Stimulated astrocytes release high-mobility group 1 protein, an inducer of LAN-5 neuroblastoma cell differentiation.

Stimulated astrocytes specifically release large amounts of high-mobility group 1 protein into the extracellular medium. The identity of the released protein has been established on the basis of its biological activity on murine erythroleukaemia cells and by its immunoreactivity against a specific monoclonal antibody. High-mobility group 1 protein also plays an essential role in differentiation of LAN-5 neuroblastoma cells which, following stimulation with retinoic acid, express high-mobility group 1 protein on to the external surface of the plasma membrane. In retinoic acid-induced LAN-5 cells, high-mobility group 1 protein is not secreted but is accumulated in a membrane-bound form, particularly at the level of neurite outgrowths. These cells can also be induced to differentiate by high-mobility group 1 protein coated on the surface of the cell culture vessels. The specific function of the protein in this process is indicated by inhibition of cell differentiation by an anti-high-mobility group 1 protein antibody. The data are consistent with a role of high-mobility group 1 protein in promoting cell-cell interactions and in the development of nerve tissues.

Quantitation of low abundance mRNAs in glial cells using different polymerase chain reaction (PCR)-based methods.

The conventional methods for mRNA quantitation such as Northern blotting or ribonuclease protection assay sometimes lack enough sensitivity to study low abundance mRNAs or to work with limited amounts of biological samples. The sensitivity of the polymerase chain reaction (PCR) linked to reverse transcription (RT-PCR) has proven useful in amplifying specific mRNAs, especially those present in low copy number. Though, the quantitation of nucleic acids by means of PCR has proven problematic. The main constraint in obtaining quantitative data is inherent in the amplification reaction. Because amplification is an exponential process, small variations in the efficiency of amplification may significantly affect the final yield of the PCR product. The variables that influence the rate of the PCR include the abundance of the mRNA present in the starting material, the concentrations of the Taq DNA polymerase, dNTPs and magnesium ions, the annealing and elongation conditions, the ramping temperatures and the formation of primer secondary structures. Moreover, with the progression of the PCR cycles, reagents are consumed and inhibitors generated, leading to non-linear synthesis of DNA. Finally, tube-to-tube variations sometimes preclude accurate quantitation. Most of the above-mentioned problems can be overcome by the choice of adequate internal controls. The present report reviews two recently developed methods for RNA quantitation, the semi-quantitative PCR and the quantitative PCR illustrated for the measurement of monoamine oxidase (MAO) A and B mRNAs and the estrogen receptor (ER) mRNA respectively, with a particular emphasis on the design of appropriate internal controls to compensate for the intra- and inter-assay variability inherent to RT-PCR.

Dopaminergic phenotype induced by oestrogens in a human neuroblastoma cell line.

Oestrogens are the key factor in the sexual differentiation of the mammalian brain and play an important role in the activity of selected areas of the mature brain. To pursue the study of oestrogen action on neural cells at the molecular level, we developed a human neuroblastoma cell line (SK-ER3) expressing the oestrogen receptor (ER). Treatment of these cells with 17beta-oestradiol causes growth arrest and morphological and biochemical differentiation. The aim of the present study was to investigate whether oestrogen-differentiated SK-ER3 neuroblastoma cells acquire the ability to synthesize a specific neurotransmitter and whether the growth arrest previously reported can be ascribed to the blockage of the cells at a specific stage of the cell cycle. The results presented here indicate that oestrogens induce accumulation of SK-ER3 cells in the G0 phase of the cell cycle, underscoring the acquisition of a mature neural phenotype upon hormonal treatment. Most importantly, we show that in the differentiated cells the content of tyrosine hydroxylase and Na+-dependent dopamine uptake is significantly augmented, proving that the oestrogen-differentiated SK-ER3 cells can synthesize and store a specific neurotransmitter. In addition, we prove that the dopamine accumulated in differentiated SK-ER3 cells can be released. These studies therefore suggest that oestrogen treatment results in the acquisition of a fully functional dopaminergic phenotype of SK-ER3 cells. Ample evidence shows a link between dopaminergic neurons and oestrogen activity in hypothalamic and non-hypothalamic areas of the mammalian brain. Our study indicates that oestrogens might play a primary role in committing undifferentiated neuroblasts towards the dopaminergic phenotype.

Monoamine oxidase B expression is selectively regulated by dexamethasone in cultured rat astrocytes.

The influence of dexamethasone on monoamine oxidase (MAO) A and B expression and activity was investigated in primary cultures of rat type 1 astrocytes cultured under serum free, defined conditions. Dexamethasone treatment resulted in a dose- and time-dependent induction of MAO-B, but not of MAO-A, activity. The selective MAO-B increase was substantially reduced by the antagonist RU 486, thus suggesting a glucocorticoid receptor-mediated action of the hormone. Kinetic analysis showed an increase in Vmax of MAO-B with no change in apparent K(m). The dexamethasone-induced selective rise in MAO-B activity appeared to be due to enhanced enzyme synthesis, since MAO-B mRNA was markedly increased by dexamethasone treatment and the recovery of MAO-B activity after its irreversible inhibition by deprenyl was more pronounced in the presence than in the absence of the hormone. Furthermore, the dexamethasone effect was abolished by the protein synthesis inhibitors actinomycin D or cycloheximide. The present study demonstrates that dexamethasone is able to selectively induce MAO-B in type 1 astrocytes and leads to speculation of a possible role for glucocorticoids in the increase in brain MAO-B associated with neurodegenerative disorders, such as Parkinson's and Alzheimer's diseases.

Influence of culture conditions on monoamine oxidase A and B activity in rat astrocytes.

Astroglial cells dispersed from newborn rat hemispheres were established in medium supplemented with 20 per cent fetal calf serum (FBS) and then grown to a confluent monolayer in the presence of 10 per cent FBS or charcoal-stripped FBS (CS). Type 1 astrocytes were subcultured and either maintained under the same conditions of the primary cultures or converted to serum-free chemically defined medium (CDM). No differences were found in either MAO A or MAO B activity of astrocytes grown in the presence of FBS or CS after 15 and 21 days in vitro (day 1 and 6 of subculture). In contrast, on day 21 both MAO A and MAO B activities were markedly higher in astrocytes subcultured in CDM compared with cells maintained in serum-supplemented medium. This difference appeared to be due to increased number of enzyme molecules, since kinetic analysis showed an increase in Vmax of both MAO isoenzymes in serum-free medium, but no change in Km. Consistently, the recovery of MAO A and MAO B activity after irreversible enzyme inhibition by clorgyline and deprenyl was faster in CDM than in FBS-supplemented medium, indicating enhanced enzyme synthesis under serum-free condition. Estimates of half-lives for the recovery of MAO A and MAO B activity indicated that, under both culture conditions, type A activity had a higher turnover rate than type B. The effect of CDM on astrocyte MAO does not appear to be due to selection of a subpopulation of cells, but rather linked to a morphological change (differentiation) with increased synthesis of both MAO isoenzymes.

Estrogenic control of monoamine oxidase A activity in human neuroblastoma cells expressing physiological concentrations of estrogen receptor.

Several lines of evidence support the hypothesis of a role played by estrogens in the manifestation of affective disorders in women. The analysis of the mechanism of action of a number of antidepressant drugs clearly demonstrated the involvement of the catecholaminergic system in the etiology of these complex behavioral pathologies. The present in vitro study was therefore undertaken to investigate the presence of a functional link between estrogen and catecholamine metabolism in cells of neural origin. The model system utilized was a human neuroblastoma cell line which was obtained by stable transfection of the estrogen receptor cDNA (SK-ER3). The present study shows that in SK-ER3 activation of the estrogen receptor correlates with a marked decrease in monoamine oxidase A activity. This effect is observed following treatment with a physiological concentration of 17 beta-estradiol and can be blocked by the specific antagonist of the steroid receptor, ICI 182,780. Dibutyryl cyclic AMP acting, like estrogens, on the state of differentiation of SK-ER3 cells did not affect monoamine oxidase A activity. The present study provides strong evidence of a strict relationship between estrogen receptor and monoamine oxidase A activity in human cells of neural origin, thus favoring the hypothesis of an antidepressive effect of estrogens exerted via inhibition of the monoamine oxidative pathway.

In vitro effects on monoamine uptake and release by the reversible monoamine oxidase-B inhibitors lazabemide and N-(2-aminoethyl)-p-chlorobenzamide: a comparison with L-deprenyl.

To investigate whether the reversible monoamine oxidase-B (MAO-B) inhibitors lazabemide and Ro 16-6491 have any additional effect on monoamine uptake and release, in vitro experiments were performed on rat forebrain synaptosomes and blood platelets. The effects of the two drugs were compared with those of L-deprenyl, the well-known irreversible MAO-B inhibitor which is reported to affect amine uptake. Both lazabemide and Ro 16-6491 behaved as weak inhibitors of [3H]monoamine uptake by synaptosomes, with a similar rank order of potency for amine uptake inhibition (noradrenaline (NA) > or = 5-hydroxytryptamine (5 HT) > dopamine (DA)). The IC50 values for lazabemide and Ro 16-6491, respectively, were: 86 microM and 90 microM for NA uptake; 123 microM and 90 microM for 5HT uptake; > 500 microM and > 1000 microM for DA uptake. L-Deprenyl (rank order of inhibitory potency: NA > DA > 5 HT) was four to 10 times more potent than either compound in inhibiting [3H]catecholamine uptake (IC50 = NA 23 microM, DA 109 microM), and two to three times less potent in inhibiting 5 HT uptake (IC50 233 microM). Lazabemide and Ro 16-6491 also differed from L-deprenyl in their ability to induce release of endogenous monoamines from synaptosomes. Thus, Ro 16-6491 (500 microM) induced a greater 5 HT release than did L-deprenyl, but was less effective than L-deprenyl in releasing DA. On the contrary, lazabemide was almost completely inactive on either 5 HT and DA release. The differential effect of the three MAO-B inhibitors on synaptosome 5 HT uptake and release was confirmed by [14C]5HT uptake and liberation experiments with isolated rat platelets. The data indicate that the reversible MAO-B inhibitors lazabemide and Ro 16-6491 at relatively high concentrations possess amine uptake-inhibiting properties. With regard to the effects examined, lazabemide markedly differs from L-deprenyl since it does not interfere with DA uptake nor induce amine release from synaptosomes.

Effect of aging on lazabemide binding, monoamine oxidase activity and monoamine metabolites in human frontal cortex.

Age-related modifications of monoamine oxidase-A and -B (MAO-A and MAO-B) and amine metabolite concentrations were studied in human frontal cortex taken postmortem from 22 subjects of various ages (21-75 years). Qualitative and quantitative analysis for MAO-B was provided by kinetic studies with a specific radioligand, [3H]lazabemide. The data demonstrated a significant (P < 0.05) positive correlation between the density of [3H]lazabemide binding sites (Bmax) and age of the subject, without showing an apparent modification in the dissociation constant (KD) of the radioligand. In parallel experiments, MAO-B but not MAO-A activity was shown to correlate with age (P < 0.05). The concentrations of the amine metabolites 4-hydroxy-3-methoxyphenylacetic acid (HVA), 5-hydroxyindole-3-acetic acid (5-HIAA), 3,4-dihydroxyphenylacetic acid (DOPAC), 4-hydroxy-3-methoxyphenylglycol (MHPG) and 3,4-dihydroxyphenylglycol (DHPG) were all devoid of a correlation with age. Neither did the concentrations of these metabolites relate to the respective subject's MAO-B enzymatic activity nor to [3H]lazabemide Bmax. A correlation, though rather weak, was obtained between MAO-A activity and MHPG concentration (P = 0.045). The MAO-A and -B enzyme characteristics in subjects who had committed suicide (n = 9) did not differ from those of subjects deceased for other causes (n = 13). Among the measured monoamine metabolites the concentrations of DOPAC and HVA were higher in the suicide versus control group (P < 0.05). The present data confirm in a direct manner that the increase in MAO-B activity in aging brain is due to an enhancement of the number of active sites of the enzyme and not through modifications of its kinetic characteristics. Furthermore, that neither the characteristics nor the activity of the enzyme are changed in the frontal cortex of suicide victims compared to control subjects.

Estrogen modulation of catecholamine synthesis and monoamine oxidase A activity in the human neuroblastoma cell line SK-ER3.

In order to assess the neuronal-like properties of a human neuroblastoma cell line obtained by stable transfection of the estrogen receptor (SK-ER3) a series of quantitative measurements of the activity of two neurotransmitter-related enzymes: tyrosine hydroxylase (TH) and monamine oxidase (MAO), and of catecholamine concentrations were performed. When compared to the parental SK-N-BE cell line, the stably transfected SK-ER3 cells show a more pronounced dopaminergic phenotype. The immunoreactivity to a TH antibody is in fact increased and the ratio between dopamine and noradrenaline concentrations is elevated. Treatment with estradiol further enhances the expression of this phenotype. Interestingly, in the transfected cell line MAO-A activity is decreased and further reduced by estrogen treatment. This finding substantiated by previous reports indicates that our model system might represent an interesting tool for the study of the pharmacological treatments of estrogen-induced pathological responses of nervous cells.

Metyrapone and adrenal response in stressed calves.

The effects on some hematochemical stress indices of previously administered metyrapone (750 mg given orally six times every 4 hours) were evaluated in calves submitted to simulated transport for 30 min. The plasma cortisol increases were significantly lower than those observed in untreated calves of the same age, sex and breed. Plasma adrenaline and NEFA increased similarly in both groups of calves during simulated transport and were unaffected by metyrapone administration. The results indicate that in cattle, as in other animals, metyrapone inhibits cortisol biosynthesis. Under our experimental conditions, the rise of NEFA, a well known stress index, seems mainly to be related to adrenaline release.

Effects of phosphatidylserine therapy in geriatric patients with depressive disorders.

The effects of phosphatidylserine (BC-PS) on cognitive, affective and behavioural symptoms were studied in a group of 10 elderly women with depressive disorders. Patients were treated with placebo for 15 days, followed by BC-PS (300 mg/day) for 30 days. The Hamilton Rating Scale for Depression, Gottfries-Bråne-Steen Rating Scale, Nurse's Observation Scale for Inpatient Evaluation and Buschke Selective Reminding Test were administered before and after placebo and after BC-PS therapy, to monitor changes in depression, memory and general behaviour. At the same time, basal plasma levels of noradrenaline, MHPG, DOPAC, HVA and 5-HIAA, and GH/beta-endorphin/beta-lipotropin responses to clonidine stimulation were measured. BC-PS induced consistent improvement of depressive symptoms, memory and behaviour. No changes in amine metabolite levels or in hormonal responses to alpha 2-adrenoceptor stimulation were observed.

Adrenal response in the calf to repeated simulated transport.

The adrenal responses in calves submitted to simulated transport on three occasions for 30 min were evaluated. Plasma adrenaline, cortisol and NEFA increased significantly during simulated transport but became less marked in successive trials. Haematological stress-related parameters (Hb, PCV) increased to the same extent on repeated exposure to simulated transport. Plasma noradrenaline, glucose and cholesterol values were unchanged throughout the study.

[3H]Ro 19-6327: a reversible ligand and affinity labelling probe for monoamine oxidase-B.

This study demonstrated the existence of specific binding sites for [3H]Ro 19-6327 in human platelet membranes. This compound is a novel, time-dependent inhibitor of monoamine oxidase type B (MAO-B) and is structurally closely related to [3H]Ro 16-6491. The density of the sites labelled with high affinity by [3H]Ro 19-6327 was similar to that observed in previous studies with [3H]Ro 16-6491 as ligand. Binding was reversible at 20 degrees C and showed a relatively slow dissociation (t1/2 = 220 min). The dissociation rate was markedly decreased (t1/2 = greater than 24h) at 0 degrees C. MAO-B, but not MAO-A inhibitors, effectively prevented the binding of [3H]Ro 19-6327. Like [3H]Ro 16-6491, [3H]Ro 19-6327 is recognized as a substrate by MAO-B, being eventually deaminated by the enzyme. Since the deaminated aldehyde derivative of Ro 19-6327 did not inhibit MAO-B, a still unidentified reversible adduct, formed at the MAO-B active site, might explain the high potency and selectivity of [3H]Ro 19-6327. Incubation of the radioligand-enzyme complex from platelet and brain membranes with NaBH3CN and acetic acid (to pH 4.5) caused the irreversible incorporation of the radioactivity into a single polypeptide as shown by SDS-PAGE analysis. This polypeptide had a molecular weight identical to that of the MAO-B subunit, i.e. 58,000. The presence of unlabelled MAO-B inhibitors in the incubation mixture prevented the covalent incorporation of [3H]Ro 19-6327. The irreversible MAO-B inhibitor, [3H] pargyline, labelled a protein with a molecular weight identical to the protein labelled by [3H]Ro 19-6327.(ABSTRACT TRUNCATED AT 250 WORDS)

Vasopressin (DDAVP) therapy in chronic schizophrenia: effects on negative symptoms and memory.

Ten chronic undifferentiated schizophrenics, 6 men and 4 women, aged 28-63, with 6- to 31-year histories of the disease were given DDAVP to observe the effects of this neuropeptide on the prevalent negative symptoms of their illness. Patients were maintained on neuroleptic therapy and first given a 20-day course of placebo followed by 20 days of DDAVP i.m., 4 micrograms Andreasen Scale for assessment of negative symptoms, the Brief Psychiatric Rating Scale, the NOSIE Rating Scale and the Luria-Nebraska Rating Scale were administered to monitor negative symptomatology, behavior and memory before the study began, after placebo and after DDAVP administration. Patients were also given a growth hormone-clonidine test and in addition plasma basal concentrations of 3-methoxy-4-hydroxyphenylglycol (MHPG), homovanillic acid, 3,4-dihydroxyphenylacetic acid (DOPAC) and 5-hydroxyindoleacetic acid (5-HIAA) were measured at the same intervals. DDAVP therapy induced a significant improvement of negative symptomatology and a trend toward improvement of short- to medium-term memory. No changes in homovanillic acid, MHPG, 5-HIAA and DOPAC, nor of growth hormone response to clonidine stimulation were observed.

[3H]Ro 16-6491, a selective probe for affinity labelling of monoamine oxidase type B in human brain and platelet membranes.

[3H]Ro 16-6491 [N-(2-aminoethyl)-p-chlorobenzamide HCl], a reversible "mechanism-based" inhibitor of monoamine oxidase (MAO) type B, binds selectively and with high affinity to the active site of MAO-B in brain and platelet membranes. Under normal conditions, the binding of [3H]Ro 16-6491 is fully reversible. However, [3H]Ro 16-6491 could be irreversibly bound (covalently) to membranes by the addition of the reducing agent NaBH3CN to the sample and adjusting to pH 4.5 with acetic acid. No irreversible labelling occurred in the absence of NaBH3CN and at neutral pH. The presence of the irreversible MAO-B inhibitor l-deprenyl completely abolished the irreversible labelling of the membranes by [3H]Ro 16-6491. The selective inactivation of MAO-B, e.g., by l-deprenyl prevented the covalent incorporation of [3H]Ro 16-6491 whereas selective inhibition of the MAO-A by clorgyline was without effect. The covalent linkage to membranes of unlabelled Ro 16-6491 and Ro 19-6327 (a selective and reversible MAO-B inhibitor closely related to Ro 16-6491) after the addition of NaBH3CN at pH 4.5 irreversibly inactivated MAO-B activity whereas MAO-A activity was unaffected. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of labelled membranes showed that [3H]Ro 16-6491 was incorporated into a single polypeptide with a molecular mass identical to the one labelled by [3H]pargyline (58 kilodaltons). Our results indicate that the polypeptide that is covalently labelled by [3H]Ro 16-6491 corresponds to one of the two MAO-B subunits.(ABSTRACT TRUNCATED AT 250 WORDS)

Serotonin and noradrenaline concentrations and serotonin uptake in platelets from hyperphenylalaninaemic patients.

In three untreated patients with phenylketonuria (PKU), three PKU and six hyperphenylalaninaemic (HPA) patients in good metabolic control, the kinetic constants of platelet in vitro uptake of [14C]serotonin (5HT) did not significantly differ from those in 12 control subjects matched for age. The platelet concentrations of endogenous 5HT and noradrenaline (NA), taken as long-term indices of the amount of these amines circulating in plasma, were lower than normal in PKU and HPA patients, whether or not they were kept on a diet. However, a reduction in plasma NA concentrations at the moment of blood collection was seen only in untreated PKU patients. These data indicate that there may be a chronic inhibition of 5HT and possibly of NA synthesis even in PKU or HPA subjects in good metabolic control, with normal psychomotor development and only slightly raised plasma phenylalanine levels.

Alcohol impairs age-dependent adaptation of human lymphocyte beta-adrenergic receptors.

Lymphocyte beta-adrenergic receptor function and norepinephrine (NE) plasma concentration have been compared in normal subjects and in ethanol-addicted patients of different ages. Direct measurement of the density and properties of beta-adrenoceptors in membrane fractions was performed using the radioligand 125I-Iodocyanopindolol (ICYP). In normal subjects beta-receptor density decreased and norepinephrine plasma concentration increased with age. There was a statistically significant negative correlation between plasma norepinephrine and beta-receptor number. In ethanol-addicted patients the age-related modification in beta-receptor number and the correlation between plasma norepinephrine and beta-receptor density were lost, in spite of the fact that the increase of NE plasma concentration was still present. The ethanol-induced effects in lymphocyte beta-receptor may have consequences on immunological function and may be qualitatively similar to alterations in other tissues not routinely accessible in humans.

Uptake, release, and subcellular localization of 1-methyl-4-phenylpyridinium in blood platelets.

The neurotoxic compound 1-[methyl-3H]-4-phenylpyridinium ([3H]MPP+) was actively taken up by human, rabbit, and guinea pig platelets incubated in plasma. In human platelets, the apparent Km of this uptake (22.6 microM) was 50 times higher than that for serotonin [5-hydroxytryptamine (5-HT]). The uptake of [3H]MPP+ by human platelets was inhibited by selective 5-HT uptake blockers [cianopramine, (-)-paroxetine, and clomipramine], by metabolic inhibitors (KCN and ouabain), and by drugs that interfere with amine storage in the 5-HT organelles (reserpine, mepacrine, and Ro 4-1284). Impairment of the transmembrane proton gradient by ionophores (monensin and nigericin) induced a marked release of radioactivity from platelets preincubated with [3H]MPP+. Fractionation of homogenates of rabbit platelets preincubated with [3H]MPP+ showed that the drug was concentrated to a great extent in the 5-HT organelle fraction. MPP+ competitively inhibited [14C]5-HT uptake by human platelets and reduced the endogenous 5-HT content of human, rabbit, and guinea pig platelets. These investigations show that MPP+ is transported into the platelets via the 5-HT carrier and is accumulated predominantly in the subcellular organelles that store 5-HT and other monoamines. It is suggested that an accumulation of MPP+ in amine storage vesicles of neurons may be involved in the effects of the drug in the CNS, e.g., by protecting other subcellular compartments from exposure to high concentrations of MPP+, by sustaining a gradual release of the toxin, or both.

Effects of diethyl ether, halothane, ketamine and urethane on sympathetic activity in the rat.

The present paper describes the effects of different general anaesthetics on plasma catecholamine (CA) concentrations taken as biochemical index of peripheral sympathetic activity. In chronically catheterized rats, diethyl ether, ketamine and urethane increased plasma adrenaline (A) and noradrenaline (NA) concentrations, indicating that these drugs stimulate both neurosympathetic and adrenomedullary functions. These effects appear to be centrally mediated, since ganglionic blockade or spinal transection completely counteracted the diethyl ether- and ketamine-induced increases in plasma CA levels. Halothane induced a transient decrease in circulating A and an increase in NA. These results support the concept that general anaesthetics may have different effects on sympathetic function. Arterial blood pressure and heart rate were also measured to look for possible correlations with peripheral sympathetic activity. The enhanced release of peripheral CAs seemed to be the determining factor for increasing blood pressure and heart rate with ketamine only. In the other instances the activation of the peripheral sympathetic system appeared to maintain homeostasis by counterbalancing the various depressive effects of anaesthetics on the cardiovascular system.