A comparison between acute exposures to ethanol and acetaldehyde on neurotoxicity, nitric oxide production and NMDA-induced excitotoxicity in primary cultures of cortical neurons

Chronic exposure of primary neuronal cultures to ethanol has been shown to potentiate N-methyl-D-aspartate (NMDA) receptor-mediated processes, such as nitric oxide (NO) formation and excitotoxicity. In the present study, we compared the effects of acute ethanol and acetaldehyde on NMDA receptor-mediated excitotoxicity and NO production in primary cultures of rat cortical neurons. The delayed cell death induced by NMDA (300 mM, 25 min) was evaluated by morphological examination and by measuring the release of the cytotoxic indicator, lactate dehydrogenase, in the culture media 24 hours after the NMDA exposure. The accumulation of nitrite, as an index of NO production, was also measured 24 hours after NMDA treatment. NMDA caused a dose-dependent cell death and nitrite accumulation, both effects were blocked by pretreatment of MK-801 (100 microM). Acute exposure to ethanol (1-1000 mM) or acetaldehyde (0.1-1 mM) for 35 minutes did not affect neuronal viability in the following 24-hr period. However, acute exposure to acetaldehyde (> or =10 mM) was neurotoxic. Neither ethanol nor acetaldehyde changed basal nitrite levels in the culture media. Acute ethanol (50-400 mM, 10 min) given before the NMDA treatment (25 min) resulted in a concentration-dependent suppression of the delayed cell death. The NMDA-induced NO production was, however, not affected by ethanol. Neither the NMDA excitotoxicity nor NO production was affected by acute ethanol given after NMDA treatment. Acute acetaldehyde (0.01-0.5 mM, 10 min) given before or after NMDA treatment had no effect on delayed NMDA neurotoxicity and NO production. Our data suggest that acute exposure to ethanol is not neurotoxic and is even protective against delayed NMDA-excitotoxicity when given before but not after NMDA treatment. Neither NO nor metabolism of ethanol to acetaldehyde is required for ethanol-mediated suppression of NMDA excititoxicity. Acetaldehyde, on the other hand, is toxic by itself at low concentrations (> or =10 mM). Furthermore, acute exposure to non-toxic concentrations of acetaldehyde could not protect cortical neurons against NMDA-induced excitotoxicity.     

Amyloid-β Decreases Nitric Oxide Production in Cultured Retinal Neurons: A Possible Mechanism for Synaptic Dysfunction in Alzheimer’s Disease?

The neurotoxicity of the amyloid-β peptide (Aβ) appears to be, at least in part, related to pathological activation of glutamate receptors by Aβ aggregates. However, the downstream signaling pathways leading to neurodegeneration are still incompletely understood. Hyperactivation of nitric oxide synthase (NOS) and increased nitric oxide (NO) production have been implicated in excitotoxic neuronal damage caused by overactivation of glutamate receptors, and it has been suggested that increased NO levels might also play a role in neurotoxicity in Alzheimer’s disease. We have examined the effect of blockade of NO production on the neurotoxicity instigated by Aβ₄₂ and by elevated concentrations of glutamate in chick embryo retinal neurons in culture. Results showed that l-nitroarginine methyl ester, a potent inhibitor of all NOS isoforms, had no protective effect against neuronal death induced by either Aβ₄₂ (20 μM) or glutamate (1 mM). Surprisingly, at short incubation times both Aβ and glutamate decreased NO production in retinal neuronal cultures in the absence of neuronal death. Thus, excitotoxic insults induced by Aβ and glutamate cause inhibition rather than activation of NO synthase in retinal neurons, suggesting that cell death induced by Aβ or glutamate is not related to increased NO production. On the other hand, considering the role of NO in long term potentiation and synaptic plasticity, the decrease in NO levels instigated by Aβ and glutamate suggests a possible mechanism leading to synaptic failure in AD.     

Interaction between NO and COX pathways in retinal cells exposed to elevated glucose and retina of diabetic rats

A nonselective inhibitor of cyclooxygenase (COX; high-dose aspirin) and a relatively selective inhibitor of inducible nitric oxide synthase (iNOS; aminoguanidine) have been found to inhibit development of diabetic retinopathy in animals, raising a possibility that NOS and COX play important roles in the development of retinopathy. In this study, the effects of hyperglycemia on retinal nitric oxide (NO) production and the COX-2 pathway, and the interrelationship of the NOS and COX-2 pathways in retina and retinal cells, were investigated using a general inhibitor of NOS [N(G)-nitro-l-arginine methyl ester (l-NAME)], specific inhibitors of iNOS [l-N(6)-(1-iminoethyl)lysine (l-NIL)] and COX-2 (NS-398), and aspirin and aminoguanidine. In vitro studies used a transformed retinal Müller (glial) cell line (rMC-1) and primary bovine retinal endothelial cells (BREC) incubated in 5 and 25 mM glucose with and without these inhibitors, and in vivo studies utilized retinas from experimentally diabetic rats (2 mo) treated or without aminoguanidine or aspirin. Retinal rMC-1 cells cultured in high glucose increased production of NO and prostaglandin E(2) (PGE(2)) and expression of iNOS and COX-2. Inhibition of NO production with l-NAME or l-NIL inhibited all of these abnormalities, as did aminoguanidine and aspirin. In contrast, inhibition of COX-2 with NS-398 blocked PGE(2) production but had no effect on NO or iNOS. In BREC, elevated glucose increased NO and PGE(2) significantly, whereas expression of iNOS and COX-2 was unchanged. Viability of rMC-1 cells or BREC in 25 mM glucose was significantly less than at 5 mM glucose, and this cell death was inhibited by l-NAME or NS-398 in both cell types and also by l-NIL in rMC-1 cells. Retinal homogenates from diabetic animals produced significantly greater than normal amounts of NO and PGE(2) and of iNOS and COX-2. Oral aminoguanidine and aspirin significantly inhibited all of these increases. The in vitro results suggest that the hyperglycemia-induced increase in NO in retinal Müller cells and endothelial cells increases production of cytotoxic prostaglandins via COX-2. iNOS seems to account for the increased production of NO in Müller cells but not in endothelial cells. We postulate that NOS and COX-2 act together to contribute to retinal cell death in diabetes and to the development of diabetic retinopathy and that inhibition of retinopathy by aminoguanidine or aspirin is due at least in part to inhibition of this NO/COX-2 axis.     

Hyperglycemia increases mitochondrial superoxide in retina and retinal cells

Oxidative stress is believed to play a significant role in the development of diabetic retinopathy. In this study, we have investigated the effects of elevated glucose concentration on the production of superoxide anion by retina and retinal cells, the cellular source of the superoxide, the effect of therapies that are known to inhibit diabetic retinopathy on the superoxide production, and the role of the superoxide in cell death in elevated glucose concentration. Superoxide release was measured from retinas collected from streptozotocin-diabetic rats (2 months) treated with or without aminoguanidine, aspirin, or vitamin E, and from transformed retinal Müller cells (rMC-1) and bovine retinal endothelial cells (BREC) incubated in normal (5 mM) and high (25 mM) glucose. Diabetes (retina) or incubation in elevated glucose concentration (rMC-1 and BREC cells) significantly increased superoxide production, primarily from mitochondria, because an inhibitor of mitochondrial electron transport chain complex II normalized superoxide production. Inhibition of reduced nicotinamine adenine dinucleotide phosphate (NADPH) oxidase or nitric oxide synthase had little or no effect on the glucose-induced increase in superoxide. Treatment of diabetic animals with aminoguanidine, aspirin, or vitamin E for 2 months significantly inhibited the diabetes-induced increase in production of superoxide in the retinas. Despite the increased production of superoxide, no increase in protein carbonyls was detected in retinal proteins from animals diabetic for 2-6 months or rMC-1 cells incubated in 25 mM glucose for 5 d unless the activities of calpain or the proteosome were inhibited. Addition of copper/zinc-containing superoxide dismutase to the media of rMC-1 and BREC cells inhibited the apoptotic death caused by elevated glucose. Diabetes-like glucose concentration increases superoxide production in retinal cells, and the superoxide contributes to impaired viability and increased cell death under those circumstances. Three therapies that inhibit the development of diabetic retinopathy all inhibit superoxide production, raising a possibility that these therapies inhibit retinopathy in part by inhibiting a hyperglycemia-induced increase in superoxide production.     

Requirement for Nitric Oxide in Retinal Neuronal Cell Death Induced by Activated Müller Glial Cells

Retinal Müller glial cells express the inducible isoform (-2) of nitric oxide (NO) synthase (NOS) in vitro after stimulation by lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) or in vivo in some retinal pathologies. Because NO may have beneficial or detrimental effects in the retina, we have used cocultures of retinal neurons with retinal Müller glial (RMG) cells from mice disrupted for the gene of NOS-2 [NOS-2 (-/-)] to clarify the role of NO in retinal neurotoxicity. We first demonstrated that NO produced by activated RMG cells was not toxic for RMG cells themselves. Second, the NO released from LPS/IFN-gamma-stimulated RMG cells induced neuronal cell death, because no neuronal cell death has been observed in cocultures with RMG cells from NOS-2 (-/-) mice and because inhibition of NOS-2 induction by transforming growth factor-beta or blockade of NO release by different NOS inhibitors prevented neuronal cell death. Addition of urate, a peroxynitrite scavenger, or superoxide dismutase partially prevented neuronal cell death induced by NO, whereas the presence of a poly(ADP-ribose) synthetase inhibitor, caspase inhibitors, or a guanylate cyclase inhibitor had no significant effect on cell death. These results demonstrated that a large release of NO from RMG cells is responsible for retinal neuronal cell death in vitro, suggesting a neurotoxic role for NO and peroxynitrite during retinal inflammatory or degenerative diseases, where RMG cells were activated.     

Docosahexaenoic Acid Is Required for the Survival of Rat Retinal Photoreceptors In Vitro

Abstract: The effect of docosahexaenoic acid (DHA) on neuronal survival was studied in cultured cells isolated from newborn rat retina. In vivo, the content of DHA in the retina increased nearly fourfold from days 2 to 12 after birth, whereas in retinal cells in culture it remained constant. Unlike amacrine cells, the photoreceptor cells in control cultures underwent a selective degeneration, starting at day 7, that led to their massive death by day 11. The addition of DHA at day 7 led to its active incorporation by the cultures, increasing from 6 to 21% of total fatty acids in cell lipids, and completely prevented photo-receptor cell death. When other fatty acids were tested, both neuronal fatty acid composition and photoreceptor death were the same as in control cultures. These results indicate that DHA is specifically required for the survival of retinal photoreceptors.     

The nitric oxide-cGKII system relays death and survival signals during embryonic retinal development via AKT-induced CREB1 activation

During early neurogenesis, retinal neuronal cells display a conserved differentiation program in vertebrates. Previous studies established that nitric oxide (NO) and cGMP accumulation regulate essential events in retinal physiology. Here we used pharmacological and genetic loss-of-function to investigate the effects of NO and its downstream signaling pathway in the survival of developing avian retinal neurons in vitro and in vivo. Six-day-old (E6) chick retinal cells displayed increased calcium influx and produced higher amounts of NO when compared with E8 cells. L-arginine (substrate for NO biosynthesis) and S-nitroso-N-acetyl-D,L-penicillamine (SNAP; a nitrosothiol NO donor) promoted extensive cell death in E6 retinas, whereas in E8 both substances decreased apoptosis. The effect of NO at both periods was mediated by soluble guanylyl cyclase (sGC) and cGMP-dependent kinase (cGK) activation. In addition, shRNA-mediated cGKII knockdown prevented NO-induced cell death (E6) and cell survival (E8). This, NO-induced cell death or cell survival was not correlated with an early inhibition of retinal cell proliferation. E6 cells also responded differentially from E8 neurons regarding cyclic AMP-responsive element-binding protein (CREB) activation in the retina in vivo. NO strongly decreased nuclear phospho-CREB staining in E6 but it robustly enhanced CREB phosphorylation in the nuclei of E8 neurons, an effect that was completely abrogated by cGKII shRNAs at both embryonic stages. The ability of NO in regulating CREB differentially during retinal development relied on the capacity of cGKII in decreasing (E6) or increasing (E8) nuclear AKT (V-Akt murine thymoma viral oncogene) activation. Accordingly, inhibiting AKT prevented both cGKII shRNA-mediated CREB upregulation in E6 and SNAP-induced CREB activation in E8. Furthermore, shRNA-mediated in vivo cGKII or in vitro CREB1 knockdown confirmed that NO/cGKII dualistically regulated the downstream CREB1 pathway and caspase activation in the chick retina to modulate neuronal viability. These data demonstrate that NO-mediated cGKII signaling may function to control the viability of neuronal cells during early retinal development via AKT/CREB1 activity.     

ATP induces the death of developing avian retinal neurons in culture via activation of P2X7 and glutamate receptors

Previous data suggest that nucleotides are important mitogens in the developing retina. Here, the effect of ATP on the death of cultured chick embryo retina cells was investigated. In cultures obtained from retinas of 7-day-old chick embryos (E7) that were cultivated for 2 days (E7C2), both ATP and BzATP induced a ～30 % decrease in cell viability that was time- and dose-dependent and that could be blocked by 0.2 mM oxidized ATP or 0.3 μM KN-62. An increase in cleaved caspase-3 levels and in the number of TUNEL-positive cells was observed when cultures were incubated with 3 mM ATP and immunolabeling for cleaved-caspase 3 was observed over neurons but not over glial cells. ATP-dependent cell death was developmentally regulated, the maximal levels being detected by E7C2-3. Nucleotides were able to increase neuronal ethidium bromide and sulforhodamine B uptake in mixed and purified neuronal cultures, an effect that was blocked by the antagonists Brilliant Blue G and oxidized ATP. In contrast, nucleotide-induced cell death was observed only in mixed cultures, but not in purified cultures of neurons or glia. ATP-induced neuronal death was blocked by the glutamatergic antagonists MK801 and DNQX and activation of P2X7 receptors by ATP decreased the uptake of [³H]-d-aspartate by cultured glial cells with a concomitant accumulation of it in the extracellular medium. These results suggest that ATP induces apoptosis of chick embryo retinal neurons in culture through activation of P2X7 and glutamate ionotropic receptors. Involvement of a P2X7 receptor-mediated inhibition of the glial uptake of glutamate is suggested.     

Calcium-permeable α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid Receptors Trigger Neuronal Nitric-oxide Synthase Activation to Promote Nerve Cell Death in an Src Kinase-dependent Fashion

In the retina information decoding is dependent on excitatory neurotransmission and is critically modulated by AMPA glutamate receptors. The Src-tyrosine kinase has been implicated in modulating neurotransmission in CNS. Thus, our main goal was to correlate AMPA-mediated excitatory neurotransmission with the modulation of Src activity in retinal neurons. Cultured retinal cells were used to access the effects of AMPA stimulation on nitric oxide (NO) production and Src phosphorylation. 4-Amino-5-methylamino-2′,7′-difluorofluorescein diacetate fluorescence mainly determined NO production, and immunocytochemistry and Western blotting evaluated Src activation. AMPA receptors activation rapidly up-regulated Src phosphorylation at tyrosine 416 (stimulatory site) and down-regulated phosphotyrosine 527 (inhibitory site) in retinal cells, an effect mainly mediated by calcium-permeable AMPA receptors. Interestingly, experiments confirmed that neuronal NOS was activated in response to calcium-permeable AMPA receptor stimulation. Moreover, data suggest NO pathway as a key regulatory signaling in AMPA-induced Src activation in neurons but not in glial cells. The NO donor SNAP (S-nitroso-N-acetyl-dl-penicillamine) and a soluble guanylyl cyclase agonist (YC-1) mimicked AMPA effect in Src Tyr-416 phosphorylation, reinforcing that Src activation is indeed modulated by the NO pathway. Gain and loss-of-function data demonstrated that ERK is a downstream target of AMPA-induced Src activation and NO signaling. Furthermore, AMPA stimulated NO production in organotypic retinal cultures and increased Src activity in the in vivo retina. Additionally, AMPA-induced apoptotic retinal cell death was regulated by both NOS and Src activity. Because Src activity is pivotal in several CNS regions, the data presented herein highlight that Src modulation is a critical step in excitatory retinal cell death.     

Increased sensitivity to NMDA is involved in alcohol-withdrawal induced cytotoxicity observed in primary cultures of cortical neurones chronically pre-treated with ethanol

Severe cellular damage and neuronal cell loss were previously observed in cultures of primary cortical neurones after chronic ethanol pre-treatment followed by ethanol-withdrawal. In this study, we investigated the circumstances and the possible cellular changes leading to alcohol-withdrawal induced neuronal cell death. When cultures were pre-treated with ethanol (25-200mM) once for 24 or 72h, the amount of the subsequent 24h alcohol-withdrawal induced cell death-estimated by measuring the release of lactate dehydrogenase (LDH)-was elevated only in cultures pre-treated with 200mM ethanol for 72h. On the contrary, as little as 50mM ethanol produced significant (P<0.01) increase in the withdrawal induced LDH-release in cultures pre-treated repeatedly with ethanol once daily for three consecutive days. When ethanol was re-added to the cultures during the withdrawal period, the LDH-release was dose-dependently reduced to the level of control. In ethanol pre-treated cultures N-methyl-D-aspartate (NMDA) (0.01-1mM) induced excitotoxicity as well as NMDA evoked elevation of cytosolic calcium ion concentration was increased. In contrast, the depolarising agent veratridine (0.01-1mM) produced similar extent of neuronal injury and elevation in cytosolic calcium ion concentration in control as in ethanol pre-treated cultures. According to these observations, repeated ethanol treatment appears to cause more robust adaptive changes in cultured neurones leading to more pronounced withdrawal induced cellular damage than chronic but single treatment does. In addition, the glutamatergic neurotransmission, especially the NMDA receptor system seems to be highly involved in the adaptive changes and in the cytotoxic effect of alcohol-withdrawal.     

Glucose metabolism in transdifferentiating and glucose-blocked cultures of chick embryo neuroretinal cells: an inverse relationship between glycogen and delta-crystallin accumulation

Chick embryo neuroretinal (NR) cells transdifferentiate extensively into lens when cultured for several weeks in low-glucose (FH) medium, but fail to do so when high levels of supplementary glucose (FHG) are present. We show here that most aspects of glucose metabolism are promoted in high-glucose cultures, including lactate dehydrogenase (LDH) and glucose-6-phosphate dehydrogenase (G-6-PDH) activities, 2-deoxyglucose uptake, pentose shunt activity and lactate production. Continuous supplementation of high-glucose cultures with low levels of ouabain (FHGO) significantly lowers 2-deoxyglucose uptake, from FHG levels down towards FH levels, especially during the early stages of NR culture. Much later, extensive transdifferentiation into lentoids (with concomitant delta-crystallin accumulation) occurs in these FHGO cultures, which thus resemble FH rather than FHG controls. Another parameter strongly affected by ambient glucose levels is the accumulation of glycogen. Both glycogen itself and glycogen synthetase activity increase steadily in FHG cultures, but decrease slightly under FH conditions. Glycogen accumulation in FHG cultures is largely confined to glial-like cells, particularly those underlying clusters of neurones. Other studies have shown that glial differentiation in vitro is promoted by histotypic interactions with retinal neurones. Thus high glucose may act in concert with neuronal influences to stimulate or stabilize the normal differentiation of retinal glial cells, whose characteristic features in vivo include glycogen synthesis and storage. Furthermore, we show that supplementation of high-glucose cultures with forskolin or dibutyryl cyclic AMP (both of which promote glycogenolysis) results in a slower rate of glycogen accumulation and in enhanced transdifferentiation into lens. In both respects, the forskolin- and dibutyryl cAMP-supplemented FHG cultures are intermediate between FH and FHG controls. Thus the enhancement of normal glial differentiation in NR cultures by high glucose may inhibit or preclude subsequent transdifferentiation into lens.     

Alteration of amino acid metabolism in neuronal aggregate cultures exposed to hypoglycaemic conditions

The neuronal effects of glucose deficiency on amino acid metabolism was studied on three-dimensional cultures of rat telencephalon neurones. Transient (6 h) exposure of differentiated cultures to low glucose (0.25 mm instead of 25 mm) caused irreversible damage, as judged by the marked decrease in the activities of two neurone-specific enzymes and lactate dehydrogenase, 1 week after the hypoglycemic insult. Quantification of amino acids and ammonia in the culture media supernatants indicated increased amino acid utilization and ammonia production during glucose-deficiency. Measurement of intracellular amino acids showed decreased levels of alanine, glutamine, glutamate and GABA, while aspartate was increased. Added lactate (11 mm) during glucose deficiency largely prevented the changes in amino acid metabolism and ammonia production, and attenuated irreversible damage. Higher media levels of glutamine (4 mm instead of 0.25 mm) during glucose deprivation prevented the decrease of intracellular glutamate and GABA, while it further increased intracellular aspartate, ammonia production and neuronal damage. Both lactate and glutamine were readily oxidized in these neuronal cultures. The present results suggest that in neurones, glucose deficiency enhances amino acid deamination at the expense of transamination reactions. This results in increased ammonia production and neuronal damage.     

Viability of dopaminergic neurones is influenced by serum and astroglial cells in vitro

Transplantation of embryonic nigral grafts into the striatum of Parkinson's disease patients is not optimal, mainly due to low survival of grafted neurones. Current strategies focus on enhancing neuronal survival by transplanting enriched neuronal cell populations. There is growing evidence for the importance of astroglia in neuronal survival.To characterise the effects of glial cells on dopaminergic neurones, 5-fluoro-2′-deoxyuridine was added to embryonic rat ventral mesencephalic cultures in the presence or absence of serum. The survival and morphology of glial fibrillary acidic protein immunopositive astroglia and tyrosine hydroxylase immunopositive dopaminergic neurones was examined. In serum-containing medium, astroglial cells predominated and 5-fluoro-2′-deoxyuridine had no significant effect on either astroglia or dopaminergic neurone survival. In serum-free medium, astroglial growth was attenuated and numbers were significantly lower in 5-fluoro-2′-deoxyuridine treated compared with untreated cultures. There was no significant difference in the numbers of dopaminergic neurones between 5-fluoro-2′-deoxyuridine treated and untreated cultures. However, by the eighth day in vitro, there were differences in the morphology of these neurones between treated and untreated cultures. This study shows that the use of 5-fluoro-2′-deoxyuridine and serum-free medium can produce a neurone-enriched culture. However, the dopaminergic neurone population present in these cultures appeared to be morphologically dissimilar to those found in control cultures as neurites were retracted and the cell somas of these cells appeared enlarged. These results provide information on the effects of astrocytes on dopaminergic neurones in ventral mesencephalic cultures and thus have implications for transplantation in Parkinson's disease.     

Different pathways for iNOS-mediated toxicity in vitro dependent on neuronal maturation and NMDA receptor expression

Co-localization of activated microglia and damaged neurones seen in brain injury suggests microglia-induced neurodegeneration. Activated microglia release two potential neurotoxins, excitatory amino acids and nitric oxide (NO), but their contribution to mechanisms of injury is poorly understood. Using co-cultures of rat microglia and embryonic cortical neurones, we show that inducible NO synthase (iNOS)-derived NO aloneis responsible for neuronal death from interferon gamma (IFNgamma) +lipopolysaccharide (LPS)-activated microglia. Neurones remain sensitive to NO irrespective of maturation state but, whereas blocking NMDA receptor activation with MK801 has no effect on NO-mediated toxicity to immature neurones, MK801 rescues 60-70% of neurones matured in culture for 12 days. Neuronal expression of NMDA receptors increases with maturation in culture, accounting for increased susceptibility to excitotoxins seen in more mature cultures. We show that MK801 delays the death of more mature neurones caused by the NO-donor DETA/NO indicating that NO elicits an excitotoxic mechanism, most likely through neuronal glutamate release. Thus, similar concentrations of nitric oxide cause neuronal death by two distinct mechanisms: NO acts directly upon immature neurones but indirectly, via NMDA receptors, on more mature neurones. Our results therefore extend existing evidence for NO-mediated toxicity and show a complex interaction between inflammatory and excitotoxic mechanisms of injury in mature neurones.     

Cultured retinal neuronal cells and Müller cells both show net production of lactate

Glucose has long been considered the substrate for energy metabolism in the retina. Recently, an alternative hypothesis (metabolic coupling) suggested that mitochondria in retinal neurons utilize preferentially the lactate produced specifically by Müller cells, the principal glial cell in the retina. These two views of retinal metabolism were examined using confluent cultures of photoreceptor cells, Müller cells, ganglion cells, and retinal pigment epithelial cells incubated in modified Dulbecco's minimal essential medium containing glucose or glucose and lactate. The photoreceptor and ganglion cells represented neural elements, and the Müller and pigment epithelial cells represented non-neural cells. The purpose of the present experiments was two-fold: (1) to determine whether lactate is a metabolic product or substrate in retinal cells, and (2) to examine the evidence that supports the two views of retinal energy metabolism. Measurements were made of lactic acid production, cellular ATP levels, and cellular morphology over 4 h. Results showed that all cell types incubated with 5 mM glucose produced lactate aerobically and anaerobically at linear rates, the anaerobic rate being 2-3-fold higher (Pasteur effect). Cells incubated with both 5 mM glucose and 10 mM lactate produced lactate aerobically and anaerobically at rates similar to those found when cells were incubated with glucose alone. Anaerobic ATP content in the cells was maintained at greater than 50% of the control, aerobic value, and cellular morphology was well preserved under all conditions. The results show that the cultured retinal cells produce lactate, even in the presence of a high starting ambient concentration of lactate. Thus, the net direction of the lactic dehydrogenase reaction is toward lactate formation rather than lactate utilization. It is concluded that retinal cells use glucose, and not glial derived lactate, as their major substrate.     

The formation of the area centralis of the retinal ganglion cell layer in the chick

In adult domestic chickens, the neurones in the retinal ganglion cell layer are very unevenly disposed such that there is a sixfold increase in neurone density from the retinal edge to the retinal centre. The formation of the high ganglion-cell-density area centralis was studied on chick retinal wholemounts from the 8th day of incubation (E8) to 4 weeks after hatching (4WAH). The density of viable neurones and the number and the distribution of pyknotic neurones in the ganglion cell layer were estimated across the whole retina. Between E8 and E10, the distribution of neurones in the ganglion cell layer was anisodensitic with 53,000 mm-2 in the centre compared to 34,000 mm-2 in the periphery of the retina. Thereafter, a progressively steeper gradient of neurone density developed, which decreased from 24,000 mm-2 in the retinal centre to 6000 mm-2 at the retinal periphery by 4WAH. Neuronal pyknosis in the ganglion cell layer was observed between E9 and E17. From E11 onwards, consistently more pyknotic neurones were found in the peripheral than in the central retina. It was estimated that over the period of cell death approximately twice as many neurones died per unit area in the retinal periphery than in the centre. Retinal area measurements and estimation of neurone densities in the ganglion cell layer after the period of neurone generation and neurone death indicated differential retinal expansion, with more expansion in the peripheral than in the central retina. These observations allow us to conclude that the formation of the area centralis of the chick retina involves (1) slightly higher cell generation in the retinal centre, (2) higher rate of cell loss in the retinal periphery and (3) differential retinal expansion.     

Small molecular weight G-protein, H-Ras, and retinal endothelial cell apoptosis in diabetes

We have demonstrated that the expressions of small molecular weight G-protein, H-Ras, and its effector protein, Raf-1, are increased in the retina in diabetes, and the specific inhibitors of Ras function inhibit glucose-induced apoptosis of retinal capillary cells. This study is to examine the contributory roles for H-Ras in glucose-induced apoptosis of retinal endothelial cells by genetic manipulation of functionally active H-Ras levels. Bovine retinal endothelial cells were transfected with the plasmids of either wild type (WT), constitutively active (V12) or dominant-negative (N17) H-Ras. Glucose-induced increase in apoptosis, nitric oxide (NO) levels and activation of NF-κB and caspase-3 were determined in these genetically manipulated cells. Exposure of bovine retinal endothelial cells to 20 mM glucose significantly increased H-Ras activation as determined by Raf-1 binding assay. Overexpression of V12 in the endothelial cells further increased their glucose-induced apoptosis by 40%, NO levels by about 50%, and activated NF-κB and caspase-3 by about 30–40% compared to the untransfected cells incubated in 20 mM glucose. In contrast, overexpression of the inactive mutant, N17, inhibited glucose-mediated increases in apoptotic cell death, NO levels and NF-κB and caspase-3 activation; the values were significantly different (p < 0.02) compared to those obtained from the untransfected cells incubated under similar conditions. Our findings demonstrate that H-Ras activation is important in the activation of the specific signaling events leading to the accelerated retinal capillary cell apoptosis in hyperglycemic conditions, suggesting the possible use of H-Ras inhibitors to inhibit the pathogenesis of diabetic retinopathy.     

Cilostazol, a cyclic AMP phosphodiesterase inhibitor, stimulates nitric oxide production and sodium potassium adenosine triphosphatase activity in SH-SY5Y human neuroblastoma cells.

Deficiencies in cellular cyclic AMP (cAMP) and nitric oxide (NO) production are thought to be involved in the pathogenesis of diabetic neuropathy. We used a human neuroblastoma cell line, SH-SY5Y, to investigate the effect of cilostazol, a specific cAMP phosphodiesterase inhibitor, on NO production and Na+, K+-ATPase activity. SH-SY5Y cells were cultured under 5 or 50 mM glucose for 5-6 days, the cells were then exposed to cilostazol or other chemicals and nitrite, cAMP and Na+, K+-ATPase activity were measured. In cells grown in 50 mM glucose, cilostazol was observed to increase significantly both NO production and cellular cAMP accumulation in a time- and dose-dependent manner. Cilostazol also significantly recovered reduced levels of protein kinase A activity (PKA) in 50 mM glucose. Furthermore, a PKA inhibitor, H-89 significantly suppressed the increase in NO production stimulated by cilostazol, suggesting that cilostazol stimulates NO production by activating PKA. Cilostazol did not affect either sorbitol or myo-inositol concentrations. Dexamethasone, which is known to induce inducible NO synthase, had no effect on NO production stimulated by cilostazol, suggesting that cilostazol stimulates NO production catalyzed by neuronal constitutive NO synthase (ncNOS) in SH-SY5Y cells. L-arginine, which is an NO agonist enhanced Na+, K+-ATPase activity in cells grown in 50 mM glucose, NG-nitro-L-arginine methyl ester (L-NAME), which is an NOS inhibitor inhibited basal Na+, K+-ATPase activity in 5 mM glucose and suppressed the increased enzyme activity induced by cilostazol in 50 mM glucose. The above results confirmed our previous observation that NO regulates Na+, K+-ATPase activity in SH-SY5Y cells and suggest that cilostazol increases Na+, K+-ATPase activity, at least in part, by stimulating NO production. The present results also suggest that cilostazol has a beneficial effect on diabetic neuropathy by improving Na+, K+-ATPase activity via directly increasing cAMP and NO production in nerves.     

Sulfisoxazole, an endothelin receptor antagonist, protects retinal neurones from insults of ischemia/reperfusion or lipopolysaccharide

Endothelins exert pathological effects in the eye and much interest centres on their role in causing retinal neuronal death in ischemic diseases like glaucoma. In the present study the influence of the non-selective endothelin antagonist, sulfisoxazole on raised intraocular pressure-induced ischemia to the rat retina was investigated. Moreover, in vitro studies on primary rat retinal cultures were undertaken to see whether sulfisoxazole is able to blunt the toxic effect of lipopolysaccharide (LPS) to retinal neurones.

In order to determine whether sulfisoxazole provides protection to the retina the a- and b-wave amplitudes of the electroretinogram (ERG), the localisation of retinal choline acetyltransferase (ChAT), nitric oxide synthase (nNOS) and Thy-1 and the retinal mRNA levels of Thy-1 and FGF-2 were deduced in retinas subjected to ischemia in the absence or presence of sulfisoxazole. The results showed that the ischemia-induced changes to the a- and b-wave amplitudes of the ERG and changes associated with the localisation of ChAT, nNOS and Thy-1 to be significantly blunted by sulfisoxazole. However, while the ischemia-induced changes to Thy-1 and FGF-2 mRNAs were reduced by sulfisoxazole, the reduction was non-significant.

The in vitro studies provided support for the protective effect of sulfisoxazole. Here, it was clearly shown that sulfisoxazole attenuated the elevation of nitric oxide (deduced by measuring nitrite) and the reduction in numbers of GABA-containing neurones caused by LPS.

The present study provides evidence for the first time that endothelin antagonist can protect the retina from ischemic-like insults as occurs in glaucoma.     

Heme Oxygenase-1 Protects Retinal Endothelial Cells against High Glucose- and Oxidative/Nitrosative Stress-Induced Toxicity

Diabetic retinopathy is a leading cause of visual loss and blindness, characterized by microvascular dysfunction. Hyperglycemia is considered the major pathogenic factor for the development of diabetic retinopathy and is associated with increased oxidative/nitrosative stress in the retina. Since heme oxygenase-1 (HO-1) is an enzyme with antioxidant and protective properties, we investigated the potential protective role of HO-1 in retinal endothelial cells exposed to high glucose and oxidative/nitrosative stress conditions. Retinal endothelial cells were exposed to elevated glucose, nitric oxide (NO) and hydrogen peroxide (H(2)O(2)). Cell viability and apoptosis were assessed by MTT assay, Hoechst staining, TUNEL assay and Annexin V labeling. The production of reactive oxygen species (ROS) was detected by the oxidation of 2',7'-dichlorodihydrofluorescein diacetate. The content of HO-1 was assessed by immunobloting and immunofluorescence. HO activity was determined by bilirubin production. Long-term exposure (7 days) of retinal endothelial cells to elevated glucose decreased cell viability and had no effect on HO-1 content. However, a short-time exposure (24 h) to elevated glucose did not alter cell viability, but increased both the levels of intracellular ROS and HO-1 content. Moreover, the inhibition of HO with SnPPIX unmasked the toxic effect of high glucose and revealed the protection conferred by HO-1. Oxidative/nitrosative stress conditions increased cell death and HO-1 protein levels. These effects of elevated glucose and HO inhibition on cell death were confirmed in primary endothelial cells (HUVECs). When cells were exposed to oxidative/nitrosative stress conditions there was also an increase in retinal endothelial cell death and HO-1 content. The inhibition of HO enhanced ROS production and the toxic effect induced by exposure to H(2)O(2) and NOC-18 (NO donor). Overexpression of HO-1 prevented the toxic effect induced by H(2)O(2) and NOC-18. In conclusion, HO-1 exerts a protective effect in retinal endothelial cells exposed to hyperglycemic and oxidative/nitrosative stress conditions.