Glia Cells In Culture Research Articles

Glutathione-Induced Calcium Shifts in Chick Retinal Glial Cells.

Neuroglia interactions are essential for the nervous system and in the retina Müller cells interact with most of the neurons in a symbiotic manner. Glutathione (GSH) is a low-molecular weight compound that undertakes major antioxidant roles in neurons and glia, however, whether this compound could act as a signaling molecule in neurons and/or glia is currently unknown. Here we used embryonic avian retina to obtain mixed retinal cells or purified Müller glia cells in culture to evaluate calcium shifts induced by GSH. A dose response curve (0.1–10mM) showed that 5–10mM GSH, induced calcium shifts exclusively in glial cells (later labeled and identified as 2M6 positive cells), while neurons responded to 50mM KCl (labeled as βIII tubulin positive cells). BBG 100nM, a P2X7 blocker, inhibited the effects of GSH on Müller glia. However, addition of DNQX 70μM and MK-801 20μM, non-NMDA and NMDA blockers, had no effect on GSH calcium induced shift. Oxidized glutathione (GSSG) at 5mM failed to induce calcium mobilization in glia cells, indicating that the antioxidant and/or structural features of GSH are essential to promote elevations in cytoplasmic calcium levels. Indeed, a short GSH pulse (60s) protects Müller glia from oxidative damage after 30 min of incubation with 0.1% H2O2. Finally, GSH induced GABA release from chick embryonic retina, mixed neuron-glia or from Müller cell cultures, which were inhibited by BBG or in the absence of sodium. GSH also induced propidium iodide uptake in Müller cells in culture in a P2X7 receptor dependent manner. Our data suggest that GSH, in addition to antioxidant effects, could act signaling calcium shifts at the millimolar range particularly in Müller glia, and could regulate the release of GABA, with additional protective effects on retinal neuron-glial circuit.

Release of ATP from avian Müller glia cells in culture

ATP can be released from neurons and act as a neuromodulator in the nervous system. Besides neurons, cortical astrocytes also are capable of releasing ATP from acidic vesicles in a Ca2+-dependent way. In the present work, we investigated the release of ATP from Müller glia cells of the chick embryo retina by examining quinacrine staining and by measuring the extracellular levels of ATP in purified Müller glia cultures. Our data revealed that glial cells could be labeled with quinacrine, a reaction that was prevented by incubation of the cells with 1μM bafilomycin A1 or 2μM Evans blue, potent inhibitors of vacuolar ATPases and of the vesicular nucleotide transporter, respectively. Either 50mM KCl or 1mM glutamate was able to decrease quinacrine staining of the cells, as well as to increase the levels of ATP in the extracellular medium by 77% and 89.5%, respectively, after a 5min incubation of the cells. Glutamate-induced rise in extracellular ATP could be mimicked by 100μM kainate (81.5%) but not by 100μM NMDA in medium without MgCl2 but with 2mM glycine. However, both glutamate- and kainate-induced increase in extracellular ATP levels were blocked by 50μM of the glutamatergic antagonists DNQX and MK-801, suggesting the involvement of both NMDA and non-NMDA receptors. Extracellular ATP accumulation induced by glutamate was also blocked by incubation of the cells with 30μM BAPTA-AM or 1μM bafilomycin A1. These results suggest that glutamate, through activation of both NMDA and non-NMDA receptors, induces the release of ATP from retinal Müller cells through a calcium-dependent exocytotic mechanism.

The effect of pretreatment or combined treatment of quercetin on menadione toxicity in rat primary mixed glial cells in vitro

Neurons and glia are highly susceptible to reactive oxygen species that play a key role in various neurodegenerative diseases. Menadione, a synthetic derivative of vitamin K, induces reactive oxygen generation. Quercetin one of the most ubiquitous bioflavonoids in food of plant origin, has strong antioxidant activities on different cell types, however recent studies demonstrated that it has also prooxidant and cytotoxic potentials. We examined the action of pre- and co-treatment of quercetin on menadione induced glial toxicity. The primary mixed glial cells obtained from 1 to 3 day old rat brain were pretreated with 10, 25, 100 or 250 muM quercetin for 1 h, washed out and 10, 25, 50, 75 or 100 muM menadione was added for 6 h. The other group of cells was treated with respective doses of quercetin combined simultaneously with the same doses of menadione for 6 h. The cells were washed and incubated for additional 24 h for recovery period and the viability was measured by using MTT assay. Menadione was dose-dependently toxic to glia cells and pretreatment with respective quercetin doses for 1 h could not eliminate this toxicity. Although 10 and 25 muM quercetin combined with 10 and 25 muM menadione could not change, 100 and 250 muM quercetin together with 10 or 25 muM menadione for 6 h increased further the menadione induced toxicity. We conclude that when combined with menadione, quercetin at high doses could be toxic to primary rat glia cells in culture.

Hydrogen sulfide regulates intracellular pH in rat primary cultured glia cells

Intracellular pH (pH(i)) plays an important role in the regulation of central nervous system function. In the present study, we examined whether hydrogen sulfide (H(2)S), a recently recognized neuromodulator, regulates pH(i) in rat primary cultured glia cells. pH(i) was measured with a fluorescent sensitive dye, BCECF-AM. Activities of Cl(-)/HCO(3)(-) exchanger and Na(+)/H(+) exchanger were examined by assessing their capacities to load or extrude H(+) upon NH(4)Cl pulse load. We found that NaHS, a H(2)S donor, decreased pH(i) in a concentration-dependent manner ranging from 10 to 200muM in the primary cultured microglia. Blockade of the Cl(-)/HCO(3)(-) exchanger with, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) or Na(+)/H(+) exchanger with 5-N-methyl-N-isobutylamiloride (MIA) significantly attenuated the pH(i)-lowering effect of NaHS. Moreover, NaHS significantly increased the activity of Cl(-)/HCO(3)(-) exchanger but inhibited that of Na(+)/H(+) exchanger. The pH regulatory effect of H(2)S was also observed in primary cultured astrocytes, but not in SH-SY5Y neuronal cells. In conclusion, we found for the first time that H(2)S induced intracellular acidification in glia cells via regulation of the activities of Cl(-)/HCO(3)(-) exchanger and Na(+)/H(+) exchanger. The present study may provide new evidence for H(2)S to serve as a neuromodulator and offer a potential approach for the treatment of neurological diseases.

Identification of two novel activities of the Wnt signaling regulator Dickkopf 3 and characterization of its expression in the mouse retina

BackgroundThe Wnt signaling pathway is a cellular communication pathway that plays critical roles in development and disease. A major class of Wnt signaling regulators is the Dickkopf (Dkk) family of secreted glycoproteins. Although the biological properties of Dickkopf 1 (Dkk1) and Dickkopf 2 (Dkk2) are well characterized, little is known about the function of the related Dickkopf 3 (Dkk3) protein in vivo or in cell lines. We recently demonstrated that Dkk3 transcripts are upregulated during photoreceptor death in a mouse model of retinal degeneration. In this study, we characterized the activity of Dkk3 in Wnt signaling and cell death.ResultsDkk3 was localized to Müller glia and retinal ganglion cells in developing and adult mouse retina. Western blotting confirmed that Dkk3 is secreted from Müller glia cells in culture. We demonstrated that Dkk3 potentiated Wnt signaling in Müller glia and HEK293 cells but not in COS7 cells, indicating that it is a cell-type specific regulator of Wnt signaling. This unique Dkk3 activity was blocked by co-expression of Dkk1. Additionally, Dkk3 displayed pro-survival properties by decreasing caspase activation and increasing viability in HEK293 cells exposed to staurosporine and H2O2. In contrast, Dkk3 did not protect COS7 cells from apoptosis.ConclusionThese data demonstrate that Dkk3 is a positive regulator of Wnt signaling, in contrast to its family member Dkk1. Furthermore, Dkk3 protects against apoptosis by reducing caspase activity, suggesting that Dkk3 may play a cytoprotective role in the retina.

GABA uptake by purified avian Müller glia cells in culture

GABA is the main inhibitory aminoacid transmitter present in neurons and glial cells. Its uptake is carried out by specific high-affinity Na(+)/Cl (-) dependent transporters (GATs). It has been reported in the past that, in the avian retina, [(3)H]GABA appears to be exclusively accumulated by horizontal and amacrine cells in the inner nuclear layer, and also by ganglion cells. Purified chick Müller glia cultures were able to take up [(3)H]GABA in a Na(+) and Cl(+) dependent way. Increasing GABA concentration increases GABA uptake by these cells, reaching half-maximal transport efficiency (EC50) around 0.3 mM. [(3)H]GABA uptake by Müller glia neuronal-free cultures was not mediated by neuronal transporters since it was not blocked by NNC-711, but was inhibited by beta-alanine, a specific glial transporter inhibitor. Chick Müller glia in culture express both GAT-1 and GAT-3 GABA transporters. Although mixed neuron-glial dense cultures released GABA upon glutamate, high K[(+) or veratridine stimulation, Müller glial cells did not release [(3)H]GABA upon treatment with these agents, suggesting that different from neurons, transporter mediated GABA release is not a common mechanism operating in these cells. The data also suggest that Müller cells take up GABA unidirectionally, which may constitute an important mechanism of inactivating GABA activity mediated by neurons.

Induction of operational tolerance to discordant dopaminergic porcine xenografts1

Porcine embryonic neural tissue transplanted intracerebrally could potentially relieve the symptoms of Parkinson's disease if the immune response toward the graft could be overcome. However, conventional immunosuppressive treatments have proven inefficient in preventing rejection. An alternative is blocking the costimulatory signals for lymphocyte activation. Treatment with cytotoxic T-lymphocyte antigen 4 immunoglobulin (CTLA4Ig) and anti-CD40L has been successful in preventing rejection of xenografts in some experimental studies, but not all. Lymphocyte function antigen (LFA)-1 is an important costimulatory molecule for CD8+ T cells, and we hypothesize that blockade with anti-LFA-1 may enhance the efficacy of CTLA4Ig and anti-CD40L therapy. C57BL/6 mice received intracerebral transplants of ventral mesencephalic tissue from embryonic porcine donors. CTLA4Ig, anti-CD40L, and anti-LFA-1 were administered every other day on days 0 to 8, and the transplants were studied after 4 to 6 weeks. Grafts were histologically analyzed for size, survival of dopaminergic nerve cells, and immune responses. Recipients were challenged with cultured glia cells of donor origin or an allogeneic skin graft to evaluate tolerance induction. Mice treated with all three substances had large grafts containing high amounts of dopamine cells but a low degree of immune response. Grafts in recipients challenged with glial cells showed an increased immunologic activity but were not rejected. Triple-treated mice showed a normal rejection process of the allogeneic skin grafts. After a short course of costimulation blocking therapy, discordant neural xenografts demonstrate long-term survival, withstand immunologic challenge, yet maintain host-versus-graft reactivity. Anti-LFA-1 complements CTLA4Ig and anti-CD40L in the induction of operational tolerance to these xenografts.

Developmental roles of platelet-derived growth factors.

Platelet-derived growth factor (PDGF) was originally identified in platelets and in serum as a mitogen for fibroblasts, smooth muscle cells (SMC) and glia cells in culture. PDGF has since expanded to a family of dimers of at least four gene products, whose biological actions are mediated through two receptor tyrosine kinases, PDGFRs. The present review summarizes and discusses the biological functions of PDGFs and PDGFRs in developmental processes, mainly as revealed through genetic analysis in mice. Such studies have demonstrated multiple critical roles of PDGFs and PDGFRs in embryonic and postnatal development. PDGFs seem to act upon specific populations of progenitor cells that give rise to several different cell types with distinct functions in a variety of developmental processes. Analogies are seen between the cell functions and the developmental processes controlled by PDGFs. This suggests that ancestral PDGF and PDGFR expression patterns and functions may have been iterated in related sets of morphogenetic processes in the course of evolution.

Interactions of Nocardia asteroides with murine glia cells in culture

Astrocytes and microglia were obtained from brains of newborn mice and infected with Nocardia asteroides. Scanning electron micrographs showed nocardiae adhering to the astrocyte cell surface and entering the cytoplasm. After 6 h of incubation the intracellular nocardiae appeared as filaments, demonstrating that growth was occurring. In contrast, the microglia phagocytized nocardiae, but after 6 h the presence of coccoid cells indicated that nocardial growth was inhibited.

Transient presence and functional interaction of endogenous GABA and GABAA receptors in developing rat optic nerve.

GABA (gamma-aminobutyric acid) is a major inhibitory synaptic neurotransmitter with widespread distribution in the central nervous system (CNS). GABA can also modulate axonal excitability by activation of GABAA receptors in CNS white matter regions where synapses and neuronal cell bodies are not present. Studies on cultured glia cells have revealed the synthesis of GABA in rat optic nerve O-2A progenitor cells that give rise to oligodendrocytes and type 2 astrocytes in vitro. We report here that: (i) GABA is detected by immuno-electron microscopy in intact rat optic nerve and is localized to glia and pre-myelinated axons during the first few weeks of postnatal development, but is markedly reduced or absent in the adult; and (ii) neonatal optic nerve is depolarized by GABAA receptor agonists or by the inhibition of GABA uptake. These results demonstrate the presence of functional GABAA receptors, and GABA uptake and release mechanisms in developing rat optic nerve, and suggest that excitability of developing axons can be modulated by endogenous neurotransmitter at non-synaptic sites.

Murine glia cells in culture can be stimulated to generate reactive oxygen.

Induction of luminol-enhanced chemiluminescence (CL) indicative of reactive oxygen formation was studied in glia cell cultures from newborn mice. A burst of CL could be induced by phorbol myristate acetate, zymosan, and antibody-coated bovine red blood cells, whereas Sendai virus and several other agents known to induce CL in myeloid cells were ineffective. Sodium azide failed to inhibit CL, indicating a myeloperoxidase-independent mechanism of light emission. In parallel experiments we identified the cells binding antibody-coated erythrocytes as macrophages characterized by the reduction of nitroblue tetrazolium and phagocytosis of zymosan and latex particles. Brain macrophages may use reactive oxygen intermediates (ROI) as a mechanism of antimicrobial defence; and, on the other hand, ROI formed by these cells may contribute to immuno-pathology in the brain.

THE DETACHMENT, ATTACHMENT AND GROWTH CAPACITY OF HUMAN GLIOMA AND GLIA CELLS IN CULTURE

The detachment, attachment and growth were studied in malignant glioma and normal glia cells in culture, using the palladium-agarose cloning method. The number of cells in each individual clone was repeatedly counted throughout 10 days. All four glioma cell-lines studied detached and attached themselves at higher rates than the normal, diploid, glia cells. This dynamic behaviour seemed to be a general property of cultured, malignant, glioma cells. All the cell-lines contained clones with different properties. Some clones were growing, others were nearly constant in cell number and yet others decreased in cell number as a function of time. The differences between these types of clones were surprisingly sharp.

Biological functions of neuroblastoma growth inhibitory factor(NGIF) derived from glia cells in culture

Biological functions of neuroblastoma growth inhibitory factor(NGIF) derived from glia cells in culture

Muscarinic acetylcholine receptors on cultured glia cells

Muscarinic acetylcholine receptors (76 fmol/mg protein) were detected on cultured glia cells (astroblasts) from embryonic chicken brain by specific [ 3H]quinuclidinylbenzilate (QNB) binding at physiological conditions. The QNB binding ( K d = 9.5 × 10 −11) to the intact cells seems to be cooperative (n H= 1.98) as shown by graphical methods.

Glia cell stimulating factor (GSF): A new lymphokine: Part 2. Cellular sources and partial purification of human GSF

Glia cell proliferation is characteristic of many inflammatory and degenerative processes in the brain, however the mechanisms underlying this response are poorly understood. We described a glia cell stimulating factor (GSF), produced by murine spleen cells, which activates DNA- and RNA synthesis of cultured glia cells. In the present series of experiments, we examined the ability of Concanavalin A (ConA)-stimulated human peripheral blood mononuclear leukocytes (PBM) and two continuous cell lines derived from human lymphocytes to spontaneously release of GSF in culture. The studies presented herein demonstrate that (1) ConA-stimulated PBM of healthy subjects produced GSF, (2) GSF is produced by T lymphocytes in collaboration with monocyte-macrophages, (3) both the human T cell line (MOLT-4F) and a B cell line (RPMI 1788) spontaneously secrete GSF, (4) GSF was found to have a molecular weight of 30,000 and less than 10,000 daltons, (5) macrophage (MIF)- and leukocyte (LIF) migration-inhibiting factor activities, as well as mitogenic factor (MF)- and lymphocyte-activation factor (LAF) activities could be separated from the GSF activity by gel filtration on Biogel P-100. These findings provide further evidence for the existence of GSF as a new lymphokine, distinct from LIF, MIF, MF and LAF.

Involvement of cyclic amp in the regulation of lymphokine induced glia cell stimulation

T and B lymphocytes of human or murine origin were found to secrete a factor which increases the DNA and RNA synthesis of cultured glia cells. This factor, termed glia cell stimulating factor (GSF), is released upon stimulation of such immune cells by mitogen or antigen qualifying it as a lymphokine. In this communication we report on the role of cyclic AMP (cAMP) in regulating the effect of GSF on glia cells. Prostaglandin E 1 (PGE 1), isoproterenol and theophylline were effective in suppressing the GSF-induced increase of the glia cell proliferation. No inhibition of DNA synthesis and no decrease in cell number was observed when testing these substances on glia cells not being activated by GSF. The drugs were found to induce an increase in cAMP concentrations of glia cells. A partial desensitization of the glia cells to these drug induced elevations of cAMP was detected after pretreatment of the glia cell cultures with GSF. It is suggested that stimulated lymphocytes not only release GSF but also low molecular weight proteins such as PGE 1 which regulate the effects of GSF on glia cells by activating their adenylate cyclase.

Glia cell response to bacterial lipopolysaccharide: Effect on nucleotide synthesis, its genetic control and definition of the active principle

Cell types sensitive to lipopolysaccharides (LPS) include macrophages, polymorphonuclear leukocytes, platelets, B-lymphocytes and fibroblasts. Earlier observations that during endotoxemia or intracerebral injection of LPS morphological and functional alterations of the central nervous system develop, suggest that LPS may also interact with brain cells. The effects of bacterial products on nucleotide synthesis of cultured murine glia cells are examined in this study. LPS extracted from the outher cell wall of gram-negative bacteria were found to cause an increased RNA synthesis and an inhibition of DNA synthesis in the cultured glia cells. The inhibitory effect of LPS on DNA synthesis is not due to an LPS-mediated increase of the cAMP content in the glia cells or contact inhibition of the monolayer culture. Within the structure of LPS lipid A was found to be the active part. On the basis of the LPS-induced increase of RNA synthesis, the glia cells of C3H/HeJ mice are low responders to low dosis of LPS. We conclude that in the presence of fetal calf serum glia cells are sensitive to the lipid A part of LPS, the sensitivity being under genetic control.

Ecto-ATPase deficiency in glia of seizure-prone mice.

A DEFICIENCY of calcium-stimulated adenosine triphosphatase (Ca2+-ATPase) is seen in brains of mice which are susceptible to audiogenic seizures1. After studying five other animal models of idiopathic epilepsy we concluded that brain nucleotide metabolism is altered in the epileptic animals and that the lesion might not be confined to Ca2+-ATPase2. We speculated that the deficiency of Ca2+-ATPase, which we observed in membrane-enriched brain homogenates, was the expression of incompetent brain cell ecto-ATPases. We have suggested that the genesis of seizures was related to a protracted action of translocated cytoplasmic ATP on cell membranes3 when the latter were deficient in this enzyme. We report here a significant deletion of ecto-ATPase in cultured glia cells raised from neonatal, seizure-prone mice. Between the ages of 20 to 35 d, mice of the DBA/2N strain convulse after repeated exposure to loud noise. Before and after this period, susceptibility to induced convulsions is minimal or absent. Mice of the C57 B1/6N strain are not afflicted in this manner.

Reset of the pre-replicative phase of human glia cells in culture

Dense and sparse cultures of human glia cells blocked in G1 by density inhibition and serum deprivation, respectively, require about 12 h of continuous stimulation by growth factors to become irreversibly committed to DNA synthesis. The decrease in preparedness to DNA synthesis has been examined in dense and sparse cultures of human glia cells by giving repeated subliminal stimulatory pulses of different stimulatory agents, interposed by non-stimulatory pauses. The results indicate a gradual slip back in G1 towards the starting point in early G1. The half-life of this apparent reset was of the order 1–3 h, indicating a fairly rapid disappearance of the imprint left by even such a long pulse as 8 h of serum or EGF (epidermal growth factor) stimulation. The reset mechanism demonstrated in human glia cells is shown not to be merely the result of a gradual disappearance of stimulatory factor but is considered to reflect a regulatory mechanism preventing undue entry into the cell cycle.

Influence of epidermal growth factor (EGF) on ruffling activity, pinocytosis and proliferation of cultivated human glia cells

Normal human glia cells in culture were studied with respect to ruffling activity, macro-pinocytosis and cell proliferation under standard culture conditions with 10% serum in the medium, in serum-free medium and after addition of epidermal growth factor (EGF) or serum to previously serum-free medium. Pinocytotic uptake of droplets of medium occurred only in relation to well developed ruffling membranes. Omitting the serum from the medium led to a drastic reduction in thymidine incorporation. The cells became slender under these conditions, and soon after the change of medium their ruffling activity and pinocytosis were almost completely abolished. Following the change to a medium containing 2 ng EGF/ml a rapid reappearance of ruffling and pinocytosis was observed. DNA synthesis, however, was not demonstrated until after 20 h, showing that ruffling and pinocytosis occurred before DNA synthesis had started. Thus EGF may initially induce conformational changes of the plasma membrane, resulting in its internalization due to formation of endocytotic vacuoles. The observed relationship between occurrence of well developed ruffling membranes, macro-pinocytosis and cell multiplication indicates that one of the functions of growth-promoting factors may be stimulation of plasma membrane turnover.