High-affinity uptake of γ-aminobutyric acid in cultured glial and neuronal cells

Both glial and neuronal cells maintained in primary culture were found to accumulate [³H]GABA by an efficient high-affinity uptake system (apparentK _m=9 M,V _max=0.018 and 0.584 nmol/mg/min, respectively) which required sodium ions and was inhibited by 1 mM ouabain. Strychnine and parachloromercuriphenylsulfonate (pCS) (both at 1 mM) also strongly inhibited uptake of [³H]GABA, but metabolic inhibitors (2,4-dinitrophenol, potassium cyanide, and malonate) were without effect. Only three structural analogs of GABA (nipecotate, -alanine, and 2,4-diaminobutyrate) inhibited uptake of [³H]GABA, while several other compounds with structural similarities to GABA (e.g. glycine,l-proline, and taurine) did not interact with the system. The kinetic studies indicated presence of a second uptake (K _m=92 M,V _max=0.124 nmol/mg/min) in the primary cultures containing predominantly glioblasts. On the other hand, only one of the neuronal cell lines transformed by simian virus SV40 appeared to accumulate [³H]GABA against a concentration gradient. ApparentK _m of this uptake was relatively high (819 M), and it was only weakly inhibited by 1 mM ouabain and 1 mM pCS. The structural specificity also differed from that of the uptake observed in the primary cultures. Significantly, none of the nontransformed continuous cell lines of either tumoral (glioma, C₆; neuroblastoma, Ml; MINN) or normal (NN; I₆) origin actively accumulated [³H]GABA. It is suggested that for the neurochemical studies related to GABA and requiring homogeneous cell populations, the primary cultures offer a better experimental model than the continuous cell lines.     

Regulation of glutamate metabolism by hydrocortisone and branched chain keto acids in cultured rat retinal Müller cells (TR-MUL)

Glutamate released from retinal neurons during neurotransmission is taken up by retinal Müller cells, where much of the amino acid is subsequently amidated to glutamine or transaminated to α-ketoglutarate for oxidation. Müller cell glutamate levels may have to be carefully maintained at fairly low concentrations to avoid excesses of glutamate in extracellular spaces of the retina that would otherwise cause excitotoxicity. We employed a cultured rat retinal Müller cell line in order to study the metabolism and the role of Müller cell specific enzymes on the glutamate disposal pathways. We found that the TR-MUL cells express the glial specific enzymes, glutamine synthetase, the mitochondrial isoform of branched chain aminotransferase (BCATm) and pyruvate carboxylase, all of which are involved in glutamate metabolism and homeostasis in the retina. Hydrocortisone treatment of TR-MUL cells increased glutamine synthetase expression and the rate of glutamate amidation to glutamine. Addition of branched chain keto acids (BCKAs) increased lactate and aspartate formation from glutamate and also oxidation of glutamate to CO₂ and H₂O. The two glutamate disposal pathways (amidation and oxidation) did not influence each other. When glutamate levels were independently depleted within TR-MUL cells, the uptake of glutamate from the extracellular fluid increased compared to uptake from control (undepleted) cells suggesting that the level of intracellular glutamate may influence clearing of extracellular glutamate.     

Effects of L-glutamate/D-aspartate and monensin on lactic acid production in retina and cultured retinal Müller cells

We have investigated the dependence of the rate of lactic acid production on the rate of Na(+) entry in cultured transformed rat Müller cells and in normal and dystrophic (RCS) rat retinas that lack photoreceptors. To modulate the rate of Na(+) entry, two approaches were employed: (i) the addition of L-glutamate (D-aspartate) to stimulate coupled uptake of Na(+) and the amino acid; and (ii) the addition of monensin to enhance Na(+) exchange. Müller cells produced lactate aerobically and anaerobically at high rates. Incubation of the cells for 2-4 h with 0.1-1 mM L-glutamate or D-aspartate did not alter the rate of production of lactate. ATP content in the cells at the end of the incubation period was unchanged by addition of L-glutamate or D-aspartate to the incubation media. Na(+)-dependent L-glutamate uptake was observed in the Müller cells, but the rate of uptake was very low relative to the rate of lactic acid production. Ouabain (1 mM) decreased the rate of lactic acid production by 30-35% in Müller cells, indicating that energy demand is enhanced by the activity of the Na(+)-K(+) pump or depressed by its inhibition. Incubation of Müller cells with 0.01 mM monensin, a Na(+) ionophore, caused a twofold increase in aerobic lactic acid production, but monensin did not alter the rate of anaerobic lactic acid production. Aerobic ATP content in cells incubated with monensin was not different from that found in control cells, but anaerobic ATP content decreased by 40%. These results show that Na(+)-dependent L-glutamate/D-aspartate uptake by cultured retinal Müller cells causes negligible changes in lactic acid production, apparently because the rates of uptake are low relative to the basal rates of lactic acid production. In contrast, the marked stimulation of aerobic lactic acid production caused by monensin opening Na(+) channels shows that glycolysis is an effective source of ATP production for the Na(+)-K(+) ATPase. A previous report suggests that coupled Na(+)-L-glutamate transport stimulates glycolysis in freshly dissociated salamander Müller cells by activation of glutamine synthetase. The Müller cell line used in this study does not express glutamine synthetase; consequently these cells could only be used to examine the linkage between Na(+) entry and the Na(+) pump. As normal and RCS retinas express glutamine synthetase, the role of this enzyme was examined by coapplication of L-glutamate and NH(4) (+) in the presence and absence of methionine sulfoximine, an inhibitor of glutamine synthetase. In normal retinas, neither the addition of L-glutamate alone or together with NH(4) (+) caused a significant change in the glycolytic rate, an effect linked to the low rate of uptake of this amino acid relative to the basal rate of retinal glycolysis. However, incubation of the RCS retinas in media containing L-glutamate and NH(4)(+) did produce a small (15%) increase in the rate of glycolysis above the rate found with L-glutamate alone and controls. It is unlikely that this increase was the result of conversion of L-glutamate to L-glutamine, as it was not suppressed by inhibition of glutamine synthetase with 5 mm methionine sulfoximine. It appears that the magnitude of Müller cell glycolysis required to sustain the coupled transport of Na(+) and L-glutamate and synthesis of L-glutamine is small relative to the basal glycolytic activity in a rat retina.     

Effect of high glucose concentration on VEGF and PEDF expression in cultured retinal Müller cells

To explore the effect of high glucose concentration on the expression of vascular endothelial growth factor (VEGF) and pigment epithelium derived factor (PEDF) in the cultured rat retinal Müller cells. Rat Müller cells were cultured and RT-PCR and Western-blot analysis were used to measure the levels of VEGF and PEDF in cultured Müller cells at different high glucose concentrations. Under 10, 20, 30 mmol/L high glucose conditions, the levels of VEGF mRNA and protein increased and the levels of PEDF mRNA and protein decreased. These results suggest that the VEGF and PEDF expression in Müller cells are unbalance under high glucose concentration, which contribute to retinal neovascularization in diabetic retinopathy.     

The glutathione content of retinal Müller (glial) cells: effect of pathological conditions

Maintenance of isolated retinal Müller (glial) cells in glutamate-free solutions over 7 h causes a significant loss of their initial glutathione content; this loss is largely prevented by the blockade of glutamine synthesis using methionine sulfoximine (5 mM). Anoxia does not reduce the glutathione content of Müller cells when glucose (11 mM), glutamate and cystine (0.1 mM each) are present. In contrast, simulation of total ischemia (i.e., anoxia plus removal of glucose) decreases the glutathione levels dramatically, even in the presence of glutamate and cystine. Less severe effects are caused by high extracellular K+ (40 mM). Reactive oxygen species are generated in the retina under various conditions, such as anoxia, ischemia, and reperfusion. One of the crucial substances protecting the retina against reactive oxygen species is glutathione, a tripeptide constituted of glutamate, cysteine and glycine. It was recently shown that glutathione can be synthesized in retinal Müller glial cells and that glutamate is the rate-limiting substance. In this study, glutathione levels were determined in acutely isolated guinea-pig Müller cells using the glutathione-sensitive fluorescent dye monochlorobimane. The purpose was to find out how the glial glutathione content is affected by anoxia/ischemia and accompanying pathophysiological events such as depolarization of the cell membrane. Our results further strengthen the view that glutamate is rate-limiting for the glutathione synthesis in glial cells. During glutamate deficiency, as caused by e.g., impaired glutamate uptake, this amino acid is preferentially delivered to the glutamate-glutamine pathway, at the expense of glutathione. This mechanism may contribute to the finding that total ischemia (but not anoxia) causes a depletion of glial glutathione. In situ depletion may be accelerated by the ischemia-induced increase of extracellular K+, decreasing the driving force for glutamate uptake. The ischemia-induced lack of glutathione is particularly fatal considering the increased production of reactive oxygen species under this condition. Therefore the therapeutic application of exogenous free radical scavengers is greatly recommended.     

Mechanisms of Müller glial cell morphogenesis

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Conversion of mammalian Müller glial cells into a neuronal lineage by in vitro aggregate-culture

Mammalian Müller glial cells are major glial cells in the retina. Here we report that these glial cells can be redirected towards a neuronal lineage by an aggregate-culture in vitro. Rat and macaque Müller glial cells did not express neuronal markers except after transfer to adhesive conditions. Furthermore, this expression could only take place in the presence of platelet-derived growth factor and valproic acid. We compared a normal monolayer-culture and an aggregate-culture, and rat Müller glial cells could only differentiate into neurons under non-adhesive conditions. However, Müller glial cells did not express the photoreceptor markers in vitro. After transplantation into the subretinal space, a retina-specific niche, rat Müller glial cells expressed the photoreceptor-specific marker, opsin (RET-P1). We demonstrate the potential of mammalian Müller glial cells as a source of photoreceptors, which may possibly contribute to the treatment of degenerative retinal diseases such as retinitis pigmentosa.     

Chronically elevated glucose-induced apoptosis is mediated by inactivation of Akt in cultured Müller cells

Hyperglycemia induces apoptotic cell death in a variety of cell types in diabetes, and the mechanism remains unclear. We report here that culture of rat retinal glial Müller cells in 25 mM glucose for 72 h significantly inactivated Akt and induced apoptosis. Likewise, hyperglycemia caused a significant dephosphorylation of Akt at serine-473 in Müller cells in the retina of streptozotocin-induced diabetic rats. Inactivation of Akt was associated with dephosphorylation of BAD, increased cytochrome c release, and activation of caspase-3 and caspase-9. Upregulation of Akt activity by overexpression of constitutively active Akt inhibited elevated glucose-induced apoptosis, whereas downregulation of Akt activity by overexpression of dominant negative Akt exacerbated elevated glucose-induced apoptosis, as assessed by caspase activity and nucleic acid staining. These data suggest that apoptosis induced by chronically elevated glucose is at least in part mediated by downregulation of Akt survival pathway in cultured Müller cells. It has been reported that antiapoptotic effect of Akt requires glucose in growth factor withdrawal-induced apoptosis. Our data suggest that although acutely elevated glucose may be beneficial to the cell survival, chronically elevated glucose can cause apoptosis via downregulation of Akt survival signaling.     

Retinal pigment epithelium melanin granules are phagocytozed by Müller glial cells in experimental retinal detachment

The ability of retinal Müller glial cells to perform phagocytosis in vivo is studied in a rabbit model of experimental retinal detachment where pigment epithelial cells are occasionally detached together with the neural retina. While macrophages and/or microglial cells phagocytoze most of the cellular debris at the sclerad surface of the detached retinae, some Müller cells accumulate melanin granules. The granules are virtually intact at the ultrastructural level, and are surrounded by a membrane. They are often located close to the sclerad end of the cells, but some are distributed throughout the outer stem process up to the soma. It is concluded that rabbit Müller cells in vivo are capable of phagocytosis and of transporting the phagocytozed material within their cytoplasm.     

Ectonucleotidases in Müller glial cells of the rodent retina: Involvement in inhibition of osmotic cell swelling

Extracellular nucleotides mediate glia-to-neuron signalling in the retina and are implicated in the volume regulation of retinal glial (Müller) cells under osmotic stress conditions. We investigated the expression and functional role of ectonucleotidases in Müller cells of the rodent retina by cell-swelling experiments, calcium imaging, and immuno- and enzyme histochemistry. The swelling of Müller cells under hypoosmotic stress was inhibited by activation of an autocrine purinergic signalling cascade. This cascade is initiated by exogenous glutamate and involves the consecutive activation of P2Y₁ and adenosine A1 receptors, the action of ectoadenosine 5′-triphosphate (ATP)ases, and a nucleoside-transporter-mediated release of adenosine. Inhibition of ectoapyrases increased the ATP-evoked calcium responses in Müller cell endfeet. Müller cells were immunoreactive for nucleoside triphosphate diphosphohydrolases (NTPDase)2 (but not NTPDase1), ecto-5′-nucleotidase, P2Y₁, and A1 receptors. Enzyme histochemistry revealed that ATP but not adenosine 5′-diphosphate (ADP) is extracellularly metabolised in retinal slices of NTPDase1 knockout mice. NTPDase1 activity and protein is restricted to blood vessels, whereas activity of alkaline phosphatase is essentially absent at physiological pH. The data suggest that NTPDase2 is the major ATP-degrading ectonucleotidase of the retinal parenchyma. NTPDase2 expressed by Müller cells can be implicated in the regulation of purinergic calcium responses and cellular volume.     

Expression and high glucose-mediated regulation of K+ channel interacting protein 3 (KChIP3) and KV4 channels in retinal Müller glial cells

Normal vision depends on the correct function of retinal neurons and glia and it is impaired in the course of diabetic retinopathy. Müller cells, the main glial cells of the retina, suffer morphological and functional alterations during diabetes participating in the pathological retinal dysfunction. Recently, we showed that Müller cells express the pleiotropic protein potassium channel interacting protein 3 (KChIP3), an integral component of the voltage-gated K(+) channels K(V)4. Here, we sought to analyze the role of KChIP3 in the molecular mechanisms underlying hyperglycemia-induced phenotypic changes in the glial elements of the retina. The expression and function of KChIp3 was analyzed in vitro in rat Müller primary cultures grown under control (5.6 mM) or high glucose (25 mM) (diabetic-like) conditions. We show the up-regulation of KChIP3 expression in Müller cell cultures under high glucose conditions and demonstrate a previously unknown interaction between the K(V)4 channel and KChIP3 in Müller cells. We show evidence for the expression of a 4-AP-sensitive transient outward voltage-gated K(+) current and an alteration in the inactivation of the macroscopic outward K(+) currents expressed in high glucose-cultured Müller cells. Our data support the notion that induction of KChIP3 and functional changes of K(V)4 channels in Müller cells could exert a physiological role in the onset of diabetic retinopathy.     

BDNF regulates GLAST and glutamine synthetase in mouse retinal Müller cells

This study investigated whether brain-derived neurotrophic factor (BDNF) regulates the L-glutamate/L-aspartate transporter (GLAST) and glutamine synthetase (GS) in mouse retinal Müller cells (RMCs) under normal and hypoxic conditions. Mouse RMCs were treated with recombinant human BDNF (50, 75, 100, 125, or 150 ng/ml) for 24 h or underwent hypoxia induced by CoCl(2) (125 μM; 6, 12, 24, 48, or 72 h). An additional group underwent combined treatment with BDNF (100 ng/ml; 24, 48, 72, or 96 h) and CoCl(2) (125 μM/ml; 72 h). GLAST and GS mRNA and protein expression, L-[3,4-3H]-glutamic acid uptake, and apoptosis were assessed. BDNF dose-dependently up-regulated GLAST and GS mRNA and protein and increased glutamate uptake. Similarly, in early-stage CoCl(2)-induced hypoxia, GLAST and GS were up-regulated and glutamate uptake increased, but these decreased over time. BDNF also up-regulated GLAST and GS and increased glutamate uptake when RMCs under CoCl(2) induced hypoxic condition. However, BDNF treatment 24 h before CoCl(2) had no effect on GLAST or GS expression. CoCl(2) alone or combined with BDNF did not induce apoptosis. Hypoxia rapidly increased GLAST and GS expressions. This effect was transient, perhaps due to compensatory mechanisms that reduce GLAST and GS by 72 h. BDNF can up-regulate GLAST and GS and increase glutamate uptake during hypoxia, and these functions may underlie its neuroprotective effects.     

Genome-wide analysis of Müller glial differentiation reveals a requirement for Notch signaling in postmitotic cells to maintain the glial fate

Previous studies have shown that Müller glia are closely related to retinal progenitors; these two cell types express many of the same genes and after damage to the retina, Müller glia can serve as a source for new neurons, particularly in non-mammalian vertebrates. We investigated the period of postnatal retinal development when progenitors are differentiating into Müller glia to better understand this transition. FACS purified retinal progenitors and Müller glia from various ages of Hes5-GFP mice were analyzed by Affymetrix cDNA microarrays. We found that genes known to be enriched/expressed by Müller glia steadily increase over the first three postnatal weeks, while genes associated with the mitotic cell cycle are rapidly downregulated from P0 to P7. Interestingly, progenitor genes not directly associated with the mitotic cell cycle, like the proneural genes Ascl1 and Neurog2, decline more slowly over the first 10-14 days of postnatal development, and there is a peak in Notch signaling several days after the presumptive Müller glia have been generated. To confirm that Notch signaling continues in the postmitotic Müller glia, we performed in situ hybridization, immunolocalization for the active form of Notch, and immunofluorescence for BrdU. Using genetic and pharmacological approaches, we found that sustained Notch signaling in the postmitotic Müller glia is necessary for their maturation and the stabilization of the glial identity for almost a week after the cells have exited the mitotic cell cycle.     

NPY in rat retina is present in neurons, in endothelial cells and also in microglial and Müller cells

NPY is present in the retina of different species but its role is not elucidated yet. In this work, using different rat retina in vitro models (whole retina, retinal cells in culture, microglial cell cultures, rat Müller cell line and retina endothelial cell line), we demonstrated that NPY staining is present in the retina in different cell types: neurons, macroglial, microglial and endothelial cells. Retinal cells in culture express NPY Y(1), Y(2), Y(4) and Y(5) receptors. Retina endothelial cells express all NPY receptors except NPY Y(5) receptor. Moreover, NPY is released from retinal cells in culture upon depolarization. In this study we showed for the first time that NPY is present in rat retina microglial cells and also in rat Müller cells. These in vitro models may open new perspectives to study the physiology and the potential pathophysiological role of NPY in the retina.     

PEDF derived from glial Müller cells: a possible regulator of retinal angiogenesis

A precise balance between stimulators and inhibitors of angiogenesis, such as vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF), respectively, is essential for angiogenic homeostasis in ocular tissues. Retinal hypoxia is accompanied by some pathological conditions that may promote intraocular neovascularization. Here we demonstrate that retinal glial (Müller) cells express and release pigment epithelium-derived factor (PEDF). Decreasing oxygen concentrations cause strong attenuation of PEDF release resulting in enhanced VEGF/PEDF ratios. Exposure of Müller cells to VEGF suppressed PEDF release in a dose-dependent manner. This may represent a novel mechanism of ocular angiogenic homeostasis sufficient in the control of PEDF levels during normoxia or mild hypoxia but supplemented by other (hitherto unknown) mechanisms in cases of strong hypoxia. In spite of the enhanced VEGF/PEDF ratios resulting from hypoxia, conditioned media of Müller cells failed to stimulate additional proliferation of retinal endothelial cells. These findings suggest that in the ischemic retina, Müller cells generate a permissive condition for angiogenesis by secreting more VEGF and less PEDF, but the onset of retinal endothelial cell proliferation requires another triggering signal that remains to be identified.     

MAPK signaling during Müller glial cell development in retina explant cultures

The Müller cell is the only glial cell type generated from the retinal neuroepithelium. This cell type controls normal retina homeostasis and has been suggested to play a neuroprotective role. Recent evidence suggests that mammalian Müller cells can de-differentiate and return to a progenitor or stem cell stage following injury or disease. In vivo exploration of the molecular mechanisms of Müller cell differentiation and proliferation will add essential information to manipulate Müller cell functions. Signal transduction pathways that regulate Müller cell responses and activity are a critical part of their cellular machinery. In this study, we focus on mitogen-activated protein kinase (MAPK) signaling pathway during Müller glial cell differentiation and proliferation. We found that both MAPK and STAT3 signaling pathways are present during Müller glial cell development. Ciliary neurotrophic factor (CNTF)-stimulated Müller glial cell proliferation is associated with early developmental stages. Specific inhibition of MAPK phosphorylation significantly reduced the number of Müller glial cells with or without CNTF stimulation. These results suggested that the MAPK signal transduction pathway is important in the formation of Müller glial cells during retina development.