Possible Involvement of Interleukin-1 in Cyclooxygenase-2Induction After Spinal Cord Injury in Rats

Abstract : A standardized compression injury of rat spinal cord brought about a time-dependent biphasic production of thromboxane A₂ (detected as thromboxane B₂) and prostaglandin I₂ (detected as 6-ketoprostaglandin F_1α. Thromboxane B₂ was predominant during the first 1 h, whereas the 6-ketoprostaglandin F_1α level exceeded that of thromboxane B₂ at 8 h postinjury. As examined by inhibitor experiments and northern blotting, cyclooxygenase-1 was responsible for the first phase, and cyclooxygenase-2 was involved in the second phase. On compression injury the levels of interleukin-1α and -1β detected as mRNA and protein increased and peaked at 2-4 h. Injection of exogenous interleukin-1 α into the spinal cord resulted in an increase of cyclooxygenase-2 mRNA content and a predominant production of 6-ketoprostaglandin F_1α resembling the second phase of eicosanoid production. Concomitantly, extravascular migration of polymorphonuclear leukocytes was enhanced after the interleukin-1α injection. These cells together with vascular endothelial cells and glial cells were stained positively with an anti-cyclooxygenase-2 antibody. The results suggest that the immediate eicosanoid synthesis after spinal cord injury was due to the constitutive cyclooxygenase-1 and the delayed synthesis of eicosanoids was attributable to the induction of cyclooxygenase-2 mediated by interleukin-1 α.     

Characterization, Distribution, and Protein Kinase C-Mediated Regulation of [35S]Glutathione Binding Sites in Mouse and Human Spinal Cord

Abstract: We have characterized a high-affinity [³⁵S]-glutathione ([³⁵S]GSH) binding site in mouse and human spinal cord. [³⁵S]GSH binding sites in mouse and human spinal cord were observed largely within the gray matter in both the dorsal and ventral horns of spinal cord at cervical, thoracic, and lumbosacral segments. High-affinity [³⁵S]GSH binding was saturable, showing a B _max of 72 fmol/mg of protein and a K _D of 3.0 n M for mouse spinal cord and a B _max of 52 fmol/mg of protein and a K _D of 1.6 n M for human spinal cord. [³⁵S]GSH binding was displaceable by GSH, l -cysteine, and S -hexyl-GSH, but not by glutamate, glycine, or NMDA. These [³⁵S]GSH binding sites exhibited kinetic and saturation characteristics similar to GSH binding sites in rat brain astrocytes. To determine whether [³⁵S]GSH binding sites could be regulated by protein kinase C, we exposed human spinal cord sections to phorbol 12,13-diacetate for 1 h before ligand binding. Phorbol ester treatment increased [³⁵S]GSH binding by ∼60%, an effect that could be blocked by exposure of spinal cord sections to 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, a general protein kinase inhibitor. [³⁵S]GSH binding sites in the spinal cord of both species exhibited many of the characteristics of a receptor including saturable binding, high affinity, ligand specificity, and modulation by kinase activity. These data suggest that GSH is a neurotransmitter in the CNS.     

Cytokine-Regulated Expression of Platelet-Derived Growth Factor Gene and Protein in Cultured Human Astrocytes

Abstract: To elucidate mechanisms regulating the production of platelet-derived growth factor (PDGF) in the CNS, we analyzed the influence of a panel of cytokines on PDGF mRNA and protein levels in astrocyte-enriched cultures from the human embryonic brain and spinal cord. Using a specific ELISA, PDGF AB protein was detected in serum-free astrocyte supernatants and its levels were significantly increased after treatment of the cultures with transforming growth factor-β₁ (TGF-β₁) or tumor necrosis factor-α (TNF-α); the largest increase was detected after combined treatment with the two cytokines. Interleukin-1β (IL-1β) by itself had little or no effect but synergized with TGF-β₁ in enhancing PDGF AB production. Supernatants from human astrocyte cultures stimulated the proliferation of rat oligodendrocyte progenitors, and most of the mitogenic activity could be accounted for by PDGF. By northern blot analysis, both PDGF A- and PDGF B-chain mRNAs were detected in untreated astrocytes. PDGF B-chain mRNA levels were increased by TGF-β₁, TNF-α, TNF-α/TGF-β₁, or IL-1β/TGF-β₁, whereas PDGF A-chain mRNA levels were not consistently affected by cytokine treatments. These in vitro data indicate that TGF-β₁, TNF-α, and IL-1β are able to stimulate astrocyte PDGF production. This cytokine network could play a role in CNS development and repair after injury or inflammation.     

The Measurement of Spinal Cord Tissue pH Using a Diffusible, Lipid-Soluble, pH-Sensitive Fluorescent Indicator

Abstract: Spinal cord tissue pH was measured in cats at normocapnia, hypocapnia, hypercapnia and death from anoxia using a pH-sensitive fluorescent indicator (umbelliferone) with both molecular and ionic fluorophors. A ratio analysis of the indicator's calibrated 450 nm fluorescent tissue clearance curves from 340 and 370 nm excitation permitted direct in vivo tissue pH determinations. Fifteen animals were divided into three equal groups according to different arterial carbon dioxide tensions (P_a co₂):five animals at P_a co₂ 20, five animals P_a co₂ 40 and five animals P_a co₂ 60 torr. Spinal cord tissue pH varied linearly with arterial pH, but within narrower limits. These values (arterial versus cord pH) were: 7.46 versus 7.15; 7.21, 7.09; and 7.04, 7.00. At death from hypoxemia the arterial pH fell to 6.99 and spinal cord to 6.67. The clearance curves of umbelliferone in spinal cord varied according to P_a co₂ and appeared to reflect spinal cord blood flow.     

Pheromone of male blue gourami and its effect on vitellogcnesis, steroidogenesis and gonadotropin cells in pituitary of the female

Female Trichogaster trichopterus were exposed to aquarium water in which males had built nests. Gonadotropin cells in the pituitary gland, and exovitellogenesis and steroidogenesis in the ovary were studied. In females in which the percentage of oocytes in vitellogenesis (%V) was low initially, it rose significantly in comparison with an unexposed control group. In females in which the %V was higher initially, it increased further, and in addition a significant percentage of oocytes reached maturation. Thin layer chromatography, using the precursors ³H-pregnenolone and ¹⁴progesterone, revealed high yields of the steroids 17β-estradiol (E₂), 17α,20β-dihydroxy-4-pregnen-3-one (17,20-P), 5β-pregane,3α,17α,20β-triol (5β-P-triol) and 11-ketotestosterone (11KT) in both experimental groups. Significant differences were found in E₂, 17,20-P and 5β-P-triol between the test and control groups. The immunoresponse of GtH-producing cells in the pituitary of the females maintained in nest water was lower than in the control group, suggesting that the GtH was secreted from the cells, which would explain the vitellogenic and steroidogenic changes found in the ovary.     

Replacement of histidine in position 105 in the α₅ subunit by cysteine stimulates zolpidem sensitivity of α₅β₂γ₂ GABA(A) receptors

Zolpidem is a positive allosteric modulator of GABA_A receptors with sensitivity to subunit composition. While it acts with high affinity and efficacy at GABA_A receptors containing the α₁ subunit, it has a lower affinity to GABA_A receptors containing α₂, α₃, or α₅ subunits and has a very weak efficacy at receptors containing the α₅ subunit. Here, we show that replacing histidine in position 105 in the α₅ subunit by cysteine strongly stimulates the effect of zolpidem in receptors containing the α₅ subunit. The side chain volume of the amino acid residue in this position does not correlate with the modulation by zolpidem. Interestingly, serine is not able to promote the potentiation by zolpidem. The homologous residues to α₅H105 in α₁, α₂, and α₃ are well-known determinants of the action of classical benzodiazepines. Other studies have shown that replacement of these histidines α₁H101, α₂H101, and α₃H126 by arginine, as naturally present in α₄ and α₆, leads to benzodiazepine insensitivity of these receptors. Thus, the nature of the amino acid residue in this position is not only crucial for the action of classical benzodiazepines but in α₅ containing receptors also for the action of zolpidem.     

Glutamic Acid Decarboxylase in Sea Lamprey (Petromyzon marinus): Characterization, Localization, and Developmental Changes

Abstract: We have carried out assays for glutamic acid decarboxylase (GAD) in homogenates of brain and spinal cord from larval and adult sea lamprey ( Petromyzon marinus ). The enzyme had similar characteristics in both stages. Optimal pH was 6.8; optimal temperature was 27–30° C; K _m at 27°C was 5 mM. GAD activity was distributed uniformly along the length of the spinal cord. Specific activities for the larval cord and brain were 26 and 63 nm CO₂/mg protein/h. respectively. The specific activities for the adult cord and brain were 29 and 236 nm CO₂/mg protein/h, respectively. Thus, the activity of cord homogenates did not change significantly between larval and adult stages, but that of the brain increased about fourfold.     

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.     

Evidence for the Existence of Differential O-Glycosylated α5-Subunits of the γ-Aminobutyric AcidA Receptor in the Rat Brain

Abstract: Polyclonal antibodies were raised to synthetic peptides having amino acid sequences corresponding with the N- or C-terminal part of the γ-aminobutyric acid_A (GABA_A) receptor α₅-subunit. These anti-peptide α₅(2–10) or anti-peptide α₅(427–433) antibodies reacted specifically with GABA_A receptors purified from the brains of 5–10-day-old rats in an enzyme-linked immunosorbent assay and were able to dose-dependently immunoprecipitate up to 6.3 or 13.1% of the GABA_A receptors present in the incubation, respectively. In immunoblots, each of these antibodies reacted with the same two protein bands with apparent molecular mass of 53 or 57 kDa. After exhaustive treatment of purified GABA_A receptors with N -Glycanase, each of these antibodies identified two proteins with apparent molecular masses of 46 and 48 kDa. Additional treatment of GABA_A receptors with neuraminidase and O -Glycanase resulted in an apparently single protein with molecular mass of 47 kDa, which again was identified by both the anti-peptide α₅(2–10) and the anti-peptide α₅(427–433) antibody. These results indicate the existence of at least two different α₅-sub-units of the GABA_A receptor that differ in their carbohydrate content. In contrast to other α- or β-subunits of GABA_A receptors so far investigated, at least one of these two α₅-subunits contains O-linked carbohydrates.     

Evidence that GABAA Receptor Subunit mRNA Expression During Development Is Regulated by GABAA Receptor Stimulation

Abstract: The expression of six mRNA species (α2, α3, α5, β2, β3, and γ2) encoding for GABA_A receptor subunits was followed in cultured early postnatal cortical neurons by in situ hybridization histochemistry. In untreated control cultures it was found that these subunit mRNA expression profiles closely follow those seen during development in vivo. α3, α5, and β3 subunit expression declined, α2 expression increased, whereas β2 and γ2 subunit mRNA expression remained relatively constant. To test the hypothesis that GABA_A receptor stimulation regulates these expression profiles, we tested the effect of a GABA_A receptor positive modulator, allopregnanolone, and a GABA_A receptor noncompetitive antagonist, tert -butylbicyclophosphorothionate (TBPS). It was found that allopregnanolone augmented the rate at which the α3, α5, or β3 subunit mRNA expression declined and prevented the increase in α2 subunit mRNA expression. As well, allopregnanolone down-regulated β2 subunit mRNA expression. TBPS, on the other hand, up-regulated α3, α5, β2, and β3 subunit mRNA expression. It also down-regulated the expression of α2 subunit mRNA. Both allopregnanolone and TBPS had no effect on γ2 subunit mRNA expression. These results imply that the developmental switchover of GABA receptor subunit mRNA expression is regulated by GABA_A receptor activity.     

Levels of Benzodiazepine Receptor Subtypes and GABAA Receptor α-Subunit mRNA Do Not Correlate During Development

Abstract: Developmental changes in the pharmacological properties of the GABA_A receptor have been suggested to result from changes in the subunit composition of the receptor complex. The nicotinic acetylcholine receptor is structurally related to the GABA_A receptor and undergoes a developmental subunit switch at the neuromuscular synapse. To examine the mechanistic similarities between these systems we sought to find whether the changes in GABA_A receptor subunits are controlled by changes in messenger RNA levels, as they are for the nicotinic acetylcholine receptor. We found a 10-fold increase in the level of α1-subunit mRNA, and a small increase in levels of GABA_A/benzodiazepine receptors from day 1 to day 24 of rat cerebellar development. We also found that the levels of α1-subunit mRNA were higher than the levels of mRNA encoding other α subunits at all developmental time points. The low levels of messenger RNA for α2, α3, and α5 subunits are inconsistent with the high levels of type II benzodiazepine binding in the rat cerebellum at birth because these α subunits have been shown to form GABA_A receptors with type II benzodiazepine binding. These findings are inconsistent with simple models that would explain the developmental differences in GABA_A receptor pharmacology simply as a result of changes in α-subunit gene expression.     

Rice alpha-mannosidase digesting the high mannose glycopeptide of glutelin

α -Mannosidase (EC 3.2.1.24) from rice dry seeds was purified to homogeneity. Optimum pH and K_m for pNP- α -Man hydrolysis were pH 4.3–4.5 and 1.04 m M , respectively. The enzyme digested mannobioses such as Man α -1,2Man, Man α -1,6Man, Man α -1,3Man but Man α -1,4Man. Zn²⁺ ion was required for the activity, whereas EDTA and swainsonine inhibited the activity by 80 and 96%, respectively. The rice storage protein, glutelin was prepared and its basic subunits were shown to have high mannose-type sugar chains by two-dimensional mapping using NH₂-P and C₁₈ silica columns. They were Man₉GlcNAc₂, Man₈GlcNAc₂, Man₇GlcNAc₂, Man₆GlcNAc₂ and Man₅GlcNAc₂. All these oligosaccharides were digested by the purified α -mannosidase, and Man-GlcNAc₂ and mannose were formed. Glycopeptides, having these high mannose-type sugar chains, could also be digested by the α -mannosidase. Subunits were prepared from glutelin basic subunit and the richest subunit among them, subunit 2 (isoform 2), was digested by the α -mannosidase. Isoform 2 was digested by V8 protease only partially and slowly. However, isoform 2, pre-treated with the α -mannosidase, was rapidly and completely digested by V8 protease.     

Behavioural and electrophysiological responses to F-prostaglandins, putative spawning pheromones, in three salmonid fishes

Behavioural and electro-olfactogram (EOG) responses to synthetic F-prostaglandins (PGFs) were recorded in the three salmonids: brown trout Salmo trutta , lake whitefish Coregonus clupeaformis and rainbow trout Oncorhynchus mykiss . Exposure to 10⁻⁸ M PGF_2α and 13, 14-dihydro-PGF_2α increased swimming activity in individually exposed brown trout in a flow-through tank. Digging and nest probing behaviours were further observed in brown trout females exposed to PGF_2α. Lake whitefish exposed to 10⁻⁸ M PGF_2α and 15-keto-PGF_2α also increased their locomotion. In rainbow trout, the absence of behavioural responses to PGFs correlates with a lack of olfactory sensitivity to these chemicals. PGFs triggered behavioural responses distinct from the feeding stimulant in brown trout. EOG measurements demonstrated that brown trout were most sensitive to PGF_2α, with a threshold concentration of 10⁻¹¹ M. Lake whitefish were most sensitive to both 15-keto-PGF_2α and 13, 14-dihydro-PGF_2α. Cross-adaptation and binary mixture experiments suggest that only one olfactory receptive mechanism is involved in PGFs detection. The behavioural and olfactory responses observed with exposure to PGF_2α and its metabolites suggest these compounds function as reproductive pheromones in brown trout and lake whitefish.     

Separation of Soluble Amoebicidal and Tumoricidal Activity of Activated Macrophages

ABSTRACT. Macrophage-conditioned medium (MøCM) prepared from mouse peritoneal macrophages activated in vivo with bacillus Calmette-Guérin (BCG) or Propionibacterium acnes and triggered with lipopolysaccharide in vitro contained tumoricidal and amoebicidal activity. The murine fibroblast cell line L929 was used as the indicator of tumoricidal activity and Naegleria fowleri amoeba was used to detect amoebicidal activity in MøCM. The protease inhibitor, soybean trypsin inhibitor, decreased tumoricidal activity but had little effect on amoebicidal activity in MøCM. Anti-TNF _α antiserum inhibited tumoricidal activity in MøCM. The antiserum reduced amoebicidal activity in BCG-activated MøCM but had no effect on amoebicidal activity in P. acnes -activated MøCM. Recombinant TNF _α , rIL-1 _α , or rIL-1 _β independently did not affect cytolysis of amoebae. Also, rTNF _α had no effect on the growth of amoebae. Preparative flat-bed electrofocusing of BCG-activated MøCM yielded fractions that exhibited different amoebicidal and tumoricidal activity profiles. Three domains of activity were analyzed (acidic, neutral, and basic). Anti-TNF _α antiserum eliminated tumoricidal activity, but not amoebicidal activity, in fractions from the acidic domain. A combination of anti-TNF _α and anti-IL-1 _α antisera failed to eliminate amoebicidal activity in fractions from the basic domain. These results indicate that different factors are responsible for macrophage amoebicidal and tumoricidal activity. The amoebicidal factors in MøCM affected cytolysis of several species of amoebae.     

Chronic Ethanol Administration Regulates the Expression of GABAA Receptor α1 and α5 Subunits in the Ventral Tegmental Area and Hippocampus

Abstract: Ethanol dependence and tolerance involve perturbation of GABAergic neurotransmission. Previous studies have demonstrated that ethanol treatment regulates the function and expression of GABA_A receptors throughout the CNS. Conceivably, changes in receptor function may be associated with alterations of subunit composition. In the present study, a comprehensive (1–12 weeks) ethanol treatment paradigm was used to evaluate changes in GABA_A receptor subunit expression in several brain regions including the cerebellum, cerebral cortex, ventral tegmental area (VTA) (a region implicated in drug reward/dependence), and the hippocampus (a region involved in memory/cognition). Expression of α₁ and α₅ subunits was regulated by ethanol in a region-specific and time-dependent manner. Following 2–4 weeks of administration, cortical and cerebellar α₁ and α₅ subunit immunoreactivity was reduced. In the VTA, levels of α₁ subunit immunoreactivity were significantly decreased after 12 weeks but not 1–4 weeks of treatment. Hippocampal α₁ subunit immunoreactivity and mRNA content were also significantly reduced after 12 but not after 4 weeks of treatment. In contrast, α₅ mRNA content was increased in this brain region. These data indicate that chronic ethanol administration alters GABA_A receptor subunit expression in the VTA and hippocampus, effects that may play a role in the abuse potential and detrimental cognitive effects of alcohol.     

CaMKII associates with CaV1.2 L-type calcium channels via selected β subunits to enhance regulatory phosphorylation

Calcium/calmodulin-dependent kinase II (CaMKII) facilitates L-type calcium channel (LTCC) activity physiologically, but may exacerbate LTCC-dependent pathophysiology. We previously showed that CaMKII forms stable complexes with voltage-gated calcium channel (VGCC) β_1b or β_2a subunits, but not with the β₃ or β₄ subunits ( Grueter et al. 2008 ). CaMKII-dependent facilitation of Ca_V1.2 LTCCs requires Thr498 phosphorylation in the β_2a subunit ( Grueter et al. 2006 ), but the relationship of this modulation to CaMKII interactions with LTCC subunits is unknown. Here we show that CaMKII co-immunoprecipitates with forebrain LTCCs that contain Ca_V1.2α₁ and β₁ or β₂ subunits, but is not detected in LTCC complexes containing β₄ subunits. CaMKIIα can be specifically tethered to the I/II linker of Ca_V1.2 α₁ subunits in vitro by the β_1b or β_2a subunits. Efficient targeting of CaMKIIα to the full-length Ca_V1.2α₁ subunit in transfected HEK293 cells requires CaMKII binding to the β_2a subunit. Moreover, disruption of CaMKII binding substantially reduced phosphorylation of β_2a at Thr498 within the LTCC complex, without altering overall phosphorylation of Ca_V1.2α₁ and β subunits. These findings demonstrate a biochemical mechanism underlying LTCC facilitation by CaMKII.     

Differential Effects of 4'-Chlorodiazepam on Expressed Human GABAA Receptors

Abstract: The interactions of the atypical benzodiazepine 4'-chlorodiazepam (Ro 5-4864) with functionally expressed human GABA_A receptor cDNAs were determined. Cotransfection of human α₂, β₁, and γ₂ subunits was capable of reconstituting a 4'-chlorodiazepam recognition site as revealed by a dose-dependent potentiation of t -[³⁵S]butylbicyclophosphorothionate ([³⁵S]TBPS) binding to the GABA-activated chloride channel. This site is found on GABA_A receptor complexes containing sites for GABA agonist-like benzodiazepines and neuroactive steroids. The importance of the α subunit was further demonstrated as substitution of either α₁ or α₃ for the α₂ subunit did not reconstitute a 4'-chlorodiazepam recognition site that was capable of modulating [³⁵S]TBPS binding under the same experimental conditions. The 4'-chlorodiazepam modulatory site was shown to be distinct from the benzodiazepine site, but the phenylquinolines PK 8165 and PK 9084 produced effects similar to 4'-chlorodiazepam, consistent with the previous analysis of the 4'-chlorodiazepam site in brain homogenates. Further analysis of the subunit requirements revealed that coexpression of α₂ and β₁ alone reconstituted a 4'-chlorodiazepam recognition site. It is interesting, however, that the 4'-chlorodiazepam site was found to inhibit [³⁵S]TBPS binding to the GABA-activated chloride channel. Thus, the 4'-chlorodiazepam site may be reconstituted with only the α and β polypeptides.