Gerd Svenneby

UPTAKE AND METABOLISM OF GLUTAMATE IN ASTROCYTES CULTURED FROM DISSOCIATED MOUSE BRAIN HEMISPHERES

Abstract— Uptake kinetics of l‐glutamate in cultured, normal glia cells obtained from the brain hemispheres of newborn mice were measured together with the activities of the glutamate metabolizing enzymes, glutamic‐oxaloacetate‐transaminase, glutamate dehydrogenase and glutamine synthetase. During 3 weeks of culturing, the activities of the enzymes rose from low neonatal values toward the levels in the adult brain (206, 12.3 and 25.9 nmol. min−1. mg−1 cell protein for the three enzymes, respectively). The uptake kinetics indicated an unsaturable component together with an uptake following Michaelis‐Menten kinetics with a Km of 220 μm and a Vmax of 7.9 nmol. min−1. mg−1 cell protein. The saturable glutamate uptake was inhibited by d‐glutamate, l‐aspartate and α‐aminoadipate whereas l‐glutamine, GABA and glutarate had no effect. The uptake which was Ca2+‐independent had a Km for sodium of 18mm and it was stimulated by an increase in the external potassium concentration from 5 to 10 and 25 mm. The results suggest that glia cells are important for the uptake of glutamate from synaptic clefts and for the subsequent metabolism of glutamate.

Uptake and metabolism of GABA in astrocytes cultured from dissociated mouse brain hemispheres.

Uptake kinetics and contents of GABA in cultured, normal (i.e. nontransformed) glia cells obtained from the brain hemispheres of newborn mice were measured together with the activity of the GABA transaminase. During three weeks of culturing the activity of the transaminase rose from a low neonatal value toward the level in the adult brain. The uptake kinetics indicated an unsaturable component together with an uptake following Michaelis-Menten kinetics. Both theKm (40 μM) and theVmax (0.350 nmol×min−1×mg−1 cell protein) were reasonably comparable to the corresponding values in brain slices, and theVmax was much higher than that reported for other glial preparations. The GABA content was low (<5 nmol/mg cell protein), which is in agreement with the high activity of the GABA transaminase.

PHOSPHATE ACTIVATED GLUTAMINASE ACTIVITY AND GLUTAMINE UPTAKE IN PRIMARY CULTURES OF ASTROCYTES

Abstract— Uptake and release of glutamine were measured in primary cultures of astrocytes together with the activity of the phosphate activated glutaminase (EC 3.5.1.2). In contrast to previous findings of an effective, high affinity uptake of other amino acids (e.g. glutamate, GABA) no such uptake of glutamine was observed, though a saturable, concentrative uptake mechanism did exist (Km= 3.3 ± 0.5 mm; Vmax= 50.2 ± 12.6 nmol ± min−1± mg−1). The phosphate activated glutaminase activity in the astrocytes (6.9 ± 0.9 nmol ± min−1± mg−1) was similar to the activity found in whole brain (5.4 ± 0.7 nmol ± min −l± mg−1), which may contrast with previous findings of a higher activity of the glutamine synthetase (EC 6.3.1.2) in astrocytes than in whole brain. The observations are compatible with the hypothesis of an in vivo flow of glutamate (and GABA) from neurons to astrocytes where it is taken up and metabolized, and a compensatory flow of glutamine towards neurons and away from astrocytes although the latter cell type may be more deeply involved in glutamine metabolism than envisaged in the hypothesis.

Ontogenetic development of glutamate and gaba metabolizing enzymes in cultured cerebral cortex interneurons and in cerebral cortex in vivo

The development of the enzymes phosphate activated glutaminase (PAG), glutamate dehydrogenase (GLDH), glutamic‐oxaloacetic‐transaminase (GOT), glutamine synthetase (GS), GABA‐transaminase (GABA‐T) and ornithine‐δ‐aminotransferase (Orn‐T) was followed in mouse cerebral cortex in vivo and in cultured mouse cerebral cortex interneurons. It was found that GLDH, GOT and Orn‐T exhibited an enhanced developmental pattern in the cultured neurons compared to cerebral cortex. The activities of PAG and GABA‐T developed in parallel in vivo and in culture but the activity of GS remained low in the cultured neurons compared to the increasing activity of this enzyme found in vivo. Compared to cerebral cortex the cultured neurons exhibited higher activities of PAG, GLDH and Orn‐T, whereas the activities of GABA‐T and GOT were lower in the cultured cells. The activity of GS in the cultured neurons was only 5–10% of the activity in cerebral cortex in vivo. It is concluded that neurons from cerebral cortex represent a reliable model system by which the metabolism and function of GABAergic neurons can be conveniently studied in a physiologically meaningful way.

Ontogenetic development of glutamate metabolizing enzymes in cultured cerebellar granule cells and in cerebellumin vivo

The ontogenetic development of the enzymes phosphate activated glutaminase (PAG), glutamate dehydrogenase (GLDH), glutamic-oxaloacetic-transaminase (GOT), glutamine synthetase (GS), and ornithine-δ-aminotransferase (Orn-T) was followed in cerebellum in vivo and in cultured cerebellar granule cells. It was found that PAG, GLDH, and GOT exhibited similar developmental patterns in the cultured neurons compared to cerebellum. PAG showed, however, a more pronounced phosphate activation in the cultured granule cells compared to in vivo. The activity of GS remained low in the cultured neurons compared to the increasing activity of this enzyme found in vivo. On the other hand Orn-T exhibited an increase in its specific activity in the cultured cells as a function of time in culture in contrast to the non-changing activity of this enzyme in vivo. Compared to cerebellum the cultured neurons exhibited higher activities of GLDH, GOT, and Orn-T whereas the activity of PAG was only slightly higher in the cultured cells. The activity of GS in the cultured neurons was only 5–10% of the activity in cerebellum in vivo. It is concluded that cultured cerebellar granule cells represent a reliable model system by which the metabolism and function of glutamatergic neurons can be conveniently studied in a physiologically meaningful way.

Properties of phosphate activated glutaminase in astrocytes cultured from mouse brain

Astrocytes in primary cultures contain a relatively high activity, of phosphate activated glutaminase, although it is significantly lower than that of synaptosomal enriched preparations. The relatively high glutaminase activity in the astrocytes appears not to be caused by substrate induction, since a 10-fold variation in the glutamine concentration of the culture medium does not affect the activity. Of the reaction products, only glutamate inhibits astrocytic glutaminase whereas that of synaptosomal enriched preparations is inhibited by both glutamate and ammonia. Similar to the synaptosomal enzyme, glutaminase in astrocytes is inhibited about 50% by N-ethylmaleimide, indicating N-ethylmaleimide-sensitive and-insensitive compartments of the enzyme. Calcium activates glutaminase in astrocytes as in synaptosomes, by promoting phosphate activation. Except for the lower activity and the lack of effect of ammonia, the properties of the astroglial glutaminase has been found to be no different from that of the synaptosomal one. The relatively unrestrained astroglial glutaminase may, however, argue against the concept of a glutamine cycle operating in a stoichiometric manner.

Glutaminase in neurons and astrocytes cultured from mouse brain: Kinetic properties and effects of phosphate, glutamate, and ammonia

Phosphate activated glutaminase comprises two kinetically distinguishable enzyme forms in cultures of cerebellar granule cells, of cortical neurons and of astrocytes. Specific activity of glutaminase is higher in cultured neurons compared with astrocytes. Glutaminase is activated by phosphate in all cell types investigated, however, glutaminase in astrocytes reguires a much higher concentration of phosphate for half maximal activation. One of the products, glutamate, inhibits the enzyme strongly, whereas the other product ammonia has only a slight inhibitory action on the enzyme.

Absence of preferential glutamine uptake into neurons — an indication of a net transfer of TCA constituents from nerve endings to astrocytes?

Uptake kinetics for glutamine were studied in several different neuronal preparations (perikarya prepared by gradient centrifugation, cultured cortical neurons, cultured, presumably glutamatergic cerebellar neurons, and brain prisms). In no case were any indications found of a high affinity uptake but a rather efficient low affinity uptake did occur. A similar, equally efficient low affinity uptake is, however, found in astrocytes. Thus, no preferential glutamine uptake occurs into neurons. It is, therefore, not likely that a net flow of glutamine takes place from astrocytes to neurons, compensating for the loss of TCA constituents when glutamate and GABA are released.

[30] Glutaminase from mammalian tissues

Publisher Summary This chapter provides an overview of glutaminase from mammalian tissues. The mitochondrial phosphate activated glutaminase (PAG), which is the most important glutaminase in mammalian tissues, is described. Measurement of the reaction products ammonia or glutamate may be used for assay of PAG. Measurement of ammonia suffers from two disadvantages—the great water solubility of this compound makes it difficult to keep the background values sufficiently low and some undissociated ammonia may escape, particularly at prolonged incubations at 37 ° when the pH exceeds 8.0. Ammonia can be monitored, for example, with Nesslers reagent after microdistillation, by the ammonia electrode, or by coupling to the glutamate dehydrogenase (GDH) reaction, whereby NADH oxidation is measured. The two latter methods permit measurement of enzyme rates. The assay of PAG in isolated mitochondria, synaptosomes, cultured neurons, granulocytes, and astrocytes is reviewed in the chapter. Glutamate formed in a prefixed time is assayed following incubation of the cellular material with L-glutamine (preferably in the physiological concentration range), two concentrations of phosphate (to ensure that PAG is assayed), and inhibitors to prevent the metabolism of glutamate.

Purification of phosphate-dependent pig brain glutaminase.

Abstract— A procedure for preparing highly purified phosphate‐activated glutaminase (EC 3.5.1.2, L‐glutamine amidohydrolase) from pig brain is described. The main steps consist of extraction with acetone, followed by sodium sulphate fractionation of the solubilized acetone powder. Thereafter, solubilization by dialysis against a buffer containing tris‐HC1, mercaptoethanol, and EDTA, followed by precipitation with phosphate‐borate, is repeated twice. The final preparation contains no impurities which can be detected by polyacrylamide gel electrophoresis, isoelectric focusing, and sedimentation equilibrium centrifugation. By the latter method, molecular weight is determined to be 187,000. By polyacrylamide gel electrophoresis in sodium dodecyl sulphate, one protein band with molecular weight 64,000 is found.