Complex involvement of nitric oxide and cGMP at N-methyl-d-aspartic acid receptors regulating γ-[H]aminobutyric acid release from striatal slices

Whilst the depolarization of postsynaptic N-methyl-d-aspartic acid (NMDA) receptors leads to an influx of Ca²⁺ and subsequent synthesis of nitric oxide (NO), we examined roles for NO at striatal NMDA receptors regulating transmitter release. In superfused rat striatal slices, NMDA-evoked release of γ-[³H]aminobutyric acid ([³H]GABA) was investigated in the presence of nitrergic drugs. NMDA-induced release of [³H]GABA was attenuated by d-2-aminophosphonopentanoate, tetrodotoxin and omission of Ca²⁺. l-Arginine enhanced NMDA-evoked release of [³H]GABA, but exogenous NO donors were ineffective. Inhibitors of NO synthase (N^G-nitro- and N^G-amino-l-arginine) and guanylate cyclase (LY83583) elevated release. Since NMDA-evoked release of [³H]GABA was partially tetrodotoxin-sensitive, nitrergic-linked NMDA receptors regulating the release are both pre- and extrasynaptic. Thus not only does NO arise from multiple sites, and involve NMDA receptors with their redox site insensitive to exogenous NO donors, but the NMDA receptors are under the influence of nitrergic and cGMP-linked negative feedback mechanisms.     

Modulation of GABA release by α-amino-3-hydroxy-5-methylisoxazole-4-propionate and N-methyl-d-aspartate receptors in matrix-enriched areas of the rat striatum

Using a new in vitro superfusion device, the release of preloaded [³H]GABA was examined in microdiscs of tissues taken from sagittal slices in matrix-enriched areas of the rat striatum. Potassium (9 mM, 15 mM) stimulated the release of [³H]GABA in a concentration- and calcium-dependent manner and the veratridine (1 μM)-evoked release of [³H]GABA was completely abolished in the presence of tetrodotoxin (1 μM).The selective glutamatergic agonist α-amino-3-hydroxy-5-methylisoxazole-4-propionate (1 mM) enhanced the potassium-evoked release of [³H]GABA as well as the basal outflow of [³H]GABA. This latter effect was found to be calcium-dependent, partially diminished by tetrodotoxin (1 μM), completely blocked by 6,7-dinitro-quinoxaline-2,3-dione (0.1 mM), which is generally used as an antagonist of α-amino-3-hydroxy-5-methylisoxazole-4-propionate receptors, but not affected by ( + )-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK801, 10μM), a specific antagonist of N-methyl-d-aspartate receptors.Similarly, N-methyl-d-aspartate (1 mM) enhanced both the potassium (9mM) and the α-amino-3-hydroxy-5-methylisoxazole-4-propionate (1 mM )-evoked release of [³H]GABA but when used alone, due to the presence of magnesium in the superfusion medium, was ineffective on the basal efflux of [³H]GABA. A stimulatory effect of N-methyl-d-aspartate (1 mM) on the basal outflow of [³H]GABA was observed, however, when magnesium was omitted from the superfusion medium. The stimulatory effect of N-methyl-d-aspartate (1 mM) observed in the presence of α-amino-3-hydroxy-5-methylisoxazole-4-propionate was not potentiated by glycine (1 μM, in the presence of strychnine 1 gmM) and the N-methyl-d-aspartate-evoked response seen in the absence of magnesium was not enhanced byd-serine (1 mM). suggesting that endogenous glycine is already acting on N-methyl-d-aspartate receptors. In fact, in the absence of magnesium, 7-chloro-kynurenate (1 mM) completely abolished the stimulatory effect of N-methyl-d-aspartate on the release of [³H]GABA confirming that under our conditions, the glycine site of the N-methyl-d-aspartate receptor is saturated. N-methyl-d-aspartate-evoked responses were all blocked by MK801 (10 μM). Finally, the N-methyl-d-aspartate-evoked response seen in the absence of magnesium was markedly reduced in the presence of tetrodotoxin (1 μM). While potassium (9 mM) markedly stimulated the release of [³H]GABA from purified synaptosomes from the rat striatum, neither α-amino-3-hydroxy-5-methylisoxazole-4-propionate alone nor N-methyl-d-aspartate in the presence of α-amino-3-hydroxy-5-methylisoxazole-4-propionate or in the absence of magnesium enhanced the release of [³H]GABA. A slight but not significant stimulatory effect was observed with N-methyl-d-aspartateandd-serine using a magnesium-free superfusion medium.Altogether, these results strongly suggest that α-amino-3-hydroxy-5-methylisoxazole-4-propionate and N-methyl-d-aspartate receptors are present on dendrites and/or cell bodies of efferent GABAergic neurons but not on their nerve terminals in matrix-enriched areas of the rat striatum.     

Relation of exocytotic release of γ-aminobutyric acid to Ca2+ entry through Ca2+ channels or by reversal of the Na+/Ca2+ exchanger in synaptosomes

The specific inhibitor of the -aminobutyric acid (GABA) carrier, NNC-711, {1-[(2-diphenylmethylene) amino]oxyethyl}-1,2,5,6-tetrahydro-3-pyridinecarboxylic acid hydrochloride, blocks the Ca²⁺-independent release of [³H]GABA from rat brain synaptosomes induced by 50 mM K⁺ depolarization. Thus, in the presence of this inhibitor, it was possible to study the Ca²⁺-dependent release of [³H]GABA in the total absence of carrier-mediated release. Reversal of the Na⁺/Ca²⁺ exchanger was used to increase the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) to test whether an increase in [Ca²⁺]_i alone is sufficient to induce exocytosis in the absence of depolarization. We found that the [Ca²⁺]_i may rise to values above 400 nM, as a result of Na⁺/Ca²⁺ exchange, without inducing release of [³H]GABA, but subsequent K⁺ depolarization immediately induced [³H]GABA release. Thus, a rise of only a few nanomolar Ca²⁺ in the cytoplasm induced by 50 mM K⁺ depolarization, after loading the synaptosomes with Ca²⁺ by Na⁺/Ca²⁺ exchange, induced exocytotic [³H]GABA release, whereas the rise in cytoplasmic [Ca²⁺] caused by reversal of the Na⁺/Ca²⁺ exchanger was insufficient to induce exocytosis, although the value for [Ca²⁺]_i attained was higher than that required for exocytosis induced by K⁺ depolarization. The voltage-dependent Ca²⁺ entry due to K⁺ depolarization, after maximal Ca²⁺ loading of the synaptosomes by Na⁺/Ca²⁺ exchange, and the consequent [³H]GABA release could be blocked by 50 M verapamil. Although preloading the synaptosomes with Ca²⁺ by Na⁺/Ca²⁺ exchange did not cause [³H]GABA release under any conditions studied, the rise in cytoplasmic [Ca²⁺] due to Na⁺/Ca²⁺ exchange increased the sensitivity to external Ca²⁺ of the exocytotic release of [³H]GABA induced by subsequent K⁺ depolarization. Thus, our results show that the vesicular release of [³H]GABA is rather insensitive to bulk cytoplasmic [Ca²⁺] and are compatible with the view that GABA exocytosis is triggered very effectively by Ca²⁺ entry through Ca²⁺ channels near the active zones.     

Integrin stimulation induces calcium signalling in rat cardiomyocytes by a NO-dependent mechanism

The myocardial stretch-induced increase in intracellular [Ca²⁺] ([Ca²⁺]_i) is considered to be caused by integrin stimulation. Myocardial stretch is also associated with increased nitric oxide (NO) formation. We hypothesised that NO is implicated in calcium signalling following integrin stimulation. Integrins of neonatal rat cardiomyocytes were stimulated with a pentapeptide containing the Arg-Gly-Asp (RGD) sequence. [Ca²⁺]_i was measured with Fura2, [NO]_i was measured with DAF2 and phosphorylation of focal adhesion kinase (FAK) was monitored with immunofluorescence techniques. Integrin stimulation increased both [NO]_i and [Ca²⁺]_i, the latter response being inhibited by ryanodine receptor-2 (RyR2) blockers and by N^G-monomethyl-L-arginine (L-NMMA), an inhibitor of NOS, but resistant to GdCl₃, diltiazem and wortmannin. Integrin-induced intracellular Ca²⁺ release thus appears to be independent of the influx of extracellular Ca²⁺ and phosphatidylinositol-3 kinase activity. In addition, integrin stimulation induced phosphorylation of FAK. Our results provide evidence for an integrin-induced Ca²⁺ release from RyR2 which is mediated by NO formation, probably via FAK-induced NOS activation.     

Potassium-evoked release of [14C]GABA and [3H]taurine from rat olfactory bulb slices

Rat olfactory bulb slices were preloaded with [³H] taurine or with [¹⁴C] GABA. Upon stimulation of the slices with increasing concentrations of KCl, we observed release of [³H] taurine or [¹⁴C] GABA. Superfusion of the slices with high concentrations of K⁺ in the absence of Ca²⁺ in the perfusion medium, led to a marked decrease in the stimulated release of both [³H] taurine and [¹⁴C] GABA.     

Modulation by nitric oxide of gastric acid secretion in toads

Nitric oxide (NO) is a novel chemical messenger that mediates a variety of biological actions. This study was undertaken to investigate the effects of NO on parietal cell function. The rate of [³H]arginine conversion to [³H]citrulline, a parameter of NO synthase activity, and NO formation (as NO^?₂), were inhibited by the NO synthase inhibitor, N ^G-nitro- L -arginine methyl ester ( L -NAME), in a concentration-dependent manner in the non-stimulated toad gastric mucosa. This range of concentrations of L -NAME provoked stimulation of H⁺ secretion in a similar fashion, which was blocked by L -arginine but not by D -arginine. Pre-treatment with carbachol plus ethylene glycol-bis(β-aminoethyl ether)-N,N,N ′,N ′-tetra-acetic acid (EGTA) prevented the effect of L -NAME on H⁺ secretion and drastically reduced NO synthase activity. L -arginine had an inhibitory effect on H⁺ secretion in non-stimulated and carbachol-stimulated gastric mucosa, which was reversed by L -NAME. Carbachol and pentagastrin, but not histamine, significantly increased NO formation in the toad gastric mucosa. The results suggest that changes in NO synthesis in the gastric mucosa may modulate parietal cell function and that a calcium-dependent mechanism may be involved.     

Trans-2-carboxy-3-pyrrolidineacetic acid (CPAA), a novel agonist at NMDA-type receptors

Although 2-carboxy-3-pyrrolidineacetic acid (CPAA) analogs are associated with activity as agonists at kainate-type receptors, here we report that CPAA is an agonist at N-methyl-D-aspartic acid-type receptors. CPAA evoked the release of [3H]acetylcholine (ACh) from striatal slices with the same efficacy as NMDA, and an EC50 of 20.0 microM, compared to an EC50 of 45.8 microM for NMDA. CPAA-evoked [3H]ACh release was inhibited by CPP (IC50 = 5.1 microM), tiletamine (IC50 = 0.53 microM), MK-801 (IC50 = 0.12 microM), and MgCl2 (IC50 = 26 microM). CPAA produced a tachyphylaxis when applied continuously for 18 min or more, and a cross-tachyphylaxis to NMDA. Similarly, NMDA generated a cross-tachyphylaxis to CPAA. All of these data suggest that CPAA is an agonist at NMDA-type receptors.     

Chronic uridine treatment reduces the level of [3H]spiperone-labelled dopamine receptors and enhances their turnover rate in striatum of young rats: relationship to dopamine-dependent behaviours

The effect was studied of chronic uridine treatment on the recovery of striatal D-2 dopamine (DA) receptors after their irreversible blockade by N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) in young (40 days old) and adult (14 months old) male rats using [³H]spiperone as radioligand. Chronic uridine treatment (15 mg kg^-1 day^-1, i.p., 14 days) causes a reduction of [³H]spiperone binding sites in striatum of young rats. This treatment also produces an increase in the rate of recovery of striatal [³H]spiperone-labelled DA receptors in young, but not in adult rats. Catalepsy and exploratory locomotor activity, two behaviours associated with blockade versus activation of DA receptors, were evaluated in the same rats. The behavioural recovery from the EEDC^induced syndrome is more rapid in the young rats treated with uridine than in the saline-treated group. The behavioural recovery in old rats was not affected by chronic uridine treatment. Thus, in young rats the pyrimidine nucleoside uridine may modulate the steady state and the turnover rate of striatal D-2 DA receptors.     

K+-stimulated release of labeled γ-aminobutyrate,glycine and taurine in slices of several regions of rat central nervous system

The effect of depolarization by high K⁺ concentration (68.5 mm) on the release of [³H] γ-aminobutyrate (GABA), [¹⁴C]glycine and [³⁵S]taurine was studied in superfused slices of rat cerebellum, striatum, hypothalamus, colliculi, cerebral cortex and ventral and dorsal halves of spinal cord. The release of [³H]GABA was notably stimulated by K⁺-depolarization in all regions studied, particularly in the cerebral cortex and the hypothalamus. The Ca²⁺-dependence of this phenomenon was studied in the cortex and ventral spinal cord; in both regions the K⁺-stimulated release was abolished when Ca²⁺ was omitted from the superfusing medium. The release of [¹⁴C]glycine was also stimulated in all regions, except the cerebellum, but to a lesser extent than that of GABA. This stimulation was Ca²⁺-dependent in the ventral spinal cord but not in the cerebral cortex. The release of [³⁵S]taurine was not affected by K⁺-depolarization in any of the regions studied.These results are consistent with a widely distributed neurotransmitter role for GABA. The Ca²⁺-dependence of glycine release in the spinal cord is in agreement with a role of this amino acid as a transmitter in this region. The finding that [³⁵S]taurine release was not stimulated by K⁺-depolarization in any of the regions studied, under experimental conditions identical to those resulting in an enhancement of [³H]GABA and [¹⁴C]glycine release, argues against a neurotransmitter role of this amino acid in brain and spinal cord.     

A comparison of the effects of acute and one year's continuous neuroleptic treatment on the release of [3H]glutamate and [3H]acetylcholine from rat striatal slices

The effect of neuroleptic drugs administered acutely or continuously for 1 year on the release of [³H]glutamate and [³H]acetylcholine from striatal slices in vitro has been compared.Acute in vivo administration of haloperidol, trifluoperazine and clozapine increased the potassium-evoked release of [³H]acetylcholine from striatal slices in a dose-dependent fashion, whereas sulpiride was without effect. None of the neuroleptics given acutely had any effect on the potassium-evoked striatal release of [³H]glutamate.Potassium-evoked striatal release of [³H]acetylcholine in animals receiving 1 year's continuous administration of haloperidol, trifluoperazine or sulpiride was no different from that in age-matched control animals, but was less than controls in animals receiving clozapine for 1 year. All drugs caused a decrease in potassium-evoked striatal [³H]glutamate release following drug administration for 1 year compared to age-matched controls.The reversal of the acute action of neuroleptic drugs on striatal [³H]acetylcholine and [³H]glutamate release is consistent with a functional increase in striatal dopamine transmission following long-term neuroleptic treatment.     

Inhibition by acetylcholine of the stimulation-evoked release of [3H]acetylcholine from the guinea-pig myenteric plexus

The longitudinal muscle-myenteric plexus preparation of the guinea-pig ileum was incubated with [³H]choline and then superfused with Tyrode's solution. Exposure to [³H]choline resulted in the formation of [³H]acetylcholine which was released upon electrical field stimulation. The effects of exogenous acetylcholine, physostigmine and scopolamine on the stimulation-evoked release of [³H]acetylcholine were studied.In the absence of a cholinesterase inhibitor exogenous acetylcholine (10^?5 M) only slightly inhibited (by 26%) the evoked release of [³H]acetylcholine. If the cholinesterase activity of the preparation was reduced by about 50% in the presence of 10^?7M physostigmine, exogenous acetylcholine caused a concentration-dependent depression of the release evoked at 1 Hz. At a concentration of 10^?5 M acetylcholine the release was reduced by 76%. Scopolamine (10^?9 M) shifted the concentration-response curve for the inhibitory action of acetylcholine in a parallel manner to the right. From the dose ratio a pA₂ value of 9.8 for scopolamine against the release-inhibitory effect of acetylcholine was calculated. Physostigmine also inhibited the stimulation-evoked release of [³H]acetylcholine in a concentration-dependent manner, the maximal effect measured being an 85% reduction by 10^?5 M physostigmine. In the absence of a cholinesterase inhibitor scopolamine enhanced the evoked release of [³H]acetylcholine. The facilitatory effect was more marked at a stimulation frequency of 3 Hz (2-fold increase) than at 1 Hz (1.4-fold increase).It is concluded that extracellular acetylcholine decreases the stimulation-evoked release of neuronal acetylcholine. This inhibition is specifically mediated by a stimulation of presynaptic muscarinic receptors. The increase by scopolamine of the evoked release of [³H]acetylcholine suggests that previously liberated acetylcholine may trigger the negative feedback mechanism of acetylcholine release even if the cholinesterase activity is not inhibited, and that the presynaptic muscarinic receptors of the myenteric plexus have a physiological role in regulating the release of acetylcholine.     

N-methyl-D-aspartate-evoked release of acetylcholine from the medial septum/diagonal band of rat brain

N-Methyl-D-aspartate (NMDA)-evoked release of [3H]acetylcholine (ACh) from slices of rat brain medial septum/vertical limb of the diagonal band (ms/vdB) was examined. NMDA increased the release of tritium in a concentration-dependent manner and the specific non-competitive NMDA antagonist, MK-801, and the competitive NMDA antagonist kynurenic acid inhibited this release. Tetrodotoxin inhibited the NMDA-evoked release suggesting the [3H]ACh released arises from collaterals of the cholinergic septohippocampal neurons. Basal release of tritium was significantly increased by glycine alone and strychnine inhibited this response while having no effect on NMDA-evoked release. However, glycine, although not affecting the NMDA-evoked release, did enhance release of tritium in the presence of NMDA and blocking concentrations of Kynurenic acid. Together, these findings suggest that under the conditions of these experiments sufficient concentrations of glycine permit the full expression of NMDA-evoked modulation of [3H]ACh release, and that the predominant actions of glycine were mediated by a specific, strychnine-sensitive receptor.     

The effect of phencyclidine on [3H]GABA and [3H]fulnitrazepam binding in the brain of naive and handling-habituated rats

The effects of handling and handling combined with phencyclidine (PCP) treatment on GABAergic neurotransmission were studied in Sprague-Dawley rats. The animal material consisted of handling-habituated (HH, for 11 d), acutely handled (naive, N), handling-habituated and PCP-treated (10 mg kg^-1 i. p., HH+PCP) and acutely handled (naive) PCP-treated (N+PCP) and unhandled ‘control’ rats. The binding of [³]GANA and [³H]flunitrazepam (FLU) was studied with membrances and the release of [³H]GABA with slices prepared from the striatum and frontal cortex. In the striatum the maximal binding capacity (B_max) and the binding constant (K_D was lower in N rats. K_D constants of [³H]FLU were significantly lower in both brain areas in N rats than in HH rats. After PCP treatment both B_max and K_D for [³H]GAGA diminished. Neither handlign nor PCP had any effect on [³H]GABA release from striatal and frontal cortical slices. Handling prior to killing thus affects differently the GABAergic parameters studied and modulates the PCP-induced effects     

GABAA and strychnine-sensitive glycine receptors modulate N-methyl-D-aspartate-evoked acetylcholine release from rat spinal motoneurons: a possible role in neuroprotection

Increasing experimental and clinical evidence suggests that abnormal glutamate transmission might play a major role in a vast number of neurological disorders. As a measure of glutamatergic excitation, we have studied the acetylcholine (ACh) release induced by N-methyl-d-aspartate (NMDA) receptor stimulation in primary cultured rat ventral horn spinal neurons and we have evaluated the possibility to limit the consequences of the hyperactivation of glutamatergic receptors, by recruiting the inhibitory transmission mediated by GABA and glycine. For this purpose, we have exposed cell cultures, previously loaded with [(3)H]choline, to NMDA, which increased the spontaneous tritium efflux in a concentration-dependent manner. Tritium release is dependent upon external Ca(2+), tetrodotoxin, Cd(2+) ions and omega-conotoxin GVIA, but not on omega-conotoxin MVIIC nor nifedipine, suggesting the involvement of N-type voltage-sensitive calcium channels. NMDA-mediated [(3)H]ACh release was completely prevented by MK-801, 5,7-diclorokynurenic acid and ifenprodil, while it was strongly inhibited by a lower external pH, suggesting that the involved NMDA receptors contain NR1 and NR2B subunits. Muscimol inhibited NMDA-evoked [(3)H]ACh release and its effect was antagonized by SR95531 and potentiated by diazepam, indicating the involvement of benzodiazepine-sensitive GABA(A) receptors. Also glycine, via strychnine-sensitive receptors, inhibited the effect of NMDA. It is concluded that glutamate acts on the NMDA receptors situated on spinal motoneurons to evoke ACh release, which can be inhibited through the activation of GABA(A) and glycine receptors present on the same neurons. These data suggest that glutamatergic overload of receptors located onto spinal cord motoneurons might be decreased by activating GABA(A) and glycine receptors.     

In vivo release of [3H]gamma-aminobutyric acid in the rat neostriatum--I. Characterization and topographical heterogeneity of the effects of dopaminergic and cholinergic agents

The release of [3H]gamma-aminobutyric acid continuously synthesized from [3H]glutamine was studied in the striatum of halothane-anaesthetized rats superfused with a push-pull cannula. The levels of spontaneously released [3H]GABA were identical in all striatal regions examined, but were found to be higher at the junction between the striatum and the globus pallidus. Superfusion with a medium enriched in K+ ions induced a concentration-dependent increase in [3H]GABA release. Superfusion with a Ca2+-free medium did not affect the spontaneous outflow of [3H]GABA but sharply reduced the release of [3H]GABA evoked by 30 mM K+. Locally applied tetrodotoxin (50 microM) decreased slightly the spontaneous release of [3H]GABA (-22%). When acetylcholine (50 or 500 microM) was added to a superfusion medium containing eserine (50 microM), the spontaneous release of [3H]GABA was enhanced in the ventral but not in the dorsal region of the striatum. The local application of 2,3,4,5-tetrahydro, 7,8,-dihydroxy, 1-phenyl, 1-H, 3-benzazepine (10 microM), a dopaminergic agonist acting preferentially on D1 receptors increased the release of [3H]GABA in the dorsal striatum (+32%) but decreased it slightly (-19%) in the ventral striatum. 3-(2-(N-3 hydroxyphenylethyl)N-propylamino)ethyl-phenol (50 microM), a preferential D2 receptor agonist, decreased [3H]GABA release when it was applied dorsally (-23%) but not ventrally in the striatum. It is concluded that the regulation of the release of [3H]GABA by acetylcholine and dopaminergic drugs is different in the dorsal and ventral regions of the striatum. These differences may be related to the existence of subpopulations of GABA neurons and may well have functional implications as suggested by behavioural studies.     

Characterization of subtypes of gamma-aminobutyric acid receptors in an Ascaris muscle preparation by binding assay and binding of PF1022A,a new anthelmintic,on the receptors

 We examined the effect of PF1022A, one of the gabergic anthelmintics newly developed in Japan, on gamma-aminobutyric acid (GABA) receptors using a radioligand binding technique in isolated membrane preparations of the nematode Ascaris suum. Membrane protein was prepared from the homogenate of somatic muscle cells after ultracentrifugation. In addition to the basic binding of [2,3-³H-(N)]-GABA, the radioligand [methyl-³H]-bicuculline is used to identify the GABA_A receptor, whereas [butyl-4-³H]-baclofen is employed for GABA_B receptor sites. The dissociation constants (K _d values) and the maximal numbers of binding sites (B_max values) from Scatchard plotting for GABA receptors are close to those obtained in mammalian brain. PF1022A displaced in a concentration-dependent way the binding of [2,3-³H(N)]-GABA and [methyl-³H]-bicuculline as did other specific gabergic agents. In addition, PF1022A decreased the binding of [butyl-4-³H]-baclofen at a higher concentration, although this binding did not represent GABA_B sites. In a comparison of the inhibition constants (K_i values) of PF1022A with those of other agents, it is conclusive that PF1022A bound with GABA receptors. A direct effect of PF1022A on GABA receptors can thus be postulated. Received: 10 March 1995 / Accepted: 27 June 1995     

Presynaptic effect of L-glutamic acid on the release of dopamine in rat striatal slices

The release of [³H] dopamine ([³H] DA) was estimated in serial superfusate fractions of rat striatal slices continuously superfused with L-[3,5-³H]-tyrosine. L-glutamic acid (5 · 10^?5 M), but not the D-stereoisomer, increased the spontaneous release of [³H] DA (60%). The stimulating effect of L-glutamic acid was still observed in the presence of tetrodotoxin (5 · 10^?7 M), suggesting that the amino-acid acts at a presynaptic site. Moreover, atropine (10^?6 M) or pempidine (10^?5 M) which blocks the acetylcholine (ACh) evoked release of [³H] DA did not reduce the stimulatory effect of L-glutamic acid on [³H] DA release, thus excluding the possible intervention of striatal cholinergic neurons. The data obtained support the hypothesis of a direct control of DA release from nerve terminals by glutamatergic neurons.     

NMDA receptors and nitric oxide regulate prostaglandin D2 synthesis in the rabbit hippocampus in vivo

The aim of this in vivo microdialysis study was to characterise the regulation of prostaglandin D2 (PgD2) synthesis by NMDA receptors in the rabbit hippocampus in relation to changes in extracellular Ca2+ concentration ([Ca2+]e) and nitric oxide (NO) levels. Samples of dialysate were analysed for changes in PgD2 concentration, in [Ca2+]e and in the level of NO. The results demonstrated that a 20-min pulse application of 0.1-2.5 mM NMDA via a microdialysis probe induced a prolonged stimulation of PgD2 release that was sensitive to competitive NMDA receptor antagonists. An inhibitor of voltage-sensitive Na+ channels, tetrodotoxin, did not influence this effect but significantly suppressed basal PgD2 production, whereas a NOS inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME), prevented NMDA-evoked NO release and inhibited NMDA-induced PgD2 release in an L-arginine-sensitive manner. NO donors, S-nitroso-N-acetylpenicillamine and sodium nitroprusside, stimulated PgD2 release. NMDA-evoked decrease in [Ca2+]e was insensitive to TTX and L-NAME. These results demonstrate an in vivo NMDA receptor-mediated modulation of PgD2 synthesis in the brain, in which NO participates.     

Effect of muramyl dipeptides on postsynaptic GABA, NMDA, and AMPA receptors and presynaptic NMDA receptors in rat brain

We studied the effect of muramyl dipeptides on postsynaptic GABA, NMDA, and AMPA receptors and presynaptic NMDA receptors. L,D-Dipeptide more potently than L,L-dipeptide inhibited postsynaptic NMDA receptors, potentiated GABA and AMPA receptors, and inhibited ⁴⁵Ca²⁺ uptake in synaptosomes from of rat brain cortex. Our results indicate that muramyl dipeptides modulate function of glutamate and GABA receptors. Translated from Byulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 146, No. 9, pp. 247–249, September, 2008     

Calcium- and barium-dependent exocytosis from the rat insulinoma cell line RINm5F assayed using membrane capacitance measurements and serotonin release

Electrophysiological measurements of cell capacitance (C _m) and biochemical assays of [³H] serotonin ([³H]5-hydroxytryptamine or [³H]5-HT) release were combined to study the control of secretion in rat insulinoma RINm5F cells. Depolarizing pulses produced C _m changes (ΔC _m), indicative of exocytosis, with the same voltage and Ca²⁺ dependency as the inward Ca²⁺ currents (I _Ca). Ba²⁺ was able to substitute for Ca²⁺ in stimulating exocytosis, but not endocytosis. However, both the relative potency and kinetics of Ca²⁺-versus Ba²⁺-triggered exocytosis differed significantly. 5-HT synthesis and uptake were demonstrated in RINm5F cells. This allowed the use of [³H]5-HT to study hormone release from cell populations. [³H]5-HT was released in a depolarization-, Ca²⁺- and time-dependent manner. Ba²⁺ also substituted for Ca²⁺ in depolarization-induced [³H]5-HT release. Thapsigargin, used to deplete Ca²⁺ stores, had no effects on Ca²⁺-triggered C _m increases, but Ca²⁺-triggered [³H]5-HT release was abolished. Ba²⁺-triggered [³H]5-HT release, however, was only slightly affected by Ca²⁺ store depletion. Ba²⁺ was found to act directly as a secretagogue of [³H]5-HT in intact cells, but not in C _m measurements of voltage-clamped cells, suggesting that cell depolarization is a prerequisite for this action. Received: 18 October 1995/Received after revision and accepted: 9 January 1996