Biomedical subjects

V L Machen

Publications and source records attributed to V L Machen.

12 recordsLinked to original sources

PubMed1991

Effect of ferric nitrilotriacetate on rostral mesencephalic cells.

After murine fetal cells from the rostral mesencephalic tegmentum were isolated, prepared, and cultured; neuronal and glial cells in primary mixed cell cultures were exposed to ferric nitrilotriacetate (Fe-NTA) at varying concentrations. Studies were performed at 23 days in culture after 14 day exposure to Fe-NTA. In addition to morphologic studies, biochemical assays including specific [3H]flunitrazepam (FLU) binding, clonazepam (CLO)-displaceable [3H]-FLU binding, Ro5-4864-displaceable [3H]-FLU binding, [3H]-FLU binding, [3H]dopamine (DA) uptake, [3H]haloperidol (HAL) binding, [3H]spiperone (SP) binding, glutamine synthetase activity (GS), and protein determinations were performed. The data demonstrate that chelated ferric iron has an adverse effect on these cells. The data also demonstrate that increasing concentrations of Fe-NTA resulted in massive neuronal dropout leaving the culture population virtually all glial; however, the specific binding of [3H]HAL and [3H]SP increased. There was a concomitant decrease in both glutamine synthetase activity and overall protein content. The mechanism of enhancement in the presence of Fe-NTA of [3H]HAL and [3H]SP binding is unknown and may be unique, but may be related to the known increase in D2 receptor ligand affinity in the presence of other multivalent cations (Ca2+ and Mg2+).

Animals↗

PubMed1991

Effects of phenobarbital and phenytoin on cortical glial cells in culture.

Studies were undertaken to determine the effects of 7-day phenobarbital and phenytoin exposure on 14-day-old glial cell cultures of fetal murine cortex. Biochemical markers monitored were Ro5-4684-displaceable 3H-flunitrazepam binding, 3H-beta-alanine uptake, glutamine synthetase activity, and protein content. Phenobarbital concentrations were 30, 60, and 120 micrograms/ml and phenytoin concentrations 15, 30, 60 micrograms/ml. There were no discernible phase microscopic changes at any concentration of either drug. Phenobarbital produced no significant changes in the biochemical measures monitored. Exposure to phenytoin produced no biochemical changes at 15 micrograms/ml, but did produce significant changes at 30 and 60 micrograms/ml. There was an increase in Ro5-4684-displaceable 3H-flunitrazepam binding signifying increased binding or an increase in the number of binding sites and perhaps an increased population of glial cells although, the unchanged protein content suggests that the number of glial cells was not increased. There was a decrease with 30 and 60 micrograms/ml phenytoin of 3H-beta-alanine uptake suggesting interference with normal membrane transport of this compound. The latter effect may well mirror changes in GABA uptake in glial cells in the presence of phenytoin.

Animals↗

PubMed1990

Effect of ferric nitrilotriacetate on predominantly cortical glial cell cultures.

Cultured glial cells were exposed to ferric nitrilotriacetate (Fe-NTA) at varying concentrations. Studies of the exposed glial cells were performed at days 29 and 36 post-conceptional age (culture days 8 and 15). In addition to morphologic studies, biochemical assays including [3H]-flunitrazepam (FLU) specific binding, Ro5-4864-displaceable 3H-FLU binding, and protein determinations were performed. At day 29 post-conceptional age, significant decreases in 3H-FLU specific binding, Ro5-4864-displaceable 3H-FLU binding, and protein determinations were discernible only in the presence of 100 microM Fe-NTA. At day 36 post-conceptional age 3H-FLU specific binding was significantly decreased at 20, 60, and 100 microM Fe-NTA concentrations, while Ro5-4864-displaceable 3H-FLU binding and protein determinations were significantly reduced at 60 and 100 microM Fe-NTA concentrations. The effects of Fe-NTA exposure appear to be both concentration and duration-of-exposure related. When compared to previously reported neuronal cell culture studies utilizing 3H-FLU specific binding, Ro5-4864-displaceable 3H-FLU binding, and protein determinations, glial cells appear to be significantly more resistant to chelated iron exposure.

Acetates↗

PubMed1989

Effect of ferric nitrilotriacetate on predominantly cortical neuronal cell cultures.

Predominately neuronal cell cultures were produced as described in previous communications. Neuronal cells were exposed to ferric nitrilotriacetate (Fe-NTA) at varying concentrations. Studies of the neuronal cells were performed at 13 and 20 days in culture. In addition to morphologic studies, biochemical assays including choline acetyltransferase (ChAT) activity, specific [3H]flunitrazepam (FLU) binding, clonazepam (CLO)-displaceable [3H]FLU binding, Ro5-4864-displaceable [3H]FLU binding, high-affinity [3H]GABA uptake, and protein determinations were performed. The data demonstrate that chelated ferric iron has an adverse effect on predominately neuronal cultures after 7 days of exposure as measured by choline acetyltransferase activity, while other measures remained unaffected; however, after 14 days of exposure all measures were significantly decreased. The effects of Fe-NTA exposure appear to be both concentration and duration-of-exposure related.

Acetates↗

PubMed1987

Benzodiazepine receptor affinity alterations at physiologic temperature after chronic clonazepam exposure.

Cerebral cortical cell cultures obtained from fetal mice were exposed to 200 nM clonazepam (CZP) for 14 days and benzodiazepine (BDZ) receptor binding was measured on intact cells in situ at 37 degrees C. Total, specific, and CZP-displaceable BDZ binding were significantly reduced from control values immediately after drug removal (77.7 +/- 1.4%, 75.1 +/- 3.0%, and 40.9 +/- 6.0% of control, respectively) but Ro5-4864-displaceable binding was not affected (87.6 +/- 5.1%). Binding returned to control values within 48 hours. Saturation analysis of the binding data indicated that a high-affinity binding site could not be detected in CZP-exposed cultures immediately after drug removal (162 nM versus 49 nM in controls, p less than .001), but was present 24 hours later.

Animals↗

PubMed1986

Transferrin binding by mammalian cortical cells.

Predominantly neuronal (neuronal) or non-neuronal (glial) cerebral cortical cell cultures were employed to study the kinetics and changes with maturation of 125I-diferric-transferrin uptake. The diferric-transferrin association curve of neuronal cultures at 37 degrees C was nonphasic and indicated equilibrium at 90 minutes. Dissociation was completed by 70 minutes. Diferric-transferrin specific uptake (80% of total) in neuronal cells (evaluated at days 6, 9, 13, 16, and 23 in culture) increased with maturation. Scatchard transformation of the data revealed increasing Bmax from day 6 to day 16 in culture (1626 to 2740 fmoles/mg protein). However, the K uptake was statistically unchanged over time and equaled 48.7 +/- 13.9 nM (mean +/- SD). In contrast, association studies of glial cultures documented equilibrium by 45 minutes and dissociation by 40 minutes. The concentration curves for diferric-transferrin uptake in glial cells, evaluated at days 11, 15, and 18 in culture, revealed virtually identical uptake at the three ages studied, but the percent specific uptake (58%) was less than for neurons (88%). Scatchard transformation of the data revealed no statistical alteration of Bmax or K uptake from days 11 to 18 in culture. Bmax ranged from 595 to 751 fmol/mg protein; overall K uptake was 48.3 +/- 13.2 nM (mean +/- SD).

Animals↗

PubMed1986

Chloroquine reduces neuronal and glial iron uptake.

The effect of chloroquine, a lysosomotropic agent, on iron uptake into neuronal and glial cell cultures is reported. Chloroquine significantly inhibited iron uptake in both neuronal and glial cells. These findings suggest that iron transport into both neuronal and glial cells is mediated by the transferrin-iron complex.

Animals↗

PubMed1986

Autoradiographic localization of benzodiazepine receptor binding in dissociated cultures of fetal mouse cerebral cortex.

Autoradiography utilizing photoaffinity labelling with [3H]flunitrazepam was used in living cultures of fetal mouse cerebral cortex in situ to localize benzodiazepine receptor binding sites. There was a predominant localization of silver grains over neurons; however, substantial labelling also occurred over nonneuronal background cells. Clonazepam (0.1 microM) and Ro 5-4864 (0.1 microM) displaced substantial numbers of silver grains over neurons and background cells, respectively. In addition, clonazepam displaced 58-68% of specific grains over background cells and Ro 5-4864 displaced 30% of grains over neurons, suggesting that multiple cell types in the CNS may participate in the neuropharmacologic actions of the benzodiazepines.

Affinity Labels↗

PubMed1985

The effect of iron on mammalian cortical neurons in culture.

We added iron in the ferric form to predominantly neuronal, cortical cell cultures, and determined clonazepam-displaceable [3H]diazepam binding, choline acetyltransferase activity, high-affinity [3H]GABA uptake, and glutamic acid decarboxylase activity. Chronic exposure (14 days) to low concentrations (0.01, 0.04, and 0.1 micrograms/ml) of added ferric iron resulted in a significant decrease in each of the measures studied.

Animals↗

PubMed1985

Iron uptake by glial cells.

Dynamic studies of iron metabolism in brain are generally unavailable despite the fact that a number of neurologic conditions are associated with excessive accumulation of iron in central nervous tissue. Cortical non-neuronal (glial) cultures were prepared from fetal mouse brain. After 13 days the cultures were exposed to radiolabeled iron. Brisk and linear total iron uptake and ferritin iron uptake occurred over 4 hours. When methylamine or ammonium chloride was added, (both known inhibitors of transferrin iron release because of their lysosomotropic properties), total iron uptake was diminished. Further studies indicated that methylamine inhibits glial cell ferritin iron incorporation. Glial cell iron transport is similar to previously reported neuronal cell iron transport (1) but glial cell iron uptake proceeds at a faster rate and is more susceptible to the inhibition of certain lysosomotropic agents. The data reinforces the likelihood that iron uptake by nervous tissues is transferrin-mediated.

Ammonium Chloride↗

PubMed1984

Iron uptake by mammalian cortical neurons.

Although iron accumulates in the brain in a number of pathological conditions, including Hallervorden-Spatz syndrome, Parkinson's disease, and neurosyphilis, studies of brain iron metabolism have been performed only rarely. Neuronal-enriched cultures were prepared from fetal mouse brain. After 18 days the cells were exposed to radiolabeled iron. Total iron uptake and incorporation into ferritin were rapid and linear over four hours. The addition of either methylamine or ammonium chloride, both known blockers of transferrin-iron release through their lysosomotropic properties, inhibited total iron uptake. Methylamine also inhibited the rate of ferritin-iron incorporation, most likely by interfering with transferrin-iron release. The data suggest that neuronal iron transport, much like that in other mammalian tissues, is transferrin mediated and that blockers of transferrin-iron release may be of value in conditions in which there is brain iron overload.

Animals↗

PubMed1984

Properties of [3H]diazepam binding sites on cultured murine glia and neurons.

[3H]Diazepam binding was assayed in situ on living cultures of fetal mouse cerebral cortex or glia in an attempt to further characterize the high and low affinity binding sites. Mixed neuronal-glial cultures were found to have a high (Kd approximately equal to 10 nM) as well as a low (Kd approximately equal to 240 nM) affinity binding site. Glial cultures also had a similarly high affinity site (Kd 13 nM). In both types of cultures, the high affinity site was Ro 5-4864 sensitive and clonazepam resistant. Since Ro 5-4864 has particular affinity for non-neuronal elements and clonazepam for neuronal elements, the data suggest that the high affinity binding site may be localized to glial elements and the low affinity site primarily neuronal.

Animals↗