Calcium-independent release of [H]spermine from chick retina

Spermine has been shown to influence NMDA receptor function through an interaction at the coagonist site for glycine in the central nervous system (CNS) and the retina. In order to support a role for spermine as neurotransmitter or neuromodulator in the chick retina, specific stimulated-release of spermine should be demonstrated. Isolated chick retinas, preloaded with [³H]spermine, were stimulated with 1 mM NMDA and other glutamate agonists at ionotropic receptors, in a continuous superfusion system. [³H]spermine was released from the retina by depolarization with 50 mM KCl, in a Ca²⁺-independent manner. Inhibition of Na⁺/K⁺-ATPase by ouabain or digitoxigenin also induced spermine release following 36 min in the presence of the drugs; such effect seems unrelated to changes in Na⁺ electrochemical gradients, since nigericin and veratrine did not induce release in Na⁺ containing medium. The lack of effect of glutamate, NMDA and kainate at 1 mM concentration, suggests that release of spermine in the retina is mediated by the reversal of uptake and not necessarily linked to EAA-receptor activation.     

GABA release mechanism in the golden hamster retina.

High K+ medium and glutamate elicited a significant [3H]-GABA release in the golden hamster retina. High K+ -induced GABA release was largely calcium-dependent, while the effect of glutamate was Ca2+ -independent. After replacing Na+ by Li+, glutamate-evoked [3H]-GABA release was abolished, while high K+ -evoked release remained unchanged. The effect of glutamate was completely blocked by DNQX but not by APV. Furthermore, kainate induced [3H]-GABA release, whereas NMDA was ineffective. Assessment of endogenous GABA efflux further confirmed results obtained for [3H]-GABA. GABA-like immunoreactivity was observed in amacrine cells, in neurons localized in ganglion cell layer, as well as in fibers and terminals at the inner plexiform layer. In addition a few horizontal cells showed GABA-like immunolabeling. The present results suggest the existence of at least two pools of GABA in the hamster retina, compatible with both vesicular and carrier-mediated mechanisms of transmitter release, being the amacrine cells the main gabaergic source in this tissue.     

Excitatory amino acid-evoked release of [3H]GABA from hippocampal neurons in primary culture

We investigated the release of gamma-[2,3-3H(N)]aminobutyric acid ([3H]GABA) from hippocampal neurons in primary cell culture. [3H]GABA release was stimulated by the excitatory amino acid neurotransmitter glutamate as well as by N-methyl-D-aspartate (NMDA) and kainate. Cell depolarization induced by raising [K+]o or by veratridine also stimulated [3H]GABA release. NMDA-induced release was completely blocked by 3-((+/-)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP+), Mg2+ and Zn2+ whereas the release induced by glutamate and kainate was much less susceptible to inhibition by these substances. Furthermore, removal of external Ca2+ inhibited NMDA-induced release, but not that induced by glutamate, kainate, veratridine or 50 mM K+. Removal of external Na+ reduced [3H]GABA release evoked by all stimuli, but to different extents. All of the excitatory amino acids tested increased [Ca2+]i within hippocampal neurons as assessed by fura-2 based microspectrofluorimetry. This increase in [Ca2+]i was completely dependent on the presence of external Ca2+. These results suggest that Ca2+-dependent and -independent forms of GABA release from hippocampal interneurons may occur. [3H]GABA release evoked by glutamate, kainate, veratridine or 50 mM K+, appeared to be mediated by the reversal of electrogenic, Na+-coupled GABA uptake. Release was inhibited by nipecotic acid, an inhibitor of the Na+-coupled GABA uptake system. However, release induced by NMDA may also include a Ca2+-dependent component.     

Glutamate release evoked by glutamate receptor agonists in cultured chick retina cells: Modulation by arachidonic acid

We studied the effect of ionotropic glutamate receptor agonists on the release of endogenous glutamate or of [³H]D -aspartate from reaggregate cultures (retinospheroids) or from monolayer cultures of chick retinal cells, respectively. Kainate increased the fluorescence ratio of the Na⁺ indicator SBFI and stimulated a dose-dependent release of glutamate in low (0.1 mM) Ca²⁺ medium, as measured using a fluorometric assay. Under the same experimental conditions, the release evoked by N-methyl-D -aspartate (NMDA; 400 μM) was about half of that evoked by the same kainate concentration; α-amino-3-hydroxy-5-methyl-4-isoxasolepropionic acid (AMPA; 400 μM) did not trigger a significant response. In the presence of 1 mM CaCl₂, all of the agonists increased the [Ca²⁺]_i, as determined with the fluorescence dye Indo-1, but the glutamate release evoked by NMDA and kainate was significantly lower than that measured in 0.1 mM CaCl₂ medium. Inhibition by Ca²⁺ of the kainate-stimulated release of glutamate was partially reversed by the phospholipase A₂ inhibitor oleiloxyethyl phosphorylcholine (OPC), suggesting that the effect was mediated by the release of arachidonic acid, which inhibits the glutamate carrier. Accordingly, kainate, NMDA, and AMPA stimulated a Ca²⁺-dependent release of [³H]arachidonic acid, and the direct addition of the exogenous fatty acid to the medium decreased the release of glutamate evoked by kainate in low (0.1 mM) CaCl₂ medium. In monolayer cultures, we showed that NMDA, kainate, and AMPA also stimulated the release of [³H]D -aspartate, but in this case release in the presence of 1 mM CaCl₂ was significantly higher than that evoked in media with no added Ca²⁺. The ranking order of efficacy for stimulation of Ca²⁺-dependent release of [³H]D -aspartate was NMDA ≪ kainate < AMPA. © 1996 Wiley-Liss, Inc.     

Glutamate increases the [Ca]i but stimulates Ca-independent release of [H]GABA in cultured chick retina cells

The effect of glutamate of [Ca²⁺]_i and on [³H]γ-aminobutyric acid (GABA) release was studied on cultured chick embryonic retina cells. It was observed that glutamate (100 μM) increases the [Ca²⁺]_i by Ca²⁺ influx through Ca²⁺ channels sensitive to nitrendipine, but not to ω-conotoxin GVIA (ω-Cg Tx) (50%), and by other channels insensitive to either Ca²⁺ channel blocker. Mobilization of Ca²⁺ by glutamate required the presence of external Na⁺, suggesting that Na⁺ mobilization through the ionotropic glutamate receptors is necessary for the Ca²⁺ channels to open. The increase in [Ca²⁺]_i was not related to the release of [³H]GABA induced by glutamate, suggesting that the pathway for the entry of Ca²⁺ triggered by glutamate does not lead to exocytosis. In fact, the glutamate-induced release of [³H]GABA was significantly depressed by Ca_o²⁺, but it was dependent on Na_o⁺, just as was observed for the [³H]GABA release induced by veratridine (50 μM). The veratridine-induced release could be fully inhibited by TTX, but this toxin had no effect on the glutamate-induced [³H]GABA release. Both veratridine- and glutamate-induced [³H]GABA release were inhibited by 1-(2-(((diphenylmethylene)amino)oxy)ethyl)-1,2,5,6-tetrahydro-3-pyridine-carboxylic acid (NNC-711), a blocker of the GABA carrier. Blockade of the NMDA and non-NMDA glutamate receptors with MK-801 and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), respectively, almost completely blocked the release of [³H]GABA evoked by glutamate. Continuous depolarization with 50 mM K⁺ induced maximal release of [³H]GABA of about 1.5%, which is much smaller than the release evoked by glutamate under the same conditions (6.0–6.5%). Glycine (3 μM) stimulated [³H]GABA release induced by 50 mM K⁺, and this effect was blocked by MK-801, suggesting that the effect of K⁺ on [³H]GABA release was partially mediated through the NMDA receptor which probably was stimulated by glutamate released by K⁺ depolarization. We conclude that glutamate induces Ca²⁺-independent release of [³H]GABA through reversal of the GABA carrier due to Na⁺ entry through the NMDA and non-NMDA, TTX-insensitive, channels. Furthermore the GABA carrier seems to be inhibited by Ca²⁺ entering by the pathways open by glutamate. This Ca²⁺ does not lead to exocytosis, probably because the Ca²⁺ channels used are located at sites far from the active zones.     

Effect of oxidative stress on the release of [H]GABA in cultured chick retina cells

The effect of ascorbate (1.5 mM)/Fe²⁺ (7.5 μM)-induced oxidative stress on the release of pre-accumulated [³H]γ-aminobutyric acid ([³H]GABA) from cultured chick retina cells was studied. Depolarization of control cells with 50 mM K⁺ increased the release of [³H]GABA by 1.01 ± 0.16% and 2.5 ± 0.3% of the total, in the absence and in the presence of Ca²⁺, respectively. Lipid peroxidation increased the release of [³H]GABA to 2.07 ± 0.31% and 3.6 ± 0.39% of the total, in Ca²⁺-free or in Ca²⁺-containing media, respectively. The inhibitor of the GABA carrier, 1-(2-(((diphenylmethylene)amino)oxy)ethyl)-1,2,5,6-tetrahydro-3-pyridine-carboxylic acid hydrochloride (NNC-711) blocked almost completely the release of [³H]GABA due to K⁺-depolarization in the absence of Ca²⁺, but only 65% of the release occurring in the presence of Ca²⁺ in control and peroxidized cells. Under oxidative stress retina cells release more [³H]GABA than control cells, being the Ca²⁺-independent mechanism, mediated by the reversal of the Na⁺/GABA carrier, the most affected. MK-801 (1 μM), a non-competitive antagonist of the NMDA receptor-channel complex, blocked by 80% the release of [³H]GABA in peroxidized cells, whereas in control cells the inhibitory effect was of 40%. The non-selective blocker of the non-NMDA glutamate receptors, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), inhibited the release of [³H]GABA by 30% and 70% in control and peroxidized cells, respectively. Glycine (5 μM) stimulated [³H]GABA release evoked by 50 mM K⁺-depolarization in control but not in peroxidized cells. The release of -[³H]aspartate (a non-metabolized analog of -glutamate) evoked by 50 mM K⁺, in the absence of Ca²⁺, was significantly higher in peroxidized cells (6.76 ± 0.64% of the total) than in control cells (3.79 ± 0.27% of the total). The results suggest that oxidative stress induced by ascorbate/Fe²⁺ causes an excessive release of endogenous excitatory amino acids upon K⁺-depolarization. The glutamate released may activate NMDA and non-NMDA receptors, raising the intracellular Na⁺ concentration and consequently stimulating the release of [³H]GABA by reversal of the Na⁺/GABA carrier.     

In vitro effects of D-2-hydroxyglutaric acid on glutamate binding, uptake and release in cerebral cortex of rats

Neurological dysfunction is common in patients with D-2-hydroxyglutaric aciduria (DHGA). However, the mechanisms underlying the neuropathology of this disorder are far from understood. In the present study, we investigated the in vitro effects of D-2-hydroxyglutaric acid (DGA) at various concentrations (0.1-1.0 mM) on various parameters of the glutamatergic system, namely the basal and potassium-induced release of L-[3H]glutamate by synaptosomal preparations, Na(+)-dependent L-[3H]glutamate uptake by synaptosomal preparations and Na(+)-independent L-[3H]glutamate uptake by synaptic vesicles, as well as of Na(+)-independent and dependent L-[3H]glutamate binding to synaptic plasma membranes from cerebral cortex of male adult Wistar rats. We observed that DGA significantly increased synaptosomal L-[3H]glutamate uptake, without altering the other parameters. Although these findings do not support a direct excitotoxic action for DGA since the metabolite did not affect important parameters of the main neurotransmission system, they do not exclude a direct action of DGA on NMDA or other glutamate receptors. More comprehensive studies are therefore necessary to evaluate the exact role of DGA on neurotransmission.     

Subtypes of excitatory amino acid receptors involved in the stimulation of [3H]dopamine release from cell cultures of rat ventral mesencephalon

N-methyl-D-aspartic acid (NMDA), quisqualic acid (QUIS), and kainic acid (KAIN), respective agonists for three excitatory amino acid (EAA) receptor subtypes, stimulated [3H]dopamine ([3H]DA) release from dissociated cell cultures of fetal rat ventral mesencephalon. Release evoked by all three agonists was Ca2(+)-dependent and inhibited by broad-spectrum antagonists (D,L-cis-2,3-piperidine dicarboxylic acid [PDA] and kynurenic acid [KYN]). However, both of these antagonists were more potent against KAIN than against QUIS and only KAIN-evoked release was blocked by gamma-D-glutamyl-aminomethyl sulfonic acid (GAMS, IC50 700 microM). NMDA-stimulated [3H]DA release was selectively inhibited by competitive (3-[2-carboxypiperazine-4-yl]propyl-1-phosphonic acid [CPP] and D,L-2-amino-5-phosphonovaleric acid [APV]) and non-competitive (phencyclidine and MK-801) NMDA receptor antagonists. In 1.2 mM Mg2+, NMDA-stimulated [3H]DA release was Na(+)-dependent and inhibited by tetrodotoxin (TTX, 2 microM) or by the local anaesthetic, lidocaine (200 microM). However, in 0 Mg 2+, NMDA-evoked release was not inhibited by TTX or lidocaine. Thus, TTX-sensitivity of the NMDA response in 1.2 mM Mg2+ apparently occurs because Na(+)-action potentials are required to alleviate a Mg2+ blockade. Neither QUIS- nor KAIN-evoked release was affected by Mg2+ or TTX. When extracellular NaCl was replaced by sucrose or Na2SO4, the QUIS response was increased. KAIN-evoked release was unaffected by the sucrose substitution and was attenuated in the Na2SO4-containing buffer. It is concluded that NMDA and QUIS/KAIN release [3H]DA via separate receptor subtypes.     

Glutamate receptor agonists release [3H]GABA preferentially from horizontal cells

A total of 5-6 different cell types in vertebrate retinas accumulate [3H]gamma-aminobutyric acid (GABA). In frog retina, specific populations of cells in the horizontal, amacrine and ganglion cell layers are labeled autoradiographically after a 15-min in vitro incubation with [3H]GABA. Cells which may be bipolar or interplexiform cells are also labeled. Similar autoradiographic patterns are observed in chick retina except for the absence of labeled bipolar or interplexiform cells. In rat retinas, [3H]GABA uptake is limited primarily to Muller and amacrine cells. Depolarizing glutamate receptor agonists (glutamate, aspartate and kainic acid) applied in an in vitro perfusion system, stimulated massive release of [3H]GABA from frog and chick retina but not from rat retina. Under these conditions, autoradiographic labeling of horizontal cells was virtually depleted, while labeling of other cell types remained robust. In contrast, potassium caused release of the label from all 3 types of retina, and loss of autoradiographic labeling occurred uniformly in all cell types. We conclude that [3H]GABA-accumulating horizontal cells possess depolarizing glutamate receptors and that activation of these receptors leads to a release of GABA stores. On the other hand, Muller cells and the various subclasses of [3H]GABA-accumulating amacrine, bipolar and/or interplexiform cells, do not release GABA in response to glutamate receptor stimulation and thus appear to be relatively insensitive to excitatory amino acids.     

Exposure to N-methyl-D-aspartate increases release of arachidonic acid in primary cultures of rat hippocampal neurons and not in astrocytes

The release of [3H]arachidonic acid (ARA) was investigated from prelabelled primary cultures of hippocampal neurons and astroglial cells. The activation of N-methyl-D-aspartate (NMDA) subtype of glutamate receptors resulted in a dose-dependent stimulation of [3H]ARA release. The half maximal effect was obtained at about 15 microM NMDA, whereas the maximum concentration (50 microM NMDA) produced about a 2-fold increase in 7-day-old cultures. This elevation in [3H]ARA release was blocked in a dose-related manner by the NMDA receptor antagonist, 2-amino-5-phosphonovaleric acid (APV), and by Mg2+ which blocks NMDA receptor-linked Ca2+ ion channels. The removal of external Ca2+ inhibited NMDA-induced release, whereas treatment with calcimycin (A 23187, a Ca2+ ionophore) greatly increased the [3H]ARA release. The inhibitors of phospholipase A2, nordihydroguaiaretic acid and mepacrine, decreased the NMDA-dependent [3H]ARA release in a dose-related manner, maximum inhibition reaching to about 90% at high doses. Entry of Ca2+ brought about by opening the voltage-sensitive channels by high K+ had no effect on the release of [3H]ARA, indicating that NMDA gated channels are situated in a part of the neuron where Ca2+ entry through this route is more efficiently coupled to the activation of phospholipase A2. Treatment with NMDA had no significant effect on [3H]ARA release in hippocampal astroglial cells as opposed to neurons. This was not due to inability of astrocytes to release ARA, for ATP still evoked [3H]ARA release, and this was markedly inhibited by mepacrine. It is suggested that ARA act as both intracellular and intercellular messengers in the functioning of NMDA receptors in synaptic transmission and plasticity in the hippocampus.     

Ca-dependent release of [H]GABA in cultured chick retina cells

Depolarization by K+ (50 mM) of cultured chick retina cells released 1.14 +/- 0.28% of the accumulated [3H] gamma-aminobutyric acid (GABA) in the absence of Ca2+, but when 1.0 mM Ca2+ was present, the internal free calcium ion concentration [Ca2+]i rose by about 750 nM and the [3H]GABA release about doubled to a value of 2.22 +/- 0.2% of the total [3H]GABA. Nitrendipine (0.1 microM), a blocker of the L-type Ca2+ channels, blocked the [Ca2+]i response to K+ depolarization by about 65%, and the omega-Conotoxin GVIA (omega-CgTx) (0.5 microM), a blocker of the N-type of Ca2+ channels, inhibited by 27% the [Ca2+]i rise due to K+ depolarization. Parallel experiments showed that nitrendipine inhibits [3H]GABA release to the level observed in the absence of Ca2+, whereas omega-CgTx did not inhibit significantly the release of [3H]GABA. The results also show that the release of [3H]GABA due to K(+)-depolarization in the absence of Ca2+ can be totally blocked by 1-(2-(((Diphenylmethylene) amino)oxy)ethyl)-1,2,5,6-tetrahydro-3-pyridine-carboxylic acid hydrochloride (NNC-711), an inhibitor of the GABA carrier. However, in the presence of Ca2+, NNC-711 blocks the release only by about 66%, corresponding to the Ca(2+)-independent release. Thus, it is concluded that [3H]GABA is released in chick retina cells by the exocytotic mechanism, which is Ca(2+)-dependent, and by reversal of the carrier, which is Ca(2+)-independent, in much the same way as has been found for other GABAergic neurons.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibitory effect of taurine on veratridine-evoked D-[3H]aspartate release from murine corticostriatal slices: involvement of chloride channels and mitochondria

We have previously shown that the inhibitory neuromodulator taurine attenuates the release of preloaded D-[3H]aspartate from murine corticostriatal slices evoked by ischemic conditions or by application of the sodium channel agonist veratridine. The release of D-[3H]aspartate (a non-metabolized analog of glutamate) was used as an index of glutamate release. The aim of the present study was to reveal the molecular mechanisms responsible for this inhibitory effect of taurine. It was shown that 10 mM taurine suppresses D-[3H]aspartate release evoked by 0.1 mM veratridine, but does not affect the high-K+ -(50 mM) or ouabain- (0.1 mM) evoked release. Taurine had no effect in Ca2+ -free medium when the synaptic exocytosis of D-[3H]aspartate was inhibited. Nor did it suppress the release from slices preloaded with the competitive glutamate uptake blocker DL-threo-beta-hydroxyaspartate (THBA), which inhibits D-[3H]aspartate release mediated by the reverse action of glutamate transporters. Omission of Cl- from the incubation medium reduced the effect of taurine, signifying the involvement of a Cl- channel. The glycine receptor antagonist strychnine and the GABA(A) receptor antagonist bicuculline did not block the taurine effect, although picrotoxin, a less specific blocker of agonist-gated chloride channels, completely prevented the effect of taurine on veratridine-induced D-[3H]aspartate release. The respiratory chain blocker rotenone or mitochondrial protonophore carbonyl cyanide 3-chlorophenylhydrazone (CCCP) in combination with the mitochondrial ATPase inhibitor oligomycin, which inhibits the mitochondrial Ca2+ uniporter, also reduced the effect of taurine. The results obtained in the present study show that taurine acts specifically on the release of preloaded D-[3H]aspartate evoked by veratridine, but not on that evoked by other depolarizing agents, and affects the release mediated both by synaptic exocytosis and the reverse action of glutamate transporter. Taurine may attenuate D-[3H]aspartate release by regulation of mitochondrial Ca2+ sequestration and by activation of a chloride channel, but not that governed by GABA(A) or strychnine-sensitive glycine receptors.     

Release of [3H]gamma-aminobutyric acid from glial (Müller) cells of the rat retina: effects of K+, veratridine, and ethylenediamine

In several neural systems, glial cells appear to take up and release gamma-aminobutyric acid (GABA) upon depolarization. We have studied the release of [3H]GABA from Müller (glial) cells in the rat retina by a double isotope-labeling technique in which Müller cells are preloaded with 3H-GABA while a population of neurons is prelabeled with [14C]glycine. By autoradiography, we have confirmed that [3H]GABA is taken up by the radially oriented Müller cells, whereas [3H]glycine is accumulated by a subset of amacrine cells (neurons). Using the double-labeling procedure, we have examined the effects of two depolarizing agents, high K+ and veratridine, and the GABA mimetic, ethylenediamine, on transmitter release from glial cells and neurons simultaneously. We found the following. (1) Depolarization with 56 mM K+ released both [3H]GABA and [14C]glycine. About 70 to 80% of this release was blocked in Ca2+-free medium. (2) Veratridine (10 microM) also released both of the transmitters. This release was strongly inhibited by 100 nM tetrodotoxin or 1mM procaine. Under Ca2+-free conditions, less than 20% isotope release was observed. (3) Ethylenediamine released [3H]GABA readily, whereas little [14C]glycine release was observed. Removal of Ca2+ had no significant effect on transmitter release. Furthermore, in Na+-free medium ethylenediamine failed to induce [3H] GABA or [14C]glycine release. These results suggest that high K+ and veratridine release [3H]GABA from Müller cells by a Ca2+-dependent process. Ethylenediamine, on the other hand, appears to induce [3H]GABA release by a Ca2+-independent, carrier-mediated exchange mechanism.     

Effects of amygdaloid kindling on NMDA receptor function and regulation

These studies were conducted to determine whether amygdaloid kindling results in the long-term alteration of NMDA receptors which could explain the persistent reduction in seizure threshold seen in this phenomenon. NMDA-induced [3H]norepinephrine (NE) release, NMDA-sensitive L-[3H]glutamate binding, and NMDA and glycine-enhanced [3H]TCP binding were measured in brain tissue from kindled rats and nonstimulated control rats 3 to 6 weeks after the last seizure. There was no difference in the ability of NMDA to induce [3H]NE release from kindled or control slices of amygdala or hippocampus. There was also no difference in the ability of phencyclidine (PCP) or Mg2+ to inhibit [3H]NE release induced by 100 microM NMDA. Equilibrium saturation experiments of NMDA-sensitive L-[3H]glutamate binding revealed no differences in KD or Bmax values between control and kindled cortex, amygdala, and hippocampus. The Ki values for NMDA displacement of L-[3H]glutamate binding also did not differ in kindled tissue. NMDA-enhanced [3H]TCP binding was similar in cortex, amygdala, and hippocampus of kindled and control tissues. Finally, glycine-enhanced [3H]TCP binding was not different in control or kindled tissues. These studies suggest that the NMDA recognition site and the modulation of the NMDA receptor/ion channel complex by magnesium, PCP, and glycine are not altered several weeks after the last seizure. Even though NMDA-mediated electrophysiological responses are reportedly enhanced in kindled tissue at that time, the mechanism(s) underlying the enhancement remains to be determined.     

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)     

Development and regulation of excitatory amino acid receptors involved in the release of arachidonic acid in cultured hippocampal neural cells

Release of [3H]arachidonic acid mediated by excitatory amino acid (EAA) receptors was investigated from prelabelled primary cultures of hippocampal neurons and astroglial cells. Treatment with N-methyl-D-aspartate (NMDA), quisqualate (QA) and kainate resulted in age- and dose-dependent stimulation of [3H]arachidonic acid release. During development, the maximum response for NMDA was observed relatively earlier (at 7 days) than those for QA and kainate (at 14 days) in the hippocampal neuronal cultures. The half maximal effects were obtained at about 15 microM NMDA at all ages studied and about 0.5 microM QA at 14 and 20 days. At optimum concentrations NMDA- and QA-induced releases were additive. Unlike with neurons, treatment with all the 3 EAA receptor agonists, NMDA, QA and kainate, had no significant effect on [3H]arachidonate release in hippocampal astroglial cells. In cultured 14-day-old neurons, the increases in NMDA- and QA-mediated [3H]arachidonic acid release were completely blocked by the NMDA receptor antagonist, 2-amino-5-phosphonovaleric acid, and the ionotropic QA receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione, respectively. But the iontropic QA receptor agonist alpha-amino-3-hydroxy-5-methyl-isoxazole-4- propionic acid (AMPA) had no significant effect on [3H]arachidonate release, indicating that interaction between ionotropic QA and metabolotropic QA receptors may be essential for optimal QA-mediated arachidonic acid release. At physiological concentrations of Mg2+ (1.2 mM), AMPA was found to potentiate NMDA-induced release of [3H]arachidonic acid; the effect appeared to be related to a removal of Mg2+ blockade mediated by mild depolarisation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Localization and Quantitative Autoradiography of Glutamatergic Ligand Binding Sites in Chick Brain

The anatomical localization of glutamate receptor subtype-selective ligand binding sites was investigated in 1-day-old chick brain using quantitative autoradiography. Under the conditions used, the regional distributions of [3H]glutamate, [3H]AMPA (a selective quisqualate receptor ligand) and [3H]kainate binding sites are manifestly different. [3H]l-glutamate binding is densely localized in the telencephalon, particularly in the neostriatum (2.8 pmol/mg protein). In addition, [3H]l-glutamate labels the thalamus, the nucleus mesencephalicus lateralis pars dorsalis, the superficial layers of the optic tectum and the molecular layer of the cerebellum. [3H]AMPA binding sites are most densely localized in the hippocampus (0.90 pmol/mg protein), with an otherwise relatively uniform distribution of binding within the telencephalon. [3H]AMPA also labels the striatum griseum et fibrosum superficiale of the optic tectum and the molecular layer of the cerebellum. [3H]Kainate binding sites are extremely densely packed in the molecular layer of the cerebellum (10 pmol/mg protein). Other regions of [3H]kainate binding include the hyperstriatum and the thalamus. The binding of the NMDA receptor channel blocker [3H]MK-801 is increased in the presence of 1 mM l-glutamate. [3H]MK-801 binding is generally widespread in the telencephalon but is notably absent from the ectostriatum. No evidence of [3H]MK-801 binding sites was detected in the cerebellum, even in the presence of 1 mM l-glutamate. The relatively high densities and the well-defined localizations of the glutamate receptor subtype binding sites suggest that chick brain provides a useful system for the further study of excitatory amino acid receptors.     

3H]Spermine binding to synaptosomal membranes from the chick retina.

The binding of [3H]spermine to synaptosomal membranes from chick retina was examined. Saturable specific binding of [3H]spermine to synaptosomal membranes from plexiform layers of retina (P1 and P2) has been characterized, and found to concentrate in the inner plexiform layer compared to the outer plexiform layer (Bmax=9.3 and 37 pmol/mg protein for P1 and P2, respectively). Kinetics of specific [3H]spermine binding yield a sigmoidal saturation curve, indicating positive cooperativity (nH: 2.4 and 3.2 for P1 and P2, respectively) with high affinity: Kapp=61 and 67 nM for P1 and P2. The time required to attain equilibrium at room temperature was less than 5 min in both fractions. Dose-response curves for spermine, spermidine, and diethylene-triamine (DET) show different potencies for inhibiting [3HDET. Our results support a role for polyamines (PA) as neurotransmitters or neuromodulators in the vertebrate retina.     

Inhibition of N-methyl-D-aspartate evoked sodium flux by MK-801

The inhibition of N-methyl-D-aspartate (NMDA) stimulated 22Na+ efflux from rat hippocampal slices was studied using competitive and non-competitive receptor antagonists. There was a good correlation between the abilities of the competitive antagonists to block NMDA evoked 22Na+ efflux and their potencies as inhibitors of L-[3H]glutamate binding. The recently reported novel NMDA receptor antagonist, (+)-5-methyl-16,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801) was shown to non-competitively inhibit NMDA stimulated 22Na+ efflux with an IC50 value of 0.4 microM. Relatively high (10 microM) concentrations of MK-801 had no effects on quisqualic acid, alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA), or kainic acid stimulated efflux. However, MK-801 was able to block 22Na+ efflux induced by ibotenic acid and L-homocysteic acid, amino acids that act as NMDA receptor agonists. MK-801, (-)-MK-801, and non-competitive NMDA receptor antagonists of the arylcyclohexylamine and dioxolane classes inhibited NMDA stimulated 22Na+ efflux with potencies that reflected their abilities to compete for [3H]MK-801 binding sites in rat cortical membranes. These results indicate the utility of the 22Na+ efflux assay in studying the properties of NMDA receptors and confirm the nature and selectivity of the inhibition of NMDA receptor linked ion channel activation by MK-801.     

Regulation of NMDA-stimulated [14C]GABA and [3H]acetylcholine release by striatal glutamate and dopamine receptors.

Striatal function is heavily influenced by glutamatergic and dopaminergic afferent input. To ultimately better understand how the N-methyl-D-aspartate (NMDA) antagonist, phencyclidine (PCP), alters striatal function, we sought to determine how NMDA receptor function is influenced by activation of other glutamatergic receptors and by dopaminergic receptors. To this end, we used NMDA-stimulated efflux of [14C]GABA and [3H]acetylcholine (ACh) from striatal slices to assess the influence of these receptors on NMDA function. NMDA-stimulated [14C]GABA release was more sensitive to NMDA and glycine antagonists than was [3H]ACh release, suggesting that different NMDA receptors regulate the release of these neurotransmitters. Furthermore, NMDA-stimulated [3H]ACh release was inhibited by a D2 receptor mechanism whereas NMDA-stimulated [14C]GABA release was enhanced by D1 receptor activation. NMDA and (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid hydrobromide (AMPA) interact additively to evoke [3H]ACh release, and synergistically to evoke [14C]GABA release. An additive effect of NMDA and kainate (KA) was found on [14C]GABA release, but NMDA and KA acted in a less than additive manner in evoking [3H]ACh release. KA-stimulated [3H]ACh release was largely blocked by NMDA antagonists, suggesting mediation through activation of NMDA receptors, probably secondary to KA-induced glutamate release. A selective group II metabotropic receptor agonist inhibited NMDA-stimulated [14C]GABA and [3H]ACh release. On the other hand, NMDA-stimulated [14C]GABA release was potentiated by activation of group I metabotropic receptors. Thus, in addition to the differential modulation by D1- and D2-like receptors, the release of striatal neurotransmitters by NMDA receptor activation depends on the extent to which the other glutamate receptors, both ionotropic and metabotropic, are activated.