Biomedical subjects

B Hamprecht

Publications and source records attributed to B Hamprecht.

At least 73 records · Page 4Linked to original sources

PubMed1994

Immunocytochemical demonstration of glycogen phosphorylase in Müller (glial) cells of the mammalian retina.

Glycogen phosphorylase (GP) was immunocytochemically detected in Müller cells of the rabbit and rat retina using a monoclonal antibody raised against bovine brain GP. Immunofluorescence and immunoenzymatic procedure were applied on isolated, Müller cells and sections of paraformaldehyde-fixed, paraffin-embedded retinas. All methods used revealed positive immunostaining. GP immunoreactivity was most intense in the Müller cell endfeet and the pericarya, corresponding to the nerve fibre layer and the inner nuclear layer in the retina. The presence of GP in Müller cells stresses the important role of these glial cells in the energy metabolism of the mammalian retina.

Animals↗

PubMed1994

Utilization of mannose by astroglial cells.

Uptake and metabolism of mannose were studied in astroglia-rich primary cultures derived from neonatal rat brains. A saturable component of mannose uptake was found with half-maximal uptake at 6.7 +/- 1.0 mM mannose. In addition, a non-saturable component dominated the uptake at high concentrations of mannose. Glucose, cytochalasin B, or phloretin in the incubation buffer inhibited the carrier-mediated uptake of mannose. Within the astroglial cells mannose is phosphorylated to mannose-6-phosphate. In cell homogenates, the KM value of mannose-phosphorylating activity was determined to be 24 +/- 7 microM. The Vmax value of this activity is only 40% that of glucose-phosphorylating activity. Mannose-6-phosphate was converted to fructose-6-phosphate by mannose-6-phosphate isomerase. The specific activity of this enzyme in homogenates of astroglial cultures was higher than that of hexokinase. Two products of mannose utilization in astroglial cells are glycogen and lactate. The amounts of each of these products increased with increasing concentrations of mannose. In contrast to the generation of lactate, that of glycogen from mannose was enhanced in the presence of insulin. In conclusion, we suggest that mannose is taken up into the cells of astroglia-rich primary cultures by the glial glucose transporter and is metabolized to fructose-6-phosphate within the astroglial cells.

Animals↗

PubMed1993

Uptake of L-lactate by cultured rat brain neurons.

The uptake of L-lactate was investigated in neuronal primary cultures derived from embryonic rat brain with a radioactive tracer method. After preincubation of the cells in glucose-free buffer for 30 min, uptake increased with time for at least 10 min. A saturable component of uptake was found with half-maximal uptake at 10 mM lactate. This saturable component was abolished in the presence of 10 mM alpha-cyano-4-hydroxcinnamic acid. In addition, a non-saturable component dominated the uptake at high concentrations of lactate. Uptake was accelerated with decreasing pH, and was inhibited considerably by pyruvate. It is concluded that neurons are endowed with a lactate transport system which resembles in its properties the monocarboxylate carrier of peripheral tissues.

Animals↗

PubMed1993

Glycogen in astrocytes: possible function as lactate supply for neighboring cells.

In order to contribute to the elucidation of the function of astrocyte glycogen in brain, studies on the fate of the glucosyl residues of glycogen were carried out on astroglia-rich primary cultures derived from the brains of newborn rats. On glucose deprivation astroglial cells rapidly deplete their glycogen. In contrast to the situation with hepatocytes, only lactate, but not glucose, is detectable in the medium surrounding the astroglial cells. Besides glucose, astroglial cultures can also use mannose as a substrate for the synthesis of glycogen and the generation of lactate. Although mannose-fed astroglial cells contain glucose-6-phosphate, they do not release a measurable amount of glucose into the culture medium. Instead of glucose the astroglial cells release high amounts of lactate into the culture medium. Gluconolactone or 2-deoxyglucose which prevent glycogen breakdown in astroglial cells after glucose deprivation, allow to discriminate between lactate generated from glycogen and lactate from other sources. The amount of lactate found in the medium in the absence of gluconolactone (or 2-deoxyglucose) exceeds the amount found in the presence of either compound by the lactate equivalents calculated to be contained in the cellular glycogen. In conclusion, glycogen in astrocytes can be considered as a store for lactate rather than for glucose.

Animals↗

PubMed1993

Differences in glycogen metabolism in astroglia-rich primary cultures and sorbitol-selected astroglial cultures derived from mouse brain.

Recently it has become possible by chemical selection using sorbitol instead of glucose in the culture medium to produce pure astroglial cultures from astroglia-rich primary cultures from mouse brain. The glycogen-degrading enzyme glycogen phosphorylase in brain is localized in astrocytes and ependymal cells. In view of this fact it appeared necessary to study the influence of glucose and other hexoses on the glycogen metabolism in these cultures lacking the influence of other cell types in comparison to the astroglia-rich primary cultures containing several types of cells. The sorbitol-fed selected cultures and the glucose-deprived astroglia-rich primary cultures contain less than 10% of the glycogen encountered in glucose-fed primary cultures. During incubation with glucose the glycogen content of the selected cultures and the glucose-deprived primary cultures increases by more than one order of magnitude. Nevertheless, not all cells are found to have accumulated glycogen. The time course of the replenishment of glycogen is similar in both types of culture, although maximal levels reached in the selected cultures are 3 times those in the astroglia-rich primary cultures. This difference might be explained by the fact that the ratio of the maximal activities of glycogen synthase and glycogen phosphorylase in selected cultures was found to be twice that in the unselected cultures. During glucose deprivation the glycogen content is reduced in both culture systems with half-maximal contents being reached at 15 min (primary culture) and 45 min (selected culture). Both types of culture can also utilize mannose for the synthesis of glycogen and the production of lactate.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Glycogen phosphorylase activity and immunoreactivity during pre- and postnatal development of rat brain.

Catalytic activity and immunoreactivity of glycogen phosphorylase were studied in pre- and postnatal rat brain. The catalytic activity was assayed in brain homogenates; immunoreactivity was investigated by immunoblot analysis using a monoclonal anti-bovine brain glycogen phosphorylase antibody. The cellular localization and intensity of immunoreactivity were analysed on paraffin-embedded sections utilizing the same monoclonal antibody. The catalytic activity increased 10-fold from embryonic day 16 to adult; immunoreactivity became detectable on embryonic day 16 and increased in intensity as the enzyme activity rose to adult values. The first cellular elements to be stained immunohistochemically were ependymal cells lining the ventricles, ependymal cells of the choroid plexus, meningeal cells and a selected population of neurons in the brain stem. The immunoreactivity of plexus cells and meningeal cells was reduced or absent in the adult rat brain. The earliest appearance of glycogen phosphorylase immunoreactivity in astroglial cells was seen at postnatal day 9 in the hippocampus. The staining pattern of the adult brain was reached at day 22 post partum. The developmental changes in glycogen deposition and in glycogen phophorylase activity and immunoreactivity may indicate a variable physiological role of glycogen metabolism for different cell types in the pre- and postnatal periods.

Animals↗

PubMed1993

Purification of cytosolic malic enzyme from bovine brain, generation of monoclonal antibodies, and immunocytochemical localization of the enzyme in glial cells of neural primary cultures.

Cytosolic malic enzyme (EC 1.1.1.40) was purified from bovine brain 5,600-fold to a specific activity of 47 U/mg. The enzyme is a homotetramer with a subunit molecular mass of 60 kDa and an isoelectric point of 6.2. Mouse monoclonal antibodies raised against this enzyme were purified and shown to be monospecific, as indicated by immunoblotting. Immunocytochemical examination of rat astroglia-rich primary cultures at the light microscopic level revealed colocalization of cytosolic malic enzyme with the astroglial marker glial fibrillary acidic protein. Also, a colocalization with the oligodendroglial marker myelin basic protein was found. Neurons in rat neuron-rich primary cultures did not show positive staining. The data suggest that cytosolic malic enzyme is a glial enzyme and is lacking in neurons.

Animals↗

PubMed1993

Inhibition by 2-deoxyglucose and 1,5-gluconolactone of glycogen mobilization in astroglia-rich primary cultures.

The presence of glycogen in astroglia-rich primary cultures derived from the brains of newborn rats depends on the availability of glucose in the culture medium. On glucose deprivation, glycogen vanishes from the astroglial cultures. This decrease of glycogen content is completely prevented if 2-deoxyglucose in a concentration of > 1 mM or 1,5-gluconolactone (20 mM) is present in the culture medium. 2-Deoxyglucose itself or 3-O-methylglucose, a glucose derivative that is not phosphorylated by hexokinase, does not reduce the activity of glycogen phosphorylase purified from bovine brain or in the homogenate of astroglia-rich rat primary cultures. In contrast, deoxyglucose-6-phosphate strongly inhibits the glycogen phosphorylase activities of the preparations. Half-maximal effects were obtained at deoxyglucose-6-phosphate concentrations of 0.75 (phosphorylase a, astroglial culture), 5 (phosphorylase b, astroglial culture), 2 (phosphorylase a, bovine brain), or 9 mM (phosphorylase b, bovine brain). Thus, the block of glycogen degradation in these cells appears to be due to inhibition of glycogen phosphorylase by deoxyglucose-6-phosphate rather than deoxyglucose itself. These results suggest that glucose-6-phosphate, rather than glucose, acts as a physiological negative feedback regulator of the brain isoenzyme of phosphorylase and thus of glycogen degradation in astrocytes.

3-O-Methylglucose↗

PubMed1993

Incorporation of radioactivity from [14C]lactate into the glycogen of cultured mouse astroglial cells. Evidence for gluconeogenesis in brain cells.

A pure population of astroglial cells was selected from heterogeneous astroglia-rich primary cultures in a medium containing sorbitol instead of glucose. It was shown that astroglial cells synthesize glycogen when they are returned to a glucose-containing medium, and that when [14C]lactate is also present the synthesized glycogen is radioactively labelled. Compared with the degree of incorporation of radioactivity in the presence of tritiated glucose, the incorporation of radioactivity from lactate was small but significant. After incubation of astroglial cells with radioactively labelled lactate, the glycogen was isolated and enzymatically hydrolysed to glucose, which was found to be radioactively labelled. Astrocytes are therefore able to convert lactate to glucosyl residues, a metabolic pathway known as gluconeogenesis. It is proposed that astrocytic gluconeogenesis may consume lactic acid formed in neighboring cells such as neurons, during anaerobic glycolysis at times of high energy demand.

Animals↗

PubMed1992

Immunohistochemical co-localization of glycogen phosphorylase with the astroglial markers glial fibrillary acidic protein and S-100 protein in rat brain sections.

Immunofluorescence double-labelling and immunoenzyme double-staining methods were used to examine the location of glycogen phosphorylase brain isozyme with the astrocyte markers glial fibrillary acidic protein (GFAP) and S-100 protein in formaldehyde-fixed, paraffin-embedded slices from adult rat brain. Astrocytes in the cerebellum and the hippocampus, which express GFAP or S-100 protein immunoreactivity, show glycogen phosphorylase immunoreactivity. Regional intensity and intracellular distribution of the three antigens vary characteristically. In ependymal cells, glycogen phosphorylase immunoreactivity is co-localized with S-100 protein immunoreactivity, but not with GFAP immunoreactivity. These findings confirm that glycogen phosphorylase in the rat brain is exclusively localized in astrocytes and ependymal cells. All astrocytes, as far as they express GFAP or S-100 protein, do contain glycogen phosphorylase.

Animals↗

PubMed1992

Glucose, insulin, and insulin-like growth factor I regulate the glycogen content of astroglia-rich primary cultures.

The glycogen content of astroglia-rich primary cultures derived from the brains of newborn rats depends on the concentration of glucose in the culture medium. After administration of culture medium lacking glucose, the glycogen content decreases with a half-time of 7 min. Readdition of glucose results in replenishment of the glycogen stores within 2-3 h, but fully only if glucose is present in a concentration of at least 4 mM. Insulin, or the more potent insulin-like growth factor I, increases the content of glycogen approximately 1.7-fold, with the half-maximal effects being attained at concentrations of 10 and 0.5 nM, respectively. These results suggest that (a) glucose or a metabolite of it and (b) insulin-like growth factor I or a closely related peptide, but not insulin, are likely to be physiological regulators of the level of glycogen in astrocytes.

Animals↗

PubMed1991

Replacement of glucose by sorbitol in growth medium causes selection of astroglial cells from heterogeneous primary cultures derived from newborn mouse brain.

Primary cultures derived from the brains of newborn mice are quantitatively dominated by astroglial cells, but contain also oligodendroglial, phagocytic and ependymal cells. When confluent cultures are fed with glucose-free growth medium containing 25 mM sorbitol for 14 days, oligodendroglial, phagocytic and ependymal cells are eliminated from the culture, as judged by morphological and immunocytochemical criteria. The remaining cells stain positively for vimentin and glial fibrillary acidic protein and, therefore, can be considered as astroglial cells. Inoculation of freshly dissociated mouse brain cells in the absence of glucose in a sorbitol-containing medium is not possible; however, feeding of the cultures from day 2 on with sorbitol instead of glucose results in a pure astroglial culture at confluency. Therefore glucose-free growth medium supplemented with sorbitol can be considered a selective medium for astroglial cells in primary mouse glial cultures.

Animals↗

PubMed1990

Activation of a K+ conductance by bradykinin and by inositol-1,4,5-trisphosphate in rat glioma cells: involvement of intracellular and extracellular Ca2+.

Extracellular application of bradykinin and injection of inositol-1,4,5-trisphosphate (Ins-P3) induced a hyperpolarization in polyploid rat glioma cells. Ins-1,4,5-P3 and Ins-2,4,5-P3 were effective but not Ins-4,5-P2, Ins-1,3,4,5-P4 and Ins-1,3,4,5,6-P5. The reversal potential of the hyperpolarizing response induced by bradykinin or by Ins-P3 increased to a comparable degree with increasing the extracellular K+ concentration. Certain blockers of K+ channels, for example charybdotoxin (5-50 nM), Ba2+ (5-20 mM), 4-aminopyridine (5-10 mM) and quinidine (0.1-0.5 mM) reversibly suppressed the membrane potential response to bradykinin or to Ins-P3; however, apamin (1 microM) and D-tubocurarine (0.5 mM) had no effect. Intracellular injection of EGTA made the glioma cells unresponsive to bradykinin. Superfusion of the cells with Ca2(+)-free medium gradually and reversibly abolished the response to bradykinin, but only slightly reduced the effect of Ins-P3. The Ca2+ channel blockers Co2+ (1-5 mM), Mn2+ (2-6 mM) and nifedipine (1-20 microM), but not desmethoxyverapamil (100 microM) inhibited the hyperpolarizing effect of bradykinin. The hyperpolarization induced by Ins-P3, however, was not influenced by Mn2+ (1-5 mM) or by Co2+ (7 mM). Injection of Ca2+ into the glioma cells induced a hyperpolarization susceptible to Ba2+ and quinidine. Treatment of glioma cells with an activator or with inhibitors of protein kinase C or with pertussis toxin did not affect the response to bradykinin. Incubation of the cells with the Ca2+ ionophore A23187 (0.1-1 microM) made the cells unresponsive to bradykinin and, somewhat less, to Ins-P3. At these concentrations the Ca2+ ionophore primarily depletes intracellular Ca2+ stores. In summary, bradykinin, via B2-receptors (blocked by [Thi5,8, D-Phe7]-bradykinin) activates a K+ conductance in glioma cells following a rise of cytosolic Ca2+ activity most likely due to Ins-P3-mediated release of Ca2+ from internal stores. Entry of extracellular Ca2+ appears also to be involved in this process.

Animals↗

PubMed1990

Sorbitol pathway activity and utilization of polyols in astroglia-rich primary cultures.

Astroglia-rich rat primary cultures can be grown in a glucose-free medium containing 25 mM sorbitol. After 10 days under these conditions, the total number of cells and DNA content are reduced to 50-60% of those of control cultures, but remain constant thereafter. The specific activities of the sorbitol pathway enzymes, sorbitol dehydrogenase and aldose reductase, are increased 2.5-fold and unchanged, respectively, if the cells are grown in the presence of sorbitol instead of glucose. Treatment with the aldose reductase inhibitor sorbinil does not decrease the number of cells cultured in the glucose-free medium in the presence of sorbitol. Fructose is as good a substrate for the glial cells as sorbitol, whereas out of a number of other polyols tested only xylitol can support the primary cultures for more than 3 days. Neither neuron-rich rat brain primary cultures nor rat glioma cells can be cultured in the sorbitol-containing medium in the absence of glucose. With sorbitol substituting for glucose in the culture medium, effects of glucose deprivation on cellular functions like sugar transport and metabolism can be investigated in glial cultures for an extended period of time.

Aldehyde Reductase↗

PubMed1990

Immunohistochemical demonstration of glycogen phosphorylase in rat brain slices.

Paraffin-embedded sections from paraformaldehyde-fixed rat brain were stained immunocytochemically for glycogen phosphorylase brain isozyme BB, using a monoclonal mouse antibody and the biotin-strept-avidin method, with either horseradish peroxidase or beta-galactosidase as marker enzymes. Two cell types showed strong glycogen phosphorylase-immunoreactivity: Astrocytes and ependymal cells. Most intensive staining was observed in the cerebellar cortex, the neocortex and the hippocampus. Astrocytes in the cerebellar white matter stained positively. The choroid plexus cells stained poorly or not at all. Neurons throughout the brain were negative, as well as oligodendrocytes and bundles of myelinated nerve fibers. These data are consistent with the immunocytochemical localization of glycogen phosphorylase in astroglia-rich primary cultures derived from rat brain.

Animals↗

PubMed1990

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.

Animals↗

PubMed1989

Serotonin raises the cyclic GMP level in a neuronal cell line via 5-HT3 receptors.

Serotonin (5-HT) induced a transient rise of the cyclic GMP level in neuroblastoma X glioma hybrid cells, half-maximally at 1 microM 5-HT. 2-Methyl-5-HT displayed an about 5 times lower potency but equal efficacy. alpha-Methyl-5-HT and 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) were completely ineffective at concentrations up to 30 microM. Antagonists specific for 5-HT3 receptors, ICS 205-930, GR 38032 F and MDL 72222, blocked the response to 5-HT at nanomolar concentrations but antagonists directed towards 5-HT1 and 5-HT2 receptors, ketanserin and methysergide, had no effect at concentrations up to 1 microM. Thus, 5-HT3 receptors are responsible for activating guanylate cyclase in the hybrid cells.

8-Hydroxy-2-(di-n-propylamino)tetralin↗

PubMed1989

Substance P and serotonin act synergistically to activate a cation permeability in a neuronal cell line.

Both substance P and, to a lesser degree, serotonin activate cation permeability in neuroblastoma x glioma hybrid cells, as determined by measurement of [14C]guanidinium uptake. Serotonin potentiates the action of substance P by shifting the concentration-effect curve of substance P to the left. The EC50 value for the synergistic effect of serotonin was around 0.3 microM. Dopamine and noradrenaline displayed comparable activity, albeit only at 50 and 130 times higher concentrations, respectively. The order of potency of various substance P-analogues was not changed by serotonin, indicating that the specificity of the substance P site on the hybrid cells was not affected by serotonin. Various other neurotransmitters and peptides had no effect on the response of the hybrid cells to substance P. The serotonin receptor interacting with the substance P receptor may be classified as a 5-HT3-receptor since methysergide, cimetidine, and ketanserin were ineffective, but two inhibitors specific for 5-HT3-receptors, ICS 205-930 (3 alpha-tropanyl-1H-indole-3-carboxylic acid ester) and MDL 72222 (1 alpha H,3 alpha,5 alpha H-tropan-3-yl-3,5-dichlorobenzoate), blocked the effect of serotonin at nanomolar concentrations. However, the two serotonin antagonists might also be blocking the ion permeability, since at higher concentrations they fully inhibited the stimulation of guanidinium uptake by substance P or by substance P plus serotonin. The synergism between substance P and serotonin on the hybrid cells offers the opportunity to study the mechanism of interaction of neurotransmitter receptors on a permanent neuronal cell line.

Catecholamines↗