The calcium-sensitive large-conductance potassium channel (BK/MAXI K) is present in the inner mitochondrial membrane of rat brain

Large-conductance voltage- and calcium-sensitive channels are known to be expressed in the plasmalemma of central neurons; however, recent data suggest that large-conductance voltage- and calcium-sensitive channels may also be present in mitochondrial membranes. To determine the subcellular localization and distribution of large-conductance voltage- and calcium-sensitive channels, rat brain fractions obtained by Ficoll-sucrose density gradient centrifugation were examined by Western blotting, immunocytochemistry and immuno-gold electron microscopy. Immunoblotting studies demonstrated the presence of a consistent signal for the alpha subunit of the large-conductance voltage- and calcium-sensitive channel in the mitochondrial fraction. Double-labeling immunofluorescence also demonstrated that large-conductance voltage- and calcium-sensitive channels are present in mitochondria and co-localize with mitochondrial-specific proteins such as the translocase of the inner membrane 23, adenine nucleotide translocator, cytochrome c oxidase or complex IV-subunit 1 and the inner mitochondrial membrane protein but do not co-localize with calnexin, an endoplasmic reticulum marker. Western blotting of discrete subcellular fractions demonstrated that cytochrome c oxidase or complex IV-subunit 1 was only expressed in the mitochondrial fraction whereas actin, acetylcholinesterase, cadherins, calnexin, 58 kDa Golgi protein, lactate dehydrogenase and microtubule-associated protein 1 were not, demonstrating the purity of the mitochondrial fraction. Electron microscopic examination of the mitochondrial pellet demonstrated gold particle labeling within mitochondria, indicative of the presence of large-conductance voltage- and calcium-sensitive channels in the inner mitochondrial membrane. These studies provide concrete morphological evidence for the existence of large-conductance voltage- and calcium-sensitive channels in mitochondria: our findings corroborate the recent electrophysiological evidence of mitochondrial large-conductance voltage- and calcium-sensitive channels in glioma and cardiac cells.     

Morphology of mitochondrial permeability transition: morphometric volumetry in apoptotic cells

Here we report on the mitochondrial permeability transition (MPT), which refers to the morphology of mitochondria whose inner membrane has lost its selective permeability. In all types of apoptotic cells so far examined, we found outer mitochondrial membranes that had been ruptured. These mitochondria present a swollen matrix covered by an inner membrane herniating into the cytoplasm through the breached outer membrane. Similarly ruptured outer mitochondrial membranes have been reported in studies on mitochondrial fractions induced to undergo MPT, carried out by others. Our observations were made on five types of rat tissue cells and six different cultured cell lines in the early stages of apoptosis. Samples from the cell lines HL-60, HeLa, WEHI-164, and a special batch of PC-12 cells were subjected to various apoptogenic agents and analyzed morphometrically. Nonapoptotic companion cells with unaltered nuclear structure (CUNS) were also analyzed. The mitochondrial volume in microm(3) and the volume fraction of the cytoplasm occupied by mitochondria in cells with typical nuclear signs of apoptosis and also in CUNS were evaluated. The volume of the mitochondria with ruptured membrane represents at least 69% (47-89%) of the total mitochondrial volume of the apoptotic cells. Thus, a considerable fraction of the cellular mitochondrial mass is or was in the state of permeability transition and probably involved in enhancement of the apoptotic program. In all samples, a fraction of the cells with normal nuclei possessed mitochondria with breached outer membranes as described above. In these cells, MPT occurred before the appearance of the typical nuclear phenotype of the apoptotic cells.     

Studies of hepatic mitochondrial structure and function: morphometric and biochemical evaluation of in vivo perturbation by arsenate.

Ultrastructural morphometric and biochemical studies were conducted on hepatic mitochondria from control rats and rats treated in vivo with arsenate to examine changes in interrelationships between mitochondrial structure and biochemical functions. Morphometric analysis disclosed an over-all 1.2-fold increase in the relative mitochondrial volume density and 1.4-fold increase in the surface density of the inner mitochondrial membrane of arsenate-exposed rats. These structural changes were associated with a 1.5-fold increase in 14C-leucine incorporation into all mitochondrial proteins, which was primarily associated with the acid-insoluble membranous fraction. Mitochondria from arsenate-treated rats showed a marked disruption of normal conformational behavior with depression of nicotinamide adenine dinucleotide (NAD)-linked substrate oxidation and a resulting in vivo increase in the mitochondrial [NAD] to [NADH] ratio. Observed changes in mitochondrial membranes from arsenate exposure also resulted in 1.5- to 2-fold increases in the specific activities of the membrane marker enzymes monoamine oxidase, cytochrome oxidase, and Mg2+-ATPase. Activity of malate dehydrogenase, which is localized in the mitochondrial matrix, was unchanged. The results of this study demonstrate a positive quantitative in vivo correlation between mitochondrial structure and function and indicate a marked dependency upon membrane integrity for normal maintenance of the specific biologic activities performed by this organelle in vivo.     

Exocrine cell mitochondria of the rat pancreas after lead intoxication.

The aim of the study was to evaluate alterations in exocrine cell mitochondria of the rat pancreas after lead acetate intoxication. The experiment used 45 rats divided into 2 experimental groups receiving lead acetate to drink, of lead concentration 50 and 500 mg/dm3 (ppm), and a control group given tap water. The animals from the experimental group were decapitated after 2, 4, 6 and 8 weeks, 5 rats from the control group after 8 weeks of the experiment. Rats from experimental groups decapitated after 8 weeks had lead administration stopped after six weeks and then, for two weeks tap water was given. Pancreatic sections were examined with biochemical methods for the activity of cytochrome oxidase and succinic dehydrogenase. Ultrastructural and morphometric examinations were also performed. It was demonstrated that: a) exocrine cell mitochondria are particularly predisposed to lead effect, b) intoxication of rats with lower lead doses (50 ppm) causes reversible adaptative or compensatory changes in these organelles, c) intoxication of rats with higher lead doses (500 ppm) induces irreversible ultrastructural alterations in numerous mitochondria, including damage to inner and to outer mitochondrial membranes, d) structural changes in the mitochondria in the course of lead intoxication are the morphological expression of the impairment of metabolic processes, associated with the inhibited activity of the respiratory enzymes: succinic dehydrogenase and cytochrome oxidase.     

Low level Pb(2+) exposure affects hippocampal protein kinase C gamma gene and protein expression in rats

We examined the effect of chronic exposure to lead (Pb(2+)) on protein kinase C (PKC) in 50-day-old rat hippocampus. Cytosolic and membrane fractions of hippocampus from Pb(2+)-exposed rats showed reduced expression of PKC gamma protein. In contrast, a significant elevation of PKC gamma mRNA was observed in pyramidal and dentate granule cell layers. Protein expression of alpha, beta I, beta II and epsilon isoenzymes were unchanged in Pb(2+)-exposed rats, as was [(3)H]phorbol 12,13 dibutyrate (PDBu) binding in tissue slices. Differences were not observed in Ca(2+)-dependent or -independent PKC activity, or in PKC-specific back-phosphorylation of hippocampal homogenates from Pb(2+)-exposed rats. Reduced subcellular levels of PKC gamma in Pb(2+)-exposed rats suggest that signal transduction in the hippocampus may be selectively altered and may be important in manifesting Pb(2+)-induced impairments of synaptic plasticity, learning and memory.     

Stereology of lavaged populations of alveolar macrophages: effects of in vivo exposure to tobacco smoke

A multi-level morphometric and stereologic study was performed on alveolar macrophages obtained by bronchopulmonary lavage of unexposed control rats and rats experimentally exposed to whole tobacco cigarette smoke for 90 consecutive days. Measurements of macrophage profile diameters made at the light microscope were used to derive cell size distribution curves, with a mean cell volume in the control group of 623 μm³. At the electron microscope level, the volume density and numerical density of mitochondria in unexposed control cells were lower than the values reported for peritoneal macrophages, but the mean volume of individual mitochondria was five times higher in the alveolar than in the peritoneal macrophage. The volume density of lysosomes was slightly higher in alveolar than in peritoneal macrophages. Exposure to smoke resulted in changes in several subcellular compartments, including a 2-fold increase in mean cell volume, a 19-fold increase in the volume density of lipid inclusions, an increase in the surface density of mitochondrial inner membrane and a decrease in the surface density of rough endoplasmic reticulum. The significance of these morphologic changes in terms of cell function is discussed.     

Inverse correlation between expression of the Wolfs Hirschhorn candidate gene Letm1 and mitochondrial volume in C. elegans and in mammalian cells

Deletion of the Letm1 gene correlates with the occurrence of epilepsy in patients with Wolf-Hirschhorn syndrome. The Letm1 gene encodes a mitochondrial protein that is homologous to yeast Mdm38. Yeast Mdm38 is localized to the mitochondrial inner membrane where it was proposed to act as a K+/H+ antiporter or alternatively as a chaperone for selected mitochondrial inner membrane proteins. Here, we present cellular and biochemical analysis of Letm1 in mammalian cells and an analysis of a C. elegans mutant that could serve as a model for Wolf-Hirschhorn syndrome. We localized the Letm1 protein to the mitochondrial inner membrane of mammalian cells, where it exists in a 550-kDa complex. We show that Letm1 can bind to itself in vitro, raising the possibility that it can form higher order multimers in vivo. Reduced levels of Letm1 in human cells and in C. elegans lead to swellings along the lengths of mitochondria, consistent with the phenotype observed in yeast. Electron micrographs show mitochondria with swollen matrices that are less electron-dense than matrices in normal mitochondria. The opposite effect is achieved by overexpression of Letm1. Overexpression increases the electron density of the mitochondrial matrix and swelling of cristae. Our results are therefore consistent with a protein that regulates the volume of the mitochondrial matrix.     

Ultrastructure of hepatic mitochondria in a child with hyperornithinemia,hyperammonemia, and homocitrullinuria

Ultrastructural studies of hepatic tissue obtained at biopsy from a nine year old severely retarded boy with hyperornithinemia, hyperammonemia, and homocitrullinuria showed mitochondria of bizarre shapes and unusual internal features. Among the latter were tubules extending throughout the length of the large mitochondria that on cross section had a rosette-like arrangement; the presence of a periodic, approximately 300thick, sievelike membrane interposed between the tubules and the inner mitochondrial membrane; and “bulges” of mitochondrial matrix occasionally formed between these two membranes.Since to be metabolized ornithine must enter the mitochondria, the hyperornithinemia is regarded as a reflection of its inability to reach the mitochondrial interior. It is speculated that among other possible causes, the unusual sievelike membrane may be the barrier to ornithine's access to the mitochondrion.     

The effects of antibodies on cells: III. Studies on the interaction of rat liver mitochondria and lysosomes with antibody and complement

Complement-fixing and precipitating antibodies to mitochondrial and lysosomal fractions of rat liver were induced in rabbits. The incubation of these subcellular fractions with ferritin-labelled antibody revealed localization of the antibody on mitochondria and lysosomes, but no specific morphological effect of antibody and complement. Incubation of the cell fractions with antibody and complement did not impair the efficiency of mitochondrial oxidative phosphorylation or affect the latency and sedimentation characteristics of lysosomal acid phosphatase. The results are discussed in relation to the action of antibody and complement on cells and to possible mechanisms of tissue damage in auto-immunity.     

Books received

Subunit c is normally present as an inner mitochondrial membrane component of the F₀ sector of the ATP synthase complex, but in the late infantile form of neuronal ceroid-lipofuscinosis (NCL) it was also found in lysosomes in high concentrations. The rate of degradation of subunit c as measured by pulse–chase and immunoprecipitation showed a marked delay of degradation in patients' fibroblasts with late infantile form of NCL. There were no significant differences between control cells and cells with disease in the degradation of cytochrome oxidase subunit IV, an inner membrane protein of mitochondria. Measurement of labeled subunit c in mitochondrial and lysosomal fractions showed that the accumulation of labeled subunit c in the mitochondrial fraction can be detected before lysosomal appearance of radioactive subunit c, suggesting that subunit c accumulated as a consequence of abnormal catabolism in the mitochondrion and is transferred to lysosomes through an autophagic process. The biosynthetic rate of subunit c and mRNA levels for P1 and P2 genes that code for it were almost the same in both control and patient cells. These findings suggest that a specific failure in the degradation of subunit c after its normal inclusion in mitochondria and its consequent accumulation in lysosomes. © 1995 Wiley-Liss, Inc.     

The transplacental toxicity of methyl mercury to fetal rat liver mitochondria. Morphometric and biochemical studies.

Ultrastructural morphometric and biochemical changes in liver mitochondria of fetal rats whose mothers were exposed to methyl mercury hydroxide in their drinking water at concentrations of 0, 3, 5, or 10 p.p.m. for 4 weeks prior to mating and through day 19 of pregnancy are described. A dose-related decrease in the volume density of mitochondria was observed in fetal hepatocytes of dams given the 5- and 10-p.p.m. dose levels. This finding was associated with decreased mitochondrial protein synthesis, which appeared to result primarily from decreased synthesis of mitochondrial structural proteins. Loss of respiratory control was observed in mitochondria from animals in the 3-p.p.m. dose group whereas state 3 respiration was abolished in animals exposed to the 5- and 10-p.p.m. dose levels. The specific activities of monoamine oxidase and cytochrome oxidase, outer and inner mitochondrial membrane marker enzymes respectively, showed dose-related decreases of up to 62 and 78 per cent of control, respectively. delta-Aminolevulinic acid synthetase, which is loosely bound to the inner mitochondrial membrane, also showed dose-related decreases of up to 68 per cent of control. Malate dehydrogenase, a mitochondrial matrix marker enzyme, showed no change in activity at any dose level tested. These observations were correlated with dose-related tissue concentrations of methyl and inormpaired mitochondrial biogenesis and functional development is one basis for explaining the sensitivity of fetal animals to methyl mercury toxicity.     

Glutamine transport by mitochondria isolated from normal and acidotic rats.

The transport of L-glutamine by isolated rat renal mitochondria was studied by means of a rapid-filtration (Millipore Filter Corp.) technique. The movement of glutamine from the incubation medium into the inner mitochondrial compartment (matrix) was inhibited by structural analogues (6-diazo-5-oxo-L-norleucine and glutamic acid), sulghydryl-binding agents (p-chloromercuri-benzoate and mersalyl), and inhibitors of mitochondrial oxidative metabolism (azide, antimycin A, and uncouplers of oxidative phosphorylation). These results suggest that glutamine is transported across the inner membrane of renal mitochondria by a carrier-mediated system that is linked to the processes of oxidative metabolism. The transport of glutamine by isolated renal mitochondria was increased two- to threefold by chronic (5-7 days) metabolic acidosis. However, short-term metabolic acidosis did not increase the glutamine transport capacity of isolated mitochondria. A hypothesis is presented for the regulation of mitochondrial glutamine transport, in vivo, during short-term and chronic acidosis.     

The localization of fumarase and malic enzyme in muscle mitochondria of Ascaris suum

The distribution of fumarase and malic enzyme in Ascaris suum muscle mitochondria was investigated by employing digitonin fractionation techniques. The ability of these procedures to resolve the various submitochondrial fractions (intracristal space, inner membrane, matrix and inner membrane-matrix particles) was verified by electron microscopy and the distribution of appropriate marker enzymes. From the data obtained, it is concluded that fumarase in Ascaris muscle mitochondria is located solely within the matrix compartment and is not present within the intracristal space as reported earlier by other authors (Rew and Saz (1974) J. Cell. Biol. 63, 125-135). In agreement with previous findings malic enzyme in the nematode organelle appears to be associated with both soluble compartments. The implications of these findings to the parasites' mitochondrial metabolism are discussed.     

Comparison of lymphoblast mitochondria from normal subjects and patients with Barth syndrome using electron microscopic tomography

Barth syndrome (BTHS) is a mitochondrial disorder that is caused by mutations in the tafazzin gene, which affects phospholipid composition. To determine whether this defect leads to alterations in the internal three-dimensional organization of mitochondrial membranes, we applied electron microscopic tomography to lymphoblast mitochondria from BTHS patients and controls. Tomograms were formed from 50 and 150 nm sections of chemically fixed lymphoblasts and the data were used to manually segment volumes of relevant structural details. Normal lymphoblast mitochondria contained well-aligned, lamellar cristae with slot-like junctions to the inner boundary membrane. In BTHS, mitochondrial size was more variable and the total mitochondrial volume per cell increased mainly due to clusters of fragmented mitochondria inside nuclear invaginations. However, mitochondria showed reduced cristae density, less cristae alignment, and inhomogeneous cristae distribution. Three-dimensional reconstruction of BTHS mitochondria revealed zones of adhesion of the opposing inner membranes, causing obliteration of the intracrista space. We found small isolated patches of adhesion as well as extended adhesion zones, resulting in sheets of collapsed cristae packaged in multiple concentric layers. We also found large tubular structures (diameter 30-150 nm) that appeared to be derivatives of the adhesion zones. The data suggest that mitochondrial abnormalities of BTHS involve adhesions of inner mitochondrial membranes with subsequent collapse of the intracristae space.     

Ultrastructural Changes in Cardiomyocyte Mitochondria during Regenerative and Plastic Insufficiency of the Myocardium

Daunomycin-induced regenerative and plastic insufficiency of the myocardium was accompanied by accumulation of cardiomyocytes with unstable mitochondrial membranes containing enlarged mitochondria with lightened matrix and fragmented cristae. Total destabilization of mitochondrial membranes was found in cardiomyocytes with most pronounced ultrastructural signs of impaired protein synthesis. These changes in mitochondria were permanent, which suggested that swelling and destruction of cristae were related to intravital decrease in mitochondrial membrane stability.     

Ultrastructural and biochemical studies of the brain and other organs in rat after chronic ethanol administration. III. Influence of ethanol intoxication on oxidative phosphorylation of the rat brain mitochondria with ultrastructural and morphometric evaluation of mitochondrial fraction)

The effect of chronic ethanol intoxication on oxidative phosphorylation in the rat brain mitochondrial fraction was examined. Moreover, electron microscopy was used to verify the quantitative composition of the fraction and for examination of ultrastructural changes in the mitochondria. The experiments were carried out with 60 rats receiving, beside the normal diet, ethyl alcohol according to a modified RATCLIFFE model. In isolated rat brain mitochondria the NAD-dependent oxidation of substrates (glutamate + malate) was decreased. The phosphorylation index ADP/0 and the respiratory control ratio (RCR) in rat brain mitochondria from ethanol-treated rats were unchanged in the presence of both succinate and glutamate + malate. Chronic ethanol feeding did not induce any changes of succinate dehydrogenase and cytochrome oxidase activities in solubilised mitochondria fractions of rat brain. Electron microscopy studies revealed that mitochondria from control animals retained their outer and inner membranes, whereas those from rats given ethanol were almost always swollen and some were disrupted. In mitochondrial fractions isolated from ethanol-intoxicated rats an increase was observed of contaminating elements i.e. axons and synaptosomes of various sizes. It should be stressed that the mitochondria located inside synaptosomes and axons were unchanged. The composition of the fractions was quantitatively evaluated and confirmed the diminution of "free" mitochondria in the experimental fractions in favour of "bound" mitochondria which mainly occurred in the synaptosomes with preserved metabolic activity. On the basis of electron microscopy studies it could be suggested that ethanol intoxication causes the damage of some mitochondria, which become more sensitive to mechanical destruction during isolation procedure, and they do not sediment together with the fraction of normal ones. The absence of "free" mitochondria in pellets explains the spurious lack of disturbances in the energy metabolism of brain mitochondria after chronic ethanol intoxication.     

Age-related alterations in cultured human fibroblast membrane structure and function

Membrane enzyme activities, lipid composition, and fluorescence probe characteristics in isolated plasma membranes, microsomes and mitochondria of cultured human fibroblasts were used to determine if structural alterations occurred as a function of donor age. The cells were sex matched and allowed to undergo approximately 8 population doublings under identical culture conditions. Plasma membrane (Na⁺,K⁺)-ATPase, microsomal NADPH cytochrome c reductase, and mitochondrial succinate cytochrome c activities showed variation as a function of increasing donor age but these changes were not statistically significant. At the same time the cholesterol/phospholipid molar ratio was unaltered in plasma membranes, decreased 50% in microsomes, and unchanged in mitochondria with increasing donor age. The phosphatidylcholine/phosphatidylethanolamine ratio increased in all three membrane fractions with increasing age of the fibroblast donor. The ratio of unsaturated/saturated fatty acids decreased in the phospholipids of microsomes but not of plasma membranes or mitochondria. The structural properties of the membranes were determined with two different fluorescence probe molecules, trans-parinaric acid and 1,6-diphenyl-1,3,5-hexatriene. These probe molecules indicated that the fluorescence lifetime and/or fluorescence polarization of the trans-parinaric acid probe decreased in microsomes, mitochondria, and in the plasma membrane, such that the limiting anisotropy, indicative of restrictions to probe motions, was significantly lower (high fluidity) with increasing subject age in plasma membranes, microsomes and mitochondria. The trans-parinaric acid fluorescence lifetime displayed two components in plasma membranes, microsomes, and mitochondria, a finding consistent with the co-existence of fluid and solid membrane lipid areas in the cultured human fibroblast subcellular membranes. The trans-parinaric acid partitioned preferentially into solid membrane     

Translocation of Protein Kinase C to Subcellular Fractions of Human Neutrophils

The subcellular localization of protein kinase C in unstimulated human neutrophils and neutrophils stimulated by phorbol-myristate-acetate (PMA), 1-oleoyl-2-acetyl-rac-glycerol (OAG), and ionomycin was investigated in subcellular fractions obtained by nitrogen cavitation and Percoll density gradient centrifugation. Protein kinase C was found to be localized mainly in the cytosol in unstimulated cells, whereas significant translocation to fractions containing the plasma membrane was observed after stimulation by PMA, OAG, and ionomycin. At the same time, phospholipid-insensitive protein kinase activity appeared in the cytosol and the plasma membrane fractions. To determine whether binding of protein kinase C occurred to the plasma membrane or to intracellular membranes that had translocated to the plasma membrane, we investigated the ability of isolated azurophil, specific and secretory granules, and plasma membrane vesicles to bind protein kinase C in response to addition of PMA and OAG. Only fractions containing plasma membranes and secretory granules were able to bind protein kinase C. The observation explains the selective activation of plasma membrane structures by protein kinase C.     

Reconstitution of brain mitochondria inner membrane into planar lipid bilayer

Ion channels are present in the inner mitochondrial membrane. They play an important role in cellular processes. Potassium and chloride channels are involved in regulation of mitochondrial volume, membrane potential and acidification. The mitochondrial potassium channels have been suggested as triggers and end effectors in cytoprotection. In our study we measured single channel activities after reconstitution of submitochondrial particles from rat brain mitochondria into planar lipid membranes. After incorporation, two different potassium selective currents were recorded with single channel conductance from 260 to 320 pS and from 70 to 90 pS in gradient (cis/trans) 50/450 and 50/150 mM KCl solutions, respectively. We also observed activity of the chloride ion channel. The measured single channel conductance was from 80 to 90 pS in gradient (cis/trans) 50/450 mM KCl solution. Our results suggest that various ion channels are present in the inner mitochondrial membrane of brain mitochondria.     

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.