Release of Glutamate and Aspartate from CA1 Synaptosomes: Selective Modulation of Aspartate Release by Ionotropic Glutamate Receptor Ligands

Abstract: Synaptosomes prepared from area CA1 of the rat hippocampus were used to determine (a) whether Schaffer collateral-commissural-ipsilateral associational terminals release both aspartate and glutamate in a Ca²⁺-dependent manner when reuptake of released glutamate is minimal and (b) whether autoreceptor mechanisms described in CA1 or hippocampal slices could reflect direct actions of glutamate receptor ligands on the synaptic terminal. When challenged for 1 min with either 25 m M K⁺ or 300 µ M 4-aminopyridine, CA1 synaptosomes released both glutamate and aspartate in a Ca²⁺-dependent manner. The glutamate/aspartate ratio was ∼5:1 in each case. K⁺-evoked glutamate release was unaffected by ligands active at NMDA or ( RS )-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors. Unlike glutamate release, the release of aspartate was enhanced by NMDA, and this effect was blocked by d -2-amino-5-phosphonovalerate ( d -AP5). Kainate selectively depressed and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) selectively increased the K⁺-evoked release of aspartate. AMPA enhanced aspartate release, like the antagonist CNQX. When applied in the presence of diazoxide, which blocks the desensitization of AMPA receptors, AMPA and kainate both depressed aspartate release. These findings support the view that Schaffer collateral-commissural-ipsilateral associational terminals release aspartate as well as glutamate and that these two release processes are regulated by different autoreceptor mechanisms.     

Phorbol Ester-Enhanced Noradrenaline Secretion Correlates with the Presence and Activity of Protein Kinase C-α in Human SH-SY5Y Neuroblastoma Cells

Abstract: The effect of inhibition and down-regulation of protein kinase C (PKC) subtypes α, ε, and ζ on noradrenaline (NA) secretion from human SH-SY5Y neuroblastoma cells was investigated. The PKC inhibitor Ro 31-7549 inhibited carbachol-evoked NA release (IC₅₀ 0.6 µ M ) but not 100 m M K⁺-evoked release. In addition, Ro 31-7549 inhibited the enhancement of carbachol- and K⁺-evoked release after pretreatment with 12- O -tetradecanoylphorbol 13-acetate (TPA; 100 n M ) for 8 min, with IC₅₀ values of 0.7 and 2.4 µ M , respectively. Immunoblotting studies showed that prolonged exposure (48 h) of SH-SY5Y cells to phorbol 12,13-dibutyrate (PDBu) or bryostatin-1 caused down-regulation of PKC-α and PKC-ε but not PKC-ζ. Under these conditions, the acute TPA enhancement of NA release was inhibited. Moreover, the inhibition of TPA-enhanced secretion was also apparent after only 2-h exposure to either PDBu or bryostatin-1, conditions that caused down-regulation of PKC-α, but not PKC-ε or ζ. The PKC inhibitor Gö-6976 (2 µ M ), which has been shown to inhibit selectively PKC-α and β in vitro, also inhibited the TPA enhancement of carbachol- and K⁺-evoked NA release by >50%. These data suggest that in SH-SY5Y cells, the ability of TPA to enhance carbachol- and K⁺-evoked NA secretion is due to activation of PKC-\ga.     

Presynaptic α2 Adrenoceptors Inhibit Glutamate Release from Rat Spinal Cord Synaptosomes

Abstract: The presynaptic regulation of amino acid release from nerve terminals was investigated using synaptosomes prepared from the rat spinal cord. The basal releases of endogenous glutamate (Glu), aspartate (Asp), and γ-amino-butyric acid (GABA) were 34.6, 21.5, and 10.0 pmol/min/mg of protein, respectively. Exposure to a depolarizing concentration of KCl (30 m M ) evoked 2.7-, 1.5-, and 2.9-fold increases in Glu, Asp, and GABA release, respectively. Clonidine reduced the K⁺-evoked overflow of Glu to 56% of the control overflow with a potency (IC₅₀) of 17 n M , but it did not affect K⁺-evoked overflow of Asp, GABA, and their basal releases. Similarly, noradrenaline inhibited the K⁺-evoked overflow of Glu, although phenylephrine and isoproterenol showed no effect. The inhibitory effect of clonidine was counteracted by α₂-adrenoceptor antagonists, rauwolscine, yohimbine, and idazoxan, regardless of the imidazoline structures. Because Glu is considered a neurotransmitter of primary afferents that transmit both nociceptive and nonnociceptive stimuli in the spinal cord, these data suggest that part of Glu release may be regulated by the noradrenergic system through α₂ adrenoceptors localized on the primary afferent terminals.     

Potassium and Taurine Release Are Highly Correlated with Regulatory Volume Decrease in Neonatal Primary Rat Astrocyte Cultures

Abstract: Neonatal rat primary astrocyte cultures were swollen by exposure to hypotonic buffer. Using an electrical impedance method for determination of cell volume coupled with on-line measurements of efflux of radioactive ions or amino acids, we have investigated the role of K⁺ (using ⁸⁶Rb), taurine, and d -aspartate (an analogue of glutamate) in regulatory volume decrease (RVD). Addition of 1 m M quinine, 10 µ M nimodipine, 100 µ M BAPTA-AM, 10 µ M trifluoperazine, or a calcium-free buffer significantly ( p < 0.0001) inhibited RVD. This was accompanied by inhibition of ⁸⁶Rb release but an increase in d -[³H]-aspartate release, which was proportional to the degree to which RVD was inhibited. These results support a regulatory role for calcium in RVD and show that inhibition of calcium entry from the extracellular fluid, intracellular calcium sequestration, inhibition of calcium-activated K⁺ channels, and inhibition of calmodulin all inhibit RVD. Because d -[³H]aspartate efflux profiles increase as RVD is inhibited, it is unlikely that d -aspartate release is a main determinant of RVD. In contrast, [³H]taurine release was increased by 1 m M quinine and inhibited by 10 µ M trifluoperazine. The net release of K⁺ and taurine is highly correlated with the degree of RVD, implicating a regulatory role for both K⁺ and taurine release in RVD.     

Hypoxia Enhances [3H]Noradrenaline Release Evoked by Nicotinic Receptor Activation from the Human Neuroblastoma SH-SY5Y

Abstract: We have used the human sympathetic neuronal line SH-SY5Y to investigate the effects of hypoxia on noradrenaline (NA) release evoked by either raised [K⁺]_o (100 m M ) or the nicotinic acetylcholine receptor (nAChR) agonist dimethylphenylpiperazinium iodide (DMPP). NA release was monitored by loading cells with [³H]NA and collecting effluent fractions from perfused cells kept in a sealed perifusion chamber. Cells were challenged twice with either stimulus and release was expressed as that evoked by the second challenge as a fraction of that evoked by the first. K⁺-evoked release was unaffected by hypoxia (P o ₂≅ 30–38 mm Hg), but release evoked by DMPP was significantly increased. For both stimuli, replacement of Ca²⁺_o with 1 m M EGTA abolished NA release. K⁺-evoked release was also dramatically reduced in the presence of 200 µ M Cd²⁺ to block voltage-gated Ca²⁺ channels, but DMPP-evoked release was less affected. In hypoxia, DMPP-evoked Cd²⁺-resistant NA release was dramatically increased. Our findings indicate that hypoxia increases NA release evoked from SH-SY5Y cells in response to nAChR activation by increasing Ca²⁺ influx through the nAChR pore, or by activating an unidentified Cd²⁺-resistant Ca²⁺-influx pathway. As acetylcholine is the endogenous transmitter at sympathetic ganglia, these findings may have important implications for sympathetic activity under hypoxic conditions.     

Involvement of Calcium Channels in Depolarization-Evoked Release of Adenosine from Spinal Cord Synaptosomes

Abstract: The potential involvement of L- and N-type voltage-sensitive calcium (Ca²⁺) channels and a voltage-independent receptor-operated Ca²⁺ channel in the release of adenosine from dorsal spinal cord synaptosomes induced by depolarization with K⁺ and capsaicin was examined. Bay K 8644 (10 n M ) augmented release of adenosine in the presence of a partial depolarization with K⁺ (addition of 6 m M ) but not capsaicin (1 and 10 μ M ). This augmentation was dose dependent from 1 to 10 n M and was followed by inhibition of release from 30 to 100 n M . Nifedipine and nitrendipine inhibited the augmenting effect of Bay K 8644 in a dose-dependent manner, but neither antagonist had any effect on release of adenosine produced by K⁺ (24 m M ) or capsaicin (1 and 10 μ M ) ω-Conotoxin inhibited K⁺-evoked release of adenosine in a dose-dependent manner but had no effect on capsaicin-evoked release. Ruthenium red blocked capsaicin-induced release of adenosine but had no effect on K⁺-evoked release. Although L-type voltage-sensitive Ca²⁺ channels can modulate release of adenosine when synaptosomes are partially depolarized with K⁺, N-type voltage-sensitive Ca²⁺ channels are primarily involved in K⁺-evoked release of adenosine. Capsaicin-evoked release of adenosine does not involve either L- or N-type Ca²⁺ channels, but is dependent on a mechanism that is sensitive to ruthenium red.     

Effect of Isonicotinic Acid Hydrazide on Extracellular Amino Acids and Convulsions in the Rat: Reversal of Neurochemical and Behavioural Deficits by Sodium Valproate

Abstract: We have studied the effect of isonicotinic acid hydrazide (INH), a convulsant agent, on the extracellular levels of amino acids in the hippocampus, and the effect of sodium valproate (VPA) administration in INH-treated rats. INH (250 mg/kg) caused a rapid and sustained decrease in basal levels of GABA, and during this period convulsions of increasing severity were observed. Basal levels of glutamine, taurine, aspartate, and glutamate were unchanged by INH. When VPA was coadministered with INH, basal GABA levels were increased and no convulsions were observed. When transmitter release was evoked using 100 m M K⁺, the increase in dialysate GABA observed in INH-treated animals was less than that seen in controls and convulsions increased in frequency. K⁺-evoked release of glutamate and aspartate tended to be higher following INH treatment, and in the case of aspartate, this increase was significant. VPA reversed the changes in evoked release of glutamate and aspartate, and release of GABA was considerably greater than that seen in control or INH-treated rats. No drug effect on evoked changes in taurine or glutamine level was seen. These are the first data to show decreased extracellular GABA in conjunction with convulsions in freely moving animals in vivo.     

Barium Evokes Glutamate Release from Rat Brain Synaptosomes by Membrane Depolarization: Involvement of K+, Na+, and Ca2+ Channels

Abstract: During K⁺ -induced depolarization of isolated rat brain nerve terminals (synaptosomes), 1 m M Ba²⁺ could substitute for 1 m M Ca²⁺ in evoking the release of endogenous glutamate. In addition, Ba²⁺ was found to evoke glutamate release in the absence of K⁺-induced depolarization. Ba²⁺ (1–10 m M ) depolarized synaptosomes, as measured by voltage-sensitive dye fluorescence and [³H]-tetraphenylphosphonium cation distribution. Ba²⁺ partially inhibited the increase in synaptosomal K⁺ efflux produced by depolarization, as reflected by the redistribution of radiolabeled ⁸⁶Rb⁺. The release evoked by Ba²⁺ was inhibited by tetrodotoxin (TTX). Using the divalent cation indicator fura-2, cytosolic [Ca²⁺] increased during stimulation by approximately 200 n M , but cytosolic [Ba²⁺] increased by more than 1 μ M . Taken together, our results indicate that Ba²⁺ initially depolarizes synaptosomes most likely by blocking a K⁺ channel, which then activates TTX-sensitive Na⁺ channels, causing further depolarization, and finally enters synaptosomes through voltage-sensitive Ca²⁺channels to evoke neurotransmitter release directly. Though Ba²⁺-evoked glutamate release was comparable in level to that obtained with K⁺-induced depolarization in the presence of Ca²⁺, the apparent intrasynaptosomal level of Ba²⁺ required for a given amount of glutamate release was found to be several-fold higher than that required of Ca²⁺.     

Basic and Regulatory Mechanisms of In Vitro Release of Met-Enkephalin from the Dorsal Zone of the Rat Spinal Cord

Abstract: Under control conditions, superfused slices of the dorsal half of the lumbar enlargement from adult rats released Met-enkephalin-like material (MELM) that behaved as authentic Met-enkephalin under two different chromatographic procedures (Bio-gel filtration, HPLC). MELM release increased markedly on exposure of slices to batrachotoxin (0.5 μ M ) or to an excess of K⁺ (28 and 56 m M instead of 5.6 m M ). The K ⁺ -evoked release was totally dependent on the presence of Ca²⁺ in the super-fusing fluid whereas the spontaneous efflux of MELM was only partially Ca²⁺-dependent. Further experiments performed with tissues of polyarthritic rats indicated that the increase in their MELM levels was associated with a lower fractional rate constant of MELM release, therefore suggesting that spinal Met-enkephalin turnover might be reduced in chronically suffering animals. Examination of the possible modulation of MELM release by various neuroactive compounds present within the dorsal horn revealed that cholecystokinin (10 μ M ), but not its desulphated derivative, substance P-sulphoxide (10 μ M ), and to a lesser extent substance P, enhanced the K⁺-evoked MELM release. In contrast, γ-aminobutyric acid (10 μ M ) and (–)-baclofen (1 μ M ) partially prevented the stimulatory effect of K⁺ on MELM release. Other compounds such as serotonin, somatostatin, and neurotensin altered neither the spontaneous nor the K⁺-evoked release of MELM.     

Depolarization and Activation of Dihydropyridine-Sensitive Ca2+ Channels Stimulate Inositol Phosphate Accumulation in Photoreceptor-Enriched Chick Retinal Cell Cultures

Abstract: Elevated concentrations of extracellular K⁺ increased inositol phosphate accumulation in primary cultures of chick retinal photoreceptors and multipolar neurons. K⁺-evoked stimulation of inositol phosphate accumulation was greater in photoreceptor-enriched cell cultures than in cultures where multipolar neurons were the predominant cell type. Destroying multipolar neurons, but not photoreceptors, with kainic acid and N -methyl- d -aspartate did not reduce the K⁺-evoked stimulation of inositol phosphate accumulation. Both of these observations indicate that the observed effects occur in photoreceptor cells. The K⁺-evoked stimulation of inositol phosphate accumulation was blocked by omitting Ca²⁺ from the incubation medium or by adding the dihydropyridine-sensitive Ca²⁺-channel antagonists, nitrendipine and nifedipine. Bay K 8644, a dihydropyridine agonist, stimulated inositol phosphate accumulation and enhanced the effect of K⁺. ω-Conotoxin GVIA, an inhibitor of N-type Ca²⁺ channels, had no significant effect on K⁺-stimulated inositol phosphate accumulation. Pretreatment with pertussis toxin neither blocked K⁺-evoked inositol phosphate accumulation nor altered the inhibitory effect of nifedipine. K⁺-evoked inositol phosphate accumulation appears to reflect activation of phosphatidylinositol-specific phospholipase C, as it is inhibited by U-73122. These results indicate that Ca²⁺ influx through voltage-gated, dihydropyridine-sensitive channels activates phospholipase C in photoreceptor inner segments and/or synaptic terminals.     

Dopamine Inhibition of the Release of Endogenous Acetylcholine from Corpus Striatum and Cerebral Cortex in Tissue Slices and Synaptosomes: A Presynaptic Response?

Abstract: The effect of dopamine on the release of endogenous acetylcholine from striatal slices and synaptosomes and from cerebral cortex synaptosomes was studied. K⁺ (56 m M ) and veratrine (75 μM ) increased the release of acetylcholine from striatal slices by 3.7 and 3.3 times the resting release, respectively. The effect of veratrine was completely abolished by tetrodotoxin (1 μM ). Dopamine (10⁻⁶ to 10⁻³ M ) reduced the K⁺-evoked release of acetylcholine from striatal slices in a dose-dependent manner. The resting release of acetylcholine was also significantly reduced by dopamine. Apomorphine (20 μM ) significantly reduced the K⁺-evoked release of acetylcholine, and both this effect and the inhibition due to dopamine (1 m M ) were significantly antagonised by chlorpromazine (20 μM ). Dopamine had a similar effect on the release of acetylcholine from striatal synaptosome beds; the resting release was depressed 32% by the presence of dopamine (1 m M ). A greater effect of dopamine was seen on the release of acetylcholine from cerebral cortex synaptosome beds, the resting release being reduced by 54% and the K⁺-evoked release by 29%. These results are discussed in terms of the possible role of presynaptic dopamine receptors in controlling the release of acetylcholine and the magnitude of their contribution compared with that of the postsynaptic dopamine receptor.     

Serotonin 5-HT1D and 5-HT1A Receptors Respectively Mediate Inhibition of Glutamate Release and Inhibition of Cyclic GMP Production in Rat Cerebellum In Vitro

Abstract: The K⁺-evoked overflow of endogenous glutamate from cerebellar synaptosomes was inhibited by serotonin [5-hydroxytryptamine (5-HT); pD₂ = 8.95], 8-hydroxy-2-(di- n -propylamino)tetralin (8-OH-DPAT; pD₂ = 7.35), and sumatriptan (pD₂ = 8.43). These inhibitions were prevented by the selective 5-HT_1D receptor antagonist N -[4-methoxy-3-(4-methyl-1-piperazinyl)phenyl]-2'-methyl-4'-(5-methyl-1,2,4-oxadiazol-3-yl)(1,1-biphenyl)-4-carboxamide (GR-127935). The three agonists tested also inhibited the cyclic GMP (cGMP) response provoked in slices by K⁺ depolarization; pD₂ values were 9.37 (5-HT), 9.00 (8-OH-DPAT), and 8.39 (sumatriptan). When cGMP formation was elevated by directly activating glutamate receptors with NMDA or α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA), the inhibition of the cGMP responses displayed the following pattern: 5-HT (pD₂ values of 8.68 and 8.72 against NMDA and AMPA, respectively); 8-OH-DPAT (respective pD₂ values of 9.15 and 9.00); sumatriptan (0.1 µ M ) was ineffective. The 5-HT_1A receptor antagonist ( S )-(+) N-tert -butyl-3-[4-(2-methoxyphenyl)piperazin-1-yl]-2-phenylpropionamide dihydrochloride [(+)-WAY 100135] did not prevent the inhibition of glutamate release by 5-HT but blocked the inhibition by 8-OH-DPAT of the NMDA/AMPA-evoked cGMP responses. It is suggested that presynaptic 5-HT_1D receptors mediate inhibition directly of glutamate release and indirectly of the cGMP responses to the released glutamate; on the other hand, activation of (postsynaptic) 5-HT_1A receptors causes inhibition of the cGMP responses linked to stimulation of NMDA/AMPA receptors.     

Protein Kinase FA/Glycogen Synthase Kinase-3α After Heparin Potentiation Phosphorylates τ on Sites Abnormally Phosphorylated in Alzheimer''s Disease Brain

In this work, we tested the effect of ion channel blockers and of phorbol ester treatments on [³H]dopamine ([³H]DA) release and neurotensin (NT)-induced facilitation of [³H]DA release from cultures of rat fetal mesencephalic cells. The potassium channel blockers tetraethylammonium and 4-aminopyridine increased basal [³H]DA release and decreased K⁺-evoked [³H]DA release, whereas apamin was without effect. K⁺-evoked [³H]DA release was decreased by ω-conotoxin and nifedipine, totally suppressed by cadmium, and unaffected by amiloride. These results show the differential sensitivity of [³H]DA release to blockade of various ion channels and suggest the involvement of N-type, L-type, and non-L-non-N-type, but not T-type, voltage-sensitive calcium channels in K⁺-evoked release. Phorbol 12-myristate 13-acetate increased both spontaneous and K⁺-evoked [³H]DA release, suggesting a modulatory action of protein kinase C on DA release in this system. Unexpectedly, however, the effects of the phorbol ester were not counteracted by the protein kinase C inhibitors H7, staurosporine, or polymyxin B. NT-induced facilitation of K⁺-evoked [³H]DA release was insensitive to most of the ion channel blockers, except cadmium (64% decrease in NT effect), suggesting that the corresponding potassium' and calcium channels were not involved in the effect of NT on [³H]DA release in this system. The NT effect was totally suppressed by phorbol ester treatments, indicating a possible desensitization of the corresponding transduction mechanisms after protein kinase C activation.     

Glutamate Uptake Stimulates Na+,K+-ATPase Activity in Astrocytes via Activation of a Distinct Subunit Highly Sensitive to Ouabain

Abstract: The excitatory amino acid glutamate was previously shown to stimulate aerobic glycolysis in astrocytes by a mechanism involving its uptake through an Na⁺-dependent transporter. Evidence had been provided that Na⁺,K⁺-ATPase might be involved in this process. We have now measured the activity of Na⁺,K⁺-ATPase in cultured astrocytes, using ouabain-sensitive ⁸⁶Rb uptake as an index. l -Glutamate increases glial Na⁺,K⁺-ATPase activity in a concentration-dependent manner with an EC₅₀ = 67 µ M . Both l - and d -aspartate, but not d -glutamate, produce a similar response, an observation that is consistent with an uptake-related effect rather than a receptor-mediated one. Under basal conditions, concentration-dependent inhibition of Na⁺,K⁺-ATPase activity in astrocytes by ouabain indicates the presence of a single catalytic site with a low affinity for ouabain ( K _0.5 = 113 µ M ), compatible with the presence of an α₁ isozyme. On stimulation with glutamate, however, most of the increased activity is inhibited by low concentrations of ouabain ( K _0.5 = 20 n M ), thus revealing a high-affinity site akin to the α₂ isozyme. These results suggest that astrocytes possess a glutamate-sensitive isoform of Na⁺,K⁺-ATPase that can be mobilized in response to increased neuronal activity.     

Glutamate Induces a Calcineurin-Mediated Dephosphorylation of Na+,K+-ATPase that Results in Its Activation in Cerebellar Neurons in Culture

Abstract: In primary cultures of cerebellar neurons glutamate neurotoxicity is mainly mediated by activation of the NMDA receptor, which allows the entry of Ca²⁺ and Na⁺ into the neuron. To maintain Na⁺ homeostasis, the excess Na⁺ entering through the ion channel should be removed by Na⁺,K⁺-ATPase. It is shown that incubation of primary cultured cerebellar neurons with glutamate resulted in activation of the Na⁺,K⁺-ATPase. The effect was rapid, peaking between 5 and 15 min (85% activation), and was maintained for at least 2 h. Glutamate-induced activation of Na⁺,K⁺-ATPase was dose dependent: It was appreciable (37%) at 0.1 µ M and peaked (85%) at 100 µ M . The increase in Na⁺,K⁺-ATPase activity by glutamate was prevented by MK-801, indicating that it is mediated by activation of the NMDA receptor. Activation of the ATPase was reversed by phorbol 12-myristate 13-acetate, an activator of protein kinase C, indicating that activation of Na⁺,K⁺-ATPase is due to decreased phosphorylation by protein kinase C. W-7 or cyclosporin, both inhibitors of calcineurin, prevented the activation of Na⁺,K⁺-ATPase by glutamate. These results suggest that activation of NMDA receptors leads to activation of calcineurin, which dephosphorylates an amino acid residue of the Na⁺,K⁺-ATPase that was previously phosphorylated by protein kinase C. This dephosphorylation leads to activation of Na⁺,K⁺-ATPase.     

Identification and Function of Glycine Receptors in Cultured Cerebellar Granule Cells

Abstract: Poly(A)⁺ mRNA was isolated from cultured mouse cerebellar granule cells and injected into Xenopus oocytes. This led to the expression of receptors that evoked large membrane currents in response to glycine. Current-responses were also obtained after application of β-alanine and taurine, but these were very low relative to that of glycine (maximal β-alanine and taurine responses were 8 and 3% of that of glycine, respectively). The role of glycine receptors on K⁺-evoked transmitter release in cultured cerebellar granule cells was also assayed. Release of preloaded d -[³H]aspartate evoked by 40 m M K⁺ was dose dependently inhibited by glycine, and the concentration producing half-maximal inhibition was 50 μ M. Taurine, β-alanine, and the specific GABA_A receptor agonist isoguvacine also inhibited K⁺-evoked release, and the maximal inhibition was similar for all agonists (˜40%). The EC₅₀ value was 200 μ M for taurine, 70 μ M for β-alanine, and 4 μ M for isoguvacine. Bicuculline (150 μ M ) antagonized the inhibitory effect of isoguvacine (150 μ M ) but not that of glycine (1 m M ). In contrast, strychnine (20 μ M ) antagonized the inhibitory effect of glycine (1 m M ) but not that of isoguvacine (150 μ M ). The pharmacology of the responses to β-alanine and taurine showed that these agonists activate both glycine and GABA_A receptors. The results indicate that cultured cerebellar granule cells translate the gene for the glycine receptor and that activation of glycine receptors produces neuronal inhibition.     

Tetanus Toxin Inhibition of K+ -Stimulated [3H]GABA Release from Developing Cell Cultures of the Rat Cerebellum

Abstract: The effect of tetanus toxin pretreatment on K⁺ -stimulated [³H]γ-aminobutyric acid release from neuron-enriched cerebellar cell cultures at various stages during their development in vitro was assessed. Tetanus toxin had little inhibitory effect on immature (1-3-day-old) cultures, but markedly reduced K⁺-evoked [³H]γ-aminobutyric acid release from 7- and 14-day-old cultures (∼80% inhibition). It is suggested that cerebellar neurons in culture develop tetanus toxin-sensitive transmitter release mechanisms similar to their in vivo counterparts.     

Effect of Inhibitors of Eicosanoid Metabolism on Release of [3H]Noradrenaline from the Human Neuroblastoma, SH-SY5Y

Abstract: Nordihydroguaiaretic acid (NDGA; a lipoxygenase inhibitor), LY-270766 (an inhibitor of 5-lipoxygenase), and the diacylglycerol lipase inhibitor RG 80267 completely eliminated potassium-evoked release of [³H]noradrenaline ([³H]NA) from the human neuroblastoma clone SH-SY5Y with IC₅₀ values of 10, 15, and 30 μ M , respectively. In contrast, these inhibitors only partially inhibited carbachol-evoked release and had little effect on the calcium ionophore A23187-evoked release of NA in this cell line. Arachidonic acid partially inhibited potassium- and A23187-evoked release but did not reverse the inhibition of potassium-evoked release observed in the presence of RG 80267. These studies suggest that arachidonic acid (or its lipoxygenase products) are not important intermediates in the regulation of exocytosis in SH-SY5Y. This conclusion is strengthened by our studies in which SH-SY5Y cells were grown in medium supplemented with bovine serum albumin-linoleic acid (50 μ M ). Under these conditions there was a selective increase in content of membrane polyunsaturated fatty acids of the ω6 series, including arachidonic acid; however, these changes did not effect potassium-, veratridine-, carbachol-, or calcium ionophoreevoked release of [³H]NA.     

Dual Modulation of Dopamine Release from Anterior Nucleus Accumbens Through Cholecystokinin-B Receptor Subsites

Abstract: Previous binding studies have suggested the existence of two affinity states for cholecystokinin-B (CCK-B) receptor. One study, using BC 197 and BC 264, two highly selective CCK-B agonists, has shown that BC 197 is selective for one subsite, B₁, and that BC 264 has the same affinity for the two subsites, B₁ and B₂. Therefore, the possible involvement of CCK-B subsites in the modulation of endogenous dopamine (DA) release from slices of the anterior part of the nucleus accumbens was investigated with these two agonists in order to associate a functional response with activation of each subsite. The selective B₁ agonist BC 197 produced a dose-dependent increase of 35 m M K⁺-stimulated DA release. In contrast, at a low concentration (20 n M ), BC 264 inhibited the K⁺-evoked DA release, whereas at a higher concentration (1 µ M ), it stimulated the DA release. These two opposing effects were suppressed by the CCK-B antagonist PD-134,308, but not by the CCK-A antagonist L-364,718 and were not prevented by tetrodotoxin, a Na⁺-channel blocker. Moreover, BC 264 at 20 n M , in the presence of PD-134,308 at a concentration that would block the B₂ subsites (0.1 n M ), increased the evoked DA release. All together, these results support further the existence of distinct CCK-B subsites and suggest that, in the anterior nucleus accumbens, their stimulation mediates opposite effects on K⁺-stimulated DA release via a presynaptic mechanism.     

The Effect of Chronic Treatment with the GABA Transaminase Inhibitors γ-Vinyl-GABA and Ethanolamine-O-Sulphate on the In Vivo Release of GABA from Rat Hippocampus

Abstract: The effects of chronic treatment with the specific, mechanism-based, irreversible inhibitors of 4-aminobutyrate aminotransferase (EC 2.6.1.19; GABA transaminase), ethanolamine O -sulphate (EOS), and 4-aminohexenoate [vigabatrin; γ-vinyl-GABA (GVG)] on the extracellular concentrations of GABA in the hippocampus have been studied using in vivo microdialysis in conscious animals. Oral dosing [3 mg/ml of drinking water, giving doses of GVG of 194 ± 38 mg/kg/day and of EOS of 303 ± 42 mg/kg/day (mean ± SD)] was followed by microdialysis at 2, 8, and 21 days. The basal outflow of GABA (in the range of ∼1–2 pmol/30 µl/30-min sample) after 2 and 8 days of treatment was not significantly different from that in control animals, but the 21-day treatment gave significant rises in the extracellular GABA concentration (up to ∼6–8 pmol/30 µl/30-min sample). Both inhibitors gave similar results. Depolarisation with 100 m M K⁺ gave large increases in GABA release in control (∼20–60 pmol/30 µl/30-min sample) and treated animals. The 8- and 21-day-treated animals showed significant increases in the stimulated release compared with control animals (∼80–100 pmol/30 µl/30-min sample). Excluding Ca²⁺ had no significant effect on either basal or stimulated release. The significant increases in K⁺-evoked release of GABA show that the increased intracellular pool of GABA is available for release, and this may be related to the anticonvulsant action of these compounds.