Valynda L. Machen

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 meth-ylamine 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.

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.

Chloroquine Reduces Neuronal and Glial Iron Uptake

Abstract: 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.

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 μg/ml) of added ferric iron resulted in a significant decrease in each of the measures studied.

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 of125I-diferric-transferrin uptake. The diferric-transferrin association curve of neuronal cultures at 37°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 increasingBmax from day 6 to day 16 in culture (1626 to 2740 fmoles/mg protein). However, theKuptake 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 differric-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 ofBmax orKuptake from days 11 to 18 in culture.Bmax ranged from 595 to 751 fmol/mg protein; overallKuptake was 48.3±13.2 nM (mean±SD).

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.

Autoradiographic Localization of Benzodiazepine Receptor Binding in Dissociated Cultures of Fetal Mouse Cerebral Cortex

Abstract: 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 μM) and Ro 5‐4864 (0.1 μM) 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.

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.

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]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+).

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.