Domain-dependent modulation of PDGFRbeta by ganglioside GM1

The regulation of receptor tyrosine kinases (RTKs) is important in several cellular events, including proliferation, differentiation, and apoptosis. Gangliosides are sialic acid-containing glycosphingolipids that can regulate RTK activity. The addition of ganglioside GM1 to the medium of Swiss 3T3 fibroblasts inhibits both platelet-derived growth factor (PDGF)-mediated tyrosine phosphorylation of PDGF receptor beta (PDGFRbeta) and receptor-mediated endocytosis. However, GM1 did not affect PDGF-mediated receptor phosphorylation, neuritogenesis, or endocytosis in PC12 cells stably transfected with the gene for PDGFRbeta. The ability of GM1 to modulate PDGFRbeta in 3T3 cells but not in transfected PC12 cells indicates a cell context-dependent response. We hypothesized that this inhibition of PDGFRbeta by GM1 must map to one or more domains of the receptor. Thus, a chimeric receptor was created that possessed the extracellular and transmembrane domains of the nerve growth factor (NGF) receptor TrkA and the cytoplasmic domain of PDGFRbeta (TTbeta). In 3T3 cells transfected with the TTbeta construct, GM1 did not inhibit NGF-induced tyrosine phosphorylation of the chimeric receptor or of Erk1/2 in this cell line. GM1 still inhibited PDGF-mediated tyrosine phosphorylation of endogenous PDGFRbeta and of Erk1/2 in Swiss TTbeta cells. Thus, the cytoplasmic domain of PDGFRbeta is not required for GM1-dependent inhibition of PDGFRbeta in 3T3 cells. This suggests that the inhibition of PDGFRbeta by GM1 in Swiss 3T3 fibroblasts maps to either the extracellular and/or transmembrane domain of PDGFRbeta.     

Oxidative metabolism in cultured astroglial cells from rat brain

Growth, morphology, glutamine synthetase activity, cytochrome C oxidase activity and respiratory activity of rat brain cultures enriched in astrocytes were studied during four weeks in culture. Two different polarographic methods were used for measurement of respiratory activity: one newly developed perfusion method leaving the cellular monolayer morphologically intact and still attached to the culture dish, and one traditional stirring method involving the removal of cells from the culture vessel. Regardless of the method used, a stable respiratory activity was registrated throughout the four weeks of culturing. Also the cytochrome C oxidase activity remained unchanged. In perfusion all absolute values for respiration were found to be higher than those obtained with the stirring method. The use of the stirring technique resulted in a doubling of oxygen consumption upon succinate addition. No such effect was seen in perfusion. It can thus be concluded that the removal of cultured astrocytic cells from their substratum alters their respiratory activity and their response to added substrates.     

Differentiation of oligodendrocytes cultured from developing rat brain is enhanced by exogenous GM3 ganglioside

Cultures consisting primarily of O-2A progenitor cells and immature oligodendrocytes with a few microglia and astrocytes were obtained by shaking primary cultures from neonatal rat brain after 12--14 days in vitro. Addition of 50 μg/ml exogenous Neu-NAcα2-3Galβ1-1′ ceramide (GM3 ganglioside) to the cultures resulted in an increase in the number and thickness of cell processes that stained intensely for sulfatide and galactocerebroside (galC) in comparison to control cultures without added GM3. The treated cultures also contained fewer astrocytes than control cultures as revealed by immunostaining for glial fibrillary acidic protein (GFAP). Cells that immunostained for both GFAP and sulfatide/galC were very rare in control cultures but were frequently seen in the GM3-treated cultures, suggesting that these may represent cells changing their direction of differentiation away from type II astrocytes toward oligodendrocytes under the influence of GM3. These effects on the developing rat oligodendrocytes were specific for GM3 ganglioside and were not produced by adding GM1, GM2, GD3, or GD1a to the cultures. Lactosyl ceramide and neuraminyl lactose were also ineffective. When control cultures were initially plated on polylysine and incubated with [¹⁴C]galactose, GD3 was the principal labeled ganglioside. However, as the control cells differentiated over time in culture without the addition of exogenous GM3 and produced increasing amounts of myelin-related components, the incorporation of [¹⁴C]galactose into endogenous GM3 increased to become the predominant labeled ganglioside by 6 days after plating. Metabolic labeling of the GM3-treated oligodendrocytes with [¹⁴C]galactose revealed increased incorporation into galC and sulfatide in comparison to control cultures, but a decreased labeling of endogenous GM3. Similarly, incorporation of an amino acid precursor into the myelin-associated glycoprotein (MAG) was increased by GM3 treatment, but incorporation into myelin basic protein (MBP) was not affected. Although the overall effect of added GM3 was to decrease the phosphorylation of most proteins in the oligodendrocytes, including MBP, GM3 enhanced the phosphorylation of MAG. These findings indicate that GM3 ganglioside has an important role in the differentiation of cells of the O-2A lineage toward myelin production, since differentiation is associated with increased metabolic labeling of endogenous GM3 in control cultures and is enhanced by the addition of exogenous GM3. © 1994 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Ganglioside GM1 actions on cell-substratum adhesion and DNA synthesis by cultured astroglial cells

Cultures of rat astroglial (AG) cells treated with the ganglioside GM1 in serum-free medium respond with an increase in DNA labeling and cell proliferation. However, GM1 doses above 60 microM cause decreasing DNA labeling to levels even below that determined in the absence of added GM1. Quantitative determination of cell numbers in 24-hr cultures treated with various GM1 concentrations shows no loss of cells but a progressive shift in cell morphology from the usual flat to a rounded shape, suggesting a GM1-induced progressive reduction in cell-substratum adhesion. The rounded cells, which can be readily washed off the culture wells, do not carry out measurable DNA synthesis but do appear normal by several other biochemical measurements. All the GM1-treated rounded cells can regain their flat morphology and resume DNA synthesis and cell replication if fetal calf serum is added to the medium. We conclude that modulation of cell adhesion is a major component of astroglial cell responses to exogenous ganglioside treatment.     

Dipeptidyl peptidase-II activity in cultured astroglial cells from neonatal rat brain

Astrocytic glial cells in primary culture from neonatal rat brain possess prominent dipeptidyl peptidase-II activity. This enzyme has been previously isolated and purified from whole brain tissue. The glial enzyme characteristically hydrolyzed glycyl-proline-p-nitroanilide (GPN) substrate to release glycyl-proline dipeptide plus p-nitroaniline products. At the enzyme's optimal pH of 5.4, the activities of other tested amino exopeptidases were virtually zero. At pH greater than 8, activity was less than 2% of the activity at pH 5.4, which suggested a paucity of the related enzyme, dipeptidyl peptidase-IV. No competitive inhibition was observed for glycine, proline nor their permuted dipeptides. Glial dipeptidyl peptidase-II activity was strongly inhibited by Hg2+, while other redox sulfhydryl agents were ineffective. Tested cations did not affect activity, except K+ which was mildly inhibitory. Chelating agents were not inhibitory. Of the peptidase inhibitors tested, only phenylmethylsulfonyl fluoride and puromycin were partially inhibitory. We suggest that dipeptidyl peptidase-II may play a role in glial processing of brain peptides which possess an N-terminal penultimate proline residue.     

Cholinergic deficits in aged rat spinal cord: restoration by GM1 ganglioside

Cholinergic neurons of spinal cord are central for the processing of motor, autonomic, and sensory modalities. Aging is associated with a variety of motor and autonomic symptoms that might be attributed, in part, to impaired spinal cord function. We found that cholinergic neurochemistry is diminished in the spinal cord of 22–24-month-old rats compared with 3-month-old rats. Choline acetyltransferase, high-affinity choline transport and hemicholinium-3 binding to the choline carrier were reduced in the aged spinal cord. The activity of the choline transporter and the hemicholinium-3 binding were decreased in all spinal segments, cervical, thoracic, lumbar and sacral. Hemicholinium-3 binding was reduced in ventral and dorsal horns along all spinal segments. The activity of choline acetyltransferase was decreased only in cervical and lumbar cord. Treatment of aged animals with GM1 induced the recovery of the presynaptic cholinergic markers in the aged spinal cord.     

Leukotriene production by cultured astroglial cells

Astrocytes metabolize arachidonic acid via the cyclooxygenase pathway to prostanoids. We examined whether primary culture astrocytes from neonatal rat brain can be induced to generate and release the lipoxygenase derivative leukotriene C4 (LTC4). While there was only minute constitutive production of immunoreactive LTC4 this metabolite was liberated by astroglial cells in response to calcium ionophore A23187. The phorbol diester 12-O-tetradecanoylphorbol 13-acetate (TPA) failed to precipitate leukotriene release. However, when threshold doses of A23187 were added to astrocyte cultures challenged with TPA, LTC4 was recovered from their supernatants. It is suggested that leukotriene generation by astrocytes bears relevance to immunoinflammatory responses in the central nervous system and may be involved in brain edema formation.     

GM1 ganglioside reduces glutamate toxicity to cortical cells

As an in vitro model of CNS excitatory amino acid (EAA) injury, rat cortical neuronal cultures were challenged with glutamate (0.5 or 10 mM) and the levels of released lactate dehydrogenase (LDH) were monitored at 1 h, 1, 2, and 7 d. LDH release is correlated with levels of plasma membrane damage. GM1 has been shown to be continuously distributed on the outer surface of CNS cellular membranes. By staining for the distribution of endogenous GM1 ganglioside using cholera toxin/antitoxin immunohistochemistry, we were able to assess morphologically cellular plasma membrane integrity after damage. We used these two measures (LDH and GM1 localization) to study the neuroprotective effects of exogenous GM1 ganglioside to further elucidate its mechanism. Cortical cultures derived from 15-d rat fetuses were subjected to the glutamate challenge for 30 min. Parallel cultures were either pre- or posttreated with 80 μM of GM1. Exposure to 10 mM glutamate caused a highly significant increase in LDH release at 1–48 h. Pretreatment with GM1 reduced the release, whereas post-treatment reduced the LDH release even more. Plasma membrane changes observed by the GM1 immunohistochemistry reflected the LDH release data. All cultures treated with GM1 evidenced substantial structural integrity (continuous staining of GM1 along perykarya and processes) as compared to untreated cultures. These data support our hypothesis that GM1 treatment (pre- and post-) reduces plasma membrane damage.     

GM1 ganglioside reduces edema and monoaminergic neuronal changes following experimental focal ischemia in rat brain

Seventy-two hours following a middle cerebral artery occlusion, the associated increase in water content on the ischemic side was significantly reduced by the exogenous administration of monosialoganglioside GM1 (30 mg/kg, i.p.). The levels of dopamine and serotonin on the ischemic side were approximately 50% and 80% of those on the contralateral non-ischemic side, respectively. Treatment with GM1 (5 times during the first 48 h after occlusion) produced a significant reduction in the levels of dopamine and serotonin loss. The present findings are compatible with the observed protective action of the exogenously administered GM1 following ischemic brain injury.     

Traumatic injury of spinal cord cells in vitro reduced by GM1 ganglioside

GM1 ganglioside (monosialoganglioside) is a significant endogenous component of central nervous system (CNS) cellular membranes, thereby contributing to the membranes' integrity and function. Exogenous gangliosides have been shown to be incorporated into plasma membranes and can exert neuroprotective effects on damaged neuronal tissue(s). An in vitro method of physical injury (trauma) previously described which used cultures derived from fetal mouse spinal cord [38] was adapted for these studies in order for us to assess GMl's neuroprotective efficacy. Injury was induced by uniformly crosshatching the spinal cell cultures with a 1 mm plastic pipette tip. The extent of injury and the effects of GM1 ganglioside posttreatment (80 μM) was assessed after 48 h by measuring lactate dehydrogenase (LDH) released and by observing changes in the plasma membrane surface distribution of endogenous GM1 using cholera toxin/antitoxin/fluorescent antibody immunohistochemistry. A gradient of injury, from the zone of maximum injury to partially traumatized or non-injured areas, was seen using immunohistochemistry. The primary injury zone in this gradient was characterized by areas of swollen or dead cells and abnormal or degenerating cell processes. At further distances, cells were observed to be nearly normal, with intact fibers. This gradient of injury may reflect proximate (at the locus of trauma) and distant effects of the release of neurotoxic levels of endogenous glutamate (Glu) and other excitatory amino acids. Ganglioside GM1 treatment resulted in a significantly reduced (>75%) release of LDH as well as enhanced cell and process integrity indicative of reduced tissue injury. These initial results indicate that GMl's previously documented neuroprotective effects using neuronal culture systems can be generalized to injured spinal cells in vitro wherein there is evidence for preservation (rescue) of cellular plasma membranes after injury as reflected by reduced cell loss, swelling, and process degeneration, as well as decreased LDH release.     

Effect of GM1 ganglioside on neonatally neurotoxin induced degeneration of serotonin neurons in the rat brain

The effect of exogenous GM1 ganglioside on the 5,7-dihydroxytryptamine (5,7-HT; a selective serotonin neurotoxin) induced alteration of the postnatal development of central 5-hydroxytryptamine (5-HT; serotonin) neurons has been investigated using neuro-chemical and immunocytochemical techniques. Neonatal 5,7-HT (50 mg/kg s.c.) treatment is known to lead to a marked and a permanent degeneration of distant 5-HT nerve terminal projections (e.g. in cerebral cortex, hippocampus and spinal cord), while projections close to the 5-HT perikarya in the mesencephalon and pons-medulla increase their nerve density. These regional alterations are reflected by decreases and increases, respectively, of endogenous 5-HT, [3H]5-HT uptake in vitro and number of 5-HT nerve terminals demonstrated by immunocytochemistry. Treatment of newborn rats with GM1 (4 X 30 mg/kg s.c.; 24 h interval) had no significant effect on the postnatal development of 5-HT neurons. GM1 administration had furthermore no effect on the 5,7-HT induced alteration of the regional 5-HT levels and [3H]5-HT uptake in the cerebral cortex acutely, indicating that GM1 did not significantly interfere with the primary neurodegenerative actions of 5,7-HT. At the age of 1 month a clear counteracting effect of GM1 was observed, in particular of the 5,7-HT induced 5-HT denervations. The 5-HT levels in the frontal and occipital cortex were reduced to 25 and 20% of control after 5,7-HT alone, while these values were 70 and 40%, respectively, after 5,7-HT and GM1 treatment. A similar antagonizing effect of GM1 was found in the frontal cortex when measuring [3H]5-HT uptake. GM1 treatment also caused a minor reduction of the 5,7-HT induced increase of the 5-HT levels in striatum and mesencephalon. Quantitation of 5-HT nerve terminal density in sections processed for 5-HT immunocytochemistry using an automatic image analysis system showed markedly more nerve terminals in the frontal and occipital cortex after 5,7-HT + GM1 compared to 5,7-HT treatment alone. Minor counteracting effects of GM1 were noted in the hippocampus and spinal cord (thoracic-lumbar) as evaluated by chemical 5-HT assay, although substantial counteracting effects were observed locally in these areas by quantitative immunocytochemistry.(ABSTRACT TRUNCATED AT 400 WORDS)     

TNF alpha increases activity of gamma-glutamyl transpeptidase in cultured rat astroglial cells

To investigate the presence of gamma-glutamyl transpeptidase (gammaGT) in brain cells, cultures enriched for astroglial cells, neurons, oligodendroglial cells, and microglial cells were studied. Astroglial cultures contained a specific gammaGT activity of 2.3 +/- 0.9 nmol/min/mg protein. A similar specific gammaGT activity was measured for oligodendroglial cultures, whereas microglial cells and neurons contained less than 30% of the specific gammaGT activity of astroglial cultures. The activity of gammaGT in astroglial cultures was elevated strongly by the presence of tumor necrosis factor-alpha (TNFalpha) in a time- and concentration-dependent manner. Maximal activity of gammaGT was observed after incubation of astroglial cultures for 3 days with 30 ng/mL TNFalpha. Under these conditions the specific gammaGT activity was increased by threefold compared to controls. Presence of the gammaGT-inhibitor acivicin completely inhibited gammaGT activity both in TNFalpha-treated and in control cells. In addition, the increase in astroglial gammaGT activity after application of TNFalpha was prevented completely by the presence of the protein synthesis inhibitor cycloheximide. gammaGT is involved in extracellular processing of glutathione (GSH) that is exported by astroglial cells. After TNFalpha-treatment the concentration of GSH in the medium of astroglial cells was reduced significantly compared to control cells. In conclusion, the data presented demonstrate that TNFalpha stimulates gammaGT synthesis in astroglial cells and thereby improves the capacity to process GSH exported by these cells.     

GM1 ganglioside effects on astroglial response in the rat nucleus basalis magnocellularis and its cortical projection areas after electrolytic or ibotenic lesions.

We examined the effects of chronic GM1 ganglioside injections on the astroglial response to bilateral electrolytic or ibotenic acid lesions in the nucleus basalis magnocellularis (NBM) within the NBM and in three cortical projection areas of NBM neurons. Glial fibrillary acidic protein (GFAP) immunohistochemistry was used to visualize the reactive astrocytes. Twenty-six days after injury, extensive astrogliosis was observed within the NBM after both types of lesions. An increased number of GFAP-positive cells were found in the cortex of saline-treated rats following electrolytic but not ibotenic lesions. We suggest that the loss of fibers of passage within the lesion area may account for the difference in cortical gliosis following the two types of damage. Although 17 days of GM1 injections did not affect astrocyte morphology within the NBM, ganglioside treatment reduced the number of GFAP-positive cells after electrolytic but not after ibotenic lesions. Within the cortex, a decrease in GFAP immunoreactivity, size, and number of astrocytes was only observed after electrolytic lesion. These data indicate that a decrease in the astroglial response to injury is the result of an interaction between the type of injury (electrolytic lesion) and chronic GM1 treatment.     

Morphological and biochemical maturation of rat astroglial cells grown in a chemically defined medium: Influence of an astroglial growth factor

Astroglial cells from cerebral hemispheres of newborn rats were cultured for 5 days in Waymouth's MD 705/1 medium containing 10% fetal calf serum. Thereafter, cells were grown in a chemically defined medium consisting of basal Waymouth's medium supplemented with insulin (5 μg/ml) and fatty acid free bovine serum albumin (0.5 mg/ml). The cells underwent morphological and biochemical development over a period of 28 days. The changes in the amount of glial fibrillary acidic protein indicated a development of gliofilaments. The level of S100 protein increased during the entire culture period, while glutamine synthetase activity remained low and relatively constant. The addition of an astroglial growth factor, partially purified from bovine brain soluble extract, stimulated the morphological maturation of the astroglial cells. The cells extended cytoplasmic processes and resembled mature astrocytes. At the ultrastructural level an increase in free ribosomes was observed and the intermediate filaments became organized into large bundles. The amount of glial fibrillary acidic protein was not significantly increased, but the level of S100 protein and the glutamine synthetase activity were greatly enhanced. Our results indicate that astroglial cells undergo limited maturation in the chemically defined medium and that this process is positively affected by the astroglial growth factor.     

Transplant and ganglioside GM1 mediated neuronal recovery in rats with brain lesions

Transplants of fetal brain tissue or injections of ganglioside GM1, given to rats with unilateral, left medial frontal cortex lesions, altered the concentrations of neuronal and glial marker proteins in cortex both adjacent and contralateral to the lesion. The markers were: the neural cell adhesion molecule (NCAM) and D3-protein, both present in neuronal and synaptic membranes; synaptophysin, present in synaptic vesicles; glial fibrillary acidic protein (GFAP) enriched in reactive astrocytes, and the astrocytic glutamine synthetase. After 21 days the concentrations of NCAM, D3 and synaptophysin in brain tissue adjacent to the lesions were decreased by 39, 32 and 42%, respectively, indicating neuronal damage. In the injured rats the GFAP concentration was increased 77%, indicating activation of astrocytes. However, astroglial proliferation was not altered as indicated by the nearly unchanged glutamine synthetase concentrations. The levels of the neuronal markers NCAM, D3 and synaptophysin showed significantly less decline in injured rats treated 7 days after the lesions with transplants or with daily injections of 30 mg/kg GM1. The decrease respectively constituted 23 (NCAM), 31 (D3) and 41% (synaptophysin) in rats with transplants and 23 (NCAM), 16 (D3) and 28% (synaptophysin) in rats treated with GM1. In another group of rats the efficacy of transplants was studied 34 days after lesions. NCAM and D3 in tissue adjacent to the lesions were decreased by 50 and 29%, respectively. In rats which received transplants the decrease was only 27 and 16%, respectively. Moreover, as measured by GFAP concentration, activation of astrocytes was less in rats with transplants (93% increased) compared with rats with lesions only (163%). In contralateral frontal cortex, the effects of lesions were similar but less pronounced. In this brain area also the treatments significantly counteracted the loss of neuronal and glial markers. Previous studies have demonstrated that synaptic remodelling is reflected by the ratios of NCAM to marker proteins for mature synapses. Twenty-one days after the initial injury to the brain the ratios of NCAM to D3 and synaptophysin were significantly increased in frontal cortex ipsilateral to the injury in rats with transplants (26 and 33%, respectively). In contrast, compared with values for injured rats, the calculated ratios were not changed, whereas all neuronal marker proteins were significantly increased in rats treated with GM1. The results suggest that transplants mediate neuronal recovery by inducing dendritic sprouting followed by synaptic remodelling whereas gangliosides mediate recovery by counteracting neuronal degeneration.     

Application and modulation of a permanent hydrogen peroxide-induced oxidative stress to cultured astroglial cells.

Hydrogen peroxide is often applied to cultured brain cells to investigate functions of these cells under oxidative stress. However, this peroxide might be quickly detoxified by cultured neural cells. At least cultured astroglial cells dispose of exogenous H(2)O(2) with a half-time in the minute range [R. Dringen, B. Hamprecht, Involvement of glutathione peroxidase and catalase in the disposal of exogenous hydrogen peroxide by cultured astroglial cells, Brain. Res. 759 (1997) pp. 67-75]. Therefore, application of hydrogen peroxide to astroglial cells leads only to a period of transient oxidative stress which depends on the ability of the cells to detoxify the peroxide. In order to apply a permanent H(2)O(2)-induced oxidative stress to astroglial cells the generation of H(2)O(2) by the coupled reactions of xanthine oxidase (XO) and superoxide dismutase (SOD) was studied and this system was applied to cultured astroglial cells. In the presence of astroglial cells, an almost constant steady state concentration of H(2)O(2) in the range up to 100 microM was generated, which depended on the activity of the XO applied. These steady state levels of H(2)O(2) were: (i) elevated by application of additional XO, (ii) slowly reduced by application of the XO inhibitor allopurinol, and (iii) immediately reduced to zero by application of catalase or a combination of allopurinol plus catalase. In conclusion, the method presented allows the application of an almost constant exogenous H(2)O(2) stress to cultured cells.     

Trophic effect of cholera toxin B subunit in cultured cerebellar granule neurons: Modulation of intracellular calcium by GM1 ganglioside

Survival of cerebellar granule cells (CGC) in culture was significantly improved in the presence of cholera toxin B subunit (Ctx B), a ligand which binds to GM1 with specificity and high affinity. This trophic effect was linked to elevation of intracellular calcium ([Ca²⁺]_i), and was additive to that of high K⁺. Survival was optimized when Ctx B was present for several days during the early culture period. ⁴⁵Ca²⁺ and cell survival studies indicated the mechanism to involve enhanced influx of Ca²⁺ through L-type voltage-sensitive channels, since the trophic effect was blocked by antagonists specific for that channel type. Inhibitors of N-methyl-D-aspartate receptor/channels were without effect. During the early stage of culture Ctx B, together with 25 mM K⁺, caused [Ca²⁺]_i to rise to 0.2–0.7 μM in a higher proportion of cells than 25 mM K⁺ alone. A significant change in the nature of GM1 modulation of Ca²⁺ flux occurred after 7 days in culture, at which time Ctx B ceased to elevate and instead reduced [Ca²⁺]_i below the level attained with 25 mM K⁺. GM1 thus appears to serve as intrinsic inhibitor of one or more L-type Ca²⁺ channels during the first 7 days in vitro, and then as intrinsic activator of (possibly other) L-type channels after that period. This is the first demonstration of a modulatory role for GM1 ganglioside affecting Ca²⁺ homeostasis in cultured neurons of the CNS. © 1996 Wiley-Liss, Inc.     

Riluzole increases glutamate uptake by cultured C6 astroglial cells

Riluzole is a drug approved for the treatment of amyotrophic lateral sclerosis (ALS) and may be effective for the treatment of other neurodegenerative and neuropsychiatric disorders. Riluzole exerts diverse actions on the central nervous system, including altering glutamate release and uptake, and therefore act diminishing glutamate extracellular levels, but the underlying mechanism of these actions is still unknown. Here, we demonstrate that riluzole stimulated glutamate uptake and augmented the expression of the glutamate EAAC1 transporter in C6 astroglial cell cultures. The effect of riluzole on glutamate uptake was reduced to below controls when it was co-administered with inhibitors of protein kinase C (PKC; bisindolylmaleimide II), phosphatidylinositol 3-kinase (PI3K; wortmannin) and fibroblast growth factor receptor 1 (FGFR1; PD173074). Riluzole also decreased reactive oxygen species load with no effect on glutathione levels. This study investigates three independent intracellular pathways and the mechanism of action of riluzole on glutamate metabolism.     

Reduction of cerebral edema with GM1 ganglioside

Administration of exogenous gangliosides has been reported to accelerate neurite outgrowth in vitro, and to enhance peripheral nerve regeneration and central nervous system recovery subsequent to damage. After injury, facilitation of CNS recovery with GM1 ganglioside treatment has been postulated to be due to enhanced neuronal regeneration. Since maximal recovery is achieved when experimental animals are treated before injury with GM1 ganglioside, an alternative or parallel mechanism is that gangliosides are "protecting" the CNS by limiting the extent of damage (ie, cell loss, process degeneration, membrane disruption). This may be due to a reduction in the edema subsequent to injury. In this study, rats were treated for 2 days with 20 mg/kg/day of GM1 ganglioside. On the third day they were subjected to a unilateral lesion (mechanical) of one cerebral hemisphere and given another 20 mg/kg of GM1. On the fourth day brains were removed for analysis of edema resulting from the injury. In treated animals there was a significant reduction in edema as measured either in the entire injured hemisphere (23%) or in the area of injury (33%). No effect was seen outside the damaged area. Since exogenous gangliosides can spontaneously "insert" into membranes, it is postulated that the effect of the GM1 may be due to alterations of membrane processes (eg, lipid hydrolysis, phospholipase activation, levels and membrane action of arachidonic acid, ionic permeation) that are characteristic of edema.