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H Herken

Publications and source records attributed to H Herken.

At least 19 recordsLinked to original sources
PubMed1989

Inhibition of biopterin synthesis and DOPA production in PC-12 pheochromocytoma cells induced by 6-aminonicotinamide.

Pheochromocytoma cells (clone PC-12) were treated with 6-aminonicotinamide. Tetrahydrobiopterin content and DOPA production of the cells were determined by reverse-phase HPLC and subsequent electrochemical detection. The same chromatographic system was used to determine total biopterin (tetrahydrobiopterin, dihydrobiopterin and quinoide dihydrobiopterin) by fluorescence detection. Tetrahydrobiopterin plays a decisive role as cofactor of tyrosine hydroxylase for the biosynthesis of DOPA and dopamine. Addition of 6-aminonicotinamide to the culture medium resulted in the accumulation of 6-phosphogluconate, suggesting that PC-12 cells synthesize 6-aminonicotinamide-adenine-dinucleotide-phosphate (6-ANADP) by a glycohydrolase localized in the endoplasmic reticulum. This substance is known to be a strong inhibitor of 6-phosphogluconate dehydrogenase and leads to a blockade of the pentose phosphate pathway. In our experiments, the synthesis of biopterins was depressed after application of 6-aminonicotinamide. The decrease of intracellular tetrahydrobiopterin and total biopterin by 6-aminonicotinamide at different concentrations was strongly correlated with a reduced cellular DOPA production. The decreased content of biopterin cofactor was compensated by addition of the precursor sepiapterin, indicating that the NADPH2-dependent reductases in biopterin synthesis are not inhibited by the antimetabolite. However, DOPA production remained suppressed at the same time. After application of NADH2, we observed an increased DOPA production though the decreased biopterin levels remained almost unchanged. The results imply that the first step in the synthesis of biopterin from GTP as well as the recycling pathways of the oxidized cofactor might be the site of action of the antimetabolite.(ABSTRACT TRUNCATED AT 250 WORDS)

6-Aminonicotinamide↗
PubMed1987

PC12 cells: a model system for studying drug effects on dopamine synthesis and release.

PC12 cells were used as a model system to examine drug effects on dopamine synthesis and release. It could be demonstrated that KCl treatment induced release of endogenous dopamine, simultaneously DOPA synthesis was increased. Despite of the increase in DOPA synthesis the intracellular concentration of tetrahydrobiopterin (the co-factor of tyrosine hydroxylase) remained stable, indicating that the catalytic recycling of the used tetrahydrobiopterin is much more rapid than the tetrahydrobiopterin consumption by tyrosine hydroxylation. Reserpine induced a decrease of intracellular dopamine but no dopamine reached the extracellular space, instead all dopamine was depleted into the cytoplasma and metabolized to DOPAC. Nitrendipine had no effect on intracellular dopamine storage, Bay K 8644 induced a small decrease of intracellular dopamine and a small increase in DOPA production. N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W7) induced a reserpine-like depletion at concentrations above 1 X 10(-6) M. The KCl-induced dopamine release and the stimulation of DOPA production were blocked by 1 X 10(-7) M nitrendipine and enhanced by 1 X 10(-7) M Bay K 8466, whereas 1 X 10(-6) M W7 had no effect on both parameters. These findings were compared to data obtained in other tissues reported in the literature indicating that PC12 cells are a useful model for studying drug effects on catecholamine synthesis and release.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1986

Apomorphine does not decrease tissue levels of tetrahydrobiopterin in vivo.

It was reported that R(-)apomorphine and other catechols are potent inhibitors of dihydropteridine reductase in vitro. It was suggested that decreased levels of tetrahydrobiopterin may represent a mechanism by which R(-)apomorphine inhibits catecholamine synthesis in vivo. This paper demonstrates that tetrahydrobiopterin levels are not affected either in vitro (PC12 cells) or in vivo (rat liver and corpus striatum) by treatment with R(-)apomorphine, whereas DOPA (3,4-dihydroxyphenylalanine) production (PC12 cells, corpus striatum) is reduced. This indicates that R(-)apomorphine does not inhibit DOPA production by reducing 6(R)-L-erythro-tetrahydrobiopterin) levels.

Animals↗
PubMed1985

Effect of apomorphine, alpha-methylparatyrosine, haloperidol and reserpine on DOPA production in clonal cell lines (PC-12 and N1E-115).

The effect of various drugs on DOPA production in the pheochromocytoma clone PC-12 and the neuroblastoma clone N1E-115 was studied. The N1E-115 cells contain only very low amounts of dopamine due to a lack of the aromatic L-amino acid decarboxylase, whereas the PC-12 cells are rich in dopamine. alpha-Methyl-p-tyrosine and apomorphine blocked DOPA production in both cell clones. Reserpine and haloperidol reduced the intracellular dopamine in the PC-12 cells and simultaneously induced a blockade of cellular DOPA production. The released dopamine was primarily recovered as 3,4-dihydroxyphenylacetic acid indicating a release of dopamine into the cytoplasm. This transient increase of cytoplasmic dopamine by reserpine or haloperidol brings about the inhibition of DOPA production in the PC-12 cells. Our results show that the PC-12 clone especially reacts to various drugs like other in vitro systems and may serve as an additional model for studying drug effects on catecholamine biosynthesis and metabolism.

Adrenal Gland Neoplasms↗
PubMed1985

Evaluation of neurotropic drug actions on tyrosine hydroxylase activity and dopamine metabolism in clonal cell lines.

Two clonal cell lines (the pheochromocytoma clone PC-12 and the neuroblastoma clone N1E-115) were used to compare direct and indirect drug effects on tyrosine hydroxylase and dopamine turnover. Both clones contain the cofactor of tyrosine hydroxylase, tetrahydrobiopterin, in sufficient concentrations. 2,4-Diamino-6-hydroxy-pyrimidine (DAO-Pyr), an inhibitor of GTP cyclohydrolase, which is the rate-limiting enzyme in tetrahydrobiopterin biosynthesis, lowers DOPA production indicating that cofactor supply is a limiting factor for catecholamine synthesis. DOPA synthesis in the PC-12 cells can be stimulated by incubation with the natural cofactor tetrahydrobiopterin, but also by its possible precursors sepiapterin and dihydrobiopterin or the analogs methyl-tetrahydropterin and dihydropterin. The regulating enzyme for DOPA synthesis, tyrosine hydroxylase, can be inhibited by certain drugs either directly or indirectly by increasing dopamine concentrations in the cytoplasm after release from its vesicular stores. Using the neuroblastoma clone N1E-115 which lacks DOPA decarboxylase and thus contains only low levels of dopamine the site of action of certain drugs could be determined. Drugs affecting the tyrosine hydroxylase directly (alpha-methyl-para-tyrosine, apomorphine) decreased DOPA production in both clones, while drugs acting via interference with the vesicular stores (reserpine, amphetamine, nigericin) were effective only in the PC-12 cells. After total depletion of dopamine by nigericin at high concentrations or long-term incubation with 3-hydroxybenzyl-hydrazine (NSD 1015), DOPA production increased in the PC-12 cells indicating a usually occurring regulation of tyrosine hydroxylase by cytoplasmic dopamine. Dopamine concentration in the cytoplasm was calculated to be in the range of 1 X 10(-6) mol/l.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

Isolation and characterization of internalized glioma cell membranes.

A method for isolating plasma membranes based on the ability of cultured C6-glioma cells to phagocytize inert material such as polystyrene (latex) beads is described. The beads (phi 1.1 micron) were incubated for 16 h or 5 h. After several washings and homogenization of the cells, the beads with the surrounding membranes were isolated by use of a sucrose density gradient. The membranes were analyzed morphologically and biochemically. Morphological studies by means of light- and electron microscopy confirmed the intracellular localization of the beads. Enzymatic studies revealed that the specific activity of acid phosphatase decreased with shorter incubation periods (from 268.00 +/- 38.56 U X mg protein-1 X min-1 after 16 h to 125.12 +/- 9.10 after 5 h), whereas that of Na, K-ATPase showed the opposite trend (3.63 +/- 0.41 and 4.73 +/- 0.78 mumoles phosphate X mg protein-1 X h-1, respectively), indicating a lesser contamination with lysosomes. The main advantages of this procedure for membrane studies lie in purity and definite orientation ("inside-out") of the membranes.

Acid Phosphatase↗
PubMed1984

Tetrahydrobiopterin and total biopterin content of neuroblastoma (N1E-115, N2A) and pheochromocytoma (PC-12) clones and the dependence of catecholamine synthesis on tetrahydrobiopterin concentration in PC-12 cells.

Tetrahydrobiopterin content was determined in several clonal cell lines by reversed-phase HPLC and subsequent electrochemical detection. The same chromatography system was used to determine the total biopterin (tetrahydrobiopterin and 7,8-dihydrobiopterin) by fluorescence detection. The catecholamine-producing clones neuroblastoma N1E-115 and pheochromocytoma PC-12 contained 96 and 60 ng tetrahydrobiopterin/mg protein, respectively. The corresponding amount for the neuroblastoma clone N2A was 36 ng/mg protein. The tetrahydrobiopterin content in C-6 glioma cells was below the limit of detection. The total biopterin is about 20% above the tetrahydrobiopterin content. Tetrahydrobiopterin and biopterin from the cells were identified by coelution with standard solutions and by potential-current relationship or emission and excitation spectra, respectively. Addition of 2,4-diamino-6-hydroxypyrimidine, an inhibitor of biopterin synthesis from GTP, to the culture medium of PC-12 cells resulted in a dose-dependent decrease of tetrahydrobiopterin and total biopterin content within 4 h, suggesting that the cells are capable of synthesising the biopterin which was found. A decrease in intracellular tetrahydrobiopterin levels by different concentrations of 2,4-diamino-6-hydroxypyrimidine reduces the cellular production of dihydroxyphenylalanine after inhibition of aromatic L-amino acid decarboxylase, indicating that the concentration of tetrahydrobiopterin might be a limiting factor for catecholamine synthesis in catecholamine-producing cells.

Adrenal Gland Neoplasms↗
PubMed1983

[Nerve cell clonal lines in culture--models for studying the molecular basis of neuropharmacological actions].

Nerve cell lines with stable properties are isolated from neuroblastomas, glioblastomas or pheochromocytomas by periodic cloning using defined culture media. After the action of different drugs, these cells show all morphological and biochemical signs of differentiation and maturation. Depending on the origin of the clone, the cell lines synthesise typical neurotransmitters, which are stored in vesicles. It is demonstrated on cell lines which synthesise catecholamines that noradrenergic and dopaminergic clones are particularly suitable test objects for basic research in neuropharmacology.

Astrocytoma↗
PubMed1982

Mouse neuroblastoma clone N1E-115: a suitable model for studying the action of dopamine agonists of tyrosine hydroxylase activity.

The DOPA-content in neuroblastoma clone N1E-115 is higher than the dopamine or noradrenaline content. Blockade of tyrosine hydroxylase by alpha-methyl-p-tyrosine (1 X 10(-3) M) resulted in a decrease of cellular DOPA-content to 24.9% after 4 hr. The accumulation of DOPA in these cells which is probably due to limited activity of l-aromatic amino acid decarboxylase led us to use DOPA-content as a measure of tyrosine hydroxylase (TH) activity. Dopamine and especially apomorphine were effective at low concentrations (dopamine IC50 1 X 10(-5) M, apomorphine 2 X 10(-7) M); lisuride had no effect on TH-activity. The low effective dose of apomorphine and the failure of lisuride to influence TH-activity are comparable to the observations in striatal synaptosomal preparations and make the N1E-115 clone a suitable model for studying the mechanism of TH-regulation. However, since haloperidol (1 X 10(-5) M) did not reverse the apomorphine-induced blockade of TH, a receptor-mediated blockade of TH seems to be improbable.

Animals↗
PubMed1982

Dopa-release from mouse neuroblastoma clone N 1 E-115 into the culture medium. A test for tyrosine hydroxylase activity.

Due to a lack of L-Dopa-decarboxylase, the mouse neuroblastoma clone N 1 E-115 contains a high intracellular Dopa-content compared to a low noradrenaline- and dopamine-content. Because of this decarboxylase deficiency, the N 1 E-115 clone releases more than 95% of the produced Dopa into the culture medium. After renewal of the culture medium, Dopa production of the cells can be measured by the increase of Dopa in the medium. Dopa production was linear during 2 h and varied from 50-180 micrograms/mg prot-1 x h-1 between different subcultures. Dopa release into the medium was used as an indirect measure for the tyrosine-hydroxylase activity. Several dopaminergic agonist and antagonists were tested. Dopa production could be blocked dose-dependent by apomorphine (1 X 10(-7)-1 x 10(-6) M), but not by lisuride hydrogen maleate and bromocryptine. Several dopaminergic and adrenergic antagonists failed to reverse the apomorphine induced blockade of the tyrosine-hydroxylase activity.

Animals↗
PubMed1980

Further studies on the nature of red fluorescent structures in neuroblastoma monolayer cells vitally stained with acridine orange.

The nature of red fluorescent particles in vitally acridine orange stained C 1300 neuroblastoma monolayer cells was evaluated by electron microscopy, cytofluorometry, cytopharmacological and cell fractionation studies. At the ultrastructural level the distribution of red fluorescent granules correlated with that of the Golgi complex and Golgi derived structures during various stages of differentiation, mitosis, and under colcemid treatment. Cytopharmacological studies revealed that red fluorescence was displaced in a concentration and time dependent manner with the basic drugs chloroquine and quinacrine. Subcellular fractionation studies showed that acridine orange was concentrated in fractions that also contained the highest amount of acid phosphatase and electron dense vesicles. Vital acridine orange staining of neuroblastoma cells in culture can give information on the relationship between Golgi-derived vesicles and cell functions like proliferation and differentiation. The influence of drugs on these processes can be studied.

Acid Phosphatase↗
PubMed1978

Impaired dopamine function and muscular rigidity induced by 6-aminonicotinamide in rats.

In corpus striatum, 6-aminonicotinamide (6-AN), 10 mg/kg i.p. lowered the concentration of dopamine and markedly reduced the disappearance of dopamine after synthesis inhibition with alpha-methyl-p-tyrosine. In the dopamine-rich part of the limbic system, the formation of DOPA after decarboxylase inhibition with 3-hydroxybenzylhydrazine was decreased. In diencephalon, 6-AN altered neither the steady-state level nor the utilization of noradrenaline. The data suggest that the muscular rigidity induced by 6-AN may be associated with disruption of dopaminergic transmission.

6-Aminonicotinamide↗
PubMed1978

Effects of 6-aminonicotinamide on growth and acetylcholinesterase activity during differentiation of neuroblastoma cells in vitro.

Addition of 6-AN (0.01 mg/ml) to growing cultures of C-1300 neuroblastoma cells strongly reduced cell division. This growth inhibition was accompanied by a higher cell volume and a lower protein content per cell as compared to controls. Concurrently the specific activity of AChE increased markedly incontrols and 6-AN-treated cultures. During the experimental periods the specific activity of AChE was significantly higher after 6-AN. Morphologically, 6-AN-treated cultures showed characteristic signs of differentiation, i.e. enlarged, flattened cells with long branched processes. The described effect of 6-AN on growth and differentiation of neuroblastoma cells was less pronounced if cells received the antimetabolite after a subcultivation period of 5 days.

6-Aminonicotinamide↗