Expression of Excitatory Amino Acid Receptors by Cerebellar Cells of the Type-2 Astrocyte Cell Lineage

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 μ M . Quisqualate was more effective than kainate in the lower concentration range but less effective at concentrations at which its releasing activity was maximal (∼50 μ M ). N -Methyl- d -aspartic acid and dihydrokainate (100 μ M ) did not stimulate [³H]GABA release from cultured astrocytes. l -Glutamic acid (20–100 μ M ) 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 μ M ) 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 μ M ). 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.     

Xylogliicae- and cello-oligosaccharides: Antagonists of the growth-promoting effect of H+

Xyloglucan-oligosaccharides and cello-oligosaccharides, both of which are potential products of the action of cellulase on plant cell wail polysaccharides, inhibited acid-induced elongation in pea ( Pisum sativum L. cv. Alaska) stem segments. Xyloglucan-derived nonasaccharide (XG9; Glc₄-Xyl₃)Gal-Fuc) and decasaccharide (XG10; Glc₄-Xyl₃-Gal₂-Fue) inhibited acid-induced growth at 1.0 and 0.1 n M , respectively, whereas the heptasaccharide (XG7; Glc₄-Xyl₃) and octasaccharide (XG8; Glc₄-Xyl₃-Gal)₂ which lack L-fucose, did not. XG9 at 1 n M inhibited acid-induced growth as effectively as it inhibits auxin-induced elongation. This suggests that XG9's effect as an inhibitor of auxin action is not mediated by a suppresion of H⁺-efflux, but rather that XG9 blocks some step that is common to the action of both auxin and H⁺ on growth. Cello-oligosaccharides (degree of polymerisation 4–7) also inhibited acid-induced growth at 10 n M ; these are therefore a new class of possible oligosaccha-rin. The inhibitory effect of xyloglucan- and cellooligosaccharides on acid-induced growth was rapidly reversed by washing.     

[3H]Metergoline: A New Ligand of Serotonin Receptors in the Rat Brain

Abstract: A specific binding site for [³H]metergoline characterized by a K_D of 0.5–1.0 nM was detected in microsomal and synaptic plasma membranes from various areas of the adult rat brain. Experiments with 5,7-dihydroxy-tryptamine- and kainic acid-induced lesions indicated that this specific binding site was localized post-synaptically with respect to serotoninergic neurons. The pharmacological characteristics of [³H]metergoline binding to microsomal membranes from the whole forebrain strongly suggest that this ligand labels a class of serotonin receptors. This was particularly obvious in the hippocampus in which serotonin was about 400 times more potent than dopamine and norad-renaline for displacing bound [³H]metergoline. In the striatum, serotonin was only 10 times as potent as dopamine in inhibiting [³H]metergoline binding, suggesting that this ligand may also bind to dopamine receptors. Striking similarities between the binding sites for [³H]metergoline and [³H]serotonin were observed in the hippocampus. Thus, not only the total numbers of binding sites for these two ligands in control rats but also their respective increases following intracerebral 5,7-dihydroxytryptamine treatment were very similar. Therefore, at least in the hippocampus, [³H]metergoline might well be the appropriate ligand for studying the characteristics of the 'antagonist form' of serotonin receptors postulated by Bennett and Snyder.     

Simultaneous Measurement of Nitrite and Nitrate Levels as Indices of Nitric Oxide Release in the Cerebellum of Conscious Rats

Abstract: We examined the modulation of nitric oxide production in vivo by measuring levels of nitrite (NO₂⁻) and nitrate (NO₃⁻) in the dialysate of the cerebellum in conscious rats, by using an in vivo brain microdialysis technique. The levels of both NO₂⁻ and NO₃⁻ were decreased by the intraperitoneal injection of N ^G-nitro- l -arginine methyl ester, an inhibitor of nitric oxide synthase, whereas N ^G-nitro- d -arginine methyl ester had no effect. l -Arginine by itself increased NO₂⁻ and NO₃⁻ levels and diminished the reduction of their levels caused by N ^G-nitro- l -arginine methyl ester. Direct infusion of l -glutamate, N -methyl- d -aspartate, or KCl into the cerebellum through a dialysis probe resulted in an increase in NO₂⁻ and/or NO₃⁻ levels. The effects of N -methyl- d -aspartate and KCl were dependent on extracellular calcium. Furthermore, the stimulatory effects of l -glutamate and N -methyl- d -aspartate were inhibited by N ^G-nitro- l -arginine methyl ester and (±)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP), an N -methyl- d -aspartate receptor antagonist. These results suggest that NO₂⁻ and NO₃⁻ levels may be related to nitric oxide production in vivo.     

The Phosphorylation of Proteins in Hippocampal Slices: Effects of Noradrenaline and of Pretreatment with Kainic Acid

Abstract: Hippocampal slices were incubated in the presence of [³²P]P_i, and protein phosphorylation was examined by means of sodium dodecyl sulfate-gel electrophoresis. Incubation for at least 30 min with 300 μCi of [³²P)P_i/brain slice gave rise to the phosphorylation of 8–10 protein bands. Most of these bands showed enhanced phosphorylation in response to noradrenaline. The basal phosphorylation of kainic acid-pretreated hippocampal slices was enhanced two- to threefold compared with controls. There was also an additional increase in kainic acid-pretreated slices in the response to noradrenaline. 8-Br-Cyclic AMP and phosphodiesterase inhibitors, such as papaverine or isobutylmethyl-xanthine, had no effect on the phosphorylation patterns.     

Homocysteate-Evoked Release of Acetylcholine from the Rabbit Retina

Abstract: The cholinergic amacrine cells of the rabbit retina can be labeled with [³H]choline and the activity of the cholinergic population monitored by following the release of [³H]acetylcholine. It has been proposed that l -homocysteate may be the main endogenous transmitter released onto cholinergic amacrine cells by bipolar cells. Therefore, we have examined the effects of the isomers of homocysteate on the release of [³H]acetylcholine. In magnesium-free medium, d -homocysteate was slightly more potent than the l -isomer. The addition of magnesium, which blocks responses mediated by NMDA receptors, preferentially reduced but did not eliminate, the response to l -homocysteate. 2-Amino-7-phosphonoheptanoate, a potent NMDA antagonist, preferentially blocked l -homocysteate evoked responses. 6,7-Dinitroquinoxaline-2,3-dione, a potent kainate antagonist, preferentially blocked d -homocysteate-evoked responses. Therefore, in the rabbit retina, l -homocysteate is an NMDA-preferring agonist, whereas d -homocysteate is a kainate-preferring agonist. In addition, we found that l -homocysteate can activate the physiologically activated kainate receptor but only when used in millimolar concentrations and under conditions that minimize NMDA-receptor activation. However, the low potency of l -homocysteate combined with low affinity for the glutamate transporter, lack of immunocytochemical localization in bipolar cells, and low retinal content place serious limitations on the role of l -homocysteate at the bipolar-to-cholinergic amacrine cell synapse.     

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.     

Lupin peroxidases. II. Binding of acidic isoperoxidases to cell walls

Extracellular acidic isoperoxidases (EC 1.11.1.7), isolated from both the cell walls and intercellular spaces of lupin ( Lupinus albus L. cv. multolupa) hypocotyls, bound to water-insoluble pectins of wall fragments also isolated from the hypocotyls. The binding was sáturable by increasing the isoenzyme concentration in the assay medium and it was dependent on the pH; neutral pH (6.0–7.0) favoured release, while acidic pH (4.0–5.0) favoured the attachment to the cell wall. Binding of acidic isoperoxidases to wall fractions was correlated with the in vitro acid-induced growth of hypocotyl segments, and both were modulated in the same direction by the Ca²⁺/H⁺ ratio in the incubation media, although the two responses were clearly separated when the Ca²⁺/H⁺ ratio varied. Binding of acidic isoperoxidases of cell walls could operate as a fine control of the activity of these cell wall enzymes, although its physiological role in the cell wall stiffening remains unclear. Some aspects of Ca²⁺ on the control of peroxidase activity at this level are also discussed.     

Enumeration of some microbial groups in thermophilic poultry waste digesters and enrichment of a feather-degrading culture

Methane-producing, cellulolytic, feather-degrading, and total anaerobic microbial populations were enumerated in four laboratory-scale (l l) thermophilic (50°C) poultry waste digesters over a 40d period. Four different operation conditions were: 5 d retention time (RT), 6% volatile solids (VS); 5 d RT, 3% VS; 10 d RT, 6% VS; and 10 d RT, 3% VS. Laying hen manure was the sole source of substrate and micro-organisms. At theoretical steady state (day 40) the biogas volumetric rate was near 3.0 l/l digester volume (l/l/d) in all but the 10 d RT, 3% VS digester which was 2 l/l/d. The total viable anaerobic population was > 10⁶ cfu/ml digester fluid at the first sampling and stabilized at 10⁷–10⁸ cfu/ml between days 20 and 40 in all digesters. Methane-producing bacteria increased from ≤ 10/ml early in the sampling period to 10⁵/ml at steady state in all but the 5 d RT, 3% VS digester which was highest at 10⁷/ml. Cellulolytic micro-organisms were low throughout the 40 d, generally less than 10/ml. Feather-degrading micro-organisms ranged from near 10²–10⁵ at steady state and were decreasing in number near day 40 in all but the 10 d RT, 6% VS digester which maintained 10⁵/ml after day 20. A feather-degrading culture was enriched from this digester and subsequently adapted to grow in a medium with feather as the sole source of carbon. Results of this study provide information regarding potential biological upgrading of poultry waste digesters for increased operational efficiency and potential industrial application of a feather-hydrolytic micro-organism.     

Effects of Non-NMDA Receptor Modulators on [3H]Dopamine Release from Rat Mesencephalic Cells in Primary Culture

Abstract: The effects of AMPA and kainate on [³H]dopamine release from fetal (embryonic day 15) rat mesencephalic neurons in primary culture were enhanced markedly in a dose-dependent fashion by cyclothiazide, a recently described inhibitor of AMPA receptor desensitization. The EC₅₀ value for cyclothiazide was 2.2 ± 0.8 µ M . The release of [³H]dopamine induced by both AMPA (or kainic acid) and the combination of AMPA (or kainic acid) with cyclothiazide was antagonized by specific antagonists like 6-cyano-7-nitroquinoxaline-2,3-dione or the noncompetitive benzodiazepine GYKI 52466. Unlike cyclothiazide, the lectin concanavalin A did not stimulate [³H]dopamine release. These results established the involvement of AMPA-preferring receptors on [³H]dopamine release from rat mesencephalic neurons in primary culture and provided further evidence for the existence of regulatory allosteric sites on AMPA receptor subunits.     

Neuronal Localization of Ca2+-dependent Protein Phosphorylation in Brain

Abstract: The cellular localization of two Ca²⁺-dependent protein phosphorylation systems was investigated using the kainic acid lesioning technique for the selective destruction of neurons. In one of these systems, a crude synaptosomal (P₂) fraction was preincubated with ³²P_j for 30 min; the phosphorylation of several proteins was increased during a short subsequent incubation with veratridine plus Ca²⁺. In the second system, crude synaptosomal membranes isolated from the P₂ fraction were incubated with [γ-³²P]ATP; in this system, the phosphorylation of several proteins was increased in the presence of a "calcium-dependent regulator" plus Ca²⁺. Kainic acid lesioning greatly reduced the amount of Ca-⁺-dependent protein phosphorylation in both systems. The results indicate a predominantly neuronal localization for both Ca²⁺-dependent protein phosphorylation systems.     

Effects of Kainic Acid in Rat Brain Synaptosomes: The Involvement of Calcium

Abstract: The effects of kainic acid were investigated in preparations of rat brain synaptosomes. It was found that kainic acid inhibited competitively the uptake of d -[³H]aspartate, with a K _i of approximately 0.3 m m . Kainic acid also caused release of two excitatory amino acid neurotranstnitters, aspartate and glutamate, in a time- and concentration-dependent manner, but had no effect on the content of γ-aminobutyric acid. Concomitant with the release of aspartate and glutamate, depolarization of the synaptosomal membrane and an increase in intracellular calcium were observed, with no measurable change in the concentration of internal sodium ions. The increase in intrasynaptosomal calcium and decrease in transmem-brane electrical potential were prevented by the addition of glutamate, whereas the kainate-induced release of ra-dioactive aspartate was substantially inhibited by lowering the concentration of calcium in the external medium. It is postulated that kainic acid reacts with a class of glutamate receptors located in a subpopulation of synaptosomes, presumably derived from the glutamatergic and aspartatergic neuronal pathways, which possesses high-affinity uptake system(s) for glutamate and/or aspartate. Activation of these receptors causes opening of calcium channels, influx of calcium into the synaptosomes, and depolarization of the synaptosomal plasma membrane with consequent release of amino acid neurotransmitters.     

Nodulation and nitrogenase activity in nitrogen-fixing woody plants stimulated by CO2 enrichment of the atmosphere

The responses of three species of nitrogen-fixing trees to CO₂ enrichment of the atmosphere were investigated under nutrient-poor conditions. Seedlings of the legume, Robinia pseudoacacia L. and the actinorhizal species, Alnus glutinosa (L.) Gaertn. and Elaeagnus angustifolia L. were grown in an infertile forest soil in controlled-environment chambers with atmospheric CO₂ concentrations of 350 μl ⁻¹ (ambient) or 700 μl ⁻¹. In R. pseudoacacia and A. glutinosa , total nitrogenase (N₂ reduction) activity per plant, assayed by the acetylene reduction method, was significantly higher in elevated CO₂, because the plants were larger and had more nodule mass than did plants in ambient CO₂. The specific nitrogenase activity of the nodules, however, was not consistently or significantly affected by CO₂ enrichment. Substantial increases in plant growth occurred with CO₂ enrichment despite probable nitrogen and phosphorus deficiencies. These results support the premises that nutrient limitations will not preclude growth responses of woody plants to elevated CO₂ and that stimulation of symbiotic activity by CO₂ enrichment of the atmosphere could increase nutrient availability in infertile habitats.     

A Novel Photoaffinity Ligand for Kainate Sites Labels Two Polypeptides with Molecular Mass 45 and 33.5 kDa in Chick Cerebellum

Abstract: The synthesis of (2 S ,3 S ,4 S )-4-[1-(4-azidobenzamidomethyl)ethenyl]-2-carboxy-3-pyrrolidineacetic acid (ABCPA) is described. This novel kainic acid analogue, bearing a photolabile functionality on the isopropenyl side chain, was proven to be a good inhibitor of [³H]CNQX and [³H]kainic acid binding on chick cerebellar membranes. [³H]ABCPA was photoaffinity cross-linked on the membrane fraction of chick cerebellum. Electrophoretic analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two major radioactive bands with apparent molecular masses of 45 and 33.5 kDa. [³H]ABCPA incorporation in both bands was completely blocked by 2 m M CNQX. When photoaffinity labeling was performed in the presence of 2 m M kainic acid, incorporation of [³H]ABCPA was blocked by ∼70% in the 45-kDa band and by 18% in the 33.5-kDa band. Incorporation of radioactivity in both bands was blocked by ∼30% with 10 m M glutamate.     

l-Aspartate as an amino acid neurotransmitter: mechanisms of the depolarization-induced release from cerebrocortical synaptosomes

The role of l -aspartate as a classical neurotransmitter of the CNS has been a matter of great debate. In this study, we have characterized the main mechanisms of its depolarization-induced release from rat purified cerebrocortical synaptosomes in superfusion and compared them with those of the well-known excitatory neurotransmitter l -glutamate. High KCl and 4-aminopyridine were used as depolarizing agents. At 15 mM KCl, the overflows of both transmitters were almost completely dependent on external Ca²⁺. At 35 and 50 mM KCl, the overflows of l -aspartate, but not those of l -glutamate, became sensitive to dl -threo-β-benzyloxyaspartic acid ( dl -TBOA), an excitatory amino acid transporter inhibitor. In the presence of dl -TBOA, the 50 mM KCl-evoked release of l -aspartate was still largely external Ca²⁺-dependent. The dl -TBOA insensitive, external Ca²⁺-independent component of the 50 mM KCl-evoked overflows of l -aspartate and l -glutamate was significantly decreased by the mitochondrial Na⁺/Ca²⁺ exchanger blocker CGP 37157. The Ca²⁺-dependent, KCl-evoked overflows of l -aspartate and l -glutamate were diminished by botulinum neurotoxin C, although to a significantly different extent. The 4-aminopyridine-induced l -aspartate and l -glutamate release was completely external Ca²⁺-dependent and never affected by dl -TBOA. Superimposable results have been obtained by pre-labeling synaptosomes with [³H] d -aspartate and [³H] l -glutamate. Therefore, our data showing that l -aspartate is released from nerve terminals by calcium-dependent, exocytotic mechanisms support the neurotransmitter role of this amino acid.     

Purification and properties of a novel polyol dehydrogenase of bacterial origin

Abstract A bacterium, as yet unidentified, has been isolated from floor dust by direct selection on minimal agar using l -glucitol ( d -gulitol) as the sole carbon energy source. The bacterium possesses a constitutive enzyme which catalyzes the reaction: l -glucitol + NAD⁺→ d -sorbose + NADH + H⁺. A new species of enzyme has been induced by l -arabinitol or ribitol, but not l - or d -glucitol, and the induction is only partially counteracted by the glucose-repression effect. The constitutive enzyme was purified by fractionation on Sephadex G-200 gel and chromatography on DEAE Biogel A. The enzyme required NAD⁺, but not NADP⁺, as a cofactor. It oxidizes also ribitol, xylitol and l -arabinitol, but not d -arabinitol, lactitol or a variety of other commercially available alditols. The enzyme is not inhibited by 10 mM sodium azide but is totally inhibited by 0.1 mM potassium ferricyanide.     

Streptococcus pyogenes grown under sublethal concentrations of penicillin G accumulates close to the septum pentapeptide subunits of peptidoglycan

Abstract The accumulation of non-crosslinked pentapeptide subunits of peptidoglycan was localized close to the septum in cells of Streptococcus pyogenes , grown under sublethal concentrations of penicillin G. Pentapeptide subunits were detected in the cell wall by the indirect immunoferritin technique, employing monospecific antibodies to the sequence R- d -Ala- d -Ala-OH. Antibodies of this specificity were obtained by immunization with synthetic immunogen albumin-(CH₂CO-Gly- l -Ala₂- d -Ala- d -Ala-OH)₃₉.     

Characterization of l-Arginine and Aminoguanidine Uptake into Isolated Rat Choroid Plexus: Differences in Uptake Mechanisms and Inhibition by Nitric Oxide Synthase Inhibitors

Abstract: Recent reports suggest that nitric oxide (NO) may contribute to several neurodegenerative diseases, e.g., focal cerebral ischemia, N -methyl- d -aspartate-mediated neurotoxicity, and experimental autoimmune encephalomyelitis. Accordingly, an understanding of the CNS transport processes of NO synthase (NOS) inhibitors has important therapeutic implications. The objective of the present study was to characterize the in vitro transport processes governing the uptake of l -[¹⁴C]arginine and the NOS inhibitor [¹⁴C]aminoguanidine in rat choroid plexus tissue. Consistent with previous reports, the uptake of l -[¹⁴C]arginine was mediated by both saturable and nonsaturable processes and was inhibited by the NOS inhibitors N ^G-methyl- l -arginine, N ^G-amino- l -arginine, and N ⁵-imidoethyl- l -ornithine. l -[¹⁴C]Arginine uptake was not inhibited by aminoguanidine or N ^G-nitro- l -arginine. Because aminoguanidine is an organic cation that bears some structural similarity to l -arginine, aminoguanidine might be transported by either an organic cation transporter or by the basic amino acid transporter governing arginine uptake. However, there was no evidence of a saturable uptake process for [¹⁴C]aminoguanidine in isolated rat choroid plexus, in contrast to that observed for l -[¹⁴C]arginine.     

Stimulatory Effects of Protein Kinase C and Calmodulin Kinase II on N-Methyl-d-Aspartate Receptor/Channels in the Postsynaptic Density of Rat Brain

Abstract: To clarify the regulatory mechanism of the N -methyl- d -aspartate (NMDA) receptor/channel by several protein kinases, we examined the effects of purified type II of protein kinase C (PKC-II), endogenous Ca²⁺/calmodulin-dependent protein kinase II (CaMK-II), and purified cyclic AMP-dependent protein kinase on NMDA receptor/ channel activity in the postsynaptic density (PSD) of rat brain. Purified PKC-II and endogenous CaMK-II catalyzed the phosphorylation of 80–200-kDa proteins in the PSD and l -glutamate-(or NMDA)-induced increase of (+)-5-[³H]methyl-10, 11-dihydro-5 H -dibenzo[a, d]cyclohepten-5, 10-imine maleate ([³H]MK-801; open channel blocker for NMDA receptor/channel) binding activity was significantly enhanced. However, the pretreatment of PKC-II-and CaMK-II-catalyzed phosphorylation did not change the binding activity of l -[³H]glutamate, cis -4-[³H](phospho-nomethyl)piperidine-2-carboxylate ([³H]CGS-19755; competitive NMDA receptor antagonist), [³H]glycine, α-[³H]-amino-3-hydroxy-5-methyl-isoxazole-4-propionate, or [³H]-kainate in the PSD. Pretreatment with PKC-II-and CaMK-II-catalyzed phosphorylation enhanced l -glutamate-induced increase of [³H]MK-801 binding additionally, although purified cyclic AMP-dependent protein kinase did not change l -glutamate-induced [³H]MK-801 binding. From these results, it is suggested that PKC-II and/or CaMK-II appears to induce the phosphorylation of the channel domain of the NMDA receptor/channel in the PSD and then cause an enhancement of Ca²⁺ influx through the channel.