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Notes: Abstract: The activation of kainic acid and quisqualic acid receptors in cultured cerebellar granule cells stimulated the release of preaccumulated d-[3H]aspartate. The effect of kainate could be distinguished from that of quisqualate by its sensitivity to the antagonists kynurenic acid and 2,3-cis-piperidine dicarboxylic acid. At a concentration of kainic acid (50 μM) close to its half-maximal releasing effect, simultaneous addition of quisqualic acid (10–50 μM) resulted in a significant dose-dependent inhibition of the kainate-induced component of d-[3H]aspartate release, which was monitored by the progressive decrease in sensitivity of the evoked release to kynurenic acid. In contrast, when kainic acid was used at a subeffective concentration (10 μM), addition of low doses of quisqualate (2–5 μM) resulted in a synergistic effect on d-[3H]aspartate release. Under these conditions, the effect of the two agonists was sensitive to kynurenic acid. Kainic acid (50–100 μM) also caused a dose-dependent, kynurenic acid-sensitive accumulation of cyclic GMP (cGMP) in granule cell cultures. Quisqualic acid was, by itself, ineffective and prevented, in a dose-dependent manner, the kainate-induced cGMP formation (IC50= 5 μM). Finally, the guanylate cyclase activator sodium nitroprusside greatly enhanced cGMP formation but had no effect on d-[3H]aspartate release. Together, these results demonstrate the existence of complex interactions between quisqualic and kainic acids and indicate that the effects of the two glutamate agonists on d-[3H]aspartate release and on cGMP accumulation are independent.

Notes: Abstract: We have used postnatal rat cerebellar astrocyte-enriched cultures to study the excitatory amino acid receptors present on these cells. In the cultures used, type-2 astrocytes (recognized by the monoclonal antibodies A2B5 and LB1) selectively took up γ-[3H]aminobutyric acid ([3H]GABA) and released it when incubated in the presence of micromolar concentrations of kainic and quisqualic acids. The releasing effect of kainic acid was concentration dependent in the range of 5–100 μM. Quisqualate was more effective than kainate in the lower concentration range but less effective at concentrations at which its releasing activity was maximal (∼50 μM). N-Methyl-d-aspartic acid and dihydrokainate (100 μM) did not stimulate [3H]GABA release from cultured astrocytes. l-Glutamic acid (20–100 μM) stimulated [3H]GABA release as effectively as kainate. The stimulatory effects of kainate and quisqualate on [3H]GABA release were completely Na+ dependent; that of kainate was also partially Ca2+ dependent. Kynurenic acid (50–200 μM) selectively antagonized the releasing effects of kainic acid and also that of l-glutamate; quisqualate was unaffected. Quisqualic acid inhibited the releasing effects of kainic acid when both agonists were used at equimolar concentrations (50 μM). d-[3H]aspartate was taken up by both type-1 and type-2 astrocytes, but only type-2 astrocytes released it in the presence of kainic acid. Excitatory amino acid receptors with a pharmacology similar to that of the receptors present in type-2 astrocytes were also expressed by the immature, bipotential progenitors of type-2 astrocytes and oligodendrocytes.

Notes: Using cerebellar, neuron-enriched primary cultures, we have studied the glutamate receptor subtypes coupled to neurotransmitter amino acid release. Acute exposure of the cultures to micromolar concentrations of kainate and quisqualate stimulated D-[3H]aspartate release, whereas N-methyl-D-aspartate, as well as dihydrokainic acid, were ineffective. The effect of kainic acid was concentration dependent in the concentration range of 20–100 μM. Quisqualic acid was effective at lower concentrations, with maximal releasing activity at about 50 μM. Kainate and dihydrokainate (20–100 μM) inhibited the initial rate of D-[3H]aspartate uptake into cultured granule cells, whereas quisqualate and N-methyl-DL-aspartate were ineffective. D-[3H]Aspartate uptake into confluent cerebellar astrocyte cultures was not affected by kainic acid. The stimulatory effect of kainic acid on D-[3H]aspartate release was Na+ independent, and partly Ca2+ dependent; the effect of quisqualate was Na+ and Ca2+ independent. Kynurenic acid (50–200 μM) and, to a lesser extent, 2,3-cis-piperidine dicarboxylic acid (100–200 μM) antagonized the stimulatory effect of kainate but not that of quisqualate. Kainic and quisqualic acid (20–100 μM) also stimulated γ-[3H]aminobutyric acid release from cerebellar cultures, and kynurenic acid antagonized the effect of kainate but not that of quisqualate. In conclusion, kainic acid and quisqualic acid appear to activate two different excitatory amino acid receptor subtypes, both coupled to neurotransmitter amino acid release. Moreover, kainate inhibits D-[3H]aspartate neuronal uptake by interfering with the acidic amino acid high-affinity transport system.

Notes: Abstract: In a previous study we noted that the release of d-[3]aspartate evoked by non-N-methyl-d-aspartate (non-NMDA) receptor agonists in cultured rat cerebellar granule cells was enhanced in the absence of extracellular Na+. To explain this apparent paradox, we tried in the present investigation to correlate the effect of Na+ removal on the kainate (KA)- and quisqualate (QA)-induced d-[3H]aspartate release with that on KA- and QA-induced 45Ca2+ accumulation. The releasing activity of KA, which was only partially Ca2+ dependent in the presence of Na+, became totally Ca2+ dependent in its absence. Moreover, the releasing activity of QA, which was Ca2+ independent in the presence of Na+, became 50% Ca2+ dependent in the absence of the monovalent cation. The releasing action of both agonists was in all cases antagonized by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), and that induced by KA was also sensitive to kynurenic acid. When glutamate was tested as an agonist in the presence of Na+, it was found that its d-[3H]aspartate releasing action was Ca2+ independent and was largely due to heteroexchange. The evoked release was Ca2+ independent, scarcely sensitive to CNQX, and insensitive to NMDA antagonists. In Na+-free medium, the glutamate-evoked d-[3H]aspartate release was lower (due to the abolishment of heteroexchange), but was totally Ca2+ dependent and antagonized by CNQX and kynurenate. KA (30 μM–1 mM) stimulated the accumulation of 45Ca2+ in a dose-dependent and CNQX-sensitive way, the effect being progressively higher as the Na+ concentration in the medium was decreased. Li+ affected KA-induced 45Ca2+ accumulation in a way similar to Na+, although 45Ca2+ uptake was somewhat lower in Li+-containing medium. The voltage-activated calcium channel antagonists La3+ and (–)-202-791 caused only a limited inhibition of the KA-induced 45Ca2+ influx both in the presence and in the absence of Na+. Under all the conditions tested [presence and absence of Na+ and of (–)-202-791], the kainate-induced 45Ca2+ uptake was scarcely sensitive to the NMDA antagonist 2-amino-5-phosphonovalerate. QA and α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid also stimulated 45Ca2+ influx in a CNQX-sensitive way, the effect being enhanced in Na+-free media. These agonists were, however, less effective than KA. It is concluded that in cultured cerebellar granule cells the influx of Ca2+ through non-NMDA receptor channels is inhibited by Na+ and similar monovalent cations, and that the potentiated KA- and QA-induced d-[3H]aspartate release observed in Na+-free medium can be accounted for by the increased accumulation of Ca2+ occurring under this condition.

Notes: Abstract: Amino acid release studies were performed by an HPLC procedure using differentiated rat cerebellar granule cell cultures. Kainic acid (KA; 50 μM) caused an increase (about threefold) in the release of endogenous glutamate and a lesser, but statistically significant, increase in the release of glutamine, glycine, threonine, taurine, and alanine. Quisqualic acid (QA) and, to a lesser degree, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) (both 50 μM) enhanced the release of the following amino acids in the order glutamate 〉 aspartate ≥ taurine, whereas the release of other amino acids was either unaffected or affected in a statistically nonsignificant way. The release of glutamate induced by KA was partially (43%) Ca2+ dependent. The other release-inducing effects of KA and QA were not Ca2+ dependent. In all cases, the evoked release could be prevented by the non-N-methyl-D-aspartate (non-NMDA) receptor antagonist 6-cyano-2,3-hydroxy-7-nitroquinoxaline, and thus appeared to be receptor mediated. NMDA (5 and 50 μM) had no releaseinducing activity. The KA-, QA-, and AMPA-evoked release of newly synthesized [3H]glutamate and [3H]aspartate (formed in the cells exposed to [3H]glutamine) was very similar to the evoked release of endogenous glutamate and aspartate. On the other hand, the release of preloaded D-[3H]aspartate (purified by HPLC in the various fractions analyzed, before radioactivity determination) induced by 50 μM KA was twice as high as that of endogenous glutamate. In the case of high [K+] depolarization, in contrast, the release of preloaded D-[3H]aspartate was ∼30% lower than that of endogenous glutamate. The reasons for the above differences in the susceptibility of the various glutamate pools to being released may be the following: Non-NMDA receptor agonists cause essentially a carrier-mediated efflux of glutamate from a cytoplasmic pool that is preferentially labeled by exogenous D-[3H]aspartate, whereas depolarization with high [K+] causes mainly an exocytotic-like release from a vesicular pool to which exogenous D-[3H]aspartate has a more limited access.

Notes: Abstract: Kainic acid (KA), quisqualic acid (QUIS), and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) stimulated D-[3H]aspartate release from cultured cerebellar granule cells in a concentration-dependent way. The EC50 values were 50 μM for KA (Gallo et al., 1987) and 20 μM for both QUIS and AMPA, but the efficacy of QUIS appeared to be greater than that of AMPA. The release of D-[3H]aspartate induced by KA, QUIS, and AMPA was blocked, in a dose-dependent way, by the new glutamate receptor antagonist 6-cyano-2,3-dihydroxy-7-nitroquinoxaline (CNQX); IC50 values were 0.7 μM in the case of AMPA (50 μM) and 1 μM in the case of KA (50 μM). AMPA (50–300 μM) inhibited the effect of 50 μM KA on D-[3H]aspartate release. At 300 μM AMPA, the effect of KA plus AMPA was not antagonized by the KA receptor antagonist kynurenic acid (KYN). In contrast, when KA was used at an ineffective concentration (10 μM), the addition of AMPA at concentrations below the EC50 value (10–20 μM) resulted in a synergistic effect on D-[3H]aspartate release. In this case, the evoked release of D-[3H]aspartate was sensitive to KYN. KA stimulated the formation of cyclic GMP, whereas QUIS, AMPA, and glutamate were ineffective. The accumulation of cyclic GMP elicited by KA (100 μM) was prevented not only by the antagonists CNQX (IC50= 1.5 μM) and KYN (IC50= 200 μM), but also by the agonists AMPA (IC50= 50 μM), QUIS (IC50= 3.5 μM), and glutamate (IC50= 100 μM). We conclude that AMPA, like QUIS, may act as a partial agonist at KA receptors. Moreover, CNQX effectively antagonizes non-N-methyl-D-aspartate receptor-mediated responses in cultured cerebellar granule cells.

Notes: In previous studies we have shown that the depolarization-induced release of preaccumulated acidic amino acids and newly synthesized glutamate from cerebellar synaptosomal preparations is potentiated by γ-aminobutyric acid (GABA) agonists through a GABAergic presynaptic mechanism. Here we report a systematic analysis of the ionic requirements of the potentiating effect of muscimol on the high K+-evoked release of d-[3H]aspartate. Our studies show that: Ca2+, Na+, and Mg2+ are not required for muscimol to exert its effect; a depolarizing concentration of K+ is a necessary, but not sufficient, condition to observe the presynaptic effect in question; and a minimal Cl- concentration (50–70 mM) is also required. A possible model based on these findings is proposed.

Notes: Abstract— Several parameters of GABA Auxes across the synaptosomal membrane were studied using synaptosomes prepared from the brain of immature (8-day-old) rats. The following aspects of GABA carrier-mediated transport were similar in immature and mature synaptosomes: (1) magnitude of [3H]GABA accumulation; (2) GABA homoexchange in normal ionic conditions; (3) GABA homoexchange in the presence of cationic fluxes (Na+ and Ca2+ influx, K+ efflux) characteristic of physiological depolarization. As in adult synaptosomes (Levi & Raiteri, 1978), in these conditions the stoichiometry of GABA homoexchange was in the direction of net outward transport (efflux 〉 influx).The essential differences between the behaviour of 8-day-old and adult synaptosomes were the following: (1) β-alanine (a glial uptake inhibitor) inhibited GABA uptake in immature synaptosomes (the inhibition being greater in crude than in purified preparations) and was without a significant effect in adult synaptosomes. DABA and ACHC (two neuronal uptake inhibitors) depressed GABA uptake more efficiently in purified than in crude immature synaptosomes, but were as effective in crude and purified nerve endings from adult animals. The data suggest a greater uptake of GABA in the‘gliosomes’contaminating the synaptosomal preparations from immature animals. (2) In immature synaptosomes prelabelled with [3H]GABA the specific radioactivity of the GABA released spontaneously or by heteroexchange (with 300 μm-OH-GABA) was the same as that present in synaptosomes, while in adult synaptosomes OH-GABA released GABA with increased specific radioactivity. The data suggest a homogeneous distribution of the [3H]GABA taken up within the endogenous GABA pool in immature, but not in mature synaptosomes. (3) In immature synaptosomes the release of GABA (radioactive and endogenous) induced by depolarization with high KC was not potentiated by Ca2+, unless the synaptosomes had been previously depleted of Na+ These data suggest that, although a Ca2+ sensitive pool of GABA may be present, this pool is not susceptible to being released in normal conditions, probably because the high intrasynaptosomal Na+ level prevents a sufficient depolarization. The possible significance of these findings in terms of functional activity of GABAergic neurotransmission in the immature brain is discussed.

Notes: Abstract: The aim of the present paper was to determine whether the release of glutamate from putative “glutamergic” terminals in the cerebellum is influenced by γ-aminobutyric acid (GABA). In a group of preliminary experiments, we present biochemical evidence in favour of a neurotransmitter role of glutamate in the cerebellum: (1) endogenous glutamate was released from depolarized cerebellar synaptosomal preparations in a Ca2+-dependent way; (2) [14C]glutamate was synthesized from [14C]glutamine in cerebellar synaptosomes, and the newly synthesized [14C]glutamate was released in a Ca2+-dependent way; (3) the elevation of cyclic GMP elicited by depolarization of cerebellar slices in the presence of Ca2+ was partly reversed by the glutamate antagonist glutamic acid diethyl ester, which probably prevented the interaction of endogenously released glutamate with postsynaptic receptors.GABA and muscimol at low concentrations (2-20 μM) potentiated the depolarization-induced release of D-[3H]aspartate (a glutamate analogue which labels the glutamate “reuptake pool”) from cerebellar synaptosomes. The effect was concentration dependent and was largely prevented by two GABA antagonists, bicuculline and picrotoxin. The stimulation of D-[3H]aspartate release evoked by muscimol was linearly related to the logarithm of K+ concentration in the depolarizing medium. GABA did not affect the overall release of endogenous glutamate, but potentiated, in a picrotoxin-sensitive manner, the depolarization-evoked release of [14C]glutamate previously synthesized from [14C]glutamine. Since nerve endings are the major site of glutamate synthesis from glutamine, GABA and muscimol appear to exert their stimulatory effect at the level of “glutamergic” nerve terminals, probably after interacting with presynaptic GABA receptors. The possible functional significance of these findings is briefly discussed.

Notes: Ten patients are reported suffering from oral lichen planus (OLP) associated with chronic liver diseases linked to HCV. All patients were affected by varieties of erosive oral lichen planus. In six of these 10 patients the diagnosis of HCV was made as a result of the OLP diagnosis and four of them had unknown, but severe, chronic liver disease. These preliminary data support the possible existence of a relationship between oral erosive lichen planus and HCV infection.