+: possible action at the dopamine transporter.

1. The NMDA-type glutamate receptor antagonist, dizocilpine (MK-801) can protect against neurotoxicity associated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its principal metabolite, the 1-methyl-4-phenylpyridinium ion (MPP+). It has been suggested that these neurotoxic effects may be mediated by release of excitatory amino acids, but possible alternative mechanisms have been little investigated. 2. MPTP and MPP+ (0.1-1000 microM) were tested in superfused rat striatal synaptosomes preloaded with [3H]-dopamine. Both MPTP (10 microM and higher) and MPP+ (1 microM and higher) evoked an immediate and concentration-dependent release of [3H]-dopamine. The maximal effect exceeded that achievable with nicotine. For subsequent experiments, submaximal concentrations of MPTP (50 microM) and MPP+ (10 microM) were tested. 3. MK-801 (0.1-100 microM) inhibited responses to MPTP (50 microM) and MPP+ (10 microM) in a concentration-dependent manner. However, further tests of NMDA-type glutamate receptor involvement proved negative. Responses to MPTP or MPP+ were unaffected by the omission of Mg2+ or Ca2+ and were not reduced by the NMDA receptor antagonists, AP-7 (200 microM) and kynurenic acid (300 microM). In this assay, N-methyl-D-aspartate (even in the absence of Mg2+ and with added glycine and strychnine) did not evoked [3H]-dopamine release. 4. In crude membrane preparations of rat cerebral cortex, MPTP and MPP+ inhibited high-affinity [3H]-nicotine binding to nicotinic cholinoceptors (IC50 1.8 microM and 26 microM, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

地佐环平对缺血突触体氨基酸释放和游离钙的影响(英文)

目的　初步探讨地佐环平 (MK 80 1)对脑缺血的保护作用与兴奋性氨基酸 (EAA)和抑制性氨基酸(IAA)释放的关系。方法　营养液中去除糖和氧 ,建立大鼠脑突触体缺血模型 ,检测静息及高钾去极化状态下 ,缺血突触体EAA和IAA释放量及游离钙浓度。结果　大鼠突触体缺血状态致天冬氨酸和甘氨酸、牛磺酸、γ 氨基丁酸释放量明显增加 ,而谷氨酸亦有增加趋势。缺血 + 5 0mmol·L- 1K+刺激后 ,上述EAA及IAA释放量均进一步增加。静息及去极化状态下的缺血突触体游离钙浓度显著增加。MK 80 10 .1mmol·L- 1可显著阻遏静息及去极化状态缺血突触体释放EAA及IAA ,但对EAA的抑制作用更明显 ;同时亦明显降低两种状态下缺血突触体内游离钙浓度。结论　MK 80 1的抗脑缺血致脑损伤作用 ,至少部分与其对抗缺血引起的突触体内游离钙浓度升高和EAA释放量增加的作用有关。     

Role of the sigma receptor in the inhibition of [3H]-noradrenaline uptake in brain synaptosomes and adrenal chromaffin cells.

1. Rat brain synaptosomes and cultured bovine adrenal chromaffin cells were used to monitor the inhibitory effects of phencyclidine (PCP) and sigma (sigma)-receptor ligands on the uptake of [3H]-noradrenaline ([3H]-NA). 2. A Na(+)-dependent high affinity uptake was observed in synaptosomes (30 degrees C) and chromaffin cells (37 degrees C) with Km of 0.22 and 0.56 microM and Vmax of 2.5 pmol min-1 mg-1 protein and 0.7 pmol min-1 per 10(6) cells, respectively. 3. PCP and haloperidol inhibited the high affinity uptake with IC50 of 0.17 and 0.42 microM, respectively in synaptosomes and 0.24 and 0.47 microM, respectively in adrenal chromaffin cells. 4. A close correlation (r = 0.96) was established between the ability of various PCP and sigma-receptor ligands to inhibit [3H]-NA uptake in both systems: PCP greater than TCP greater than haloperidol greater than 3-(+)-PPP greater than MK-801 greater than or equal to (-)-butaclamol greater than (+)-SKF-10047 greater than DTG. Spiperone and opioid receptor ligands were ineffective at 20 microM. 5. These results indicate that the central and peripheral inhibitory effects of PCP and sigma-receptor ligands on [3H]-NA uptake involves a receptor (sigma 1-like) which is distinct from that (PCP2) recognized for the inhibition of [3H]-dopamine uptake by PCP.     

Inhibition of neuronal Ca(2+) influx by gabapentin and subsequent reduction of neurotransmitter release from rat neocortical slices

Cytosolic calcium ion concentrations ([Ca(2+)](i)) were measured in rat neocortical synaptosomes using fura-2, and depolarization of synaptosomal membranes was induced by K(+) (30 mM). The release of the endogenous excitatory amino acids glutamate and aspartate was evoked by K(+) (50 mM) and determined by HPLC. The release of [(3)H]-noradrenaline from rat neocortical synaptosomes or slices was evoked by K(+) (15 and 25 mM) and measured by liquid scintillation counting. Gabapentin produced a concentration-dependent inhibition of the K(+)-induced [Ca(2+)](i) increase in synaptosomes (IC(50)=14 microM; maximal inhibition by 36%). The inhibitory effect of gabapentin was abolished in the presence of the P/Q-type Ca(2+) channel blocker omega-agatoxin IVA, but not by the N-type Ca(2+) channel antagonist omega-conotoxin GVIA. Gabapentin (100 microM) decreased the K(+)-evoked release of endogenous aspartate and glutamate in neocortical slices by 16 and 18%, respectively. Gabapentin reduced the K(+)-evoked [(3)H]-noradrenaline release in neocortical slices (IC(50)=48 microM; maximal inhibition of 46%) but not from synaptosomes. In the presence of the AMPA receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 2, 3-dioxo-6-nitro-1,2,3,4-tetrahydro[f]quinoxaline-7-sulphonamide (NBQX), gabapentin did not reduce [(3)H]-noradrenaline release. Gabapentin did, however, cause inhibition in the presence of the NMDA receptor antagonist DL-(E)-2-amino-4-methyl-5-phosphono-3-pentanoic acid (CGP 37849). Gabapentin is concluded to reduce the depolarization-induced [Ca(2+)](i) increase in excitatory amino acid nerve terminals by inhibiting P/Q-type Ca(2+) channels; this decreased Ca(2+) influx subsequently attenuates K(+)-evoked excitatory amino acid release. The latter effect leads to a reduced activation of AMPA receptors which contribute to K(+)-evoked noradrenaline release from noradrenergic varicosities, resulting in an indirect inhibition of noradrenaline release.     

D2-dopamine receptor-mediated inhibition of intracellular Ca2+ mobilization and release of acetylcholine from guinea-pig neostriatal slices.

The effect of dopamine receptor activation on electrically- or high K+ (30 mM)-evoked neurotransmitter release and rise in intracellular Ca2+ concentration was investigated using slices of guinea-pig neostriatum. A specific D2-dopamine receptor agonist, LY-171555 (a laevorotatory enantiomer of LY-141865: N-propyl tricyclic pyrazole) at 10(-6) M inhibited electrical stimulation- and high K+-evoked release of [3H]-acetylcholine ([3H]-ACh) to 47.7 +/- 6.0% and 54.1 +/- 5.0% of control, respectively. The maximal inhibition by LY-171555 at 10(-5) M was 54.8 +/- 5.1% reduction of the control. The half-maximal effective concentration (EC50) of LY-171555 for the inhibition of [3H]-ACh release was 2.3 X 10(-7) M. A specific D2-dopamine receptor antagonist, (-)-sulpiride (10(-7) M) reversed the inhibition of [3H]-ACh release induced by LY-171555. A specific D1-dopamine receptor agonist, SK&F 38393 (2,3,4,5-tetrahydro-7,8-dihydroxy-1-phenyl-1H-benzazepine) (10(-5) M) had no effect on the release of [3H]-ACh. LY-171555 (10(-6) M) also inhibited the high K+-evoked endogenous glutamate release, by 47% of control. This inhibitory effect was reversed by (-)-sulpiride (10(-7) M). We used a fluorescent, highly selective Ca2+ indicator, 'quin 2' to measure intracellular free Ca2+ concentrations ([Ca2+]i). Electrical stimulation of slices preloaded with quin 2 led to an elevation of relative fluorescence intensity and this response was reduced by the removal of Ca2+ from the bathing medium. These results indicate that the enhanced elevation in fluorescence intensity in the quin 2-loaded slices reflects the increase of intracellular free Ca2+ concentration, [Ca2+]i. The mixed D1- and D2-receptor agonist, apomorphine and LY-171555 inhibited the increase of [Ca2+]i induced by electrical stimulation or high K+ medium, in a concentration-dependent manner, while SK&F 38393 did not affect the increase of [Ca2+]i. The maximal inhibitory effect of LY-171555 at 3 X 10(-5) M was 35 +/- 3% reduction in control values. The inhibitory effect of LY-171555 was antagonized by (-)-sulpiride (10(-7) M). There was a high correlation (r = 0.997, P less than 0.05) between the D 2-receptor-mediated inhibition of the stimulated rise of [Ca2+]i and [3H]-ACh release. When the slices were superfused with the Ca2+-free medium containing EGTA (10(-4) M) for 5 min, the rise in [Ca2+]i was markedly suppressed to 18.0% of control by LY-171555 (10(-6) M). These data indicate that activation of the D2-dopamine receptor suppresses the elevation of [Ca2+]i induced by depolarizing stimuli.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of long term amineptine treatment on pre- and postsynaptic mechanisms in rat brain.

The effect of amineptine and its two metabolites on monoamine uptake, release and receptor binding was studied in vitro. Amineptine and its two metabolites did not displace labelled ligands for known neurotransmitters and drug receptor sites. Amineptine and its two metabolites did not influence [3H]-5-hydroxytryptamine ([3H]-5-HT) uptake or release by rat brain synaptosomes. Amineptine inhibited [3H]-dopamine and [3H]-noradrenaline ([3H]-NA) accumulation, with IC50 values of 1.4 and 10 microM, respectively. The effect was retained, though with lower efficacy, by the two metabolites. Amineptine released [3H]-dopamine from preloaded synaptosomes. Metabolite 1 had no effect on catecholamine release, and metabolite 2 was about half as active as the parent compound on [3H]-dopamine release. The releasing effect of amineptine on [3H]-dopamine was potentiated by reserpine pretreatment, suggesting that the drug acts on the cytoplasmic neurotransmitter pool. Chronic treatment with amineptine (20 mg kg-1, twice daily for 15 days followed by a 3 days drug withdrawal period) resulted in a decrease of [3H]-spiperone binding sites in striatum, and of [3H]-dihyroalprenolol and [3H]-clonidine in cortex. Chronic treatment with amineptine reduced basal [3H]-dopamine accumulation in striatal synaptosomes, without affecting [3H]-NA or [3H]-5-HT accumulation. The adaptive changes in the pre- and postsynaptic dopamine mechanisms observed after long term treatment with amineptine are consistent with the drug acting as an indirect dopamine agonist. The down regulation of beta- and alpha 2-noradrenoceptors observed after long term amineptine treatment may play a role in the antidepressant activity of the drug.     

Excessive release of [3H]noradrenaline and glutamate in response to simulation of ischemic conditions in rat spinal cord slice preparation: effect of NMDA and AMPA receptor antagonists

In the present study we investigated the effects of NMDA and non-NMDA glutamate receptor antagonists on the ischemia-evoked release of [3H]noradrenaline from rat spinal cord slices. An in vitro ischemia model (oxygen and glucose deprivation) was used to simulate the ischemic conditions known to cause neuronal injury. Spinal cord slices were loaded with [3H]noradrenaline and superfused with Krebs solution in a micro-organ bath. Both axonal stimulation and ischemia increased the release of [3H]noradrenaline, but the release in response to glucose and oxygen deprivation was [Ca2+]o independent. Dizocilpine (MK-801), an NMDA receptor antagonist, suppressed the release of [3H]noradrenaline produced by ischemia, while it enhanced the release of [3H]noradrenaline evoked by electrical field stimulation. In contrast, LY300168 (GYKI-53655) [(+/-)-3-N-methylcarbamyde-1-(4-aminophenyl)-4-methyl-1.8-methylen e-dioxy-5H-2.3-benzodiazepine] and its (-)isomer LY303070 (GYKI-53784) [(-)-3-N-methylcarbamyde-1-(4-aminophenyl)-4-methyl-1.8-methylene- dioxy-5H-2.3-benzodiazepine] AMPA receptor antagonists, had no effect on the release of [3H]noradrenaline evoked by either electrical stimulation or ischemia. Desipramine, a noradrenaline uptake inhibitor, potentiated the release of [3H]noradrenaline evoked by ischemia, while in the absence of [Ca2+]o but under conditions when [3H]noradrenaline release was further increased, it reduced the release. Dizocilpine also decreased glutamate and aspartate release, measured by high performance liquid chromatography, during ischemia. It is concluded that glutamate release and NMDA receptors, but not AMPA receptors, are involved in the acute effect of oxygen and glucose deprivation on the excessive release of noradrenaline and that this release is not related to physiological axonal conduction.     

Spermidine enhancement of [3H]MK-801 binding to the NMDA receptor complex in human cortical membranes

The effects of spermidine on the binding of [3H]MK-801 to the N-methyl-D-aspartate (NMDA) receptor complex was studied in human cerebral cortical membranes. [3H]MK-801 binding was increased from 56 +/- 5 fmol/mg protein (mean +/- S.E.M., n = 7) to 319 +/- 71 fmol/mg protein in the presence of 200 microM spermidine. The ED50 for spermidine stimulation of [3H]MK-801 binding was 89 +/- 22 microM (mean +/- S.E.M., n = 6). In the presence of glutamate (1 microM) plus glycine (1 microM) the ED50 was reduced to 5.5 +/- 0.7 microM. The increase in binding in the presence of spermidine was characterised by an increase in the rate of association of [3H]MK-801. In the presence of spermidine. [3H]MK-801 was inhibited by AP5. 7-chlorokynurenic acid and ifenprodil with IC50 values of 0.5 +/- 0.3 24 +/- 19 and 91 +/- 28 microM, respectively. None of these antagonists was a competitive inhibitor of the spermidine stimulation of [3H]MK-801 binding. Thus spermidine modulates the NMDA receptor complex in human brain, providing further evidence that the complex is similar in rat and human cortex.     

Effects of riluzole on electrically evoked neurotransmitter release

1. The main purpose of the present study was to investigate the effects of the neuroprotective agent riluzole on the electrically evoked release of [(3)H]-glutamate ([(3)H]-Glu) in mouse neocortical slices. The reported selectivity of riluzole for excitatory amino acids was tested in release experiments with further neurotransmitters. Also distinct species, mouse, rat and man were compared. 2. [(3)H]-Glu was formed endogenously during incubation of slices with [(3)H]-glutamine ([(3)H]-Gln). Released [(3)H]-Glu and tissue [(3)H]-Glu was separated by anion exchange chromatography. Electrically evoked [(3)H]-Glu release was strongly diminished by tetrodotoxin (TTX) and Ca(2+)-withdrawal. 3. Riluzole (100 microM) depressed the release of [(3)H]-Glu up to 77% (IC(50)=19.5 microM). Riluzole was also able to inhibit strongly the electrically evoked release of [(3)H]-acetylcholine ([(3)H]-ACh) (at 100 microM by 92%, IC(50)=3.3 microM, and [(3)H]-dopamine ([(3)H]-DA) (at 32 microM by 72%, IC(50)=6.8 microM). However, the release of [(3)H]-serotonin ([(3)H]-5-HT) was less diminished (at 100 microM by 53%, IC(50)=39.8 microM). Riluzole up to 100 microM did not affect [(3)H]-noradrenaline ([(3)H]-NA) release. 4. Between species, i.e. in mouse, rat and human neocortex, no significant differences between the effects of riluzole could be observed. 5. The NMDA-receptor blocker MK-801 (1 microM) and the AMPA/Kainate-receptor blocker NBQX (1 microM) did neither affect the electrically evoked [(3)H]-ACh release nor its inhibition by riluzole, indicating that effects of riluzole on transmitter release were neither due to modulation of ionotropic Glu receptors, nor due to indirect inhibition of Glu release through these receptors. 6. Taken together, riluzole inhibits the release of distinct neurotransmitters differently, but is not selective for the excitatory amino acid Glu.     

Carbamazepine inhibits L-type Ca2+ channels in cultured rat hippocampal neurons stimulated with glutamate receptor agonists.

In order to better understand the mechanism(s) of action of carbamazepine (CBZ), we studied its effects on the increase in [Ca2+]i and [Na+]i stimulated by glutamate ionotropic receptor agonists, in cultured rat hippocampal neurons, as followed by indo- or SBFI fluorescence, respectively. CBZ inhibited the increase in [Ca2+]i stimulated either by glutamate, kainate, alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA), or N-methyl-D-aspartate (NMDA), in a concentration-dependent manner. In order to discriminate the effects of CBZ on the activation of glutamate receptors from possible effects on Ca2+ channels, we determined the inhibitory effects of Ca2+ channel blockers on [Ca2+]i changes in the absence or in the presence of CBZ. The presence of 1 microM nitrendipine, 0.5 microM omega-conotoxin GVIA (omega-CgTx GVIA), or of both blockers, inhibited the kainate-stimulated increase in [Ca2+]i by 51.6, 32.9 or 68.7%, respectively. In the presence of both 100 microM CBZ and nitrendipine, the inhibition was similar (54.1%) to that obtained with nitrendipine alone, but in the presence of both CBZ and omega-CgTx GVIA, the inhibition was greater (54%) than that caused by omega-CgTx GVIA alone. However, CBZ did not inhibit the increase in [Na+]i stimulated by the glutamate receptor agonists, but inhibited the increase in [Na+]i due to veratridine. Tetrodotoxin, or MK-801, did not inhibit the influx of Na+ stimulated by kainate, indicating that Na+ influx occurs mainly through the glutamate ionotropic non-NMDA receptors. Moreover, LY 303070, a specific AMPA receptor antagonist, inhibited the [Na+]i response to kainate or AMPA by about 70 or 80%, respectively, suggesting that AMPA receptors are mainly involved. Taken together, the results suggest that CBZ inhibits L-type Ca2+ channels and Na+ channels, but does not inhibit activation of glutamate ionotropic receptors.     

Protective effects of fangchinoline and tetrandrine on hydrogen peroxide-induced oxidative neuronal cell damage in cultured rat cerebellar granule cells

The present study was performed to examine the neuroprotective effects of fangchinoline (FAN) and tetrandrine (TET), bis-benzylisoquinoline alkaloids, which exhibit the characteristics of Ca 2+ channel blockers, on H2O2 -induced neurotoxicity using cultured rat cerebellar granule neurons. H2O2 produced a concentration-dependent reduction of cell viability, which was blocked by (5 R,10 S)-(+)-5-methyl-10,11-dihydro-5 H-dibenzo[ a,d]cyclohepten-5,10-imine (MK-801), an N-methyl- D-aspartate (NMDA) receptor antagonist, verapamil, an L-type Ca 2+ channel blocker, and NG-nitro- L-arginine methyl ester (L-NAME), a nitric oxide synthase (NOS) inhibitor. Pretreatment with FAN and TET over a concentration range of 0.1 to 10 microM significantly decreased the H2O2 -induced neuronal cell death as assessed by a trypan blue exclusion test, a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay and the number of apoptotic nuclei. In addition, FAN and TET inhibited the H2O2 -induced elevation of glutamate release into the medium, elevation of the cytosolic free Ca 2+ concentration ([Ca 2+] c ), and generation of reactive oxygen species (ROS). These results suggest that FAN and TET may mitigate the harmful effects of H2O2 -induced neuronal cell death by interfering with the increase of [Ca 2+] c, and then by inhibiting glutamate release and generation of ROS. Abbreviations. AP5:D(-)-2-amino-5-phosphonopentanoic acid DMSO:dimethyl sulfoxide FAN:fangchinoline H 2 DCF-DA:2',7'-dichlorodihydrofluorescin diacetate MK-801:(5 R,10 S)-(+)-5-methyl-10,11-dihydro-5 H-dibenzo[ a,d]cyclohepten-5,20-imine MTT:3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide L-NAME: NG-Nitro- L-arginine methyl ester NMDA: N-methyl- D-aspartate TET:tetrandrine     

Both enantiomers of 1-aminocyclopentyl-1,3-dicarboxylate are full agonists of metabotropic glutamate receptors coupled to phospholipase C.

We tested the effects of two enantiomers of a glutamate analogue, (trans)-1-aminocyclopentyl-1,3-dicarboxylate (t-ACPD), in striatal and cerebellar neurons in primary culture, as well as in Xenopus oocytes injected with cerebellar rat RNA. In the presence of MK-801, to avoid N-methyl-D-aspartate receptor activation, and 3 microM tetrodotoxin, both enantiomers [(1R,3S)- and (1S,3R)-t-ACPD] stimulated inositol phosphate (InsP) formation both in striatal neurons after 9-11 days in vitro [EC50, 3.7 +/- 1.1 microM, three experiments, and 33 +/- 7.5 microM, three experiments; maximal stimulatory effects, 252 +/- 15%, 13 experiments, and 269 +/- 15% of basal InsP formation, 14 experiments, for (1R,3S)- and (1S,3R)-t-ACPD, respectively] and in cerebellar granule cells after 9-11 days in vitro [EC50, 50 +/- 18 microM, four experiments, and 307 +/- 92 microM, four experiments; maximal stimulatory effects, 401 +/- 71%, eight experiments, and 423 +/- 75% of basal InsP formation, eight experiments, for (1R,3S)- and (1S,3R)-t-ACPD, respectively]. These effects were not additive, indicating that both enantiomers acted at the same receptor molecule. When we monitored t-ACPD-induced increases in intracellular Ca2+ concentration ([Ca2+]i) with fura-2 ratio-imaging, we found that both enantiomers could elicit similar increase in [Ca2+]i, in the presence of 1 microM MK-801 and 3 microM tetrodotoxin; these effects were also observed in the absence of external Ca2+. Moreover, in Xenopus oocytes injected with adult rat cerebellar RNA, both drugs elicited oscillatory increases of a Ca(2+)-dependent chloride conductance, with similar efficacy, with (1R,3S)-t-ACPD being the more potent isomer. These data are in contradiction to previous reports showing that, in "immature" cerebellar neurons and adult hippocampal slices, (1S,3R)-t-ACPD was either the only active enantiomer or a full agonist of metabotropic receptors, with (1R,3S)-t-ACPD being ineffective or a partial agonist. However, performing these experiments in immature (2-3 days in vitro) striatal or cerebellar neurons, we found that only (1S,3R)-t-ACPD was active in stimulating [Ca2+]i.     

Group I mGlu receptors potentiate synaptosomal [3H]glutamate release independently of exogenously applied arachidonic acid

In the current study, we have characterized group I metabotropic glutamate (mGlu) receptor enhancement of 4-aminopyridine (4AP)-evoked [3H]glutamate release from rat cerebrocortical synaptosomes. The broad spectrum mGlu receptor agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid ((1S,3R)-ACPD, 10 microM) increased 4AP-evoked [3H]glutamate release (143.32+/-2.73% control) only in the presence of exogenously applied arachidonic acid; an effect reversed by the inclusion of bovine serum albumin (BSA, fatty acid free). In contrast, the selective group I mGlu receptor agonist (S)-3,5-dihydroxyphenylglycine (DHPG) potentiated (EC50 = 1.60+/-0.25 microM; Emax = 147.61+/-10.96% control) 4AP-evoked [3H]glutamate release, in the absence of arachidonic acid. This potentiation could be abolished by either the selective mGlu1 receptor antagonist (R,S)-1-aminoindan-1,5-dicarboxylic acid (AIDA, 1 mM) or the selective PKC inhibitor (Ro 31-8220, 10 microM) and was BSA-insensitive. The selective mGlu5 receptor agonist (R,S)-2-chloro-5-hydroxyphenylglycine (CHPG, 300 microM) was without effect. DHPG (100 microM) also potentiated both 30 mM and 50 mM K+ -evoked [3H]glutamate release (121.60+/-12.77% and 121.50 +/-4.45% control, respectively). DHPG (100 microM) failed to influence both 4AP-stimulated 45Ca2+ influx and 50 mM K+ -induced changes in synaptosomal membrane potential. Possible group I mGlu receptor suppression of tonic adenosine A1 receptor, group II/III mGlu receptors or GABA(B) receptor activity is unlikely since 4AP-evoked [3H]glutamate release was insensitive to the selective inhibitory receptor antagonists 8-cyclopentyl-1,3-dimethylxanthine, (R,S)-alpha-cyclopropyl-4-phosphonophenylglycine or CGP55845A, respectively. These data suggest an 'mGlu1 receptor-like' receptor potentiates [3H]glutamate release from cerebrocortical synaptosomes in the absence of exogenously applied arachidonic acid. This PKC dependent effect is unlikely to be via modulation of synaptosomal membrane potential or voltage-activated Ca2+ channels and not via a suppression of tonically active inhibitory adenosine A1 receptor, group II/III mGlu receptors or GABA(B) receptors.     

Protection from inorganic mercury effects on the in vivo dopamine release by ionotropic glutamate receptor antagonists and nitric oxide synthase inhibitors

The possible role of ionotropics glutamate receptors on the HgCl(2)-induced dopamine (DA) release from rat striatum was investigated by using in vivo brain microdialysis technique after administration of selective NMDA and AMPA/Kainate receptors antagonists dizocilpine (MK-801), D (-)-2-amino-5-phoshonopentanoic acid (AP5), and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Moreover, we have also studied the effects of nitric oxide synthase (NOS) inhibitors L-nitro-arginine methyl ester (L-NAME) and 7-nitro-indazol (7-NI) on HgCl(2)-induced DA release. Intraestriatal infusion of 1mM HgCl(2) increased striatal DA to 1717.2+/-375.4% respect to basal levels. Infusion of 1mM HgCl(2) in 400 microM MK-801 pre-treated animals produced an increase on striatal DA levels 61% smaller than that induced in non-pre-treated animals. In the case of AP5, this treatment reduced 92% the increase produced by HgCl(2) as compared to non-pre-treated rats. Nevertheless, the administration of CNQX did not produce any effect on HgCl(2)-induced dopamine release. Intrastriatal infusion of 1mM HgCl(2) in 100 microM L-NAME pre-treated animals produced an increase on extracellular DA levels 82% smaller than produced by HgCl(2) alone. In addition, the pre-treatment with 7-NI reduced 90% the increase produced by infusion of HgCl(2) alone in rats. Thus, HgCl(2)-induced DA release could be produced at last in part, by overstimulation of NMDA receptors with NO production, since administration of NMDA receptor antagonists and NOS inhibitors protected against HgCl(2) effects on DA release.     

Storage of biogenic amines in guinea-pig brain synaptosomes: influence of proton gradient and membrane potential

1 The effects of K(+), NaCN and the ionophores monensin, nonactin and carbonyl-cyanide-p-trifluoro-methoxyphenylhydrazone (FCCP) on the contents of [(3)H]-5-hydroxytryptamine ([(3)H]-5-HT), [(3)H]-dopamine and [(3)H]-noradrenaline ([(3)H]-NA) in guinea-pig synaptosomes preloaded with these amines were measured.2 In the presence of Ca(2+), K(+) markedly reduced the amine content of the synaptosomes, indicating an acceleration of spontaneous amine release. In the absence of Ca(2+), K(+) had much less effect.3 Monensin, nonactin and FCCP caused a release of all the three labelled amines. This release was considerably faster and more marked than that induced by K(+) and showed no dependence on Ca(2+). The ionophores did not release lactate-dehydrogenase from synaptosomes.4 NaCN, a blocker of oxidative energy production, did not enhance the spontaneous release of [(3)H]-5-HT nor did it influence the monensin-induced release of [(3)H]-5-HT.5 It is concluded that (a) the intragranular storage of 5-HT, dopamine and NA is dependent on the maintenance of a pH-gradient across the granular membrane as well as on the granular membrane potential; (b) the ionophores cause a non-exocytotic release of granular amines, and (c) blood platelets are partial models for aminergic brain neurones as far as intragranular amine storage is concerned.     

Effects of inorganic mercury on [3H]dopamine release and calcium homeostasis in rat striatal synaptosomes

Inorganic mercury (Hg2+) in vitro increases spontaneous transmitter release from nerve terminals. The mechanisms of action are not well understood but may involve alterations in intraterminal Ca2+ dynamics. In this study we describe actions of Hg2+ in vitro on isolated mammalian CNS striatal nerve terminals (synaptosomes). Cobalt (2 mM) completely blocked the effect of 2 microM Hg2+ on spontaneous [3H]dopamine release. Cadmium (100 microM) was equipotent to Co2+ in blocking depolarization-dependent [3H]dopamine release, but did not alter the 2 microM Hg2(+)-induced spontaneous [3H]dopamine release. Depolarization-dependent [3H]dopamine release was not altered by 5 microM Hg2+. It appears that the site of action of Hg2+ on spontaneous [3H]dopamine release is not the Ca2+ channel. The effects of Hg2+ on intraterminal ionized Ca2+ [( Ca2+]i) were evaluated using the Ca2(+)-specific fluorescent probe, fura-2. Hg2+ (1-8 microM) had no effect on [Ca2+]i in 1.2 mM Ca2(+)-containing buffers. In nominal Ca2+ media, 4 and 8 microM Hg2+ significantly decreased [Ca2+]i. Following exposure to 4 and 8 microM Hg2+ the quenching of extrasynaptosomal fura-2 by Mn2+ was increased, suggesting that Hg2+ facilitated the leakage of fura-2. This apparent leakage was probably due to a nonspecific increase in membrane permeability since 2 microM Hg2+ produced a Co2(+)-insensitive increase in [3H]deoxyglucose phosphate efflux. Hg2+ did not increase the leakage of either lactate dehydrogenase or soluble protein from synaptosomes. Hg2+ produced a concentration-dependent (1-8 microM) increase in 45Ca2+ efflux from superfused synaptosomes which was insensitive to blockade either by 2 mM Co2+ or by 100 microM Cd2+. These data suggest that the transmitter releasing action of Hg2+ involves interactions with sites that also interact with Co2+ but not with Cd2+. Furthermore, Hg2+ may have direct transmitter releasing actions (i.e., Ca2(+)-mimetic properties), as well as nonspecific actions on plasma membrane permeability which may not necessarily be linked to [3H]dopamine release.     

Reduction in the MK-801 binding sites of the NMDA sub-type of glutamate receptor in a mouse model of congenital hyperammonemia: prevention by acetyl-L-carnitine

Our earlier studies on the pharmacotherapeutic effects of acetyl-L-carnitine (ALCAR), in sparse-fur (spf) mutant mice with X linked ornithine transcarbamylase deficiency, have shown a restoration of cerebral ATP, depleted by congenital hyperammonemia and hyperglutaminemia. The reduced cortical glutamate and increased quinolinate may cause a down-regulation of the N-methyl-D-aspartate (NMDA) receptors, observed by us in adult spf mice. We have now studied the kinetics of [3H]-MK-801 binding to NMDA receptors in spf mice of different ages to see the effect of chronic hyperammonemia on the glutamate neurotransmission. We have also studied the Ca2+-dependent and independent (4-aminopyridine (AP) and veratridine-mediated) release of glutamate and the uptake of [3H]-glutamate in synaptosomes isolated from mutant spf mice and normal CD-1 controls. All these studies were done with and without ALCAR treatment (4 mmol/kg wt i.p. daily for 2 weeks), to see if its effect on ATP repletion could correct the glutamate neurotransmitter abnormalities. Our results indicate a normal MK-801 binding in 12-day-old spf mice but a significant reduction immediately after weaning (21 day), continuing into the adult stage. The Ca2+-independent release of endogenous glutamate from synaptosomes was significantly elevated at 35 days, while the uptake of glutamate into synaptosomes was significantly reduced in spf mice. ALCAR treatment significantly enhanced the MK-801 binding, neutralized the increased glutamate release and restored the glutamate uptake into synaptosomes of spf mice. These studies point out that: (a) the developmental abnormalities of the NMDA sub-type of glutamate receptor in spf mice could be due to the effect of sustained hyperammonemia, causing a persistent release of excess glutamate and inhibition of the ATP-dependent glutamate transport, (b) the modulatory effects of ALCAR on the NMDA binding sites could be through a repletion of ATP, required by the transporters to efficiently remove extracellular glutamate.     

Neuroprotective effect of Sanguisorbae radix against oxidative stress-induced brain damage: in vitro and in vivo

Sanguisorbae radix (SR), the root of Sanguisorba officinalis L. (Rosaceae), has been traditionally used for its anti-inflammatory, anti-infectious and analgesic activities in Korea. Previous work has shown that SR prevents neuronal cell damage induced by Abeta (25--35) in cultured rat cortical neurons. The present study was carried out to further investigate the neuroprotective effect of SR on oxidative stress-induced toxicity in primary culture of rat cortical neurons, and on ischemia-induced brain damage in rats. SR, over a concentration range of 10--50 microg/ml, inhibited H2O2 (100 microM)-induced neuronal death, which was significantly inhibited by MK-801 (5 microM), an N-methyl-D-aspartate (NMDA) receptor antagonist, and verapamil (20 microM), an L-type Ca2+ channel blocker. Pretreatment of SR (10-50 microg/ml), MK-801 (5 microM), and verapamil (20 microM) inhibited H2O2-induced elevation of intracellular Ca2+ concentration ([Ca2+]i) measured by a fluorescent dye, Fluo-4 AM. SR (10-50 microg/ml) inhibited H2O2-induced glutamate release into medium measured by HPLC, and generation of reactive oxygen species (ROS) measured by 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA). In vivo, SR prevented cerebral ischemic injury induced by 2-h middle cerebral artery occlusion (MCAO) and 24-h reperfusion. The ischemic infarct and edema were significantly reduced in rats that received SR (10, 30 mg/kg, orally), with a corresponding improvement in neurological function. Catechin isolated from SR inhibited H2O2-induced neuronal death in cultures. Taken together, these results suggest that SR inhibits H2O2-induced neuronal death by interfering with the increase of [Ca2+]i, and inhibiting glutamate release and generation of ROS, and that the neuroprotective effect of SR against focal cerebral ischemic injury is due to its anti-oxidative effects. Thus SR might have therapeutic roles in neurodegenerative diseases such as stroke.     

Pre-synaptic histamine H3 receptors regulate glutamate, but not GABA release in rat thalamus

We have investigated the presence of histamine H(3) receptors (H(3)Rs) on rat thalamic isolated nerve terminals (synaptosomes) and the effect of their activation on glutamate and GABA release. N-alpha-[methyl-(3)H]histamine ([(3)H]-NMHA) bound specifically to synaptosomal membranes with dissociation constant (K(d)) 0.78+/-0.20 nM and maximum binding (B(max)) 141+/-12fmol/mg protein. Inhibition of [(3)H]-NMHA binding by histamine and the H(3)R agonist immepip fit better to a two-site model, whereas for the H(3)R antagonist clobenpropit the best fit was to the one-site model. GTPgammaS (30 microM) decreased [(3)H]-NMHA binding by 55+/-4% and made the histamine inhibition fit better to the one-site model. Immepip (30 nM) induced a modest, but significant increase (113+/-2% of basal) in [(35)S]-GTPgammaS binding to synaptosomal membranes, an effect prevented by clobenpropit (1 microM) and by pre-treatment with pertussis toxin. In thalamus synaptosomes depolarisation-induced, Ca(2+)-dependent glutamate release was inhibited by histamine (1 microM, 25+/-4% inhibition) and immepip (30 nM, 38+/-5% reduction). These effects were reversed by clobenpropit (1microM). Conversely, immepip (up to 1 microM) had no effect on depolarisation-evoked [(3)H]-GABA release. Extracellular synaptic responses were recorded in the thalamus ventrobasal complex by stimulating corticothalamic afferents. H(3)R activation reduced by 38+/-7% the glutamate receptor-mediated field potentials (FPs), but increased the FP2/FP1 ratio (from 0.86+/-0.03 to 1.38+/-0.05) in a paired-pulse paradigm. Taken together, our results confirm the presence of H(3)Rs on thalamic nerve terminals and show that their activation modulates pre-synaptically glutamatergic, but not GABAergic neurotransmission.