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Biomedical subjects

B Hamprecht

Publications and source records attributed to B Hamprecht.

At least 109 records · Page 6Linked to original sources
PubMed1984

Cellular heterogeneity in primary cultures of brain cells revealed by immunocytochemical localization of glutamine synthetase.

The co-localization of glutamine synthetase, glial fibrillary acidic protein, galactocerebroside and fibronectin was investigated by immunofluorescence double staining in primary cultures of dissociated brain cells from newborn mice. In cultures, grown in serum-free medium, containing dibutyryl-cAMP, glutamine synthetase was found in about half of the glial fibrillary acidic protein containing astroblasts. After addition of dexamethasone to the cultures, glutamine synthetase appeared also in another cell type, in which no glial fibrillary acidic protein, but fibronectin was detectable. This demonstrates that these cells, which are present in substantial amounts, are not of glial nature. In cultures treated with dibutyryl-cAMP no other cell type was found positive for fibronectin. For cultures grown in serum-containing medium lacking dibutyryl-cAMP, evidence was obtained that glutamine synthetase was induced by dexamethasone only in part of all cells staining for fibronectin. This suggests the presence of two different populations of fibronectin-positive cells. Oligodendrocytes, revealed by staining for galactocerebroside, never contained detectable amounts of glutamine synthetase, irrespective of the presence of dexamethasone. This also holds for cultures grown in serum-free medium containing dibutyryl-cAMP. However, under these conditions, oligodendroblasts are seen only very rarely. Our results demonstrate an unexpected heterogeneity in the cellular composition of primary cultures from newborn mouse brain.

Animals↗
PubMed1984

Bradykinin regulates the level of guanosine 3',5'-cyclic monophosphate (cyclic GMP) in neural cell lines.

The nonapeptide, bradykinin, elevated the level of cyclic GMP in two neural cell lines, neuroblastoma X glioma hybrid cells (clone 108CC15) and glioma cells (clone C6-4-2). In the hybrid cells the half-maximal stimulation occurred at 0.1 nM and the maximum was reached at 10 nM bradykinin. As soon as 30 s after the addition of bradykinin to the cultured cells, the intracellular concentration of cyclic GMP had increased maximally. The subsequent decline to the original level proceeded more slowly and lasted around 10 min. Hybrid cells incubated for 10 min in the presence of bradykinin and washed thereafter, did not respond at all to a subsequent 1 min challenge incubation with bradykinin. This nearly complete desensitization lasted for a period of 20 min. One hour after removal of bradykinin the original response to the peptide was restored. Modified and partial sequences of bradykinin were also investigated for their ability to induce the cyclic GMP response in the hybrid cells. Removal of amino acids from either terminus of bradykinin led to an almost complete loss of activity. The data are discussed with respect to our previous observation that bradykinin causes a slow hyperpolarization response in these cell lines and that on prolonged exposure to the peptide the membrane potential response of the cells is lost due to desensitization.

Animals↗
PubMed1984

Cell culture as models for studying neural functions.

Two cell culture systems were used for studies of neural functions in vitro. A neuronal hybrid cell line (neuroblastoma x glioma hybrid cells) and primary glial-rich cultures of newborn murine brain. The level of cyclic AMP in both systems is regulated by two groups of hormones, those that stimulate and those that inhibit formation of cyclic AMP. Among the inhibitory hormones active on the hybrid cells are opioids. Therefore the cells are being used in the elucidation of action of opioids. The list of stimulating and inhibitory hormones regulating the primary glial-rich cultures includes several peptide hormones such as the gastrointestinal peptides secretin and vasoactive intestinal peptide, the calcaemic hormones parathyrin and calcitonin, adrenocorticotropin and melanotropins, and somatostatin. Noradrenaline (via alpha- and beta-adrenergic receptors) and adenosine (via A1 and A2 receptors) inhibit and stimulate cyclic AMP synthesis in the primary glial-rich cultures. Bradykinin slowly hyperpolarizes the hybrid cells and elicits formation of cyclic GMP. Both responses desensitize rapidly. Substance P increases the permeability of hybrid cells for Na+, as measured by using 14C-guanidinium as substitute for Na+. Hybrid cells actively accumulate taurine, an amino acid that appears to fulfill important functions in the nervous system. The transport of taurine across the plasma membrane is highly specific for and strictly dependent on Na+. The pumped station hypothesis of taurine action in the nervous system views taurine gradient plus taurine carrier as a transport system for the elimination of sodium from neurons during phases of high neuronal activity.

Animals↗
PubMed1983

Heterogeneous distribution of enkephalin-degrading peptidases between neuronal and glial cells.

Cultured neurones, astroblasts and astrocytes from murine brain have been screened with specific tests for the presence of peptidases capable of degrading enkephalin. Bestatin-sensitive aminopeptidases represent the major enkephalin-degrading activity in all cases. The dipeptidylaminopeptidasic activity is much higher in the neuronal than the glial cultures, whereas the opposite is true for the metallopeptidase called "enkephalinase". Only trace amounts of the dipeptidylcarboxypeptidase "angiotensin-converting enzyme" have been found. We conclude that bestatin-sensitive aminopeptidases on nerve cells are probable candidates for enkephalin-inactivating enzymes, whereas the "enkephalinase" on glial cells more likely serves a scavenger function.

Aminopeptidases↗
PubMed1983

Sodium-channels in non-excitable glioma cells, shown by the influence of veratridine, scorpion toxin, and tetrodotoxin on membrane potential and on ion transport.

Veratridine induces membrane potential oscillations in non-excitable glioma cells, which are not affected by ouabain (2 mM) or by D600 (0.1 mM). In the presence of veratridine, scorpion toxin causes depolarization of the glioma cells to a positive value of the membrane potential. These effects of veratridine and of scorpion toxin are observed in Na+ but not in choline medium and are inhibited by tetrodotoxin. The response of the glioma cells to bradykinin has also been studied during these experiments. Previously bradykinin has been shown in these cells to induce a hyperpolarizing response caused by an increase in K+ conductance. This response to bradykinin can still be seen during the veratridine-induced oscillations of the membrane potential. In the glioma cells the uptake of guanidinium, a substitute for Na+, is enhanced by veratridine plus scorpion toxin. This stimulation is tetrodotoxin-sensitive. However, in the excitable neuroblastoma X glioma hybrid cells studied for comparison, veratridine causes membrane potential oscillations accompanied at the rising phase by one action potential or a train of action potentials. The results demonstrate that in non-excitable glioma cells tetrodotoxin-sensitive Na+ channels can be activated by veratridine and by scorpion toxin.

Animals↗
PubMed1983

Vasoactive intestinal polypeptide: presence in neuroblastoma X glioma hybrid cells.

Extracts of neuroblastoma X glioma hybrid cells 108CC15 and their parental lines were investigated for the presence of vasoactive intestinal polypeptide (VIP). With the aid of a radioimmunoassay and a receptor binding assay, VIP activity was found in the hybrid cells, to a lesser extent in neuroblastoma cells, but not in glioma cells. These results suggest a neuronal function of VIP. Since the hybrid cells also contain acetylcholine and opioids, they may be useful in studies of co-release of neurohormones.

Animals↗
PubMed1983

Tetrodotoxin-sensitive ion channels characterized in glial and neuronal cells from rat brain.

Ion channels were studied in primary neuronal and in primary glial cultures from rat brain by measuring the uptake of guanidinium, an ion that can permeate the Na+ channel. Neuronal cells exhibit a veratridine-stimulated (EC50 30 microM) guanidinium uptake, which is blocked by tetrodotoxin (IC50 30nM). This demonstrates the presence of a voltage-dependent Na+ channel. In glial cells veratridine + scorpion toxin, but not veratridine or scorpion toxin alone can stimulate a tetrodotoxin-sensitive ion uptake, thus indicating a 'silent' Na+ channel in the glial cells. Phentolamine, propranolol and various local anesthetic drugs (e.g. tetracaine, dibucaine) blocked the two different kinds of Na+ channels in the two cell populations investigated.

Anesthetics, Local↗
PubMed1983

Irreversible activation of the opiate receptor of neuroblastoma X glioma hybrid cells by an alkylating benzomorphan derivative.

The benozomorphan derivative (-)-2-[2-(p-bromoacetamidophenyl)ethyl]-5,9 alpha-dimethyl-2'-hydroxy-6,7-benzomorphan (BAB), capable of reacting with nucleophilic groups, acts on neuroblastoma X glioma hybrid cells as a potent, irreversible opiate agonist. Its potency in inhibiting the increase in cellular cyclic AMP, evoked by prostaglandin E1, is comparable to that of Leu-enkephalin. This also applies to its capacity to compete with [3H]D-Ala2-Met-enkephalinamide ([3H]DAEA) in binding on cell membrane preparations. The comparatively lower potency of (-)-2-[2-(p-acetamidophenyl)-ethyl]-5,9 alpha-dimethly-2'-hydroxy-5,7-benzomorphan (AB), which differs from BAB in the substitution of the bromoacetamido group by an acetamido group, is of the same order of magnitude as that of morphine. The covalent interaction of BAB with the opiate receptors is deduced from the observations that (1) it is not possible to wash away this compound from the receptors, (2) the potency of BAB in inhibiting the specific binding of [3H]DAEA increases with prolonged preincubation time, and (3) AB behaves as a reversible agonist.

Affinity Labels↗
PubMed1983

Corticotropin peptides and melanotropins elevate the level of adenosine 3':5'-cyclic monophosphate in cultured murine brain cells.

Cell cultures derived from mouse and rat brain and consisting mainly of astroblasts are known to respond to several hormones by increasing or decreasing their intracellular concentration of cyclic AMP. In the present study these cultures were analyzed for their susceptibility to various additional hormonal and other neuroactive compounds. Only the peptides of the corticotropin (ACTH)/melanotropin (MSH) family were found active. Their potency for elevating the intracellular level of cyclic AMP decreases in the sequence (values for the half-maximally stimulating concentrations, EC50, in parentheses) ACTH-(1-24) (10 m) greater than alpha-,beta-MSH (30 nm) greater than ACTH (greater than or equal to 100 nm) gamma-MSH, ACTH-(1-10), -(4-10), -(4-11) (greater than or equal to 0.5 microM). The lack of additivity of the maximal effects of the peptides suggests that they all act at the same receptor. The stimulation exerted by these peptides is partially suppressed by hormones known to inhibit cyclic AMP formation in that culture, i.e., noradrenaline (acting via an alpha-adrenergic receptor), adenosine (acting via an A1 receptor), and somatostatin. It is concluded that the receptors for the ACTH/MSH peptides and the inhibitory hormones are located on the same cells, presumably the astroblasts. The maximal response to ACTH and alpha- and beta-MSH depends strongly on the age of culture. The results are discussed in view of the facts that (1) peptides of the ACTH/MSH family affect behavior and learning in animals, and (2) ACTH and alpha-MSH occur in brain.

Adrenocorticotropic Hormone↗
PubMed1983

Blockade by neurotransmitter antagonists of veratridine-activated ion channels in neuronal cell lines.

The voltage-dependent Na+ ionophore of various neuronal cells is permeable not only to Na+ ions but also to guanidinium ions. Therefore, the veratridine- (or aconitine-)stimulated influx of [14C]guanidinium in neuroblastoma x glioma hybrid cells was measured to characterize the Na+ ionophore of these cells. Half-maximal stimulation of guanidinium uptake was seen at 30 microM veratridine. At 1 mM guanidinium, the veratridine-stimulated uptake of guanidinium was lowered to 50% by approximately 60 mM Li+, Na+, or K+ and by a few millimolar Mn2+, Co2+, or Ni2+. The basal, as well as the veratridine-stimulated, uptake of guanidinium was inhibited by the cholinergic antagonists (+)-tubocurarine (Ki = 50 to 500 nM) and atropine (Ki = 5 to 30 microM) and the adrenergic antagonists phentolamine (Ki = 5 microM) and propranolol (Ki = 60 microM). The specificity of the inhibitory effects of these agents is stressed by the ineffectiveness of various other neurotransmitter antagonists. However, the corresponding ionophore in neuroblastoma cells (clone N1E-115) seems to be regulated differently. While phentolamine and propranolol inhibit the veratridine-activated uptake as in the hybrid cells, (+)-tubocurarine and atropine exert only a slight effect.

Animals↗
PubMed1982

Bradykinin induces hyperpolarizations in rat glioma cells and in neuroblastoma X glioma hybrid cells.

The effect of the nonapeptide bradykinin on the membrane potential of permanent cell lines from neural origin was studied. A hyperpolarizing response of 10-30 s duration was produced when bradykinin was iontophoretically applied onto polyploid rat glioma cells (clone C6-4-2). Starting from the resting membrane potential the peak value of the hyperpolarizing response was reached within 0.5-1.5 s. Then the potential returned more slowly to the original value. The hyperpolarization was associated with an approximately 50% decrease in membrane resistance. Neither Na+ nor Cl- seemed to be important for the hyperpolarizing response, since bradykinin elicited similar hyperpolarizations in cells exposed to media in which Na+ or Cl- were replaced by choline or isethionate, respectively. Ca2+ fluxes are unlikely to be involved, since the addition of D600 did not affect the hyperpolarizations induced by bradykinin. However, a 10-fold increase in the concentration of K+ in the medium reduced the amplitude of the hyperpolarization by 40 mV. Thus, the hyperpolarization induced by bradykinin is associated with decrease in membrane resistance which is likely to be caused by an increased K+-conductance. The glioma cells showed a desensitization upon repeated application of bradykinin. However, the sensitivity of the cells to bradykinin was restored after 3-8 min of incubation in the absence of bradykinin. Since an antagonist of bradykinin is not known, the specificity of the action of bradykinin is difficult to assess. Nevertheless, the hyperpolarizing response to bradykinin appears to be specific insofar as other peptides, i.e. lutoliberin, thyroliberin, neurotensin, substance P and apamin, exerted no effect on the membrane potential of the glioma cells. Bradykinin-elicited hyperpolarizations with characteristics similar to those described above could also be demonstrated in neuroblastoma X glioma hybrid cells, but not in multinucleated fibroblast cells.

Animals↗
PubMed1982

Biosynthesis of carnosine and related peptides by glial cells in primary culture.

Synthesis of carnosine (beta-alanylhistidine) and related peptides by glial cells in primary culture could be demonstrated. After incubation with [3H]beta-alanine, the radiolabeled dipeptides could be isolated from the cell extract and the culture medium. With gamma-amino[3H]butyric acid, however, rapid degradation of the tracer without significant synthesis of homocarnosine (gamma-aminobutyrylhistidine) was observed. Aminooxyacetic acid, a known inhibitor of gamma-aminobutyryl:alpha-ketoglutarate aminotransferase, inhibits the degradation of gamma-amino[3H]butyric acid very strongly and of [3H]beta-alanine partially. After preincubation of the cells with this inhibitor, incorporation of gamma-amino[3H]butyric acid into homocarnosine and related peptides could be demonstrated.

Alanine↗
PubMed1982

Parathyrin and calcitonin stimulate cyclic AMP accumulation in cultured murine brain cells.

Despite the key role Ca2+ plays in the nervous system, biochemical actions on neural tissue of the Ca2+-regulating peptide hormones parathyrin and calcitonin were unknown. Until a few years ago only neurons, but not glial cells, were considered as targets for peptide hormones. Our recent observation that peptide hormones do indeed act on glial cells is extended by the present report that these cells respond to the calcaemic peptide hormones parathyrin and calcitonin. In cultured murine brain cells mainly consisting of glioblasts, parathyrin stimulates the accumulation of cyclic AMP. The half-maximal effect is elicited at 30 nM parathyrin. With rat brain cells the effects are three times those observed with mouse brain cells. Calcitonin, which is less potent than parathyrin, elevates the concentration of cyclic AMP only in rat brain cells. If properly occupied, the inhibitory receptors present on the cells lower the increase in the level of cyclic AMP evoked by parathyrin and, to some extent, that elicited by calcitonin. The results suggest that: (i) these or closely related hormones might exert regulatory functions in brain; and (ii) glial cells must be considered in discussions of the targets of the calcaemic and other peptide hormones.

1-Methyl-3-isobutylxanthine↗