Pharmacological characterization of metabotropic glutamate receptors in cultured cerebellar granule cells

A detailed pharmacological characterization of metabotropic glutamate receptors (mGluR) was performed in primary cultures of cerebellar granule cells at 6 days in vitro (DIV). The rank order of agonists induced polyphosphoinositide (PPI) hydrolysis (after correcting for the ionotropic component in the response) was as follows: in terms of efficiency, Glu>quisqualate (quis)=ibotenate (ibo)>(1_S,3_R)-1-amino-cyclopentane-1,3-dicarboxylic acid (ACPD)>-methyl-amino-l-alanine (BMAA) and in terms of potency, quis>ACPD>Glu>ibo=BMAA. Ionotropic excitatory amino acid (EAA) receptor agonists, such as -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) were relatively inactive (in the presence of Mg²⁺). Quis and ACPD-induced PPI hydrolysis was unaffected by ionotropic Glu receptor antagonists, but was inhibited, in part by L-2-amino-3-phosphonopropionate (AP3). In contrast, Glu-or ibo- induced PPI hydrolysis was reduced, in part, by both AP3 and NMDA receptor antagonists. Characteristic interactions involving different transmitter receptors were noted. PPI hydrolysis evoked by quis and 1_S,3_R-ACPD was not additive. In contrast, PPI hydrolysis stimulated by quis/ACPD and carbamylcholine was additive (indicating different receptors/transduction pathways). In the presence of Mg²⁺, the metabotropic response to quis/AMPA and NMDA was synergistic (this being consistent with AMPA receptor-induced depolarization activating NMDA receptor). On the other hand, in Mg²⁺-free buffer the effects of quis and NMDA, at concentrations causing maximal PPI hydrolysis, were additive (indicating that PPI hydrolysis was effected by two different mechanisms). Thus, in cerebellar granule cells EAAs elicit PPI hydrolysis by acting at two distinct receptor types: (i) metabotropic Glu receptors (mGluR), with pharmacological characteristics suggesting the expression of a unique mGluR receptor that shows certain similarities to those observed for the mGluR1 subtype (Aramori and Nakanishi, 1992) and (ii) NMDA receptors. The physiological agonist, Glu, is able to stimulate both receptor classes.Abbreviations ACPD (1_S,3_R)-1-amino-cyclopentane-1,3-dicarboxylic acid - AMPA -amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid - AP₃ L-2-amino-3-phosphono-propionate - AP₅ D-2-amino-5-phosphonopentenoate - BMAA -methyl-amino-L-alanine - DIV days in vitro - DNOX 6,7-dinitroouinoxoline-2,3-dione - EAA excitatory amino acids - Glu glutamate - InsP inositol monophosphate - mGluR metabotropic glutamate receptors - MK-801 (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohept-5,10-imine hydrogen maleate - NMDA N-methyl-D-aspartate - PPI polyphosphoinositide - quis quisqualate     

Non-NMDA excitatory amino acid receptors in a subcellular fraction enriched in cerebellar glomeruli

In the internal granular layer of the cerebellar cortex the polysynaptic complexes called glomeruli consist mainly of homogeneous populations of glutamatergic and GABAergic synapses, both located on granule cell dendrites. A subcellular fraction enriched in glomeruli was prepared from rat cerebellum, and the distribution of the different types of NMDA and non-NMDA glutamate binding sites was studied in the membranes derived from this fraction (fraction G) as compared to that in the membranes prepared from a total cerebellar homogenate (fraction T). Cl^–/Ca²⁺ independent [³H]glutamate binding sites were not abundant and could be reliably measured only in fraction G. Cl^– dependent/Ca²⁺ activated [³H]glutamate binding sites were more abundant and exhibited a single K _d in both fractions G and T. Quisqualate, NMDA, kainate, L-AP4 andtrans-ACPD inhibited [³H]glutamate binding to different extents in the two membrane fractions. Quisqualate sensitive sites were predominant in all cases but more abundant in fraction T than in fraction G. An opposite distribution was observed for the NMDA sensitive binding sites while kainate sensitive binding sites were scarce everywhere.Trans-ACPD, a ligand presumed selective for metabotropic glutamate binding sites, displaced [³H]glutamate from fraction T but nor from fraction G, suggesting the absence of these sites from glomeruli. Similarly, no L-AP4 sensitive sites were present in fraction G while they were abundant in fraction T. Binding sites associated with ionotropic receptors of the quisqualate type were determined by measuring [³H]AMPA binding. The density of the high affinity [³H]AMPA binding sites in fraction T was twice as high as in fraction G, indicating that these sites are abundant in structures other than glomeruli. High-affinity [³H]kainate binding sites are more abundant in fraction G than in fraction T; the same, but with smaller differences, occurs for the distribution of the low affinity [³H]kainate binding sites. The density of the latter sites is close to that of the high affinity [³H]AMPA binding sites confirming the presence of quisqualate/kainate receptors on granule cells, as previously hypothesized (for review, see Gallo et al., 1990). Taken together, these results indicate a segregation of the glutamate binding sites types at specialized synapses or neuronal cell types in the cerebellar network.Abbreviations AMPA (RS)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid - DL-AP4 dl-2-amino-4-phosphonobutyric acid - D-AP5 d-2-amino-5-phosphonovaleric acid - EAA excitatory amino acid - EGTA ethylene glycol-bis(-aminoethyle ether) N,N,N,N-tetracetic acid - NMDA N-methyl-D-aspartate - Quisqualate -[3,5-dioxo-1,2,4-oxadiazolidin-2-yl]-L-alanine - trans-ACPD trans-1-amino-cyclopentyl-1,3-dicarboxylic acid     

Characterization of quisqualate recognition sites in rat brain tissue usingDl-[3H]α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and a filtration assay

The binding of [³H]AMPA (Dl--amino-3-hydroxy-5-methylisoxazole-4-propionic acid), a ligand for the putative quisqualate excitatory amino acid receptor subtype, was evaluated using centrifugation and filtration receptor binding techniques in rat brain crude synaptosomal membrane preparations. Maximal specific binding of [³H]AMPA occurred in Triton X-100 treated membranes in the presence of the chaotropic agent potassium thiocyanate (KSCN). The effects of KSCN on binding were reversible and optimal at 100 mM. Supernatant obtained from detergent-treated membranes inhibited specific [³H]AMPA and [³H]kainic acid binding, suggesting the presence of an inhibitory agent which was tentatively identified as glutamate. Using centrifugation, saturation analysis revealed two distinct binding sites in both the absence and presence of KSCN. The chaotrope was most effective in increasing binding at the low affinity binding site, enhancing the affinity (K _d) without a concommitant change in the total number of binding sites. Using filtration, a single binding site was detected in Triton-treated membranes. Like the data obtained by centrifugation, KSCN enhanced the affinity of the receptor (K _d value=10 nM) without altering the number of binding sites (B _max=1.2 pmol/mg protein). The rank order of potency of various glutamate analogs in the [³H]AMPA binding assay was quisqualate > AMPA > l-glutamate > kainate > d-glutamate, consistent with the labeling of a quisqualate-type excitatory amino acid receptor subtype.l-glutamic acid diethylester, and 2-amino-7-phosphonoheptanoic acid (AP₇) were inactive. The present technique provides a rapid, reliable assay for the evaluation of quisqualate-type excitatory amino acid agonists and/or antagonists that may be used to discover more potent and selective agents.     

Effects of dorsal root section and occlusion of dorsal spinal artery on the neurotransmitter candidates in rat spinal cord

In order to obtain further evidence of putative neurotransmitters in primary sensory neurons and interneurons in the dorsal spinal cord, we have studied the effects of unilateral section of dorsal roots and unilateral occlusion of the dorsal spinal artery on cholinergic enzyme activity and on selected amino acid levels in the spinal cord. One week after sectioning dorsal roots from caudal cervical (C₇) to cranial thoracic (T₂) levels, the specific activity of choline acetyltransferase (ChAT) was significantly decreased and acetylcholinesterase (AChE) showed a tendency to decrease in the dorsal quadrant on the operated side of the spinal cord. Dorsal root sectioning had little effect on the levels of free glutamic acid or other amino acids in the dorsal spinal cord. These results suggest that primary sensory neurons may include some cholinergic axons, and that levels of putative amino acid transmitters are not regulated by materials supplied by axonal transport from the dorsal root ganglia. By contrast, one week following unilateral occlusion of the dorsal spinal artery, the activities of ChAT and AChE were unchanged in the operated quadrant of the spinal cord, while decreases of Asp, Glu, and GABA, and an increase in Tau were detected. These findings are consistent with the proposals that such amino acids, but not ACh, may function as neurotransmitter candidates in interneurons of the dorsal spinal cord.Abbreviation used ACh acetylcholine - AChE acetylcholinesterase - Asp aspartic acid - ChAT choline acetyltransferase - GABA -aminobutyric acid - Glu glutamic acid - Gly glycine - SP substance P - Tau taurine     

Support for Radiolabeling of a Glycine Recognition Domain on the N-Methyl-d-Aspartate Receptor Ionophore Complex by 5,7-[3H]Dichlorokynurenate in Rat Brain

Abstract: Pretreatment with Triton X-100 more than doubled the binding of radiolabeled 5,7-dichlorokynurenic acid (DCKA), a proposed antagonist at a glycine (Gly) recognition domain on the N-methyl-d -aspartate (NMDA) receptor ionophore complex, in rat brain synaptic membranes. The binding exhibited an inverse temperature dependency, reversibility, and saturability, the binding sites consisting of a single component with a high affinity (27.5 nM) and a relatively low density (2.87 pmol/mg of protein). The binding of both [³H]DCKA and [³H]Gly was similarly displaced by numerous putative agonists and antagonists at the Gly domain in a concentration-dependent manner at a concentration range of 100 nM to 0.1 mM. Among the 24 putative ligands tested, DCKA was the second most potent displacer of the binding of both radioligands with no intrinsic affinity for the binding of [³H]kainic acid and α-amino-3-hydroxy-5-[³H]methylisoxazole-4-propionic acid (AMPA) to the non-NMDA receptors. In contrast, the other proposed potent Gly antagonist, 5,7-dinitroquinoxaline-2,3-dione, was active in displacing the binding of [³H]glutamic ([³H]Glu) and D,L-(E)-2-amino-4-[³H]propyl-5-phosphono-3-pentenoic acids to the NMDA recognition domain with a relatively high affinity for the non-NMDA receptors. In addition, the proposed antagonist at the AMPA-sensitive receptor, 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline, not only displaced weakly the binding of both [³H]- Gly and [³H]DCKA, but also inhibited the binding of (+)-5-[³H]methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine ([³H]MK-801) to an ion channel associated with the NMDA-sensitive receptor in the presence of added Glu alone in a manner sensitive to antagonism by further added Gly. Clear correlations were seen between potencies of the displacers to displace [³H]DCKA binding and [³H]Gly binding, in addition to between the potencies to displace [³H]-DCKA or [³H]Gly binding and to potentiate or inhibit [³H]MK-801 binding. All quinoxalines tested were invariably more potent displacers of [³H]DCKA binding than [³H]Gly binding, whereas kynurenines were similarly effective in displacing the binding of both [³H]Gly and [³H]-DCKA. These results undoubtedly give support to the proposal that [³H]DCKA is one useful radioligand available in terms of its high selectivity and affinity for the Gly domain in the brain. Possible multiplicity of the Gly domain is suggested by the differential pharmacological profiles between the binding of [³H]Gly and [³H]DCKA.     

Brain development:1H magnetic resonance spectroscopy of rat brain extracts compared with chromatographic methods

We compared in vitro¹H magnetic resonance spectroscopy (MRS) measurements of rat brain extracts (rats: 2–56 days old) with chromatographic measurements and in a further step also with results of in vivo MRS. The following substances can be reliably measured in brain extracts by in vitro MRS: N-acetylaspartate (NAA), total creatine (Cr), phosphorylethanoloamine (PE), taurine (Tau), glutamate (Glu), glutamine (Gln), -aminobutyrate (GABA) and alanine (Ala). Two different methods of MRS data evaluation compared with chromatographic data on Cr and NAA are shown. During development of the rat from day 2–56 brain concentrations of PE, Tau and Ala decrease, those of NAA, Cr, Glu and Gln increase, while GABA does not change. The developmental patterns of these substances are the same, whether measured by in vitro MRS or by chromatographic methods. Quantification of NAA, Cr, Tau, GABA and PE leads to the same results with both methods, while Glu, Gln and Ala concentrations determined by in vitro MRS are apparently lower than those measured chemically. The NAA/Cr ratios of 7 to 35-day-old rats were determined by in vivo¹H MRS. These results correlate with chromatographic and in vitro data. Using appropriate methods in the in vivo and in vitro MR-technique, the obtained data compare well with the chromatographic results.     

Neurochemical,pharmacological, and developmental studies on cerebellar receptors for dicarboxylic amino acids

Specific binding ofl-[³H]glutamate ([³H]Glu) andl-[³H]asparate ([³H]Asp) to cerebellar membranes represented a time-, temperature- pH- and protein-dependent interaction which was both saturable and reversible. Binding sites for both radioligands appeared maximally enriched in synaptosomal fractions isolated by gradient centrifugation. Kinetically derived dissociation constant (K _off/K _on=K _d) for [³H]Glu binding to this fraction indicated high-affinity (443 nM). Competition experiments employing analogs of excitatory amino acids, including new antagonists, helped identify binding sites for [³H]Glu and [³H]Asp as receptors with differential pharmacological, specificities. Membrane freezing reduced numbers of both receptor types, but binding activity could be recovered partially by incubation at 37°C. Glu receptors exhibited a pronounced deleterious sensitivity to thiol modifying reagents andl-Glu (50–1000 M) provided protection, against these compounds during co-incubation with cerebellar membranes. It is suggested that cold storage may induce partially reversible receptor inactivation by promoting sulfhydryl group/bond modification. Rat cerebellar glutamatergic function (endogenous Glu content, Glu uptake and receptor sites) exhibited an apparent ontogenetic peak between days 8–12 postpartum with a plateauing profile from day 30 to adulthood. The accelerated development (days 8–12) coincides with the first demonstrable Glu release and kainic acid neurotoxicity, as described previously.     

Ethanol modulates calmodulin-dependent Ca2+-activated ATPase in synaptic plasma membranes

The effects of ethanol in vitro on calmodulin-dependent Ca²⁺-activated ATPase (CaM–Ca²⁺-ATPase) activity were studied in synaptic plasma membranes (SPM) prepared from the brain of normal and chronically ethanol-treated rats. In SPM from normal animals, ethanol at 50–200 mM inhibited the Ca²⁺-ATPase activity. Lineweaver-Burk analysis indicates that the inhibition was the result of a decreased affinity of the enzyme for calmodulin, whereas the maximum activity of the enzyme was not changed. Arrhenius analysis indicates that the enzyme activity was influenced by lipid transition of the membranes, and ethanol in vitro resulted in a shift of the transition temperature toward a lower value. From animals receiving chronic ethanol treatment (3 weeks), the SPM were resistant to the inhibitory effect of ethanol on the enzyme activity. The resistance to ethanol inhibition was correlated with a higher enzyme affinity for calmodulin and a higher transition temperature, as compared with normal SPM. Since the calmodulin-dependent Ca²⁺-ATPase in synaptic plasma membranes is believed to be the Ca²⁺ pump controlling free Ca²⁺ levels in synaptic terminals, its inhibition by ethanol could therefore lead to altered synaptic activity.Abbreviations used ATPase adenosine triphosphatase - CaM calmodulin - CaM–Ca²⁺-ATPase calmodulin-dependent Ca²⁺-activated ATPase - EGTA ethylene-bis(oxyethylenenitrilo)tetraacetic acid - EtOH ethanol - Hepes N—2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - SPM synaptic plasma membranes - TFP trifluoperazine - Tris tris(hydroxymethyl)aminomethane - K_m Michaelis constant - T_d transition temperature - V_max maximum velocity     

[3H]Thienylcyclohexylpiperidine Binding Activity in Brain Synaptic Membranes Treated with Triton X-100

Binding activity of [3H]thienylcyclohexylpiperidine was examined using rat brain synaptic membranes treated with Triton X-100. This compound is proposed to be a noncompetitive antagonist for the N-methyl-D-aspartate (NMDA)-sensitive subclass of brain excitatory amino acid receptors. The activity decreased in proportion to increasing concentrations of the detergent up to 0.08%. In vitro addition of L-glutamate (Glu) partially restored the decreased activity caused by this Triton treatment, whereas further addition of glycine (Gly) entirely reversed the loss of activity to the level found in membranes extensively washed but not treated with a detergent. These stimulatory effects were found to be due to the acceleration of the association of ligand. The rank order of potentiation of the activity coincided well with that of the affinity for the NMDA-sensitive subclass among numerous Glu analogs. The potentiation by Gly as well as Glu was invariably prevented by competitive NMDA antagonists, such as DL-2-amino-5-phosphonovalerate and (+/-)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonate, but not by strychnine. No significant difference was observed between pharmacological profiles of the activities in synaptic membranes treated and not treated with Triton X-100, except haloperidol. The potency of this sigma-ligand to inhibit the activity was greatly reduced by the Triton treatment in the presence of both Glu and Gly. These results suggest that the regulatory properties of Triton-treated synaptic membranes remain unchanged in terms of the interaction within the NMDA receptor complex.     

Effects of Prostaglandin E2 and Capsaicin on Behavior and Cerebrospinal Fluid Amino Acid Concentrations of Unanesthetized Rats: A Microdialysis Study

Abstract: Prostaglandin E₂ (PGE₂) delivered to the spinal cord produces an increased sensitivity to noxious (hyperalgesia) and innocuous (allodynia) stimuli. The mechanisms that underlie this effect remain unknown, but a PGE₂-evoked enhancement of spinal neurotransmitter release may be involved. To address this hypothesis, we examined the effect of PGE₂ on CSF concentrations of amino acids and also the modulatory effect of PGE₂ on capsaicin-evoked changes of spinal amino acid concentrations using a microdialysis probe placed in the lumbar subarachnoid space. Amino acids were quantified using HPLC with fluorescence detection. Addition of 1 mM, but not 10 or 100 µM, PGE₂ to the perfusate for a 10-min period (flow rate, 5 µl/min) evoked an immediate increase (80–100%) in glutamate (Glu), aspartate (Asp), taurine (Tau), glycine (Gly), and γ-aminobutyric acid (GABA) concentrations. Similarly, capsaicin infusion (0.1–10 µM) induced a dose-dependent increase in Glu, Asp, Tau, Gly, GABA, and ethanolamine levels. Significant increases in amino acid levels evoked by PGE₂ or capsaicin were associated with a touch-evoked allodynia. The combination of PGE₂ (10 µM) and capsaicin (0.1 or 1.0 µM) at concentrations that individually had no effect together evoked a significant increase (60–100%) in Glu, Asp, Tau, Gly, and GABA concentrations and produced tactile allodynia. These data demonstrate that spinally delivered PGE₂ or capsaicin substantially elevates CSF concentrations of both excitatory and inhibitory amino acids. The capacity of PGE₂ to enhance and prolong capsaicin-evoked amino acid concentrations may be one of the mechanisms by which spinal PGE₂ produces hyperalgesia and allodynia.     

Levels of glutamate,aspartate, GABA,and taurine in different regions of the cerebellum after X-irradiation-induced neuronal loss

The levels of glutamate (Glu), aspartate (Asp), -amino-n-butyric acid (GABA), and taurine (Tau) were determined in the cortex, molecular layer, and deep nuclei of cerebella of adult rats exposed to X-irradiation at 12–15 days following birth (to prevent the acquisition of late-forming granule cells; 12–15x group) and 8–15 days following birth (to prevent the acquisition of granule and stellate cells; 8–15x group). Also, the levels of the four amino acids were measured in the crude synaptosomal fraction (P₂) isolated from the whole cerebella of the control, 12–15x, and 8–15x groups. The level of Glu was significantly decreased by (1) 6–20% in the cerebellar cortex; (2) 15–20% in the molecular layer; and (3) 25–50% in the P₂ fraction of the X-irradiated groups relative to control values. The content of Glu in the deep nuclei was not changed by X-irradiation treatment. Regional levels of Asp were unchanged by X-irradiation, while its level in P₂ decreased by 15–30% after treatment. The levels of GABA and Tau in the molecular layer, deep nuclei, or P₂ were not changed in the experimental groups. However, there was a 15% increase in the levels of GABA and Tau in the cerebellar cortex of the 8–15x group relative to control values. The data support the proposed role of glutamate as the excitatory transmitter released from the cerebellar granule cells but are inconclusive regarding a transmitter role for either Tau or GABA from cerebellar stellate cells.     

Enhanced taurine release in cultured cerebellar granule cells in cell-damaging conditions

Summary The release of taurine from cultured cerebellar granule neurons was studied in different cell-damaging conditions, including hypoxia, hypoglycemia, ischemia, oxidative stress and in the presence of free radicals. The effects of both ionotropic and metabotropic glutamate receptor agonists on the release were likewise investigated. The release of [³H]taurine from the glutamatergic granule cells was increased by K⁺ (50mM) and veratridine (0.1 mM), the effect of veratridine being the greater. Hypoxia and ischemia produced an initial increase in release compared to normoxia but resulted in a diminished response to K. Hypoglycemia, oxidative stress and free radicals enhanced taurine release, and subsequent K^– treatment exhibited a correspondingly greater stimulation. A common feature of taurine release in all the bove conditions was a slow response to the stimulus evoked by K⁺ and particularly to that evoked by veratridine. All ionotropic glutamate receptor agonists potentiated taurine release, but only the action of kainate seemed to be receptor-mediated. Metabotropic receptor agonists of group I slightly stimulated the release. The prolonged taurine release seen in both normoxia and cell-damaging conditions may be of importance in maintaining homeostasis in the cerebellum and reducing excitability for a longer period than other neuroprotective mechanisms.Abbreviations AIDA (RS)-1-aminoindan-1,5-dicarboxylate - AMPA 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate - CNOX 6-cyano-7-nitroquinoxaline-2,3-dione - DCG IV (2S,2R,3R)-2-(2,3-dicarboxycyclo-propyl)glycine - DHPG (S)-3,5-dihydroxyphenylglycine - EGLU (2S)-2-ethylglutamate - L-AP3 L(+)-2-amino-3-phosphonopropionate - L-AP4 L(+)-2-amino-4-phosphonobutyrate - L-SOP o-phospho-l-serine - NBOX 6-nitro-7-sulphamoyl[f]quinoxaline-2,3-dione - NMDA n-methyl-d-aspartate - trans-ACPD (1S,3S)-1-aminocyclopentane-1,3-dicarboxylate     

The primary structure of the COOH-terminal half of cholera toxin subunit A1 containing the ADP-ribosylation site

The sequence of 96 amino acid residues from the COOH-terminus of the active subunit of cholera toxin, A₁, has been determined as PheAsnValAsnAspVal LeuGlyAlaTyrAlaProHisProAsxGluGlu GluValSerAlaLeuGlyGly IleProTyrSerGluIleTyrGlyTrpTyrArg ValHisPheGlyValLeuAsp GluGluLeuHisArgGlyTyrArgAspArgTyr TyrSerAsnLeuAspIleAla ProAlaAlaAspGlyTyrGlyLeuAlaGlyPhe ProProGluHisArgAlaTrp ArgGluGluProTrpIleHisHisAlaPro ProGlyCysGlyAsnAlaProArg(OH). This is the largest fragment obtained by BrCN cleavage of the subunit A₁ (M_r 23,000), and has previously been indicated to contain the active site for the adenylate cyclase-stimulating activity. Unequivocal identification of the COOH-terminal structure was achieved by separation and analysis of the terminal peptide after the specific chemical cleavage at the only cysteine residue in A₁ polypeptide. The site of self ADP-ribosylation in the A₁ subunit [C. Y. Lai, Q.-C. Xia, and P. T. Salotra (1983) Biochem. Biophys. Res. Commun.116, 341–348] has now been identified as Arg-50 of this peptide, 46 residues removed from the COOH-terminus. The cysteine that forms disulfide bridge to A₂ subunit in the holotoxin is at position 91.     

Insulin: Its binding to specific receptors and its stimulation of DNA synthesis and 2′,3′-cyclic nucleotide phosphohydrolase activity in cerebral cells cultured from embryonic mouse brain

The presence and specificity of insulin receptors was investigated in cultured cells obtained from 15–16 days old embryonic mouse cerebra. Developmental studies suggested that the maximum insulin binding occurred at about 11 days in vitro (DIV). Scatchard analysis of binding data revealed two types of binding sites. One type of receptor was the high affinity type (K _d=7.77×10^–9 M; number of receptor sites,B _max=350 fmol/mg protein) while the other type was of low affinity type (K _d=5.75×10^–8 M;B _max=1150 fmol/mg protein). The specificity of receptors for insulin was also confirmed by showing that [¹²⁵I]insulin was displaced by non-radioactive insulin but not by glucagon or growth hormone. Insulin displayed a clear dose-dependent stimulation of thymidine incorporation. It also stimulated the activity of the enzyme 2,3-cyclic nucleotide phosphohydrolase (CNPase), which is specifically associated with myelin produced from oligodendroglia. Thus insulin has a positive influence on the proliferation and differentiation of brain cells.     

Involvement of metabotropic glutamate receptors in taurine release in the adult and developing mouse hippocampus

Summary The inhibitory amino acid taurine has been held to function as an osmoregulator and modulator of neural activity, being particularly important in the immature brain. lonotropic glutamate receptor agonists are known markedly to potentiate taurine release. The effects of different metabotropic glutamate receptor (mGluR) agonists and antagonists on the basal and K⁺-stimulated release of [³H]taurine from hippocampal slices from 3-month-old (adult) and 7-day-old mice were now investigated using a superfusion system. Of group I metabotropic glutamate receptor agonists, quisqualate potentiated basal taurine release in both age groups, more markedly in the immature hippocampus. This action was not antagonized by the specific antagonists of group I but by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 6-nitro-7-sulphamoylbenzo[f]quinoxaline-2,3-dione (NBQX), which would suggest an involvement of ionotropic glutamate receptors. (S)-3,5-dihydroxyphenylglycine (DHPG) potentiated the basal release by a receptor-mediated mechanism in the immature hippocampus. The group II agonist (2S, 2R, 3R)-2-(2,3-dicarboxycyclopropyl)glycine (DCG IV) markedly potentiated basal taurine release at both ages. These effects were antagonized by dizocilpine, indicating again the participation of ionotropic receptors. Group III agonists slightly potentiated basal taurine release, as did several antagonists of the three metabotropic receptor groups. Potassium-stimulated (50 mM K⁺) taurine release was generally significantly reduced by mGluR agents, mainly by group I and II compounds. This may be harmful to neurons in hyperexcitatory states. On the other hand, the potentiation by mGluRs of basal taurine release, particularly in the immature hippocampus, together with the earlier demonstrated pronounced enhancement by activation of ionotropic glutamate receptors, may protect neurons against excitotoxicity.Abbreviations ACPD (1±)-1-aminocyclopentane-trans-1,3-dicarboxylate - AIDA (RS)-1-aminoindan-1,5-dicarboxylate - AMPA 2-amino-3-hydroxy05-methyl-4-isoxazolepropionate - CNQX 6-cyano-7-nitroquinoxaline-2,3-dione - CPPG (RS)-2-cyclopropyl-4-phosphonophenylglycine - DCG IV (2S,2R,3R)-2-(2,3-dicarboxycyclopropyl)glycine - DHPG (S)-3,5-dihydroxyphenylglycine - EGLU (2S)-2-ethylglutamate - L-AP3 L(+)-2-amino-3-phosphonopropionate - L-AP4 L(+)-2-amino-4-phosphonobutyrate - L-AP6 L(+)-2-amino-6-phosphonohexanoate - L-SOP O-phospho-L-serine - MPPG (RS)-2-methyl-4-phosphonophenylglycine - MSOP (RS)-2-methylserine-O-phosphate - MSOPPE (RS)-2-methylserine-O-phosphate monophenyl ester - MTPG (RS)-2-methyl-4-tetrazolylphenylglycine - NBQX 6-nitro-7-sulphamoyl[f]quinoxaline-2,3-dione - NMDA N-methyl-D-aspartate - QA quisqualate - S-3C4H-PG (S)-3-carboxy-4-hydroxyphenylglycine - S-4C-PG (S)-4-carboxyphenylglycine; - S-MCGP (S)-2-methyl-4-carboxyphenylglycine     

Pharmacological and biochemical characteristics of partially purified GABAB receptor

Pharmacological and biochemical characteristics of the partially purified -aminobutyric acid (GABA)_B receptor using baclofen affinity column chromatography have been examined. The Scatchard analysis of [³H]GABA binding to the purified GABA_B receptor showed a linear relationship and the K_D and B_max values were 60 nM and 118 pmol/mg of protein, respectively. Although GTP and Mg²⁺ did not affect on the GABA_B receptor binding, Ca²⁺ significantly increased [³H]GABA binding to the purified GABA_B receptor in a dose-dependent manner and showed its maximum effect at 2 mM. The enhancement of the binding by Ca²⁺ was found to be due to the increase of Bmax by the Scatchard analysis. The treatments with pronase and trypsin significantly decreased the binding of [³H]GABA, but phospholipase A₂ had no significant effect on the binding. In addition, treatment with glycosidases such as glycopeptidase A and -galactosidase significantly decreased the binding of [³H]GABA to the purified GABA_B receptor. These results suggest that purification of the solubilized GABA_B receptor by the affinity column chromatography may result in the functional uncoupling of GABA_B receptor with GTP-binding protein. Furthermore, the present results suggest that cerebral GABA_B receptor may be a glycoprotein and membrane phospholipids susceptible to phospholipase A₂ treatment may not be involved in the exhibition of the binding activity.Special issue dedicated to Dr. Eugene Roberts.     

The first bacterial β-1,6-endoglucanase from <Emphasis Type="Italic">Saccharophagus degradans</Emphasis> 2-40<Superscript>T</Superscript> for the hydrolysis of pustulan and laminarin

β-1,6-glucan is a polysaccharide found in brown macroalgae and fungal cell walls. In this study, a β-1,6-endoglucanase gene from Saccharophagus degradans 2-40^T, gly30B, was cloned and overexpressed in Escherichia coli. Gly30B, which belongs to the glycoside hydrolase family 30 (GH30), was found to possess β-1,6-endoglucanase activity by hydrolyzing β-1,6-glycosidic linkages of pustulan (β-1,6-glucan derived from fungal cell walls) and laminarin (β-1,3-glucan with β-1,6-branchings, derived from brown macroalgae) to produce gentiobiose and glucose as the final products. The optimal pH and temperature for Gly30B activity were found to be pH 7.0 and 40 °C, respectively. The kinetic constants of Gly30B, V _max, K _M, and k _cat were determined to be 153.8 U/mg protein, 24.2 g/L, and 135.6 s^?1 for pustulan and 32.8 U/mg protein, 100.8 g/L, and 28.9 s^?1 for laminarin, respectively. To our knowledge, Gly30B is the first β-1,6-endoglucanase characterized from bacteria. Gly30B can be used to hydrolyze β-1,6-glucans of brown algae or fungal cell walls for producing gentiobiose as a high-value sugar and glucose as a fermentable sugar.     

Effects of taurine on GABA release from synaptosomes of rat olfactory bulb

Summary Superfusion of synaptosomes prepared from rat olfactory bulb revealed constant basal release of endogenous taurine (Tau), aspartate (Asp), glutamate (Glu) and-aminobutyrate (GABA): their release rates were 110.4 ± 13.0, 30.3 ± 6.7, 93.7 ± 13.1, and 53.3 ± 8.8 pmol/min/mg protein, respectively. The depolarizing-stimulation with 30mM KCl evoked 1.17-, 2.18-, 2.55- and 1.53-fold increases, respectively. Tau release was calcium-independent. However, the perfusion of synaptosomes with Tau (10µM) inhibited the evoked increase in GABA release by 63% without changing basal release, although it did not affect release of Asp and Glu. Phaclofen (10µM, a GABA_B receptor antagonist), but not bicuculline (10µM, a GABA_A receptor antagonist), counteracted the Tau-induced reduction in GABA release. These data suggest that Tau may be abundantly released from nerve endings of rat olfactory bulb and that it may regulate GABA release through the activation of presynaptic GABA_B autoreceptors.     

Effect of the redox state of QB on electric field-induced charge recombination in Photosystem II

Electric field-induced charge recombination in Photosystem II (PS II) was studied in osmotically swollen spinach chloroplasts (blebs) by measurement of the concomitant chlorophyll luminescence emission (electroluminescence). A pronounced dependence on the redox state of the two-electron gate Q_B was observed and the earlier failure to detect it is explained. The influence of the Q_B/Q_B ^– oscillation on electroluminescence was dependent on the redox state of the oxygen evolving complex; at times around one millisecond after flash illumination a large effect was observed in the states S₂ and S₃, but not in the state S₄ (actually Z⁺S₃). The presence of the oxidized secondary electron donor, tyrosine Z⁺, appeared to prevent expression of the Q_B/Q_B ^– effect on electroluminescence, possibly because this effect is primarily due to a shift of the redox equilibrium between Z/Z⁺ and the oxygen evolving complex.Abbreviations BSA bovine serum albumin - EDTA ethylene-diaminetetraacetic acid - EL electroluminescence - FCCP carbonylcyanide p-trifluoromethyloxyphenyl-hydrazone - HEPESI 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - I primary electron acceptor - MOPS 3-(N-morpholino) propane sulfonic acid - P680 primary electron donor of Photosystem II - P700 primary electron donor of Photosystem I - Q_A and Q_B secondary and tertiary electron acceptors of Photosystem II - Z secondary electron donor (D1 Tyr 161)     

Elucidation of a Novel Extracellular Calcium-binding Site on Metabotropic Glutamate Receptor 1α (mGluR1α) That Controls Receptor Activation

Metabotropic glutamate receptor 1α (mGluR1α) exerts important effects on numerous neurological processes. Although mGluR1α is known to respond to extracellular Ca²⁺ ([Ca²⁺]_o) and the crystal structures of the extracellular domains (ECDs) of several mGluRs have been determined, the calcium-binding site(s) and structural determinants of Ca²⁺-modulated signaling in the Glu receptor family remain elusive. Here, we identify a novel Ca²⁺-binding site in the mGluR1α ECD using a recently developed computational algorithm. This predicted site (comprising Asp-318, Glu-325, and Asp-322 and the carboxylate side chain of the receptor agonist, Glu) is situated in the hinge region in the ECD of mGluR1α adjacent to the reported Glu-binding site, with Asp-318 involved in both Glu and calcium binding. Mutagenesis studies indicated that binding of Glu and Ca²⁺ to their distinct but partially overlapping binding sites synergistically modulated mGluR1α activation of intracellular Ca²⁺ ([Ca²⁺]_i) signaling. Mutating the Glu-binding site completely abolished Glu signaling while leaving its Ca²⁺-sensing capability largely intact. Mutating the predicted Ca²⁺-binding residues abolished or significantly reduced the sensitivity of mGluR1α not only to [Ca²⁺]_o and [Gd³⁺]_o but also, in some cases, to Glu. The dual activation of mGluR1α by [Ca²⁺]_o and Glu has important implications for the activation of other mGluR subtypes and related receptors. It also opens up new avenues for developing allosteric modulators of mGluR function that target specific human diseases.