Biomedical subjects

B Hamprecht

Publications and source records attributed to B Hamprecht.

At least 55 records · Page 3Linked to original sources

PubMed1997

Cellular distribution of branched-chain amino acid aminotransferase isoenzymes among rat brain glial cells in culture.

The first step in the catabolism of branched-chain amino acids (BCAA), reversible transamination, is catalyzed by one of the two isoforms of branched-chain amino acid aminotransferase (BCAT). The mitochondrial isoenzyme (BCATm) is widely distributed among tissues, whereas the cytosolic isoenzyme (BCATc) is restricted to only a few organs. Remarkably, BCATc is the prominent isoenzyme found in brain. The physiological significance of the subcellular compartmentation of BCAT is still not understood. To contribute to the elucidation of the cellular distribution of the two isoenzymes in brain, we used cultured rat glial cells in an immunocytochemical study to determine the pattern of BCAT isoenzyme expression by glial cells. Antiserum against BCATm generated a punctate staining pattern of astroglial cells, confirming the mitochondrial location of this isoenzyme. In contrast, the cytosol of galactocerebroside-expressing oligodendroglial cells and O2A progenitor cells displayed intense staining only for BCATc. In addition, subpopulations of astroglial cells exhibited BCATc immunoreactivity. The presence of BCATm in astrocytes is consistent with the known ability of these cells to oxidize BCAA. Furthermore, our results on BCATc provide support for the hypothesis that BCATs are also involved in nitrogen transfer from astrocytes to neurons.

Amino Acids, Branched-Chain↗

PubMed1996

Effect of astroglial cell swelling on pH of acidic intracellular compartments.

A variety of pathological conditions lead to swelling of astrocytes, which in turn stimulates ion release by activation of ion channels at the plasma membrane. In the present study, acridine orange and fluorescein isothiocyanate coupled to dextran (FITC-dextran) have been used to examine the effect of cell swelling on pH in acidic compartments of cultured astroglial cells. Both NH4Cl (2 mM) and chloroquine (10 microM), known to alkalinize acidic cellular compartments, led to the expected increase in acridine orange fluorescence intensity. Similar, albeit smaller, effects were elicited by a reduction of extracellular osmolarity (-80 mOsm) and treatment of the cells with glutamate (l mM), manoeuvres which enhanced cell volume. Determination of changes in the FITC-dextran fluorescence ratio (485/440 nm) allowed quantification of the pH changes in lysosomal compartments. Treatment with NH4Cl, reduced extracellular osmolarity and glutamate increased lysosomal pH by 0.65 +/- 0.07, 0.85 +/- 0.14 and 0.25 +/- 0.07, respectively. Measurement of cytosolic pH using 2',7',-bis-(2-carboxyethyl)-5- (and -6) carboxyfluorescein (BCECF) demonstrated a pronounced acidification following cell swelling, observed with both reduced extracellular osmolarity (by 0.23 +/- 0.05 pH units) and 1 mM glutamate (by 0.26 +/- 0.02 pH units). In conclusion, pH within lysosomes and possibly other acidic cellular compartments of astrocytes is increased by cell swelling, which may have important consequences for astrocyte function.

Acridine Orange↗

PubMed1996

Different preferences in the utilization of amino acids for glutathione synthesis in cultured neurons and astroglial cells derived from rat brain.

The intracellular contents of glutathione in neuron-rich and astroglia-rich primary cultures derived from the brains of embryonal and newborn rats were found to be 23.1 +/- 3.0 and 31.2 +/- 6.5 nmol/mg of protein, respectively. Deprivation of amino acids for 4 h reduced the level of glutathione in neuron-rich cultures by 24%. Glutathione was resynthesized on refeeding of cysteine, glutamine, and glycine. A maximal content of glutathione was found 4 h after refeeding, exceeding that of untreated neuron-rich cultures by 84%. Replacement of cysteine by cystine or glutamine by glutamate during the 4 h refeeding period resulted in a lower intracellular amount of glutathione. An increase in the glutathione level of neuron-rich cultures by 76% was found if the culture medium was supplemented with 250 microM cysteine. However, no such increase occurred if cystine was used instead. In contrast to neuron-rich cultures, astroglia-rich primary cultures restored a maximal content of glutathione if glutamate and cystine were refed after amino acid deprivation. These results demonstrate that cysteine is the limiting compound in the culture medium for glutathione synthesis in neuron-rich cultures and that astroglial cells and neurons in culture have different preferences for uptake and utilization of amino acids for glutathione synthesis.

Amino Acids↗

PubMed1996

Evidence that stress activates glial lactate formation in vivo assessed with rat hippocampus lactography.

Extracellular lactate of the rat hippocampus is inter alia increased by immobilization stress. The origin of lactate is, however, not well established, so it is not known whether it is mainly derived form neurons or glial cells. Dialysates were collected shortly (1 or 2 days) or with a delay (14 or 15 days) after implantation of the probe. In the short-term experiment lactate increased after stress, both with or without glucose added to the perfusate. In the long-term experiment there was marked gliosis around the dialysis probe and the stress effects were seen only in the presence of 5 mM glucose. The results are consistent with the idea that stress induces glycogenolysis and lactate export from astroglial cells via neurotransmitter or hormonal related processes.

Animals↗

PubMed1996

Ultrastructural localization of glycogen phosphorylase predominantly in astrocytes of the gerbil brain.

The localization of glycogen phosphorylase in gerbil brain was determined by immunoelectron microscopy using the pre-embedding peroxidase technique. Electron-dense granular reaction product of peroxidase activity was observed in astrocytes of all brain regions examined (cerebral cortex, striatum, cerebellar cortex, hippocampal formation, corpus callosum, mesencephalic trigeminal nucleus). The reaction product was distributed in a diffuse pattern throughout the cytoplasmic matrix of perikarya and processes; sometimes the nucleus of labeled astrocytes also contains immunopositive material. The light microscopically visible glycogen phosphorylase immunoreactivity associated with capillaries could be characterized as a staining of astrocytic endfeet ensheathing capillaries. Endothelial cells and pericytes were never labeled. In addition to astrocytes, ependymal cells also presented immunopositive material in their cytoplasm. On the other hand, no reaction product was observed in cells identified as oligodendroglia or microglia. Neurons (with the exception of neurons of the mesencephalic trigeminal nucleus), their processes, and their synaptic endings were free of reaction product. In the neuropil we frequently observed immunopositive glial processes adjacent to synaptic structures. This intimate spatial relationship may be interpreted as a morphological sign of a metabolic interaction. The data support the hypothesis that astroglia play a key role in glycogen metabolism and energization of the brain.

Animals↗

PubMed1996

Possible glial contribution of rat hippocampus lactate as assessed with microdialysis and stress.

Microdialysis for the continuous monitoring of lactate ("lactography") was applied in rat brain hippocampus in an attempt to establish whether lactate is of neuronal or glial origin. Lactate was analyzed with an electrochemical assay after enzymatic oxidation in dialysates derived from a short-term (1 or 2 days after implantation of the probe) and a long-term (14 or 15 days) preparation. In the short-term experiment the lactate levels in the dialysate were higher without glucose in the perfusate, whereas in the long-term experiment a several fold increase in lactate was observed in the presence of 5 mM glucose. During stress, increases in lactate were virtually similar both in the acute (with or without glucose in the perfusate) and in the chronic preparation (response in the presence of glucose only). In the long-term preparation presence of reactive astroglia cells was visualized immunohistochemically with antibodies against glial fibrillary acidic protein. Damage of the hippocampus and the corpus callosum was seen in the chronic preparation with silver impregnation staining. These results emphasize the importance of the presence of glucose in the perfusate and they are consistent with the idea that glial cells contribute to extracellular lactate in the rat hippocampus in vivo and that stress activates the astroglial glycogen pool.

Animals↗

PubMed1996

Glutathione content as an indicator for the presence of metabolic pathways of amino acids in astroglial cultures.

The intracellular content of glutathione in astroglia-rich primary cultures derived from the brains of newborn rats was measured to be 32.1 +/- 5.4 nmol/mg of protein. During a 24-h incubation in a minimal medium lacking amino acids and glucose, the content of glutathione in these cultures was reduced to 52% of the original content. On refeeding of glucose, glutamate, glycine, and cysteine, glutathione was resynthesized. A maximal content of glutathione was found 4 h after refeeding, exceeding the amount of glutathione of untreated cultures by 72%. Maximal glutathione synthesis was observed only if glutamate, cysteine, and glycine were present. If successively each one of these amino acids was made limiting for the synthesis of glutathione, half-maximal contents of glutathione were found at 0.2 mM glutamate, 20 microM cysteine, or 10 microM glycine. Replacement of glutamate or glycine by other amino acids revealed the potential of astroglial cells to convert glutamine, aspartate, asparagine, proline, and ornithine into glutamate, and serine into glycine. These results demonstrate that the concentration of intracellular glutathione can serve as an indicator for the presence of metabolic pathways of amino acids in cultured cells.

Amino Acids↗

PubMed1996

Studies on fructose metabolism in cultured astroglial cells and control hepatocytes: lack of fructokinase activity and immunoreactivity in astrocytes.

Astroglia-rich primary cultures derived from the brains of newborn rats can be grown in the presence of sorbitol or fructose. In the present study, evidence was obtained by enzymatic analysis and immunocytochemistry that fructose is further metabolized to fructose-6-phosphate and that fructokinase is lacking in the astrocytes. In contrast, fructose-1-phosphate as well as fructokinase immunoreactivity could be detected in cultured hepatocytes. Considerable amounts of astroglial glycogen were synthesized from fructose. Lactate release in fructose-fed cultures was still 30% that of glucose-fed cells and was abolished in the presence of 2-deoxyglucose. No glycogen was synthesized when sorbitol, which is converted intracellularly to fructose, replaced glucose in the incubation medium. However, lactate release from sorbitol-fed cultures was still significant and was not abolished by 2-deoxyglucose. The results are compatible with the idea of astroglial glycogen being a store of lactate rather than glucose. Furthermore, the results demonstrate that sorbitol is an adequate substrate for astroglial glycolysis but, in contrast to fructose, cannot be utilized for the buildup of glycogen stores.

Animals↗

PubMed1996

Colocalization of three types of intermediate filament proteins in perisinusoidal stellate cells: glial fibrillary acidic protein as a new cellular marker.

The presence and the colocalization of the three intermediate filament proteins, glial fibrillary acidic protein (GFAP) and the marker of mesenchymal liver cells, vimentin, were studied by an immunofluorescence double-labeling technique in cultures of isolated rat perisinusoidal stellate cells (PSC) and hepatocytes, in cocultures of isolated PSC and hepatocytes as well as in cryostat sections of rat liver. GFAP and vimentin immunoreactivities were localized in cultured PSC which were identified by the presence of the cellular marker desmin, another intermediate filament protein, or the stellate morphology to be seen after staining for one of three intermediate filament proteins. Both GFAP and vimentin were strongly expressed in the perinuclear region and the cell processes of cultured PSC. Staining for GFAP highly coincided with that for vimentin or desmin in cultured PSC and with that for vimentin in the liver sections. Desmin-positive cells were always also GFAP-positive. However, of the GFAP-positive cells only an estimated 50% were found desmin-positive. The coexpression of desmin and GFAP in the same cells appear to be unique, since apparently it has not been previously reported for any other cell type. Almost all of the vimentin-positive cells in hepatocyte culture were also expressing GFAP. Since desmin was not found in all of the cultured cells with PSC morphology, GFAP is suggested as a more reliable marker for PSC than desmin.

Animals↗

PubMed1996

Glial fibrillary acidic protein as a marker of perisinusoidal stellate cells that can distinguish between the normal and myofibroblast-like phenotypes.

Glial fibrillary acidic protein (GFAP) has recently been shown to provide a marker for normal perisinusoidal stellate cells (PSC) in the liver. However, nothing was known so far about the changes in the intercellular abundancy of GFAP during transformation of PSC into myofibroblasts, a process characterized by marked changes in the expression of elements of the cytoskeleton. In order to address this question, we have used double-labelling immunofluorescence techniques for detecting smooth muscle alpha-actin (SMAA) and the intermediate filament proteins, GFAP, desmin and vimentin, taking advantage of the fact that PSC present in primary cultures of rat hepatocytes proliferate and transform. GFAP and vimentin were expressed in PSC throughout cultivation, while desmin which stained only about half of the PSC in early cultures was expressed in all GFAP-positive cells later on. The intensity of staining for GFAP in PSC transiently increased till the second day of cultivation followed by a decrease. At the 6th day of cultivation, staining for GFAP was seen only in the perinuclear region and as a faint rim at the contours of the cells. In contrast, SMAA, an established marker for transformed PSC, started to be expressed at the third day. Thereafter, immunoreactivity for SMAA increased continuously, indicating an inverse expression of GFAP and SMAA during transformation. These results indicate that GFAP, due to the plasticity of its expression, can discriminate between the normal untransformed and transformed phenotypes of PSC. Furthermore, they suggest a role of GFAP not only as a reliable marker, but also in the maintenance and/or function of the normal differentiated phenotype of liver PSC.

Actins↗

PubMed1995

The 4F2hc surface antigen is necessary for expression of system L-like neutral amino acid-transport activity in C6-BU-1 rat glioma cells: evidence from expression studies in Xenopus laevis oocytes.

Mammalian cells possess a variety of amino acid-transport systems with overlapping substrate specificity. System L is one of the major amino acid-transport systems in all non-epithelial cells. Its molecular structure is not known. To clone the neutral amino acid-transporter system L, we followed an expression cloning strategy using Xenopus laevis oocytes. A cDNA library derived from C6-BU-1 rat glioma cells was used as a source, because high expression of system L activity could be demonstrated with polyadenylated RNA isolated from these cells, when injected into Xenopus laevis oocytes [Bröer, Bröer and Hamprecht (1994) Biochim. Biophys. Acta 1192, 95-100]. A single clone (ILAT) was identified, the sense cRNA of which, on injection into Xenopus laevis oocytes, stimulated sodium-independent isoleucine transport by about 100-fold. Further characterization revealed that transport of cationic amino acids was also stimulated. Sequencing of the cDNA showed that the identified clone is the heavy chain of the rat 4F2 surface antigen, a marker of tumour cells and activated lymphocytes. Uptake of neutral and cationic amino acids was not stimulated by the presence of Na+ ions. Antisense cRNA transcribed from this clone or antisense oligonucleotides, when co-injected with polyadenylated RNA from C6-BU-1 rat glioma cells, completely suppressed system L-like isoleucine-transport activity. We conclude that ILAT is necessary for expression of system L-like amino acid-transport activity by polyadenylated RNA from C6-BU-1 rat glioma cells.

Amino Acid Sequence↗

PubMed1995

Colocalization of fructose-1,6-bisphosphatase and glial fibrillary acidic protein in rat brain.

Immunofluorescence studies of rat brain sections demonstrated an exclusive colocalization of the gluconeogenic key enzyme fructose-1,6-bisphosphatase (FBPase) with the astroglial marker glial fibrillary acidic protein, indicating FBPase in brain as an astrocyte-specific enzyme. This conclusion was supported by the presence of FBPase activity in astroglia-rich but not neuron-rich primary cultures derived from rat brain.

Animals↗

PubMed1995

Significant amounts of glycogen are synthesized from 3-carbon compounds in astroglial primary cultures from mice with participation of the mitochondrial phosphoenolpyruvate carboxykinase isoenzyme.

The incorporation was studied of the gluconeogenic substrates lactate, alanine, aspartate and glutamate into glycogen of astroglial primary cultures derived from mouse brain. The incorporation was inhibited by 3-mercaptopicolinate, an inhibitor of one of the characteristic gluconeogenic enzymes, phosphoenolpyruvate carboxykinase. Only the mitochondrial isoenzyme of phosphoenolpyruvate carboxykinase was detectable in the astroglial primary cultures. After the incubation of glucose-starved cells with medium containing a mixture of [6-3H]glucose and [U-14C]glucose, the newly synthesized glycogen showed a 3H/14C ratio which was approximately 15% less than the isotope ratio for the medium. The decrease of the isotope ratio was not significantly inhibited by 3-mercaptopicolinate, indicating a cycling of approximately 15% of the glucose to the level of the triose phosphates before its incorporation into astroglial glycogen. During the initial phase of glycogen resynthesis, the contribution of the gluconeogenic substrates appeared to be higher. This was in agreement with the accumulation of fructose 2,6-bisphosphate during refeeding. A participation of gluconeogenic substrates in glycogen metabolism was also detectable when the glycogen content was not changing significantly.

Animals↗

PubMed1995

Immunocytochemical localization of glycogen phosphorylase in primary sensory ganglia of the peripheral nervous system of the rat.

Neuronal localization was investigated of glycogen phosphorylase (GP) in ganglia of the peripheral nervous system of the rat. Immunofluorescence and immunoenzymatic procedures were applied with a monoclonal anti-bovine brain GP antibody on paraformaldehyde-fixed, paraffin-embedded tissues. Immunoreactivity was only present in the somatic neurons of the mesencephalic trigeminal nucleus in the brain stem and in dorsal root ganglia (DRG), but not in the autonomic neurons of the superior cervical ganglia or in the sensory nuclei of the spinal cord. GP immunoreactivity was present as early as day 1 after birth. In the adult rat, staining was present in neurons of different sizes, and to varying intensities. No relationship was apparent between the staining intensities and morphologically distinguishable types of neurons. In DRG, the type of reactivity was the same from cervical to sacral ganglia. The selected occurrence of GP in specific neurons of the peripheral nervous system in contrast to the ubiquitous occurrence in all astrocytes of the central nervous system may indicate a different role of neuronal glycogen compared to astrocytic glycogen.

Animals↗

PubMed1995

Immunocytochemical examination of neural rat and mouse primary cultures using monoclonal antibodies raised against pyruvate carboxylase.

Pyruvate carboxylase (EC 6.4.1.1; PC) catalyzes the formation of oxaloacetate by energy-dependent fixation of CO2 to pyruvate. The aim of the present work was to generate antibodies against PC and use them to localize PC in the cells of astroglia-rich and neuron-rich primary cultures derived from the brains of rats and mice. Mouse monoclonal antibodies raised against the enzyme were shown to be monospecific as indicated by immunoblotting. The staining of the cells for PC appeared in grains. These represent mitochondria, as PC is known as a mitochondrial enzyme. Immunocytochemical examination of astroglia-rich primary cultures of rat or mouse brain cells revealed a colocalization of PC with the astroglial marker glial fibrillary acidic protein (GFAP) in many cells. However, there were GFAP-positive cells showing no specific staining for PC, and vice versa. Also, in neuron-rich primary cultures PC was found only in the approximately 10% GFAP-expressing astroglial cells contaminating the neuron-rich primary culture, whereas it was absent from the neurons identified by antibodies against neuron-specific enolase. These results suggest that PC is predominantly an astroglial enzyme and that astroglial cells play an important role in the intermediary and the energy metabolism of the brain.

Animals↗

PubMed1995

Generation of ketone bodies from leucine by cultured astroglial cells.

To elucidate the significance of branched-chain amino acids (BCAAs) for brain energy metabolism, the capacity to use BCAAs for oxidative metabolism was investigated in astroglia-rich primary cultures derived from newborn rat brain. The cells selectively removed BCAAs from the culture medium, the disappearance following first-order kinetics. The BCAAs disappeared rapidly in spite of the presence of sufficient glucose as substrate for the generation of energy. Taking into consideration that the ketogenic amino acid leucine could be degraded only to acetyl-CoA and acetoacetate, and with the knowledge that astroglial cells have the capacity to secrete ketone bodies, this amino acid was chosen for further metabolic studies. After incubation of the cells with leucine, acetoacetate, D-beta-hydroxybutyrate, and alpha-ketoisocaproate were found to have accumulated in the culture medium. Identification of the radioactive metabolites generated from [4,5-3H]leucine established that the source of the substances released was indeed leucine. These results indicate that, at least in culture, astroglial cells degrade leucine via the known metabolite alpha-ketoisocaproate, to acetoacetate, which can be further reduced to D-beta-hydroxybutyrate. It is hypothesized that upon release from brain astrocytes, the ketone bodies could serve as fuel molecules for neighboring cells such as neurons and oligodendrocytes. In view of these and other results, astrocytes may be considered the brain's fuel processing plants.

3-Hydroxybutyric Acid↗

PubMed1995

Lactate transport in cultured glial cells.

Uptake of L-lactate was investigated with a radioactive tracer method in cultured rat glioma cells and in astroglia-rich primary cultures derived from rat brain. In the glioma cells, a saturable component of uptake was identified with half-maximal uptake occurring at 1.0 +/- 0.4 mM lactate. In addition, a non-saturable component dominated the uptake at high concentrations of lactate. In astroglia-rich primary cultures, no saturable component of uptake could be detected in the concentration range studied (0.1-15 mM). In conclusion, lactate uptake at physiological concentrations is predominantly mediated in the glioma cells by a carrier-dependent process, whereas in astroglial cells, simple diffusion appears to be the dominant way of lactate transport.

Animals↗

PubMed1994

Expression of Na+-independent isoleucine transport activity from rat brain in Xenopus laevis oocytes.

Poly(A)+ RNA from C6-BU-1 rat glioma cells and rat astroglial cells induced isoleucine transport activity when injected into Xenopus laevis oocytes. The Na+-independent component of isoleucine transport was inhibited by leucine, phenylalanine and 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH) but neither by methylaminoisobutyric acid (MeAIB) nor lysine. A Km value of approx. 100 microM was determined for the Na+-independent transport of isoleucine. These data are in accordance with expression of a system L like transporter. By injection of size fractionated poly(A)+ RNA a length of approx. 1.9 kb was determined for the pertinent mRNA.

Amino Acid Transport Systems↗