GABA metabolism in cultured glial cells

GABA-transaminase has been characterized in cultured astrocytes. It is identical to the synaptosomal and perikaryal enzyme in terms of charge, molecular weight, and stability, but it differs in its affinity for GABA, which is much higher in the glial compartment. GABA-transaminase has been shown to be inducible by high GABA concentrations, which suggests that astrocytes have the possibility not only to transport GABA but also to metabolize the amino acid which is taken up.     

Structural and microspectrofluorometric studies on glial cells from the periventricular and arcuate nuclei of the rat hypothalamus

Summary Fluorescence spectroscopy and electron microscopic techniques have been employed to investigate a class of glial cells that is characterized by the presence in their cytoplasm of large fluorescing inclusions that stain with paraldehyde-fuchsin or chrome-hematoxyline-alum. In the periventricular nucleus the cells have been identified as a population of astrocytes whose inclusions emit an orange-red fluorescence. In the arcuate nucleus there are, in addition to an overwhelming majority of such astrocytes, also some microglial cells with similar characteristics. The ability of the latter to emit any kind of fluorescence has not yet been established. The fluorescence maximum of these astrocytic inclusions was found to be at 640 nm when excited at 405 nm. The data obtained suggest that the fluorescence observed is due to the presence of porphyrins in the astrocytic inclusions. In the majority of our electron microscopic pictures the inclusions lack a bounding membrane. By contrast, neuronal lipofuscin has an outer membrane. In cryostat sections, the lipofuscin emits a yellow fluorescence when excited at 400–410 Onm.     

Steroid metabolism and effects in central and peripheral glial cells.

Hormonal steroids participate in the control of a large number of functions of the central nervous system (CNS); recent data show that they may also intervene at the level of the peripheral nervous system (PNS). Both the CNS and the PNS metabolize endogenous as well as exogenous steroids; one of the major enzymatic system is represented by the 5alpha-reductase-3alpha-hydroxysteroid complex. This is a versatile system, since every steroid possessing the delta 4-3keto configuration (e.g., testosterone, progesterone, deoxycorticosterone) may be a substrate. High levels of 5alpha-reductase are found in the white matter of the CNS and in purified myelin. The observation that, in addition to neurons, glia may be a target for steroid action is an important recent finding. The effects of progesterone, testosterone, corticoids, and their respective 5alpha and 3alpha-5alpha derivatives on the expression of glial genes are presented and discussed. It has also been found that progesterone and/or its 5alpha-reduced metabolites increase the mRNA for the two major proteins of peripheral myelin, the glycoprotein Po and the peripheral myelin protein 22, in the sciatic nerve of normal and aged animals and in Schwann cells. The hypothesis has been put forward that glycoprotein Po might be under the control of progestagens acting mainly via the progesterone receptor, and that peripheral myelin protein 22 might be controlled via an interaction of steroids with the gamma-aminobutyric acid (GABA)ergic system. It is known that tetrahydroprogesterone, the 3alpha-5alpha-reduced metabolite of progesterone, interacts with the GABA(A) receptor. Our recent data show that several subunits of this receptor are present in sciatic nerve as well as in Schwann cells that reside in this nerve. These data open multiple possibilities for new therapeutic approaches to demyelinating diseases.     

Glycoprotein synthesis in the mucous cells of the vascularly perfused rat stomach. II. Differentiating mucous cells

Labeled leucine, serine, galactose, glucosamine and sulphate were administered to rat stomachs in a perfusion system. Sections of the gastric fundus were studied by light microscopic autoradiography. Five categories of mucous cells were distinguished and their glycoprotein synthetic activity was measured in autoradiographs by counting silver grains over each category. During their differentiation, while migrating from the isthmus of the fundic glands to the free luminal surface, the surface mucous cells (SMC) showed an increase in incorporation of all precursors used. Differences between the incorporation patterns of the various precursors, in cells of different ages, suggest that structural development runs ahead of functional activity, and that the latter continues up to the very moment the cell is shed from the surface. Sulphate was incorporated at a considerably lower rate by the SMC of the free surface than by the foveolar SMC, in which by cytochemical staining strongly acidic glycoproteins were shown. Since the mucous neck cells incorporated all precursors at a low rate, these cells apparently do not play an important role in gastric mucus synthesis. They did not incorporate sulphate, which is consistent with histochemical observations.     

Glycoprotein synthesis in the mucous cells of the vascularly perfused rat stomach. I. Surface mucous cells

We developed a constant-pressure vascular perfusion system of the isolated rat stomach, utilizing an artificial, fluorocarbon (FC-75)-containing medium. Perfusion could be maintained for at least six hours, as demonstrated by the ultrastructure of the mucosal cells and by the constant incorporation of [3H]-galactose in the surface mucous cells. Moreover all mucous cell types in tissue fixed after six hours of perfusion showed the same histochemical reactions for glycoproteins as in tissue fixed shortly after decapitation of the animal. The surface mucous cells of the antrum incorporated 30% less [3H]-galactose, [3H]-serine and [35S]-sulphate than those of the fundus. The amount of radioactivity incorporated per cell did not decrease during a subsequent 2 hour chase.     

Testosterone metabolism in the medial basal hypothalamus of the starved male rat

The conversion of testosterone to estradiol by aromatase and to dihydrotestosterone by 5 alpha-reductase was measured in the medial basal hypothalamus of starved and control male rats. Activities of both enzymes were significantly reduced in starved animals. Aromatase activity was 18.2 +/- 2.3 versus 29.8 +/- 5.7 fmol E2/mg protein/90 min (mean +/- SEM, P less than 0.02) and 5 alpha-reductase was 4.95 +/- 0.35 versus 5.96 +/- 0.30 pmol DHT/mg protein/90 min (P less than 0.02) for starved and control animals respectively. The results indicate that hypothalamic metabolism of testosterone is decreased during starvation. Therefore the increased sensitivity of the T-LH feedback described earlier in starved rats [4] cannot be explained by changes in central testosterone metabolism.     

Low density lipoprotein-receptors in primary cultures of rat glial cells.

Newborn rat glial cells in primary culture contain an active cholesterol side chain cleavage cytochrome P450. Cholesterol can be supplied either by biosynthesis or derive from low density lipoproteins (LDL), which bind apolipoprotein Band E (apoB,E) (LDL)-receptors and undergo receptor-mediated endocytosis. Using antibodies to purified human plasma LDL and antibodies to bovine adreno-cortical LDL-receptor, the presence of LDL-receptors was demonstrated on rat glial cells after 3-4 weeks of primary culture, by ligand blotting, immunoblotting, and indirect immunofluorescence staining. The latter approach indicated that oligodendrocytes express higher levels of LDL-receptors than astrocytes present in the same culture. The immunofluorescence staining was observed not only at the cell surface, but also within the cytoplasm, suggesting that the LDL-receptor complexes had been internalized. Western blotting of LDL-receptors extracted from glial cells indicated a band of approximately 130 kDa, the size expected for intact receptors. Their functionality was shown by the conversion of [3H]cholesterol linoleate, incorporated into reconstituted LDL and added to the cell cultures, to [3H]pregnenolone and/or its 20 alpha-hydroxy-metabolite. This is the first characterization of functional LDL-receptors on isolated, well characterized, normal brain cells.     

Glycoprotein transport in the surface mucous cells of the rat stomach

The intracellular transport of glycoproteins pulse-labeled in vitro with tritiated leucine and galactose in the surface mucous lining cells (SMC) of the fundus of the rat stomach was studied by electron microscope autoradiography. The SMC survive for several hours in pieces of the fundus incubated in a bicarbonate-buffered medium. The SMC have a normal ultrastructure for at least 4 h of incubation. Kinetic activity is normal for at least 5 h, as demonstrated by the normal nuclear incorporation of tritiated thymidine; The SMC incorporate labeled leucine and galactose at normal rates up to 4 h and 6 h, respectively. In contrast to the SMC, the cells of the gastric glands show signs of degeneration within 1 h after the start of incubation. In the SMC the secretory protein forms a smaller part of the total protein synthesized than in other secretory cells studied. The intracellular tranpsort of the leucine-labeled moiety of the glycoproteins follows the normal pathway. The RER loses 35% of its transportable labeled protein within 30 min. The Golgi complex is maximally labeled at 40 min and the mucous granules after 120 min. Galactose is attached to the glycoproteins mainly in the Golgi complex. Glycoproteins are not secreted within 2 h after synthesis of their protein moiety.     

A microprobe analysis of Gomori-positive glial cells in the rat arcuate nucleus.

Astrocytes and microglial cells in the arcuate nucleus of the rat hypothalamus contain lipofuscin-like granules which react with chrome alum gallocyanin and exhibit endogenous peroxidase activity. These granules were assessed with energy dispersive X-ray microanalysis and compared to neuronal dense bodies and glial cytoplasm. The granules are distinguished by a consistent content of sulphur and a frequent presence of calcium. The localization of other elements such as iron, copper, potassium and chlorine is impaired by methodical difficulties. The sulphur content as well as the endogenous peroxidase activity is interpreted as indicating a special variant of lipofuscin. The presence of calcium is discussed with respect to recent concepts of glia as a regulator of the ionic environment of the CNS.     

Phospholipid metabolism in neuronal and glial cells during aging

The incorporation of cytidine-containing precursors (CDP-Cho and CDP-Etn) into the main phospholipid classes of cellular fractions enriched in neurons and glial cells from whole rat brains of different ages was examined. The rate of synthesis of choline phosphoglycerides in neuronal homogenates significantly decreased with age up to 18 months; after this time no additional decrease was found. The decrease of CDP-Etn incorporation in neurons was found to be less significantly affected by age up to 18 months, but the enzymic activity decreased after 18 months of age. No changes were found in the corresponding glial activity at any age. Biochemical phenomena that occur in 18-month-old rat brain (aged animals) were compared with phenomena occurring in 2-month-old rat brain (adult animals). No significant variations of lipid composition were found in neurons from either 18-month-old or 2-month-old rat brain. These results, together with some kinetic parameters, suggest that ethanolamine and choline phosphotransferases are affected differently by aging.     

Role of retinal glial cells in neurotransmitter uptake and metabolism

In addition to photoreceptors and neurons, glial cells (in particular Müller cells) contribute to the removal and metabolization of neurotransmitters in the neural retina. This review summarizes the present knowledge regarding the role of retinal glial cells in the uptake of glutamate, N-acetylaspartylglutamate, γ-aminobutyric acid, glycine, and d-serine, as well as the degradation and removal of purinergic receptor agonists. Some major pathways of glutamate metabolism in Müller cells are described; these pathways are involved in the glutamate–glutamine cycle of the retina, in the defense against oxidative and nitrosative stress via the production of glutathione, and in the production of substrates for the neuronal energy metabolism. In addition, the developmental regulation of the major glial glutamate transporter, GLAST, and of the glia-specific enzyme glutamine synthetase is described, as well as the importance of a malfunction and even reversal of glial glutamate transporters, and a downregulation of the glutamine synthetase, as pathogenic factors in different retinopathies.     

Hormonal control of metabolism of gamma-aminobutyric acid in the rat hypothalamus and hippocampus

It has been established that hydrocortisone administration increased the amount of total, free, bound and synaptosomal GABA in the hypothalamus, glutamate decarboxylase activity in the homogenate and synaptosomes and time of the mediator turnover. ACTH administration increased the GABA content and glutamate decarboxylase activity in synaptosomes. The total amino acid content and time of its turnover got higher only with single hormone administration. In the hippocamp hydrocortisone administration increased the total and free GABA contents, its turnover time, glutamate decarboxylase activity in the homogenate and decreased GABA-aminotransferase activity in the homogenate and synaptosomes. The GABA level in synaptosomes grew only with multiple hormone administration. Single administration of ACTH decreased the total GABA content, glutamate decarboxylase activity in the homogenate, while its multiple administration increased the GABA level in synaptosomes followed by a decrease of GABA-aminotransferase activity in the homogenate and synaptosomes. The GABA turnover time fell with single hormone administration and grew with the multiple one. Adrenalectomy induced no changes in the GABA content and activity of its metabolism enzymes in the hypothalamus, however the bound GABA level decreased, while the turnover time increased. In the hippocamp adrenalectomy decreased total, free and synaptosomal GABA contents, glutamate decarboxylase activity in a homogenate and turnover time. Subsequent hydrocortisone administration only partly normalized the revealed changes of the GABA metabolism in the brain structures under adrenalectomy.     

Physiological significance of altered insulin metabolism in the conscious rat during lactation.

Uptake of radioactively labelled insulin by the mammary gland of the rat increased 12-fold in lactation compared with non-lactating controls. This uptake was decreased by the presence of unlabelled insulin, indicating that it occurred via insulin receptors. The plasma half-life of insulin is decreased in lactation from 9.4 min to 4.8 min, and the metabolic clearance rate for insulin increased from 7.26 to 13.03 ml/kg body wt. per min. The basal insulin and glucose concentrations in the plasma were decreased in lactation. Infusion of insulin at a dose which led to a small physiological rise in plasma insulin concentration increased lipogenic rates in the mammary gland by 100% without causing marked hypoglycaemia. It is concluded that the lactating mammary gland is a highly insulin-sensitive tissue and that the lower plasma insulin during lactation occurs primarily as a result of this sensitivity increasing extraction of glucose by the gland and thus producing a decrease in the plasma glucose concentration. It is suggested that a secondary result of the fall in plasma insulin concentration is the preferential direction of substrates (glucose and non-esterified fatty acids) towards the lactating mammary gland and away from adipose tissue and the liver.     

Accumulation and metabolism of norepinephrine in rat hypothalamus after exhaustive stress

—Exhaustive stress in rats is followed by a temporary reduction of hypothalamic norepinephrine (NE) together with a persistent increase in turnover during recovery. To test for persistent alterations of NE storage and metabolism produced by stress, rats were subjected to 3 h of forced running and were then injected intraventricularly with [³H]NE or [³H]dopamine (DA). The hypothalamus was assayed for [³H]NE and its metabolites at various intervals after injection. The effects of stress were compared with those of reserpine (7·5 mg/kg) or α-methyltyrosine (AMT, 300 mg/kg) pretreatment. It was found that the stress-induced reduction of endogenous NE was not accompanied by a change in the accumulation of exogenous [³H]NE either 10 or 30 min after injection, whereas the NE depletions produced by reserpine or AMT were associated with decreased or increased accumulation, respectively. However, stress did produce an increased accumulation of [³H]NE endogenously synthesized from [³H]DA. These results indicate that exhaustive stress does not adversely affect the storage of NE. They also suggest that stores of NE depleted by stress are replenished chiefly with newly synthesized NE and not through an increased uptake and binding or decreased metabolism of extraneuronal NE. The latter factors may play a role in the maintenance of brain NE stores when biosynthesis is low, i.e. after AMT. The major metabolites of exogenous [³H]NE, at 30 min after injection, were identified as conjugates of 3,4-dihydroxyphenylglycol (DOPEG) and 3-methoxy-4-hydroxyphenylglycol (MOPEG) in approximately equal amounts. The finding of high levels of conjugated DOPEG confirms a recent report (Slgden and Eccleston , 1971) that this compound is a major metabolite of brain NE. Reserpine produced marked elevations of both conjugates; AMT slightly reduced each. Prior stress increased only conjugated MOPEG, an observation suggesting that CNS levels of this metabolite may reflect NE released by nervous activity.     

Glycoprotein metabolism in rat colonic epithelial cell populations with different proliferative activities

Abstract. Epithelial cells with different proliferative activities were isolated from rat proximal and distal colon. The distribution of fucose, hexose, hexosamine, and sialic acid as well as the activities of three glycosyltransferases, eight glycosidases, and a nucleotide sugar pyrophosphatase, enzymes involved in glycoprotein metabolism, were then examined in these cells. The results of the present study demonstrate that: (1) all proximal cell populations appear to possess a higher content of hexose, fucose, and sialic acid than their distal counterparts; (2) in general, the proximal colonic populations have higher glycosyltransferase but similar glycosidase activities than their distal counterparts; (3) proliferative cells in both colonic regions have greater glycosyltransferase and glycosidase activities than non-proliferative cells, although their carbohydrate content is similar.
These findings suggest that alterations in glycoprotein metabolism exist during differentiation along the length of the rat colon. Furthermore, these data indicate that certain enzymes involved in glycoprotein metabolism may serve as markers for cellular differentiation in this organ.