Inger Schousboe

Glutamate receptor agonists up-regulate glutamate transporter GLAST in astrocytes

LONG-TERM treatment of astrocytes in primary culture with L-glutamate (0.1–3 mM) resulted in a dose-dependent increase in D-[3H]aspartate uptake. The effect was abolished by an antagonist of kainate/AMPA receptors, CNQX, and mimicked by kainate, but not by AMPA or tACPD. Both glutamate and kainate caused a dramatic up-regulation (82% and 69%, respectively) of GLAST, a predominant glutamate transporter in cultured astroglia, though the mRNA levels appeared unaffected. Long-term treatment of cultures with dBcAMP stimulated D-[3H]aspartate uptake as well as GLAST expression. Apart from glutamate, none of the agonists used was capable of increasing further the uptake capacity of the dBcAMP-treated astroglia. The glutamate receptor- dependent modulation of glutamate transport in astroglial cultures may represent a novel feedback regulatory mechanism for glutamate uptake in the brain.

Binding of β2-glycoprotein I to platelets: Effect of adenylate cyclase activity

β2-glycoprotein I was trace-labelled with 3H by reductive methylation. It was shown to bind specifically to washed human platelets. No binding was measured to erythrocytes and only insignificantly small amounts were bound to lymphocytes. Bound and free [3H]β2-glycoprotein I were measured after separation from the suspending medium by centrifugation. Maximum binding was obtained within 45 min and was proportional to the number of platelets in the incubation mixture. Equilibrium binding was established with reequilibration after dilution. Analysis of β2-glycoprotein I as a function of β2-glycoprotein I concentration indicates that platelets bind 6 × 105 β2-glycoprotein I molecules per platelet at saturation with an apparent dissociation constant of 559 nM. In the presence of Ca2+ a significantly lower amount of β2-glycoprotein I is bound with an apparent dissociation constant of 334 nM. Platelets treated with formalin bind more β2-glycoprotein I than do untreated platelets but the dissociation constant is unaffected by the formalin treatment. Neuraminidase treatment of the platelets has no effect on the binding. The presence of other serum proteins in binding assays performed at 25°C has no effect on the binding. The binding of β2-glycoprotein I to the platelets can be correlated with inhibition of adenylate cyclase activity.

Receptor expression in primary cultures of neurons or astrocytes

beta-Adrenergic receptors are abundant on astrocytes which contain mainly the beta 1 subtype. These cells also possess alpha 1 and alpha 2 receptors. Neurons display fewer beta-adrenergic receptor sites and those present are of the beta 2 subtype. Dopaminergic receptors are found on astrocytes and show a regional variation. Serotonergic receptors have been demonstrated on astrocytes but are expressed to a larger extent in neurons. Astrocytes display muscarinic receptors but have no receptor sites for the transmitter amino acids glutamate and GABA. GABA receptors are present on cerebellar granule cells, are induced by the presence of GABA agonists in the medium and mediate an evoked release of glutamate. Glutamate receptors are found on cultured cortical interneurons. Some, but not all, peptide hormones have receptors on astrocytes and/or neurons in primary cultures.

Molecular cloning and mammalian expression of human β2‐glycoprotein I cDNA

Human β2‐glycoprotein (β2gpI) cDNA was isolated from a liver cDNA library and sequenced. The cDNA encoded a 19‐residue hydrophobic signal peptide followed by the mature β2gpI of 326 amino acid residues. In liver and in the hepatoma cell line HepG2 there are two mRNA species of about 1.4 and 4.3 kb, respectively, hybridizing specifically with the β2gpI cDNA. Upon isoelectric focusing, recombinant β2gpI obtained from expression of β2gpI cDNA in baby hamster kidney cells showed the same pattern of bands as β2gpI isolated from plasma, and at least 5 polypeptides were visible

Purification, characterization and identification of an agglutinin in human serum

A serum protein named agglutinin is able to induce mitochondria to agglutinate. The protein has been purified from human serum by chromatography on DE-52. Sephadex G-200 and immunoglobulin-Sepharose 4B columns. Agglutinin is a glycoprotein that migrates electrophoretically as a gamma-globulin. Its molecular weight was determined to be 50,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Monospecific antiserum prepared against the agglutinin was found to be identical with anti-beta 2-glycoprotein I and agglutinating activity could be adsorbed on anti-beta 2-glycoprotein I-Sepharose 4B columns. Thus, the agglutinin has been identified as beta 2-glycoprotein I. The reaction between mitochondria and agglutinin shows positive cooperativity, which is independent on the stage of purification of agglutinin. The agglutinating activity could be diminished (inhibited) by acidic non-soluble lipids such as oleic acid, phosphatidic acid, phosphatidyl serine and phosphatidyl inositol.

Knockout of GAD65 has major impact on synaptic GABA synthesized from astrocyte-derived glutamine

γ-Aminobutyric acid (GABA) synthesis from glutamate is catalyzed by glutamate decarboxylase (GAD) of which two isoforms, GAD65 and GAD67, have been identified. The GAD65 has repeatedly been shown to be important during intensified synaptic activity. To specifically elucidate the significance of GAD65 for maintenance of the highly compartmentalized intracellular and intercellular GABA homeostasis, GAD65 knockout and corresponding wild-type mice were injected with [1-13C]glucose and the astrocyte-specific substrate [1,2-13C]acetate. Synthesis of GABA from glutamine in the GABAergic synapses was further investigated in GAD65 knockout and wild-type mice using [1,2-13C]acetate and in some cases c-vinylGABA (GVG, Vigabatrin), an inhibitor of GABA degradation. A detailed metabolic mapping was obtained by nuclear magnetic resonance (NMR) spectroscopic analysis of tissue extracts of cerebral cortex and hippocampus. The GABA content in both brain regions was reduced by ~20%. Moreover, it was revealed that GAD65 is crucial for maintenance of biosynthesis of synaptic GABA particularly by direct synthesis from astrocytic glutamine via glutamate. The GAD67 was found to be important for synthesis of GABA from glutamine both via direct synthesis and via a pathway involving mitochondrial metabolism. Furthermore, a severe neuronal hypometabolism, involving glycolysis and tricarboxylic acid (TCA) cycle activity, was observed in cerebral cortex of GAD65 knockout mice.

Neuron-glia interactions in glutamatergic neurotransmission: roles of oxidative and glycolytic adenosine triphosphate as energy source.

Glutamatergic neurotransmission accounts for a considerable part of energy consumption related to signaling in the brain. Chemical energy is provided by adenosine triphosphate (ATP) formed in glycolysis and tricarboxylic acid (TCA) cycle combined with oxidative phosphorylation. It is not clear whether ATP generated in these pathways is equivalent in relation to fueling of the energy‐requiring processes, i.e., vesicle filling, transport, and enzymatic processing in the glutamatergic tripartite synapse (the astrocyte and pre‐ and postsynapse). The role of astrocytic glycogenolysis in maintaining theses processes also has not been fully elucidated. Cultured astrocytes and neurons were utilized to monitor these processes related to glutamatergic neurotransmission. Inhibitors of glycolysis and TCA cycle in combination with pathway‐selective substrates were used to study glutamate uptake and release monitored with D‐aspartate. Western blotting of glyceraldehyde‐3‐P dehydrogenase (GAPDH) and phosphoglycerate kinase (PGK) was performed to determine whether these enzymes are associated with the cell membrane. We show that ATP formed in glycolysis is superior to that generated by oxidative phosphorylation in providing energy for glutamate uptake both in astrocytes and in neurons. The neuronal vesicular glutamate release was less dependent on glycolytic ATP. Dependence of glutamate uptake on glycolytic ATP may be at least partially explained by a close association in the membrane of GAPDH and PGK and the glutamate transporters. It may be suggested that these enzymes form a complex with the transporters and the Na+/K+‐ATPase, the latter providing the sodium gradient required for the transport process.

The effect of β2-glycoprotein i on the dextran sulfate and sulfatide activation of the contact system (Hageman factor system) in the blood coagulation

1. beta 2-Glycoprotein I, inhibits the initiation of the contact system in plasma accomplished by dextran sulfate. 2. The dextran sulfate induced activation could be inhibited both when dextran sulfate was preincubated with beta 2-glycoprotein I and when the amount of beta 2-glycoprotein I in plasma was increased. 3. The concentration of beta 2-glycoprotein I at which an inhibitory effect could be registered was dependent upon the concentration of negatively charged groups on the surface. Calculation of the molar ratios between beta 2-glycoprotein I and sulfate residues in dextran sulfate showed that beta 2-glycoprotein I had to be present in excess of a 1:1 stoichiometric ratio of the sulfate group in order to inhibit the activation. 4. beta 2-Glycoprotein I does not inhibit the initiation of the contact system in plasma accomplished by sulfatide, unless the sulfatide has been preincubated with beta 2-glycoprotein I.

In vitro activation of the contact activation system (Hageman factor system) in plasma by acidic phospholipids and the inhibitory effect of β2-glycoprotein I on this activation

1. Negatively charged phospholipids promote initiation of the contact activation system in the blood coagulation. 2. Neutral phospholipids were unable to activate this system. 3. The activation is inhibited by beta 2-glycoprotein I at physiological concentrations. 4. The results raise the question whether people with low concentration of beta 2-glycoprotein I are more easily exposed to blood coagulation defects, such as disseminated intravascular coagulation, than those with normal concentration of beta 2-glycoprotein I.

Characterization of the interaction between β2-glycoprotein i and mitochondria, platelets, liposomes and bile acids

Abstract 1. 1. Several different binding mechanisms appear to be involved in the binding of β2-glycoprotein I to biological membranes. 2. 2. One of these mechanisms is a hydrophilic interaction between negatively-charged phospholipids in the membrane and histidine residues in β2-glycoprotein I. This mechanism seems to be involved in binding of the protein to mitochondria but not to platelets. 3. 3. Another mechanism may involve a site on β2-glycoprotein I, which binds to the steroid ring system particularly to such steroids not having a 7-hydroxy group. This type of binding may be involved in the interaction between β2-glycoprotein I and platelets as well as mitochondria.