Factors affecting the lactate dehydrogenase isoenzyme pattern of cultured kidney-cortex cells

1. The lactate dehydrogenase isoenzyme pattern of cultured calf kidney-cortex cells was correlated to growth phase, changes in oxygen supply, mean generation time and changes in nutritional supply. 2. During culture of free cells and intact explants the lactate dehydrogenase isoenzyme pattern changed towards a dominance of isoenzymes containing the M subunit. 3. Of the shift in monomer proportion, 58% occurred during the lag phase and 42% during the initial part of the exponential growth phase. During the stationary phase the shift in monomer proportion reversed slightly. It was possible to relate the observed shift in monomer proportion to the glycolytic rate. 4. Factors that depressed glycolysis decreased the shift in monomer proportion. Oxygen was found to limit the decrease in the H subunit/M subunit ratio caused by anaerobic culture in vitro. 5. The results obtained support the view that the altered lactate dehydrogenase isoenzyme pattern of urine in renal ischaemia may be explained by anaerobic changes in the lactate dehydrogenase isoenzyme pattern of cortical tubule cells.     

Fibronectin is expressed by astrocytes cultured from embryonic and early postnatal rat brain

In early primary cultures from newborn rat brain, few glial fibrillary acidic protein (GFAP)-positive glial cells expressed intracytoplasmic immunoreactivity for fibronectin. After the second week in culture, however, fibronectin was expressed by a distinct population of GFAP-positive flat astrocytes, irrespective of which brain region was studied. In cerebellar cultures, these cells were more abundant than in cortical or neostriatal cultures and often formed a major population of the GFAP-positive cells. The difference in fibronectin expression between cerebellum and the other areas studied was statistically significant. When cultures were started from 9-day-old postnatal rat brain, fibronectin-positive astrocytes appeared earlier than in those from newborn animals, in all areas studied. Further, especially in the case of cerebellum, the number of fibronectin-positive astrocytes increased as a function of time in culture. In cultures started from whole brains of 12-day-old rat embryos, fibronectin was expressed within 24 h in culture by all the cells with morphology of flat astrocytes, positive for vimentin but negative for GFAP. These results indicate that astrocytes cultured from newborn and early postnatal rat brain are a heterogeneous population of cells: depending on the brain region studied and also depending on the age of brain tissue or the time in culture, less than 1-60% of the GFAP-positive flat astrocytes expressed fibronectin. This, together with the fact that fibronectin was present in early embryonic brain cells in culture, suggests that fibronectin may be a prerequisite for the development or interactions of brain cells.     

Expression of differentiated activities in reaggregated brain cell cultures.

Dissociated fetal mouse brain cells are allowed to reassociate in rotation culture to form aggregates. After several weeks these reaggregated brain cell cultures show markedly increased specific activities of monoamine oxidase, lactate dehydrogenase, and the brain-specific protein S-100, while catechol-O-methyltransferase activity increases slightly. Similar changes in these activities are found during mouse brain maturation. The amounts of monoamine oxidase, catechol-O-methyltransferase, and S-100 were also determined in surface cultures of fetal mouse brain cells, as well as glioma and neuroblastoma cell lines. The fetal brain and glial cell cultures possess much higher activities than the cultured neuroblastoma cells. However, lactate dehydrogenase activity was highest in the glioma and lowest in the surface cultures of fetal brain cells.     

Modifications in energy metabolism during the development of chick glial cells and neurons in culture

Developmental changes in lactate dehydrogenase (LDH), enolase, hexokinase (HK), malate dehydrogenase (MDH), and glutamate dehydrogenase (GDH) activities were measured in cultures of pure neurons and glial cells prepared from brains of chick embryos (8 day-old for neurons, 14 day-old for glial cells) as a function of cellular development with time in culture. The modifications observed in culture were compared to those measured in brain extracts during the development of the nervous tissue in the chick embryo and during the post-hatching period. A significant increase of MDH, GDH, LDH, and enolase activities are observed in neurons between 3 and 6 days of culture, whereas simultaneously a decrease of HK values occurs. In the embryonic brain between 11 and 14 days of incubation, which would correspond for the neuronal cultures to day 3 through 6, modifications of MDH, GDH, HK, and enolase levels are similar to those observed in neurons in culture. Only the increase of LDH activity is less pronounced in vivo than in cultivated cells. The evolution of the tested enzymatic activities in the brain of the chick during the period between 7 days before and 10 days after hatching is quite similar to that observed in cultivated glial cells (prepared from 14 day-old embryos) between 6 and 18 days of culture. All tested activities increased in comparable proportions. The modifications of the enzymatic profile indicate that some maturation phenomena affecting energy metabolism of neuronal and glial elements in culture, are quite similar to those occuring in the total nervous tissue. A relationship between the development of the energy metabolism of the brain and differentiation processes affecting neuroblasts and the glial-forming cells is discussed.     

Activity and Isoenzyme Pattern of Lactate Dehydrogenase in Neurons and Astroblasts Cultured from Brains of Chick Embryos

Primary cultures of neurons and glial cells (astroblasts) prepared from brains of 8-day-old and 15-day-old chick embryos, respectively, were grown for periods between 3 and 19 days. Specific activity of lactate dehydrogenase (LDH) increased in both types of cultures as a function of time and was always significantly higher in glial cells than in neurons. Glial cell extracts were found to contain predominantly the anaerobic isoenzymatic form of LDH (LDH-H4), and this pattern did not change over a period of 19 days. Cultured neurons contained predominantly the aerobic isoenzymatic form LDH-H4, and there was a progressive appearance of all other isoenzymes over an 8-day period. These results support the hypothesis of a different energy metabolism in neurons and glia.     

A Comparison of the Polypeptide Pattern Between a Brain Primary Culture and Fractions of Bulk-Isolated Glial Cells

Abstract: This paper reports on the electrophoretic protein/polypeptide pattern of a rat brain primary culture. For comparison, the polypeptide pattern of neuronal and glial enriched fractions from adult rat brain and cerebral hemispheres from newborn and adult rat have been analysed. Water-soluble and SDS-extractable polypeptide fractions appeared and/or increased in amount in the cultures until confluency. The polypeptide pattern of the cultures most resembled that of the glial cell fractions, showing some of this fraction's specificity. Removal of fetal calf serum and addition of 0.1 mM dibutyryl cyclic adenosine monophosphate (dB-cAMP) produced few changes in the electrophoretic pattern. The study thus provides evidence in favour of the astroglial nature of the brain primary culture. It also shows that the cells undergo some maturation in the culture.     

Isoenzyme spectrum of lactate dehydrogenase, malate dehydrogenase, esterase and acid phosphatase of rat brain cells at different times after external 1 Gy gamma irradiation]

The influence of external single gamma-irradiation with a dose of 1 Gy on the isoenzyme composition of lactate dehydrogenase, malate dehydrogenase, esterase and acid phosphatase in the cytoplasm of rat brain cells has been investigated. Irradiation was shown to cause differently directed changes in the ratio of the isoenzymes under study at different times after exposure. The isoenzyme spectrum of lactate dehydrogenase and malate dehydrogenase was shown to be normalized on day 30 after irradiation, whereas the isoform composition of esterase and acid phosphatase was not stabilized at that time.     

Cellular Location of Glutamine Synthetase and Lactate Dehydrogenase in Oligodendrocyte-Enriched Cultures from Rat Brain

Glial cells were isolated from 1-week-old rat brain and cultured in a serum-free medium supplemented with the hormones insulin, hydrocortisone, and triiodothyronine. After 1 week in culture the cell population consisted mainly of galactocerebroside-positive cells (GC+; oligodendrocytes), the remainder of the cells being positive for glial fibrillary acidic protein (GFAP+; astrocytes). Oligodendrocytes were selectively removed from the cultures by complement-mediated cytolysis. The activities of glutamine synthetase and of various marker enzymes were measured in the nonlysed cells remaining after complement treatment of the cultures and in the culture medium containing proteins of the lysed cells. We found that the cellular activity of glutamine synthetase decreased in parallel with the lysis of GC+ cells and that the activity of glutamine synthetase in the supernatant increased. The activity of glycerol-3-phosphate dehydrogenase, a marker enzyme for oligodendrocytes, was no longer detectable in complement-treated cultures and the activity of glutamine synthetase was markedly lowered, whereas the activity of lactate dehydrogenase was as high as in untreated cultures. The location of glutamine synthetase both in oligodendrocytes and in astrocytes was confirmed by double-label immunocytochemistry with antisera against glutamine synthetase, GC, and GFAP. We conclude that in this culture system glutamine synthetase is expressed in both types of glial cells and that the activity of lactate dehydrogenase is at least one order of magnitude higher in astrocytes than in oligodendrocytes.     

Rat brain glial cells in culture: Effects of brain extracts on the development of oligodendroglia-like cells

Cells dissociated from brains of newborn rats and grown on plastic surfaces develop into a glial culture, composed of at least three morphologically different cell types. The predominating cell type consists of astroglial cells, which form a monolayer. The second cell type, rarely observed, consists of ependymal cells. The third type consists of small cells scattered upon the astroglial layer. After 3 weeks very few of these small cells remain and the glial culture develops into a more homogenous appearance, mainly composed of astroglial cells. The effects of various brain extracts on the development of the small cell type was investigated. The treatment by either rat or chick brain extracts caused an increase in the number of these cells, which were seen to form clusters. Brain extracts from older animals have a stronger effect than brain extracts from younger animals. These data suggest that factors contained in the brain during and after the myelination period influence the development of this cell type in dissociated cultures. The small cells were tentatively identified as oligodendroglial cells by ultrastructural and histochemical criteria. They did not contain acetylcholinesterase (AChE) and did not bind tetanus toxin. Furthermore, they did not contain glial fibrillary acidic (GFA) protein. But carbonic anhydrase II (CAII) was found in them at light and electron-microscopical level. CAII was found to be localized essentially on the plasmic membrane and on the endoplasmic reticulum of these cultured oligodendroglial cells.     

Mutually exclusive expression of fibronectin and glial fibrillary acidic protein in cultured brain cells

The reported expression of the cell surface-associated, mainly mesenchymal glycoprotein fibronectin by cultured glial cells is in discrepancy with recent work on brain tissue failing to demonstrate any glial or neuronal fibronectin. We have investigated the expression of fibronectin in relation to glial fibrillary acidic protein in cultured human glial and glioma cell lines as well as in cultures derived from newborn rat brain. Using double immunofluorescence technique we found that cells containing glial fibrillary acidic protein do not express fibronectin, and vice versa. The only exception to this rule was the occasional finding of fibronectin at points of cell-to-cell adhesion also in relation to cells containing glial fibrillary acidic protein. The results were also tested by polyacrylamide gel electrophoresis of the culture media of the human cell lines, and by subcultures from the brain of newborn rat, cultures stimulated with dibutyryl cyclic AMP (db-cAMP), and by vinblastine treatment of the cells. The lack of expression of fibronectin in cells containing glial fibrillary acidic protein, a gliospecific cytoskeletal protein, is discussed with reference to glio-mesenchymal interactions and glial markers in vitro.     

Hypoxia induced metabolism dysfunction of rat astrocytes in primary cell cultures

In order to study the astroglial contribution to hypoxic injury on brain tissue metabolism, modifications of glutamine synthetase (GS) lactate dehydrogenase (LDH) enolase and malate dehydrogenase activity produced by reduced oxygen supply have been determined in primary cultures of astrocytes prepared from newborn rat cerebral cortex. Enzymatic activities were measured immediately after the hypoxic treatment (9 h) and during post injury recovery. GS level is significantly decreased in response to low oxygen pressure and increased above control value during the post hypoxic recovery period. The magnitude of GS reduction by hypoxia depends on the age of the cells in culture. Lactate dehydrogenase and enolase levels were significantly enhanced during the two periods considered. No modification of the MDH level was observed. The synthesis of LDH isoenzymes containing mainly M subunits is specifically induced by hypoxia. Our results suggest that astroglial cells may represent a particularly sensitive target toward hypoxia injury in brain tissue. Low oxygen pressure available may modify some fundamental metabolical functions of these cells such as glutamate turnover and lactic acid accumulation.     

Isoenzymes of lactate dehydrogenase in human leukemic cells in culture treated with inducers of differentiation

The human leukemic cell lines HL60 and K562, were induced to differentiate terminally by chemical agents. The isoenzyme patterns of lactate dehydrogenase (LD) in the cells before and after differentiation were determined electrophoretically on agarose gels. In general, treatment of the leukemic cells with inducers of differentiation resulted in a quantitative shift of the isoenzyme pattern towards anodic or cathodic forms. This was correlated with the conversion of the chemically treated cells to morphologically more normal cells, as verified by light microscopy and/or synthesis of hemoglobin. The LD isoenzyme patterns of the chemically differentiated cells were: (a) characteristic for the particular cell type obtained rather than for the nature of the inducer used; and (b) not similar to those of normally differentiated cells of the corresponding lineage, indicating that incomplete differentiation had occurred.     

ACTIVITY AND ISOENZYME PATTERN OF LACTATE DEHYDROGENASE IN ASTROBLASTS CULTURED FROM BRAINS OF NEWBORN MICE

Lactate dehydrogenase activity and isoenzyme distribution was determined in primary cultures of astroblasts as a function of the culture period. The specific activity increased during this period with a peak value (1.91 ± 0.18μmol x min^-1 x mg^-1 cell protein) after 2 weeks in culture. The isoenzyme pattern changed during 3 weeks in culture towards a higher proportion of the H₄ (LDH-1) isoenzyme which is analogous to the in vivo pattern. Omission of serum with or without dBcAMP (0.5 mM) in the culture medium during the third week of culture further enhanced this prominence of the H₄ isoenzyme. The specific activity (1.58 × 0.06 μmol x min^-1 x mg^-1 cell protein) of cultures grown in the presence of 0.5 mM-dBcAMP and absence of serum was close to the activity in the adult brain.     

Common myofibroblastic features of newborn rat astrocytes and cirrhotic rat liver stellate cells in early cultures and in vivo.

Double-immunolabelling techniques were employed to investigate the distribution of smooth muscle alpha-actin (actin) in glial fibrillary acidic protein (GFAP)-positive cells in rat brain during early postnatal development and maturation and in glial primary culture derived from newborn rat brain. In addition the expression of desmin was studied in the glial primary cultures as a function of the differentiation of the cells. Comparison of the cultured astroglial cells at an early age with hepatic stellate cells derived from CCl4-induced cirrhotic rat liver, revealed features of the astrocytic cytoskeleton characteristic of myofibroblastic cells, i.e., strong expression of both myofibroblastic markers, actin and desmin. In astroglial cells with an initial morphology reminiscent of fibroblasts the non-filamentous perinuclear immunoreaction of GFAP increased with time at the expense of actin and, partially, desmin. GFAP filaments were spread throughout the cytoplasm of the cells which acquired stellate morphology. The alterations in the morphology of the cells and the distribution and intensity of staining for GFAP and actin during the differentiation of astrocytes in culture were similar to those observed in astrocytes during the maturation of the brain. In astrocytes from a newborn brain as well as in cirrhotic hepatic stellate cells, the area of immunoreaction of GFAP was reduced and confined mainly to the nuclear region. In contrast, the cells expressed actin throughout the cytoplasm. These findings may hint at a similar function of these regionally specialized perivascular myofibroblastic cells in a normal brain and diseased liver and at inverse organ-specific functions which the cells fulfill under non-pathological conditions in vivo.     

Glucose metabolism in transdifferentiating and glucose-blocked cultures of chick embryo neuroretinal cells: an inverse relationship between glycogen and delta-crystallin accumulation

Chick embryo neuroretinal (NR) cells transdifferentiate extensively into lens when cultured for several weeks in low-glucose (FH) medium, but fail to do so when high levels of supplementary glucose (FHG) are present. We show here that most aspects of glucose metabolism are promoted in high-glucose cultures, including lactate dehydrogenase (LDH) and glucose-6-phosphate dehydrogenase (G-6-PDH) activities, 2-deoxyglucose uptake, pentose shunt activity and lactate production. Continuous supplementation of high-glucose cultures with low levels of ouabain (FHGO) significantly lowers 2-deoxyglucose uptake, from FHG levels down towards FH levels, especially during the early stages of NR culture. Much later, extensive transdifferentiation into lentoids (with concomitant delta-crystallin accumulation) occurs in these FHGO cultures, which thus resemble FH rather than FHG controls. Another parameter strongly affected by ambient glucose levels is the accumulation of glycogen. Both glycogen itself and glycogen synthetase activity increase steadily in FHG cultures, but decrease slightly under FH conditions. Glycogen accumulation in FHG cultures is largely confined to glial-like cells, particularly those underlying clusters of neurones. Other studies have shown that glial differentiation in vitro is promoted by histotypic interactions with retinal neurones. Thus high glucose may act in concert with neuronal influences to stimulate or stabilize the normal differentiation of retinal glial cells, whose characteristic features in vivo include glycogen synthesis and storage. Furthermore, we show that supplementation of high-glucose cultures with forskolin or dibutyryl cyclic AMP (both of which promote glycogenolysis) results in a slower rate of glycogen accumulation and in enhanced transdifferentiation into lens. In both respects, the forskolin- and dibutyryl cAMP-supplemented FHG cultures are intermediate between FH and FHG controls. Thus the enhancement of normal glial differentiation in NR cultures by high glucose may inhibit or preclude subsequent transdifferentiation into lens.     

Cellular Localization and Regulation of Glutamine Synthetase in Primary Cultures of Brain Cells from Newborn Mice

The cellular distribution of glutamine synthetase was determined by indirect immunofluorescence in cultures of dissociated brain cells from newborn mice. The enzyme could be detected in about 40% of all cells, among which cells with astrocytic morphology were clearly identified. Treatment with the glucocorticoid dexamethasone led to a strong increase in the number of positivity stained cells. Enzyme induction by dexamethasone was maximal after 36 h and at a concentration of 0.1 micrometer. Under these conditions glutamine synthetase specific activity was elevated about six fold. Steroid hormones other than corticosteroids had no effects. The basal activity in these cultures was near that found in brains of newborn mice, but far below the activity in adult brains, showing that in culture the normal development of these cells is disturbed. A comparison of glial and neuronal cell lines showed that glutamine synthetase is present in both types of cell lines at a very low specific activity. Inducibility of this enzyme by dexamethasone was found in glial but not in neuronal cell lines.     

Changes in the lactate dehydrogenase isoenzyme pattern during differentiation of rabbit bone-marrow erythroid cells.

1. Differentiation and maturation of rabbit bone-marrow erythroid cells was accompanied by a 15-fold decrease in lactate dehydrogenase activity from approx. 0.1pmol of NADH utilized/min per cell in basophilic cells to 0.007 pmol of NADH/min per cell in reticulocytes. 2. In early cells, cell division takes place with a corresponding decrease in cell volume, but the concentration of lactate dehydrogenase remains almost constant. 3. When cell division ceases, qualitative as well as quantitative changes in the lactate dehydrogenase isoenzyme pattern become apparent and reticulocytes were found to contain almost exclusively the H4 isoenzyme, whereas early erythroblasts contained also the M4 and hybrid isoenzymes. 4. Extracts from a lysosome-enriched subcellular fraction of bone-marrow erythroid cells specifically degraded the M4 isoenzyme in vitro, but the H4 form was stable. It is suggested that lysosomal enzymes are involved in bringing about the observed changes in lactate dehydrogenase isoenzyme patterns in vivo.     

Biochemical and Ultrastructural Studies of Cultured Rat Astroglial Cells

The growth of astroglial cells in primary cultures derived from newborn rat cerebral hemispheres was investigated in the absence and in the presence of newborn rat brain extract or dBcAMP. The parameters chosen were the content of DNA, total protein, and glial fibrillary acidic protein (OFA) as well as the morphologic development of gliofilaments. During the entire culture period the DNA content increased in control culture indicating a continuous cell division, whereas the cells stopped dividing after 14 or 4 days of treatment with either brain extract or dBcAMP respectively. In contrast, a constant increase of total protein was found in both control and treated cultures. Since cell divisions had stopped in treated cultures, the increase in total protein in these cultures indicates growth of the individual cells. The GFA levels increased progressively and similarly in control cultures and in cultures treated with brain extract. The values in the treated cultures remained slightly higher than those in controls. Conversely, immediately after the addition of dBcAMP a sudden increase in GFA protein occurred and the amounts were statistically significantly different from those of the controls. The GFA levels were expressed relative to total protein indicating that GFA constitutes an increasing amount of the total protein of the individual cells during culture. The changes in the amount of GFA was shown to parallel the morphologic development of gliofilaments. Indeed, when the level of GFA increased a progressive accumulation of gliofilaments was observed. The results obtained were discussed in relation to the astrocytic maturation.     

Biochemical and ultrastructural studies of cultured rat astroglial cells: effect of brain extract and dibutyryl cyclic AMP on glial fibrillary acidic protein and glial filaments

The growth of astroglial cells in primary cultures derived from newborn rat cerebral hemispheres was investigated in the absence and in the presence of newborn rat brain extract or dBcAMP. The parameters chosen were the content of DNA, total protein, and glial fibrillary acidic protein (GFA) as well as the morphologic development of gliofilaments. During the entire culture period the DNA content increased in control culture indicating a continuous cell division, whereas the cells stopped dividing after 14 or 4 days of treatment with either brain extract or dBcAMP respectively. In contrast, a constant increase of total protein was found in both control and treated cultures. Since cell divisions had stopped in treated cultures, the increase in total protein in these cultures indicates growth of the individual cells. The GFA levels increased progressively and similarly in control cultures and in cultures treated with brain extract. The values in the treated cultures remained slightly higher than those in controls. Conversely, immediately after the addition of dBcAMP a sudden increase in GFA protein occurred and the amounts were statistically significantly different from those of the controls. The GFA levels were expressed relative to total protein indicating that GFA constitutes an increasing amount of the total protein of the individual cells during culture. The changes in the amount of GFA was shown to parallel the morphologic development of gliofilaments. Indeed, when the level of GFA increased a progressive accumulation of gliofilaments was observed. The results obtained were discussed in relation to the astrocytic maturation.     

Cellular Distribution of 6-Phosphofructo-1-Kinase Isoenzymes in Rat Brain

Abstract: In the brain, all three isoenzyme types [muscle (M), liver (L), and brain (C)] of 6-phosphofructo-1-kinase (PFK; EC 2.7.1.11) occur, forming a complex mixture of homo- and heterotetramers. The PFK isoenzyme pattern of the different brain cell types is yet unknown. In the present study, we investigated the distribution of the PFK isoenzyme subunits in primary and secondary cell cultures and in bulk-isolated cells of rat brain by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotting. All three PFK isoenzymes are present in all cell types but in different proportions. The cellular distribution of the PFK isoenzymes in situ was studied immunohistochemically with different polyclonal antisera against purified rat PFKs. The monospecific antibody against M-type PFK stained preferentially the perinuclear areas of neurons and glial cells. The antibodies that in immunoblots detected mainly the L-type PFK showed a characteristic staining in only the cytoplasma and the processes of cells, whereas the C-type antibodies almost homogeneously stained whole cell bodies as well as large dendrites. Because the PFK isoenzymes differ with respect to their allosteric properties, their differential distribution in different brain cells might be of importance for the regulation of brain glycolysis in the different cellular compartments of the brain.