Rat Brain Cell Cultures Research Articles

Release of angiotensin in response to ionic stimulation: A possible role for calmodulin in the secretory event

Although the synthesis and distribution of the putative neurotransmitter angiotensin (ANG) has been well established, little is known about the mechanism for its release. In this study, we utilized dissociated cell cultures of fetal rat brain to examine the cellular and ionic properties of angiotensin release. Graded concentrations of 0–60 mM KCl in the presence of 5 mM CaCl 2 were added to the cultured cells and the resulting angiotensin release was measured by radioimmunoassay and high-performance liquid chromatography. Levels of angiotensin release increased from 13.85 ±1.53 pg/mg protein to 172.64 ± 17.49 pg/mg protein with increasing concentrations of K +. Cultures incubated with 60 mM KCl buffer that did not contain CaCl 2 released 39.87 ± 15.74 pg ANG/mg protein. To further show the link between ionic stimulation and angiotensin release and determine the potential role of extracellular sodium ions on angiotensin release, cultures were incubated with the Na +-channel blocker tetrodotoxin (300 nM TTX) prior to maximal stimulation with 60 mM KCl/5 mM CaCl 2 in the presence of the channel antagonist. Release was attenuated following incubation with stimulating buffer containing TTX (300 nM) to 5.49± 4.37 pg/mg protein. Finally, to determine the role of the calcium binding protein calmodulin in the release event, the cells were incubated with graded concentrations of W-7 (2.5–250 μM) and subsequently stimulated with 60 mM KCl/5 mM CaCl 2 in the presence of the calmodulin antagonist. W-7, which displays specificity for inhibition of the Ca 2+ calmodulin complex below 0.2 mM, decreased angiotensin release in a dose-dependent manner. These data support the hypothesis that potassium-induced angiotensin release is a calcium-dependent process that requires the activation of calmodulin, providing additional support for the role of angiotensin as a putative neurotransmitter.

Neuronal lysosomal enzyme replacement using fragment C of tetanus toxin.

Development of a strategy for efficient delivery of exogenous enzyme to neuronal lysosomes is essential to achieve enzyme replacement in neurodegenerative lysosomal storage diseases. We tested whether effective lysosomal targeting of the human enzyme beta-N-acetylhexosaminidase A (Hex A; beta-N-acetyl-D-hexosaminide N-acetylhexosaminohydrolase, EC 3.2.1.52) can be obtained by coupling it via disulfide linkage to the atoxic fragment C of tetanus toxin (TTC) that is bound avidly by neuronal membrane. TTC-Hex A conjugation resulted in neuronal surface binding and enhanced endocytosis of enzyme as observed in immunofluorescence studies with rat brain cultures. In immunoelectrophoretic quantitative uptake studies, rat neuronal cell cultures contained 16- and 40-fold greater amounts of enzyme after incubation with TTC-Hex A than with nonderivatized Hex A. In cerebral cortex cell cultures from a feline model of human GM2 gangliosidosis (Tay-Sachs and Sandhoff diseases), binding and uptake patterns of the enzymes were similar to those in the rat brain cell cultures. After exposure to extracellular concentrations of enzyme attainable in vivo, lysosomal storage of immunodetectable GM2 ganglioside was virtually eliminated in neurons exposed to TTC-Hex A, whereas a minimal effect was observed with Hex A. These findings demonstrate the usefulness of TTC adducts for effective neuronal lysosomal enzyme replacement.

Toxic effects of solstitialin a 13-acetate and cynaropicrin from Centaurea solstitialis L. (asteraceae) in cell cultures of foetal rat brain

Extracts of the aerial parts of the yellow star thistle ( Centaurea solstitialis L.) were prepared using petroleum ether, chloroform and methanol and toxicity assessed in primary cell cultures of striatum, frontal cortex and mesencephalon, containing the substantia nigra or raphe nucleus from the brian of the foetal rat. Chloroform extracts significantly reduced the percentage of live cells whereas the petroleum ether extract was inactive. The ability of the methanol extract to reduce the percentage of live cells could not be distinguished from the effects of the vehicle controls for methanol. Subsequently, the plant material was extracted with dichloromethane and 10 fractions were obtained by column chromatography. Fractions 5, 6, 7 and 8 caused concentration-dependent cell death in the culture of substantia nigra, the other fractions were not toxic at the concentrations used. In further studies, solstitialin A was isolated from fraction 9, solstitialin A 13-acetate from fraction 6 and solstitialin A-3 acetate and cynaropicrin from fraction 7. Solstitialin A 13-acetate and cynaropicrin were toxic to cultures of substantia nigra cells. It is concluded that, of the compounds identified, solstitialin A 13-acetate and cynaropicrin have toxic potential in cell cultures, containing cells from the substantia nigra of the rat, the specificity of action to cells of the substantia nigra remains to be shown, and that a toxic action in the midbrain may contribute to the nigro-pallidal encephalomalacia, caused by the ingestion of the yellow star thistle by horses.

Role of locally produced growth hormone-releasing factor in somatostatin regulation by fetal rat brain cells in culture.

To determine the possible physiological role of endogenous growth hormone-releasing factor (GRF) in the neuronal content and release of cerebral somatostatin (SS), we studied the effect of endogenous GRF blockade on the immunoreactive SS (IR-SS) content of cells and media in fetal rat cerebral cortical and hypothalamic cells in culture. Cells were cultured in minimum essential medium (MEM) with 10% fetal calf serum and 10% horse serum. After 7-10 days in vitro, media were replaced with MEM without sera containing anti-GRF immunoglobulins G (IgG) for 1, 5 or 24 h. Controls were incubated with equal amounts of IgG from normal rabbit serum (NRS). In another group of experiments, cells were incubated with GRF (10(-11) to 10(-7) M) for 1 or 24 h. Long-term exposure (24 h) to anti-GRF IgG resulted in decreased media and intracellular IR-SS content, in both cerebral cortical and hypothalamic cells. 24 h treatment with GRF caused a dose-dependent increase in the IR-SS content of cells and media, the stimulatory action being abolished by the addition of anti-GRF to plates containing 10(-7) M GRF. On the contrary, when cells were exposed to anti-GRF IgG for 1 h, IR-SS increased in the media as compared to the control group. Short-term incubation (1 h) with GRF (10(-9) to 10(-7) M) resulted in a dose-dependent inhibition of IR-SS content in the cells and media. This inhibitory action was partially prevented by the addition of anti-GRF to plates containing 10(-7) M GRF.(ABSTRACT TRUNCATED AT 250 WORDS)

119 IN VITRO NEURÖTOXICITY OF ANTIBIOTICS

Prediction, measurement, and monitoring of neurotoxicity of antibacterial agents are extremely difficult. We studied the effects of antibiotics on growth and differentiation in dissociated rat brain cell cultures. Drugs were added from 7 to 14 days in cultures (DIC) in order to attain concentrations representing 10 and 2 times the levels reached by intraventricular antibiotic administration. At DIC 14, 21 and 24 the following evaluations were performed and compared to controls: content of protein and DNA reflecting cell growth, two enzymes of brain cell differentiation and staining for oligodendrocytes and astrocytes. No effects showed penicillin G, amoxicillin, sulbactam, aztreonam and chloramphenicol. Only marginal and always reversible influences produced ceftriaxone, vancomycin and ciprofloxacin. Clear inhibition of both cell growth and cell differentiation with only partial or absent regeneration was induced by cefepime, ceftazidime, cefotaxime, cefuroxime, imipenem, temafloxacin and amikacin. These results are in accordance with other in vitro experiments (bone marrow cells, fibroblasts) and also with clinical findings. We conclude that the sensible use of this model on the cellular level can predict potential neurotoxicity of antimicrobial agents.

Insulin‐like growth factor I stimulates oligodendrocyte development and myelination in rat brain aggregate cultures

Insulin-like growth factor I (IGF-I) and high concentrations of insulin have been shown to stimulate an increase in the number of oligodendrocytes that appear in developing monolayer cultures of rat brain cells (McMorris et al., Proc Natl Acad Sci USA 83: 822-826, 1986; McMorris et al., Ann NY Acad Sci 605:101-109, 1990; McMorris and Dubois-Dalcq, J Neurosci Res 21:199-209, 1988). In the present study, we investigated whether IGF-I or insulin treatment induces a corresponding increase in the synthesis and accumulation of myelin. Aggregate cultures, established from 16-day-old fetal rat brains, were treated with either 100 ng/ml IGF-I or 5,000 ng/ml insulin and analyzed for the number of oligodendrocytes, activity of 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP), total amount of myelin, and synthesis rate of myelin proteins. Cultures treated with IGF-I beginning on day 2 after explantation contained 35-80% more oligodendrocytes and had 60-160% higher CNP activity than controls when tested on day 13, 20, or 27. By day 27, treated cultures had 35-90% more myelin than controls. Similar results were observed in response to 5,000 ng/ml insulin, a concentration at which insulin binds to IGF receptors and acts as an analogue of IGF-I. The synthesis rate of myelin proteins was measured in experiments using 5,000 ng/ml insulin. When treatment was begun at day 20 rather than day 2, cultures did not exhibit an increased number of oligodendrocytes over control during the following 4-6 days.(ABSTRACT TRUNCATED AT 250 WORDS)

Neurotrophic activity of platelet-derived growth factor (PDGF): Rat neuronal cells possess functional PDGF beta-type receptors and respond to PDGF.

The different isoforms of PDGF bind with different affinities to two different receptor types. Previously, these receptors have been demonstrated mainly on mesenchymal and glial cells. We show here that PDGF beta-type receptors are present also on rat brain neurons in vivo as well as in vitro. Immunohistochemical analysis of a sequentially sectioned brain of a newborn rat revealed the presence of PDGF beta receptors in neurons throughout the central nervous system. These beta-receptor-bearing cells were shown to contain neurofilaments by double immunofluorescence staining. Furthermore, the PDGF-BB isoform bound to cells in a neuron-enriched culture from newborn rat brains and induced c-Fos protein in these cells. Continuous PDGF-BB treatment of primary rat brain cell cultures resulted in outgrowth of neurites and prolonged survival. These results indicate a neurotrophic effect of PDGF-BB.

Dexamethasone-Induced Accumulation of Neuropeptide-Y by Aggregating Fetal Brain Cells in Culture: A Process Dependent on the Developmental Age of the Aggregates*

Neuropeptide-Y (NPY) and glucocorticoid receptors are coexpressed in many neurons in the brain. We addressed the question: Do glucocorticoids regulate the accumulation and/or secretion of immunoreactive (IR) NPY by fetal rat brain cells in culture, and if so, is the effect developmental stage dependent? Aggregates, formed from dissociated cells obtained from the hypothalamus-olfactory tubercle of 17-day-old fetuses, were cultured in serum-free medium for 23 days. On day 23, the aggregate NPY content was 6 ng/flask, and secretion (last 2 days) was approximately 12 ng/24 h. Exposure to dexamethasone (Dex; 20 nM) between days 0-23 led to a 1.9-fold increase in the aggregate content of NPY, whereas NPY secretion was not altered. When Dex exposure was limited to days 12-23, 16-23, 19-23, or 21-23, only a 12- to 23-day exposure induced NPY accumulation, and it was as effective as a 0- to 23-day exposure. The Dex-induced increase in NPY content was evident after a lag period of 4 days or more. When Dex exposure occurred on days 0-12, the aggregate NPY content on day 12 or 23 was not altered. None of these treatments altered the aggregate/medium content of immunoreactive somatostatin (SRIF) or the response to a 48-h exposure to forskolin (10 microM). Dex induction of NPY accumulation was a saturable function of the Dex concentration (maximal at 20 nM), and it was completely inhibited by RU486, a glucocorticoid/progesterone receptor antagonist; neither progesterone, 17 beta-estradiol, nor testosterone altered aggregate/medium NPY contents. Protein/DNA contents of the aggregates were either unaffected or slightly reduced by Dex. Thus, 1) Dex stimulates the accumulation of immunoreactive NPY, but not SRIF, by cultured fetal brain cells; 2) this effect requires a continuous 8-12 days of exposure to Dex during a late developmental stage in culture; 3) Dex does not potentiate or attenuate forskolin action on the NPY neuron; and 4) Dex action appears to be mediated by the glucocorticoid receptor. These results are consistent with glucocorticoid induction of production and/or decreased intracellular degradation of NPY, and with glucocorticoids regulating the NPY neuron in the perinatal brain in a developmental age-dependent manner.

Developmental Expression of Rat Insulin-Like Growth Factor Binding Protein-2 by Astrocytic Glial Cells in Culture*

Brain astrocytes were established in primary culture from postnatal and adult rats to characterize the developmental expression of secreted proteins. Astrocytes cultured from 21-day rat brain, but not 1-day rat brain, secreted a distinct group of proteins with Mr of 35,000 as determined by analysis of [35S]methionine-labeled proteins using two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. N-terminal amino acid sequence analysis of this protein group showed 100% identity to rat insulin-like growth factor binding protein-2 (rIGFBP-2), the BRL-3A IGFBP purified from a fetal rat liver cell line. An antiserum was generated against this astrocyte 35,000 Mr protein, and immunoblot analysis revealed a dramatic increase in rIGFBP-2 secretion in astrocytes cultured from 14-day, 21-day, and adult rat brain compared to astrocytes from 1-day and 7-day rat brain. Similar analysis of neonatal rat brain neurons in culture failed to show immunoreactive rIGFBP-2 in cell lysates or secreted protein. Ligand Western blot analysis demonstrated [125I]IGF-II binding to a single protein band which comigrated with a prominant rIGFBP-2 immunoreactive species in nonreduced conditioned medium from 21-day astrocytes. In comparison [125I]IGF-II binding proteins were detected only at low levels in medium from astrocytes cultured from 1-day rat brain and were undetectable in neuron-conditioned media. Northern blot analysis using a rIGFBP-2 complementary DNA revealed 5-fold greater messenger RNA levels in astrocytes from 21-day rat brain compared with astrocytes from 1-day brain, whereas neonate neurons showed no transcripts. Thus, rIGFBP-2 exhibits a pattern of developmental and cell-specific expression in cultured rat brain cells.

The effect of Ca 2+ channel antagonists (cadmium, ω-conotoxin GIVA, and nitrendipine) on the release of angiotensin II from fetal rat brain in vitro

We have shown previously that K + stimulation of dissociated cell cultures of fetal rat brain results in a graded release of angiotensin II (ANG II) that is dependent on the availability of extracellular Ca 2+. In this study, using dissociated cell cultures of fetal rat hypothalamus, thalamus, septum, and midbrain (HTSM), we further examined the role of calcium channels on ANG II release using specific channel blockers (cadmium, ω-conotoxin, and nitrendipine) and a calcium ionophore (A23187). Levels of ANG II release were quantitated by radioimmunoassay and HPLC. For control levels of ANG II release, cells were incubated in a stock buffer containing 89 mM choline chloride/58 mM KCl/2 mM CaCl 2. Pretreatment of the cells with either 100 μM Cd 2+ (to block N-, L-, and T-type calcium channels), 100 nM ω-conotoxin (to block N- and L-type calcium channels), or 500 nM nitrendipine (to block L-type calcium channels) decreased ANG II release by approximately 71%, 71%, and 22%, respectively, when compared to control levels. In contrast, pretreatment of the cells with 1.6 μM A23187 (a calcium ionophore) increased ANG II release by approximately 90% over control levels. These findings suggest that angiotensin release is dependent on the intracellular entry of Ca 2+ ions through primarily N-type channels, and to a lesser extent, L-type channels.

Intralysosomal Generation of Ammonia from Urea by Endocytosed Urease Results in Secretion of Free Lysosomal Arylsulfatase-A and Increased Activity of Membrane-Bound β-Glucosidase in Cultured Brain Cells

Hyperammonemia interferes with normal brain function. The effect of ammonia on free and membrane-bound lysosomal enzymes and on mucopolysaccharide metabolism was studied in cultured rat brain cells (ROC-1, hybridoma between C6-astrocytoma and oligodendrocytes). Intralysosomal ammoniagenesis was achieved from urea by endocytosed Jackbean urease followed by incubation of the cultures with urea. The intralysosomal location of urease was evidenced by the protective effects of leupeptin and urea on the stability of intracellular urease. Ammonia formed from urea resulted in an increased secretion of lysosomal arylsulfatase-A (AS-A), but not of the membrane-bound lysosomal beta-glucosidase into the culture medium, thus intralysosomal AS-A activity decreased. Lysosomal, membrane-bound beta-glucosidase activity increased, presumably due to intralysosomal proteolytic protection following an increased lysosomal pH. Intralysosomal ammoniagenesis temporarily impaired 35SO4-glycosaminoglycan degradation of prelabeled cells. The results support the hypothesis that hyperammonemic states may interfere with lysosomal functions in vivo as well in cultured cells.

Developmental Changes in Enzymes Involved in Dolichyl Phosphate Metabolism in Cultured Embryonic Rat Brain Cells

The rates of synthesis of dolichol-linked oligosaccharide intermediates and protein N-glycosylation increased substantially during a developmental period corresponding to glial differentiation in primary cultures of embryonic rat brain. In this study developmental changes in three enzymes involved in dolichyl phosphate (Dol-P) metabolism have been examined by in vitro assays and correlated with the induction pattern for lipid intermediate synthesis and protein N-glycosylation. Dolichyl pyrophosphate (Dol-P-P) phosphatase activity was relatively low during the first 9 days in culture, but it increased significantly between days 9 and 25. Dol-P-P phosphatase did not change appreciably between days 22 and 30 in culture. A kinetic analysis of the developmental change in Dol-P-P phosphatase activity revealed that the Vmax increased 10-fold between days 4 and 22, and there was also a significant change in the apparent Km for Dol-P-P. Dolichol kinase activity increased during the period (9-15 days) when there was a significant induction in oligosaccharide-lipid synthesis and protein N-glycosylation, and then declined in parallel with lipid intermediate synthesis and protein N-glycosylation. Dol-P phosphatase activity was present at relatively low levels for the first 9 days in culture, but it increased steadily between days 9 and 30. A kinetic comparison of the activity in membrane fractions from brain cells cultured for 9 and 25 days indicated that there was a 10-fold increase in enzyme protein with unaltered affinity for Dol-P.(ABSTRACT TRUNCATED AT 250 WORDS)

Hyaluronan synthesis by astrocytes in newborn rat brain cell cultures

Hyaluronan synthesis by astrocytes in newborn rat brain cell cultures

Cloning and Expression of a Developmentally Regulated Protein That Induces Mitogenic and Neurite Outgrowth Activity

A heparin binding mitogenic protein isolated from bovine uterus shares NH2-terminal amino acid sequence with a protein isolated from newborn rat brain. The cDNA's of the bovine, human, and rat genes have been isolated and encode extraordinarily conserved proteins unrelated to known growth or neurotrophic factors, although identity of nearly 50 percent has been found with the predicted sequence of a retinoic acid induced transcript in differentiating mouse embryonal carcinoma cells. Lysates of COS-7 cells transiently expressing this protein were mitogenic for NRK cells and initiated neurite outgrowth from mixed cultures of embryonic rat brain cells. RNA transcripts encoding this protein were widely distributed in tissues and were developmentally regulated. This protein, previously designated as heparin binding growth factor (HBGF)-8, is now renamed pleiotrophin (PTN) to reflect its diverse activities. PTN may be the first member of a family of developmentally regulated cytokines.

Effects of Ethanol on Hexose Uptake by Cultured Rat Brain Cells

The effects of ethanol on hexose uptake by glial cells was investigated using primary cultures prepared from term rat fetuses. Specific 3H 2-deoxy-D-glucose (2DG) uptake was significantly reduced by a 4-hr exposure to ethanol at concentrations of 25, 50, and 100 mM, but not 200 or 300 mM. The inhibitory effect of 50 mM ethanol increased with the duration of exposure, with 2DG uptake inhibited by 36% after 18 hr. Astrocytes cultured from the brains of term fetuses of rats fed ethanol during pregnancy showed essentially the same 2DG uptake response to in vitro ethanol treatment. Kinetics of 2DG uptake showed a significant decrease of Vmax in the presence of ethanol. No interaction was found between ethanol and insulin, which stimulated 2DG uptake and protein content of the cultures. The data suggest that ethanol can modulate hexose uptake by astrocytes cultured from fetal rat brain. However, insulin actions on glial cells were not affected by ethanol.

Effects of methyl isocyanate on rat brain cells in culture.

Since the disaster in Bhopal, India, people exposed to methyl isocyanate (MIC) have complained of various disorders including neuromuscular dysfunction. In an attempt to get information about such dysfunction we have previously shown that MIC can affect muscle cells in culture. The present communication reports investigations into the effect of MIC on brain cells in culture. MIC was toxic to brain cells and the response was dose related. The observations were supported by light and electron microscopy.

Differential effect of μ, δ, and κ ligands on G protein α subunits in cultured brain cells

Rat and guinea pig fetal brain cell cultures and immunoblotting techniques were used to study the effect of receptor selective opioids on the level of the membrane-bound alpha i and alpha o GTP binding protein subunits. Incubation of rat hindbrain cultures with the mu selective peptide DAGO decreased the amount of both alpha proteins. The reduction observed was equivalent to 36% in alpha o and 41% in alpha i. On the other hand, incubation of rat forebrain cultures with this peptide had an opposite effect, increasing the alpha o and alpha i levels by 66% and 68%, respectively. This differential effect of the peptide on the G proteins at the two brain areas may reflect the selective interaction at the receptor level; DAGO induced a fast and effective receptor down-regulation (50% decrease in Bmax) in hindbrain but not in forebrain cultures. Moreover, delta and mu selective ligands differed in their effect, as indicated by the finding that the delta selective peptide DPDPE increased the amount of both alpha proteins in hindbrain cultures by 40%. Similar experiments conducted with guinea pig brain aggregate cultures indicated that the kappa selective agonist U50,488 decreased the amount of the membrane bound alpha i protein subunit by 56%. The results thus indicate that opioid agonists, interacting selectively with the three types of opioid receptors, induce a complex repertoire of changes in the immunoreactive levels of the membrane-bound alpha GTP binding protein subunits in various CNS structures.

Characterization of the dynamic nature of microglial cells

Dynamic properties of ramified microglia were investigated in tissue cultures of rat brain cells. Microglia were identified in viable cultures based on previously determined aspects of their morphology and location, and this identification was confirmed through immunohistochemical staining for type-three complement receptors. When living microglial cells were monitored using time-lapse video recording, an extremely dynamic nature was revealed. These cells exhibited morphological alteration, surface and cytoplasmic activity, and migration. All of these properties were both constant and rapid, with individual cells being capable of assuming a completely different overall morphology within a period as short as 5–10 min. These dynamic properties were consistently observed in microglia and appeared specific to this cell type. Thus, ramified microglia appear to possess a unique and highly dynamic nature, and this feature is proposed as a potential marker for these cells. Additionally, the significance of microglial cellular dynamics in the functional role of this cell population in vivo is considered.

Characterization of oxytocin-binding sites in primary rat brain cell cultures

Detection and characterization of oxytocin-binding sites in dissociated brain cell cultures were performed, using a highly selective iodinated oxytoxin antagonist ([ 125I]OTA). Dissociated cells derived from hypothalamus and extrahypothalamic forebrain of 16-day-old fetal rats were maintained in chemically defined medium in order to enrich the cultures in neuronal cells. Specific binding of [ 125I]OTA, demonstrated in both hypothalamic and forebrain cell cultures, was temperature- and time-dependent; maximal binding was obtained by incubating the iodinated ligand for 60 min at 37 °C. The binding parameters were shown to be identical in both cell type cultures. The Scatchard plot analysis suggested the presence of a single class of binding sites of high affinity ( K d about 0.06 nM) and low binding capacity ( B max about 4 fmol/dish). The specificity of these binding sites tested in competition experiments revealed that the unlabelled OT antagonist was the most potent in inhibiting specific [ 125I]OTA binding ( K i = 0.1nM). A lower affinity, of the nM range was demonstrated for oxytocin (OT), arginine-vasopressin (AVP) and the V 1 antagonist, whereas the V 2 AVP agonist poorly competed for [ 125I]OTA binding sites ( K i about 250 nM). In conclusion, the [ 125I]OTA binding characteristics, identical in both hypothalamic and forebrain cultures, fulfil the classical conditions required for the existence of receptor sites since the binding was reversible, saturable and specific. As these characteristics were similar to those already described in the adult rat, at the central level in hippocampus, and at the periphery in the mammary gland, it could be postulated that [ 25I]OTA binds to an OT receptor site. Therefore, fetal rat brain cell cultures may provide a relatively simplified in vitro model for studying the OT receptor and its regulation.

Purification of Glycogen Phosphorylase from Bovine Brain and Immunocytochemical Examination of Rat Glial Primary Cultures Using Monoclonal Antibodies Raised Against This Enzyme

The physiological function in brain of glycogen and the enzyme catalyzing the rate-limiting step in glycogenolysis, glycogen phosphorylase (EC 2.4.1.1), is unknown. As a first step toward elucidating such a function, we have purified bovine brain glycogen phosphorylase isozyme BB 1,700-fold to a specific activity of 24 units/mg protein. When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequent silver staining, a single major protein band corresponding to an apparent molecular mass of 97 kDa was observed. Mouse monoclonal antibodies raised against the enzyme were purified and shown to be monospecific as indicated by immunoblotting. Immunocytochemical examination of astroglia-rich primary cultures of rat brain cells revealed a colocalization of glycogen phosphorylase with the astroglial marker glial fibrillary acidic protein in many cells. The staining for the enzyme appeared at two levels of intensity. There were other cells in the culture showing no specific staining under the experimental conditions employed. Neurons in neuron-rich primary cultures did not show positive staining. The data suggest that glycogen phosphorylase may be predominantly an astroglial enzyme and that astroglia cells play an important role in the energy metabolism of the brain.