Ingeborg Aasland Torgner

Postembedding immunogold labelling reveals subcellular localization and pathway-specific enrichment of phosphate activated glutaminase in rat cerebellum

Phosphate activated glutaminase is a key enzyme in glutamate synthesis. Here we have employed a quantitative and high-resolution immunogold procedure to analyse the cellular and subcellular expression of this enzyme in the cerebellar cortex. Three main issues were addressed. First, is phosphate activated glutaminase exclusively or predominantly a mitochondrial enzyme, as biochemical data suggest? Second, to what extent is the mitochondrial content of glutaminase dependent on cell type and transmitter identity? Third, can individual neurons maintain a subcellular segregation of mitochondria with different glutaminase content? An attempt was also made to disclose the intramitochondrial localization of glutaminase, and to correlate the content of this enzyme with that of glutamate and glutamine in the same mitochondria (by use of triple labelling). Antisera to the N- and C-termini of glutaminase revealed strong labelling of the putatively glutamatergic mossy fibre terminals. The vast majority of gold particles (approximately 96%) was associated with the mitochondria. Equally high labelling intensities were found in mitochondria of perikarya and dendrites in the pontine nuclei, a major source of mossy fibres. The level of glutaminase immunoreactivity in parallel and climbing fibres (which like the mossy fibres are thought to use glutamate as transmitter) was only about 20% of that in mossy fibres, and similar to that in non-glutamatergic neurons (Purkinje and Golgi cells). Glial cell mitochondria were devoid of specific glutaminase labelling and revealed a much lower glutamate:glutamine ratio than did the mitochondria of mossy fibres. As to the submitochondrial localization of glutaminase, immunogold particles were often found to be aligned with the cristae, suggesting an association of the enzyme with the inner mitochondrial membrane. However, the existence of a glutaminase pool in the mitochondrial matrix could not be excluded. The outer mitochondrial membrane was unlabelled. The present study provides quantitative evidence for a substantial heterogeneity in the mitochondrial content of glutaminase. This heterogeneity applies not only to neurons with different transmitter signatures, but also to different categories of glutamatergic pathways. In terms of the routes involved, the synthesis of transmitter glutamate may be less uniform than previously expected.

Brain mitochondria contain aquaporin water channels: evidence for the expression of a short AQP9 isoform in the inner mitochondrial membrane

Aquaporins are a family of water channels found in animals, plants, and microorganisms. A subfamily of aquaporins, the aquaglyceroporins, are permeable for water as well as certain solutes such as glycerol, lactate, and urea. Here we show that the brain contains two isoforms of AQP9—an aquaglyceroporin with a particularly broad substrate specificity—and that the more prevalent of these isoforms is expressed in brain mitochondria. The mitochondrial AQP9 isoform is detected as an ∼25 kDa band in immunoblots. This isoform is likely to correspond to a new AQP9 mRNA that is obtained by alternative splicing and has a shorter ORF than the liver isoform. Subfractionation experiments and high‐resolution immunogold analyses revealed that this novel AQP9 isoform is enriched in mitochondrial inner membranes. AQP9 immunopositive mitochondria occurred in astrocytes throughout the brain and in a subpopulation of neurons in the substantia nigra, ventral tegmental area, and arcuate nucleus. In the latter structures, the AQP9 immunopositive mitochondria were located in neurons that were also immunopositive for tyrosine hydroxylase, as demonstrated by double labeling immunogold electron microscopy. Our findings suggest that mitochondrial AQP9 is a hallmark of astrocytes and midbrain dopaminergic neurons. In physiological conditions, the flux of lactate and other metabolites through AQP9 may confer an advantage by allowing the mitochondria to adjust to the metabolic status of the extramitochondrial cytoplasm. We hypothesize that the complement of mitochondrial AQP9 in dopaminergic neurons may relate to the vulnerability of these neurons in Parkinsons disease. Amiry‐Moghaddam, M., Lindland, H., Zelenin, S., Roberg, B. A., Gundersen, B. B., Petersen, P., Rinvik, R., Torgner, I. A., Ottersen, O. P. Brain mitochondria contain aquaporin water channels: evidence for the expression of a short AQP9 isoform in the inner mitochondrial membrane. FASEB J. 19, 1459–1467 (2005)

[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.

Increased expression of phosphate-activated glutaminase in hippocampal neurons in human mesial temporal lobe epilepsy

Patients with mesial temporal lobe epilepsy (MTLE) have increased basal concentrations of extracellular glutamate in the epileptogenic versus the non-epileptogenic hippocampus. Such elevated glutamate levels have been proposed to underlie the initiation and maintenance of recurrent seizures, and a key question is what causes the elevation of glutamate in MTLE. Here, we explore the possibility that neurons in the hippocampal formation contain higher levels of the glutamate synthesizing enzyme phosphate-activated glutaminase (PAG) in patients with MTLE versus patients with other forms of temporal lobe epilepsy (non-MTLE). Increased PAG immunoreactivity was recorded in subpopulations of surviving neurons in the MTLE hippocampal formation, particularly in CA1 and CA3 and in the polymorphic layer of the dentate gyrus. Immunogold analysis revealed that PAG was concentrated in mitochondria. Double-labeling experiments indicated a positive correlation between the mitochondrial contents of PAG protein and glutamate, as well as between PAG enzyme activity and PAG protein as determined by Western blots. These data suggest that the antibodies recognize an enzymatically active pool of PAG. Western blots and enzyme activity assays of hippocampal homogenates revealed no change in PAG between MTLE and non-MTLE, despite a greatly (>50%) reduced number of neurons in the MTLE hippocampal formation compared to non-MTLE. Thus, the MTLE hippocampal formation contains an increased concentration and activity of PAG per neuron compared to non-MTLE. This increase suggests an enhanced capacity for glutamate synthesis—a finding that might contribute to the disrupted glutamate homeostasis in MTLE.

Ultrastructure of pig renal glutaminase. Evidence for conformational changes during polymer formation

Abstract Highly purified pig renal glutaminase has been examined by electron microscopy. It is demonstrated that the Tris·HCl form of the enzyme contains cylindrical particles with a diameter of about 60 A and a length of about 82 A. Treatment of the Tris·HCl form of the enzyme with sodium dodecyl sulphate and mercapto-ethanol, followed by polyacrylamide gel electrophoresis in sodium dodecyl sulphate, shows that the enzyme contains two types of sub units with molecular weights of 53,000 and 61,000. In agreement with earlier data, it is further demonstrated that the phosphate form is a simple dimer of the Tris·HCl form. Evidence that major conformational changes are involved in the formation of large, helical polymers of the enzyme described earlier, is also presented.

Phosphate activated glutaminase is concentrated in mitochondria of sensory hair cells in rat inner ear: a high resolution immunogold study

Glutamate has been implicated in signal transmission between sensory hair cells and afferent fibers in the inner ear. However, the mechanisms responsible for glutamate replenishment in these cells are not known. Here we provide evidence that phosphate activated glutaminase, which is thought to be the predominant glutamate-synthesizing enzyme in the brain, is concentrated in all types of hair cell in the organ of Corti and vestibular epithelium. By use of two different antibodies (directed to the N and C terminus, respectively) it was shown that glutaminase is largely restricted to mitochondria and that part of the enzyme pool is associated with the inner membrane of this organelle. Quantitative analysis of immunogold labelled Lowicryl sections revealed that the level of glutaminase immunoreactivity in mitochondria of supporting cells is less than 15% of that in hair cell mitochondria. Using triple labelling for glutaminase, glutamate, and glutamine, evidence was provided of a positive correlation between the glutamate/glutamine ratio and the level of glutaminase immunoreactivity, suggesting that the glutaminase antibodies identify a functional enzyme pool. Our results strengthen the idea that glutamate is a hair cell transmitter and indicate that the sensory epithelia in the inner ear show a metabolic compartmentation analogous to that in the brain.

Phosphate-dependent effects of palmityl-CoA and stearyl-CoA on phosphate-activated pig brain and pig kidney glutaminase

Phosphate-activated glutaminase (EC 3.5.1.2) purified from pig kidney [l] and brain [2] , has been shown to be activated by low concentrations of Bromothymol Blue (< 0.05 mM) [ 1,3] , and inhibited by higher concentrations. The dye has, thus, a dual and unique effect on the enzyme. Furthermore, the activation is potentiated by phosphate, and phosphate also protects against inhibition by the dye. In a search for physiological compounds with similar properties to Bromothymol Blue, acetyl-CoA has been found to be an activator of glutaminase with I(, about 0.2 mM [4]. As the activation by acetyl-CoA is diminished by phosphate and other anions such as citrate, the mode of action is different from that of Bromothymol Blue. We show here that palmityl-CoA and stearyl-CoA in principle have similar properties to Bromothymol Blue.

Type I Inositol (1,4,5) Trisphosphate Receptor and Phosphate-Activated Glutaminase are Highly Enriched in Neurons as Compared to Glial Cells in Rat Neostriatum

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized histopathologically by neuritic plaques of accumulated β-amyloid and cytoskeletal abnormalities. Although a variety of pathological processes have been proposed, including exci-totoxic effects of glutamate and disturbances in Ca2+-homeostasis (1, 2), the mechanisms responsible for this condition remain essentially unknown. We have recently observed a significant decrease in IP3-receptor levels in post mortem brain samples from AD cases compared to matched control material (3). Interestingly, in the same samples, the levels of several other brain proteins including synaptophysin and phosphate-activated glutaminase (PAG), were unchanged.