Stimulatory Effect Of N-methyl-D-aspartate Research Articles

Control of gonadotropin secretion in prepubertal male rats by excitatory amino acids.

The role of N-methyl-D-aspartate (NMDA) and non-NMDA receptors in the control of FSH and LH secretion was analysed in prepubertal male rats. In the first experiment 4, 8, 12, 16, 20 and 30-day-old males were decapitated 15 min after vehicle, NMDA or kainic acid (KA) administration. In the second experiment, 23-day-old males were sham-orchidectomized or orchidectomized. Orchidectomized males were or were not implanted with silastic capsules containing testosterone and were sacrificed on day 30 after injection with vehicle, NMDA (15 mg kg-1), LHRH (100 ng rat-1), NMDA plus LHRH, or MK801, a non-competitive NMDA antagonist, (1 mg kg-1). In the third experiment, 30-day-old intact males pretreated with Nw-nitro-L-arginine methyl ester (NAME) or NG-methyl-L-arginine (MA), blockers of nitric oxide (NO) synthase, were sacrificed after NMDA or KA administration. In the fourth experiment, the effects of NMDA, KA, LHRH and NAME were analysed in monolayer cultures of dispersed adenohypophyseal cells. We found that: (i) NMDA and KA stimulated LH and FSH secretion in intact males; (ii) the NMDA effect on LH secretion remained after orchidectomy; (iii) FSH and LH responses to LHRH were not affected by simultaneous NMDA administration; (iv) antagonization of NMDA receptors with MK801 reduced the LH secretion in intact and orchidectomized males and blunted the FSH response to orchidectomy; (v) the stimulatory effect of NMDA or KA on LH secretion was not affected by pretreatment with blockers of NO synthase; (vi) the in vitro LH secretion remained unchanged after administration of NMDA, KA or NAME. We conclude that NMDA and non-NMDA receptors play a physiological role in the control of the basal and post-orchidectomy secretion of gonadotropins, and that this effects is independent of changes in NO generation and does not include changes in pituitary responsiveness to LHRH.

NMDA receptor-mediated control of GABA release from neurointermediate lobes of female and male rats

The effect of glutamate (GLUT) and its ionotropic receptor agonists on K +-evoked GABA release from the neurointermediate lobe (NIL) was investigated in diestrus, ovariectomized, ovariectomized–estrogenized female rats and intact male rats. GLUT and N-methyl- d-aspartate (NMDA) increased K +-evoked GABA release from the NIL in all the experimental groups. This stimulatory effect of NMDA was blocked by specific NMDA receptor antagonists but not by non-NMDA receptor antagonists. However, kainate did not modify evoked GABA release from the NIL in any of these groups. Neither GLUT nor NMDA modified nitric oxide synthase activity. These results indicate that GLUT, acting through NMDA receptors, stimulates evoked GABA release from the NIL of female and male rats. This effect is not influenced by gonadal status and does not appear to be mediated by nitric oxide production.

Modulation of striatal acetylcholine concentrations by NMDA and the competitive NMDA receptor-antagonist AP-5: an in vivo microdialysis study.

The effects of local perfusion with the competitive NMDA receptor antagonist 2-amino-5-phosphonovalerate (AP-5) and the glutamate receptor agonist N-methyl-D-aspartate (NMDA) on release of extracellular acetylcholine (ACh) and choline (Ch) in the dorsolateral striatum were studied using in vivo microdialysis in freely moving rats. AP-5 caused a dose-dependent decrease in ACh release that was counteracted by the addition of NMDA. Perfusion with AP-5 also decreased Ch levels. Local perfusion with NMDA induced an elevation of ACh release in low (10(-5) M), but not high (10(-2) M and 10(-3) M) concentrations, that were associated with massive cellular death. These inhibitory effects of AP-5 and the stimulatory effect of NMDA in non-neurotoxic dosages on ACh release provide further evidence for a tonic stimulation of striatal cholinergic interneurons by glutamatergic neurons via NMDA receptors.

Divalent cation entry in cultured rat cerebellar granule cells measured using Mn2+ quench of fura 2 fluorescence.

In this study the rate of Mn2+ quench of fura-2 fluorescence evoked by glutamatergic and cholinergic agonists, depolarization and Ca2+ store modulators was measured in cultured cerebellar granule cells, in order to study their effects on Ca2+ entry in isolation from effects on Ca2+ store release. The rate of fluorescence quench by 0.1 mM Mn2+ was markedly increased by 25 mM K(+)-evoked depolarization or by 200 microM N-methyl-D-aspartate (NMDA), with a significantly greater increase occurring during the rapid-onset peak phase compared to the plateau phase of the K(+)- or NMDA-evoked [Ca2+]i response. The stimulatory effect of NMDA on Mn2+ quench was abolished by dizocilpine (10 microM), but nitrendipine (2 microM), while decreasing the rate of basal quench, did not affect NMDA-stimulated Mn2+ entry. This suggests that nitrendipine may not act on NMDA channels in granule cells, at least under these conditions, and that voltage-operated Ca2+ channels are involved in control quench whereas the NMDA-evoked quench is dependent on entry through the receptor channel. The t1/2 of quench was unaffected by alpha-amino-hydroxyisoxazole propionic acid (200 microM) and carbamyl choline (1 mM). Neither thapsigargin (10 microM) nor dantrolene (30 microM) significantly affected the rate of quench under control or NMDA- or K(+)-stimulated conditions, which confirms that the previously reported inhibitory effects on [Ca2+]i elevations evoked by these agents are due to actions on Ca2+ stores. However, thapsigargin elevated [Ca2+]i in the presence of normal [Ca2+]o but not in nominally Ca(2+)-free medium, indicating that it evokes Ca2+ entry in cerebellar granule cells, probably subsequent to store depletion, which appears to be either too small to be detected by Mn2+ quench or to occur via Mn(2+)-impermeant channels.

Effects of l- and d-enantiomers of Nω-nitro-arginine on NMDA-evoked striatal dopamine overflow

The microdialysis method was utilized to study the effects of N ω- nitro- l-arginine (L-NNA) and N ω- nitro- d-arginine (D-NNA), an inhibitor of nitric oxide (NO) synthesis from l-arginine and its d-enantiomer, on spontaneous and N- methyl- d-aspartate (NMDA)-evoked overflow of striatal extracellular do-pamine (DA) in unanesthetized, freely moving rats. Perfusion of 3–10 mM NMDA through the microdialysis probe resulted in a dose-dependent increase in striatal DA overflow. Perfusion of 500 μM L-NNA alone did not change the DA level significantly, but when added to 3 mM NMDA, it substantially suppressed the NMDA-evoked increase of DA. Perfusion of 500 μM D-NNA altered neither the basal nor the NMDA-evoked DA level. Data suggest that the stimulatory effect of NMDA in DA release involves activation of the l-arginine-NO pathway in the striatum.

Stimulatory effect of [formula omitted] on somatostatin gene expression in cultured hypothalamic neurons

The aim of the present study was to determine whether N- methyl- d-aspartate (NMDA) stimulates somatostatin gene function in primary cultures of hypothalamic neurons. Neurons were either shortly (for 3, 8, 24 and 72 h) or chronically (for 11 days) exposed to NMDA (20 μM). Medium and cellular somatostatin contents were determined by radioimmunoassay, and steady-state preprosomatostatin mRNA levels by Northern blot analysis with an oligonucleotide probe. DNA content was measured as a cellular viability control. After 8 h incubation, NMDA induced a significant 2-fold increase in somatostatin mRNA accumulation, with a maximal 4-fold increase after 24 h incubation. A significant and dose-dependent (1.7-fold and 2.5-fold at 20 and 100 μM, respectively) stimulatory effect was also observed after chronic treatment. The kinetic patterns for medium and cellular somatostatin contents were similar to those obtained for somatostatin mRNA levels. Total DNA content was not modified under any experimental condition. The augmentations in cellular somatostatin and somatostatin mRNA determined after 24 h or chronic exposure to NMDA were blocked by (+)-5-methyl-10.11-dihydihydro-5 H-dibenzo( a, d′)cyclohepten-5,10-imine hydrogen maleate (MK-801), an NMDA receptor antagonist. MK-801 alone significantly ( P < 0.05) reduced somatostatin mRNA. The stimulatory effect of NMDA on somatostatin mRNA was specific since it was not accompanied by any change in glyceraldehyde-3-phosphate deshydrogenase (GAPDH) mRNA. After immunostaining with a specific antibody against somatostatin, no difference was observed in the number of immunostained neurons detected in control and NMDA exposed groups. However, further analysis of immunostained neurons revealed qualitative and quantitative staining differences between the two groups, with maximal immunostaining intensity detected in perikarya of neurons exposed to NMDA for 24 h. These results demonstrate that NMDA induces specific stimulation of somatostatin gene expression and somatostatin biosynthesis in vitro in hypothalamic neurons.

Stimulation of noradrenaline release in human cerebral cortex mediated by N-methyl-D-aspartate (NMDA) and non-NMDA receptors.

1. Human brain cortical slices from patients undergoing neurosurgery for treatment of epilepsy resistant to antiepileptic drugs were used to identify and characterize N-methyl-D-aspartate (NMDA) and non-NMDA receptors mediating stimulation of noradrenaline release. The slices preincubated with [3H]-noradrenaline were superfused with Krebs-Henseleit solution with or without Mg2+ (1.2 mmol l-1) and were stimulated by 2-min exposure to NMDA, kainic acid or (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). 2. In slices superfused without Mg2+, NMDA induced a concentration-dependent tritium overflow. 3. The NMDA-evoked tritium overflow was almost abolished by tetrodotoxin (TTX), Mg2+ or by omission of Ca2+ from the superfusion fluid. 2-Amino-5-phosphonopentanoic acid (AP5; a competitive NMDA receptor antagonist) or dizocilpine (formerly MK-801; an antagonist at the phencyclidine receptor within the NMDA-gated ion channel) inhibited the NMDA-evoked tritium overflow. The stimulatory effect of NMDA was not significantly enhanced by glycine added to the superfusion fluid but was reduced by 7-chlorokynurenic acid (an antagonist at the glycine site coupled to the NMDA receptor). 4. In slices superfused with solution containing Mg2+, kainic acid or AMPA induced a concentration-dependent tritium overflow which was susceptible to blockade by TTX. 5. The kainic acid-evoked tritium overflow was not affected by DL-(E)-2-amino-4-methyl-5-phosphono-3-pentanoic acid (CGP37849; a competitive NMDA receptor antagonist), but was inhibited by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; an antagonist at glutamate receptors of the non-NMDA type). 6. The AMPA-evoked tritium overflow was also inhibited by CNQX.2ń

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 exposur) 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 Ca 2+ from the superfusion fluid and was considerably reduced in the presence of 1.2 mM Mg 2+. 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 phenyclidine, 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 μM Ca 2+ into the superfusion fluid. This Ca 2+ -evoked overflow was also not altered by tetrodotoxin and dizocilpine, but was inhibited by the inorganic Ca 2+ channel antagonist Cd 2+. Three main conclusions can be drawn: (1) NMDA receptors mediating stimulation of NE release are also located on the cortical noradrenergic varicosities (and not only on so far unknown excitatory interneurones within the cortex); (2) these receptors exhibit the characteristic pharmacological features of the NMDA receptor system; (3) ethanol selectively inhibits the NE release evoked by stimulation of the presynaptic NMDA receptors, leaving the Ca 2+-evoked release promoted by high K + unaffected. This finding is compatible with the suggestions that the NMDA receptor system itself is a site of action of ethanol.

Immunocytochemistry of cGMP in the Cerebellum of the Immature, Adult, and Aged Rat: the Involvement of Nitric Oxide. A Micropharmacological Study.

In this study we describe the localization of formaldehyde-fixed cGMP-immunoreactivity (cGMP-IR) in rat cerebellar tissue slices incubated in vitro. In the absence of phosphodiesterase inhibition, cGMP-immunofluorescence was of low intensity in tissue slices prepared from immature cerebella. Addition of isobutylmethylxanthine (IBMX) to the incubation medium resulted in the appearance of cGMP-IR in clusters of astrocytes in the internal granular layer. Addition of N-methyl-d-aspartate (NMDA), kainic acid, atrial natriuretic factor (ANF), or sodium nitroprusside (SNP) gave an intense cGMP-IR in Bergmann fibres, Bergmann cell bodies, and astrocytes in the internal granular layer. Astrocytes in the white matter showed cGMP-IR after incubation of the slice in the presence of ANF or nitroprusside, but not after NMDA or kainic acid. In addition, after SNP stimulation of cGMP production, cGMP-IR was found in fibres which were not positive for glial fibrillary acidic protein (GFAP). In the adult cerebellar slice, intense basal cGMP-immunostaining was observed in Bergmann fibres, Bergmann cell bodies, and astrocytes in the granular layer. No cGMP-IR was observed in Purkinje cells. Stimulation of the cGMP-content in the glial structures by NMDA, ANF, or SNP, was suggested by the immunocytochemical results. However, when measured biochemically, only the effect of SNP was statistically significant, and immunocytochemistry showed that SNP clearly stimulated cGMP synthesis in neuronal cell structures. In the cerebellum of the aged rat a reduced cGMP-IR was found compared to the adult, in the same structures which showed cGMP-IR in the adult. Basal cGMP-immunostaining was reduced in the presence of haemoglobin, methylene blue, by inhibiting nitric oxide synthesis with NG-monomethyl-l-arginine (NGMAr), or by depletion of external Ca2+. Also the stimulatory effect of NMDA and of ANF (partly) on the cGMP-IR was inhibited by these compounds. cGMP-IR after stimulation of guanylate cyclase by SNP was reduced by the concomitant presence of haemoglobin or methylene blue, but not by NGMAr, or by omission of Ca2+. Our results point to an important role for cGMP in the functioning of glial tissue in the cerebellum and also suggest a role for nitric oxide as an intercellular mediator in the functioning of glutamate and ANF in the cerebellum.

Veratridine and other depolarizing agents counteract the inhibitory effect of Mg2+ ions on N-methyl-D-aspartate (NMDA)-induced noradrenaline release in vitro

Rat brain cortex slices preincubated with 3H-noradrenaline were superfused with Krebs-Henseleit solution with or without Mg2+. In the absence of Mg2+ ions, NMDA evoked 3H-noradrenaline overflow above basal efflux; this effect was concentration-dependently inhibited by Mg2+ (IC50: 19 mumol/l). Despite the presence of 1.2 mmol/l Mg2+, which is known to block cation influx through the ion channel coupled to the NMDA receptor, NMDA evoked 3H-noradrenaline release if the membrane was permanently kept depolarized by 20 or 25 mmol/l K+, 1 mumol/l veratridine or 200 mumol/1 3,4-diaminopyridine; the stimulant effect of NMDA was counteracted by 2-amino-5-phosphonovaleric acid (2-APV), a competitive antagonist at the NMDA receptor and by (+)-5-methyl-10,11-dihydro-5H-dibenzo(a,d)cyclohept-5,10-imine hydrogen maleate (MK 801), an antagonist acting at the cation channel associated with the NMDA receptor. In contrast, no stimulatory effect of NMDA in the presence of 1.2 mmol/l Mg2+ was observed when the membrane of the nerve terminals was intermittently depolarized by electrical impulses of 2 ms duration at a frequency of 1-3 Hz. It is concluded that continuous depolarization of the nerve membrane counteracts the blocking effect of Mg2+ on cation influx through the NMDA receptor-associated ion channel. Under this condition, noradrenaline release can be stimulated by NMDA receptor activation even in the presence of physiological Mg2+ concentrations.