GABA metabolism in cultured glial cells

GABA-transaminase has been characterized in cultured astrocytes. It is identical to the synaptosomal and perikaryal enzyme in terms of charge, molecular weight, and stability, but it differs in its affinity for GABA, which is much higher in the glial compartment. GABA-transaminase has been shown to be inducible by high GABA concentrations, which suggests that astrocytes have the possibility not only to transport GABA but also to metabolize the amino acid which is taken up.     

Microgravity-induced apoptosis in cultured glial cells

Apoptosis is a form of naturally occurring cell death that plays fundamental roles during embryonic developement. In adults, it neatly disposes of cells damaged by injuries provoked by external causes such as UV radiation, ionisation and heat shock. Alteration of the gravity vector may be one of the external apoptosis inducers. Neurophysiological impairment signs were seen during space flights in astronauts, but very few studies were carried out on the nervous system and none at the cellular level. In this study, we submitted cultured C6 glioma cells to microgravity (0xg) of varying duration, obtained by clinorotation in a Fokker three-dimensional clinostat for 15 min, 30 min, 1h, 20h or 32h. After 30 min at 0xg, numerous nuclei underwent the classical morphological alterations (chromatin condensation, nuclear fragmentation, apoptotic bodies) that lead to the programmed cell death. After 30 min at 0xg, immunostaining for the enzyme caspase-7 was present in the cytoplasm of many cells concurrently with DNA fragmentation identified by the TUNEL method. At 32h, the number of apoptotic nuclei was much reduced indicating the ability of glial cells to adapt to altered gravity.     

Synthesis and release of sulfated glycoproteins by cultured glial cells

Both primary cultured glial cells and cloned (C-6) glioma cells have been shown to synthesize and release sulfated glycoproteins. It was found that N-linked tri- and tetra-antennary glycopeptides recovered from the glycoproteins contained most of the (35S) sulfate label. C-6 glial cells showed a higher rate of oligosaccharide sulfation than the primary glial cultures. Both cell types exhibited a high rate of release of sulfated glycoproteins into the medium. The ratio of 35S/3H incorporated from (35S) sulfate and (3H) glucosamine in the released material was higher than that of the glycoproteins associated with the cell, indicating an enrichment of sulfated glycoproteins in the secreted materials. Monensin inhibited both the synthesis and the release of sulfated glycoproteins.     

Glutamate and GABA receptors in vertebrate glial cells

Glial cells of the central nervous system express receptors for the main inhibitory and excitatory neurotransmitters, GABA and glutamate. The glial GABA and glutamate receptors share many properties with the neuronal GABAA and kainate/quisqualate receptors, but are molecularly and, in some aspects, pharmacologically distinct from their neuronal counterparts. The functional role of these receptors is as yet speculative: They have been proposed to control proliferation of astrocytes, serve to balance ion changes at GABAergic synapses, or they could enable the glial cell to detect neuronal synaptic activity.     

Adenosine transport by cultured glial cells from chick embryo brain

The transport of adenosine was studied in pure cultures of glial cells from chick embryo brain. In order to avoid complications in uptake measurements due to adenosine metabolism, cultures were depleted of ATP by incubation with cyanide and iodoacetate prior to addition of [³H]adenosine. Under the 5- to 25-s periods used for the transport assay, no adenosine metabolism could be detected. Initial rates of adenosine transport under these conditions obeyed the Michaelis-Menten relationship with K_m = 370 μM and V_max = 10.3 nmol/min/mg cell protein. ATP depletion or elimination of Na⁺ from the assay medium had no significant effect on initial rates of adenosine uptake. However, when assays were carried out under conditions of significant adenosine metabolism (10-min uptake in the absence of metabolic inhibitors), a high-affinity incorporation process could be demonstrated in the glial cells (K_m = 12 μM; V_max = 0.34 nmol/ min/mg protein). The transport activity expressed in ATP-depleted glial cells was most sensitive to inhibition by nitrobenzylthioinosine, dipyridamole, and N⁶-benzyladenosine. In decreasing order of potency, N⁶-methyladenosine, 2-chloroadenosine, inosine, and thymidine also blocked adenosine translocation in glial cultures. Thus, adenosine transport by cultured glial cells occurs by means of a low-affinity, facilitated diffusion system which is similar to the nucleoside transporter in cells of nonneural origin.     

Production of ethanol by cultured insect cells

Summary Proton Nuclear Magnetic Resonance (¹H-NMR) Analysis of insect cell culture media used for cultivating insect cell lines derived from the fleshflySarcophaga peregrina, swallowtail butterflyPapilio xuthus, and cabbage armywormMamestra brassicae revealed that ethanol appeared in the medium as the cultures aged. By incorporating [¹³C-1]-glucose into the media, we pursued¹³C-NMR spectrograms to show that the ethanol was derived from glucose. Thus, it became evident that the insect cells culturedin vitro produce ethanol from glucose as a metabolite.     

The influence of membrane potential on chloride channels activated by GABA in rat cultured hippocampal neurons

Chloride currents were activated by a low concentration of GABA (0.5 m) in neonatal rat hippocampal neurons cultured for up to 14 days. Currents elicited by 0.5 m GABA in neurons, voltage-clamped using the whole-cell technique with pipettes containing 149 mm Cl^–, reversed close to 0 mV whether pipettes contained 144 mm Na⁺ or 140 mm Cs⁺, and were blocked by 100 m bicuculline. Current-voltage curves showed outward rectification. Single channel currents appeared in cell-attached patches when the pipette tip was perfused with pipette solution containing 0.5 m GABA and disappeared when a solution containing 100 m bicuculline plus 0.5 m GABA was injected into the pipette tip. The channels showed outward rectification and, in some patches, had a much lower probability of opening at hyperpolarized potentials. The average chord conductance in 10 patches hyperpolarized by 80 mV was 7.8±1.6 pS (sem) compared with a chord conductance of 34.1±3.5 pS (sem) in the same patches depolarized by 80 mV. Similar single channel currents were also activated in cell-free, inside-out patches in symmetrical chloride solutions when 0.5 m GABA was injected into the pipette tip. The channels showed outward rectification similar to that seen in cell-attached patches, and some channels had a lower probability of opening at hyperpolarized potentials. The average chord conductance in 13 patches hyperpolarized by 80 mV was 11.8±2.3 pS (sem) compared with 42.1±3.1 pS (sem) in the same patches depolarized by 80 mV.We are grateful to B. McLachlan and M. Robertson for their general assistance, to C. McCulloch and M. Smith for writing computer programs and to W. O'Hare for making the pipette injection device.     

Localization of two neurotropic molecules in cultured glial cells by ion microscopy]

The intracellular localization of two families of neurotropic drugs: flunitrazepam and flurazepam (benzodiazepine), triflupromazine and trifluoperazine (phenothiazine) has been studied by ion microscopy. The molecules have been incubated with C6 glioblastoma cells from rat origin and with astroglial primary cultures. The images of the intracellular distributions of the two drugs are easily obtained by selecting the fluorine atom of the molecules. The images obtained show that flunitrazepam and flurazepam, two drugs of the benzodiazepine group are mainly located to the nuclei, whereas triflupromazine and trifluoperazine, two phenothiazines are exclusively located inside the cytoplasm.     

Production of endothelin-1 by rat cultured mesangial cells

We investigated whether ET-1 is synthesized by and released from mesangial cells. ET-1-like immunoreactivity (LI) released into medium increased time-dependently under a serum-free condition. The amounts of ET-1-LI released into the medium was augmented in the presence of fetal calf serum. Reverse-phase HPLC profile of the conditioned media revealed a major component coeluting with standard ET-1. Northern blot analysis of poly(A) +RNA extracted from mesangial cells showed a single major band corresponding to the size of preproET-1 mRNA (2.3 kb). These findings demonstrate that ET-1 is synthesized by and released from rat mesangial cells and suggest a possibility that it acts on their own cells as an autocrine factor.     

Sodium dependency of GABA uptake into glial cells in bullfrog sympathetic ganglia

The kinetics of sodium dependency of GABA uptake by satellite glial cells was studied in bullfrog sympathetic ganglia. GABA uptake followed simple Michaelis-Menten kinetics at all sodium concentrations tested. Increasing external sodium concentration increased bothK _m andV _max for GABA uptake, with an increase in theV _max/K _m ratio. The initial rate of uptake as a function of the sodium concentration exhibited sigmoid shape at 100 M GABA. Hill number was estimated to be 2.0. Removal of external potassium ion or 10 M ouabain reduced GABA uptake time-dependently. The effect of ouabain was potentiated by 100 M veratrine. These results suggest that at least two sodium ions are involved with the transport of one GABA molecule and that sodium concentration gradient across the plasma membrane is the main driving force for the transport of GABA. The essential sodium gradient may be maintained by Na⁺, K⁺-ATPase acting as an ion pump.     

14C-leucine incorporation by the nerve and glial cells of a cultured spinal ganglion

Spinal ganglia of adult rabbits were cultured in the routine and protein synthesis precursors-enriched media. On days I and 4 of cultivation, the intensity of 14C-leucine incorporation in protein and in acid soluble fraction of nerve and glial cells was determined. The tissue of the spinal ganglion keeps incorporating 14C-amino acid, into neurons and glia, for all the tested periods of cultivation with both the media employed. The curves of incorporation into the above fractions of nerve and glial cells cultured in the routine medium display similar patterns of changes, whereas those obtained from the enriched medium observations appear to be anti-fasic. The enrichment of the medium results also in less pronounced fluctuations in the intensity of the labeled amino acid in protein and 14C-leucine pool, on the tested periods of cultivation, which may provide more stable conditions of the explant's survival.     

Phenotypic changes in satellite glial cells in cultured trigeminal ganglia

Satellite glial cells (SGCs) are specialized cells that form a tight sheath around neurons in sensory ganglia. In recent years, there is increasing interest in SGCs and they have been studied in both intact ganglia and in tissue culture. Here we studied phenotypic changes in SGCs in cultured trigeminal ganglia from adult mice, containing both neurons and SGCs, using phase optics, immunohistochemistry and time-lapse photography. Cultures were followed for up to 14 days. After isolation virtually every sensory neuron is ensheathed by SGCs, as in the intact ganglia. After one day in culture, SGCs begin to migrate away from their parent neurons, but in most cases the neurons still retain an intact glial cover. At later times in culture, there is a massive migration of SGCs away from the neurons and they undergo clear morphological changes, and at 7 days they become spindle-shaped. At one day in culture SGCs express the glial marker glutamine synthetase, and also the purinergic receptor P2X7. From day 2 in culture the glutamine synthetase expression is greatly diminished, whereas that of P2X7 is largely unchanged. We conclude that SGCs retain most of their characteristics for about 24?h after culturing, but undergo major phenotypic changes at later times.     

Arachidonic acid inhibits glycine transport in cultured glial cells.

The effects of arachidonic acid on glycine uptake, exchange and efflux in C6 glioma cells were investigated. Arachidonic acid produced a dose-dependent inhibition of high-affinity glycine uptake. This effect was not due to a simple detergent-like action on membranes, as the inhibition of glycine transport was most pronounced with cis-unsaturated long-chain fatty acids, whereas saturated and trans-unsaturated fatty acids had relatively little or no effect. Endogenous unsaturated non-esterified fatty acids may exert a similar inhibitory effect on the transport of glycine. The mechanism for this inhibitory effect has been examined in a plasma membrane vesicle preparation derived from C6 cells, which avoids metabolic or compartmentation interferences. The results suggest that part of the selective inhibition of glycine transport by arachidonic acid could be due to the effects of the arachidonic acid on the lipid domain surrounding the carrier.     

Cysteine sulfinic acid uptake in cultured neuronal and glial cells

The presence of an efficient high affinity uptake system for L-CSA has been demonstrated in cultured neuronal and glial cells of various types. In neurons and most glial cells L-CSA uptake is inhibited by acidic amino acids,L-glutamate andL-aspartate and requires sodium ions; however the sodium dependence varies from one cell type to the other. The characteristics of the uptake system change during cell maturation, especially in astroblasts. The predominance of CSA uptake in glial cells as compared to neurons, the similarity of the kinetic parameters and of the structural specficity ofL-glutamate uptake suggest that both excitatory amino acids are transported by a common system. In view of accumulating evidence, the present results are in agreement with a role of CSA as a neurotransmitter and as a precursor for taurine biosynthesis in the central nervous system.     

Clustering of extrasynaptic GABA(A) receptors modulates tonic inhibition in cultured hippocampal neurons

Tonic inhibition plays a crucial role in regulating neuronal excitability because it sets the threshold for action potential generation and integrates excitatory signals. Tonic currents are known to be largely mediated by extrasynaptic gamma-aminobutyric acid type A (GABA(A)) receptors that are persistently activated by submicromolar concentrations of ambient GABA. We recently reported that, in cultured hippocampal neurons, the clustering of synaptic GABA(A) receptors significantly affects synaptic transmission. In this work, we demonstrated that the clustering of extrasynaptic GABA(A) receptors modulated tonic inhibition. Depolymerization of the cytoskeleton with nocodazole promoted the disassembly of extrasynaptic clusters of delta and gamma(2) subunit-containing GABA(A) receptors. This effect was associated with a reduction in the amplitude of tonic currents and diminished shunting inhibition. Moreover, diffuse GABA(A) receptors were less sensitive to the GAT-1 inhibitor NO-711 and to flurazepam. Quantitative analysis of GABA-evoked currents after prolonged exposure to submicromolar concentrations of GABA and model simulations suggest that clustering affects the gating properties of extrasynaptic GABA(A) receptors. In particular, a larger occupancy of the singly and doubly bound desensitized states can account for the modulation of tonic inhibition recorded after nocodazole treatment. Moreover, comparison of tonic currents recorded during spontaneous activity and those elicited by exogenously applied low agonist concentrations allows estimation of the concentration of ambient GABA. In conclusion, receptor clustering appears to be an additional regulating factor for tonic inhibition.     

An interaction between benzodiazepines and neuroactive steroids at GABA A receptors in cultured hippocampal neurons

Neurosteroids are modulators of several receptors and ion channels and are implicated in the pathophysiology of several neuropsychiatric diseases including hepatic encephalopathy (HE). The neurosteroid, allopregnanolone, a positive allosteric modulator of GABA_A receptors, accumulates in the brains of HE patients where it can potentiate GABA_A receptor-mediated responses. Attenuation of the effects of neurosteroids on GABA-ergic neurotransmission is therefore of interest for the management of HE. In the present study, we determined the effect of the benzodiazepine partial inverse agonist, Ro15-4513, and the benzodiazepine antagonist, flumazenil on modulation of the GABA_A mediated chloride currents by allopregnanolone and on spontaneous synaptic activity in cultured hippocampal neurons using the patch-clamp technique. Allopregnanolone (0.03–0.3 μM), dose-dependently potentiated GABA-induced currents, an action significantly reduced by Ro15-4513 (10 μM). In contrast, flumazenil (10 μM) had no effect on the ability of allopregnanolone to potentiate GABA_A currents but it blocked the effects of Ro15-4513. The frequency of spontaneous synaptic activity was significantly reduced in the presence of allopregnanolone (0.1 μM) from 1.5 ± 0.7 to 0.1 ± 0.04 Hz. This action was partially reversed by Ro15-4513 (10 μM) but was not significantly influenced by flumazenil (10 μM). These findings suggest that the beneficial affects of Ro15-4513 in experimental HE result from attenuation of the effects of neurosteroids at GABA_A receptors. Our results may provide a rational basis for the use of benzodiazepine inverse agonists in the management and treatment of hepatic encephalopathy in patients with liver failure.     

Presence of insulin receptors in cultured glial C6 cells. Regulation by butyrate.

The presence of insulin receptor and its regulation by butyrate and other short-chain fatty acids was studied in C6 cells, a rat glioma cell line. Intact C6 cells bind 125I-insulin in a rapid, reversible and specific manner. Scatchard analysis of the binding data gives typical curvilinear plots with apparent affinities of approx. 6 nM and 70 nM for the low-affinity (approx. 90% of total) and high-affinity (approx. 10% of total) sites respectively. Incubation with butyrate results in a time- and dose-dependent decrease of insulin binding to C6 cells. A maximal effect was found with 2 mM-butyrate that decreased the receptor by 40-70% after 48 h. Butyrate decreased numbers of receptors of both classes, but did not significantly alter receptor affinity. Other short-chain fatty acids, as well as keto acids, had a similar effect, but with a lower potency. Cycloheximide caused an accumulation of insulin receptors at the cell surface, since insulin binding increased and receptor affinity did not change after incubation with the inhibitor. Simultaneous addition of butyrate and cycloheximide abolished the loss of receptors produced by the fatty acid. In cells preincubated with butyrate, cycloheximide also produced a large increase in receptor numbers, showing that in the absence of new receptor synthesis a large pool of receptors re-appears at the surface of butyrate-treated cells.     

Production of components of extracellular matrix by cultured rat Sertoli cells

Sertoli cells from rats aged 15, 20, and 25 days were cultured in plastic dishes and extracted with Triton X-100 (0.1 percent w/v) or sodium deoxycholate (2 percent w/v). Residues left after extraction were found to contain three proteins characteristic of extracellular matrix (fibronectin, collagen IV, and laminin). These proteins were identified by four methods: indirect immunofluorescence, co-migration with standard proteins on electrophoresis in polyacrylamide gels, immunoblotting (Western blots), and immunoprecipitation after incubating the Sertoli cells with [35S]methionine. In addition, fibronectin was identified by immunoelectron microscopy with a second antibody conjugated to colloidal gold. In the same cell residues, heparan sulfate was tentatively identified by the first of these methods. The cells used in these studies were shown, by electron microscopy, to be essentially pure cultures of Sertoli cells (greater than 95% pure). Since 100 percent of the cells examined showed positive and specific immunofluorescent staining with well-characterized antibodies to the four components of the extracellular matrix, and since studies with colloidal gold revealed the presence of fibronectin closely associated with and inside cells identified by electron microscopy as Sertoli cells, it must be concluded that Sertoli cells synthesize these four proteins and presumably heparan sulfate. Evidently, cultured Sertoli cells can synthesize and secrete some of the components of an extracellular matrix.