Norepinephrine activates ß1 -adrenergic receptors at the inner nuclear membrane in astrocytes.

Norepinephrine exerts powerful influences on the metabolic, neuroprotective and immunoregulatory functions of astrocytes. Until recently, all effects of norepinephrine were believed to be mediated by receptors localized exclusively to the plasma membrane. However, recent studies in cardiomyocytes have identified adrenergic receptors localized to intracellular membranes, including Golgi and inner nuclear membranes, and have shown that norepinephrine can access these receptors via transporter-mediated uptake. We recently identified a high-capacity norepinephrine transporter, organic cation transporter 3 (OCT3), densely localized to outer nuclear membranes in astrocytes, suggesting that adrenergic signaling may also occur at the inner nuclear membrane in these cells. Here, we used immunofluorescence and western blot to show that ß1 -adrenergic receptors are localized to astrocyte inner nuclear membranes; that key adrenergic signaling partners are present in astrocyte nuclei; and that OCT3 and other catecholamine transporters are localized to astrocyte plasma and nuclear membranes. To test the functionality of nuclear membrane ß1 -adrenergic receptors, we monitored real-time protein kinase A (PKA) activity in astrocyte nuclei using a fluorescent biosensor. Treatment of astrocytes with norepinephrine induced rapid increases in PKA activity in the nuclear compartment. Pretreatment of astrocytes with inhibitors of catecholamine uptake blocked rapid norepinephrine-induced increases in nuclear PKA activity. These studies, the first to document functional adrenergic receptors at the nuclear membrane in any central nervous system cell, reveal a novel mechanism by which norepinephrine may directly influence nuclear processes. This mechanism may contribute to previously described neuroprotective, metabolic and immunoregulatory actions of norepinephrine.

Analysis of BMAA enantiomers in cycads, cyanobacteria, and mammals: in vivo formation and toxicity of D-BMAA.

Chronic dietary exposure to the cyanobacterial toxin ß-N-methylamino-L-alanine (BMAA) triggers neuropathology in non-human primates, providing support for the theory that BMAA causes a fatal neurodegenerative illness among the indigenous Chamorro people of Guam. However, since there are two stereoisomers of BMAA, it is important to know if both can occur in nature, and if so, what role they might play in disease causation. As a first step, we analysed both BMAA enantiomers in cyanobacteria, cycads, and in mammals orally dosed with L-BMAA, to determine if enantiomeric changes could occur in vivo. BMAA in cyanobacteria and cycads was found only as the L-enantiomer. However, while the L-enantiomer in mammals was little changed after digestion, we detected a small pool of D-BMAA in the liver (12.5%) of mice and in the blood plasma of vervets (3.6%). Chiral analysis of cerebrospinal fluid of vervets and hindbrain of mice showed that the free BMAA in the central nervous system was the D-enantiomer. In vitro toxicity investigations with D-BMAA showed toxicity, mediated through AMPA rather than NMDA receptors. These findings raise important considerations concerning the neurotoxicity of BMAA and its relationship to neurodegenerative disease.

Pituitary Adenylate cyclase-activating polypeptide orchestrates neuronal regulation of the astrocytic glutamate-releasing mechanism system xc (.).

Glutamate signaling is achieved by an elaborate network involving neurons and astrocytes. Hence, it is critical to better understand how neurons and astrocytes interact to coordinate the cellular regulation of glutamate signaling. In these studies, we used rat cortical cell cultures to examine whether neurons or releasable neuronal factors were capable of regulating system xc (-) (Sxc), a glutamate-releasing mechanism that is expressed primarily by astrocytes and has been shown to regulate synaptic transmission. We found that astrocytes cultured with neurons or exposed to neuronal-conditioned media displayed significantly higher levels of Sxc activity. Next, we demonstrated that the pituitary adenylate cyclase-activating polypeptide (PACAP) may be a neuronal factor capable of regulating astrocytes. In support, we found that PACAP expression was restricted to neurons, and that PACAP receptors were expressed in astrocytes. Interestingly, blockade of PACAP receptors in cultures comprised of astrocytes and neurons significantly decreased Sxc activity to the level observed in purified astrocytes, whereas application of PACAP to purified astrocytes increased Sxc activity to the level observed in cultures comprised of neurons and astrocytes. Collectively, these data reveal that neurons coordinate the actions of glutamate-related mechanisms expressed by astrocytes, such as Sxc, a process that likely involves PACAP. A critical gap in modeling excitatory signaling is how distinct components of the glutamate system expressed by neurons and astrocytes are coordinated. In these studies, we found that system xc (-) (Sxc), a glutamate release mechanism expressed by astrocytes, is regulated by releasable neuronal factors including PACAP. This represents a novel form of neuron-astrocyte communication, and highlights the possibility that pathological changes involving astrocytic Sxc may stem from altered neuronal activity.

Thinking outside the cleft to understand synaptic activity: contribution of the cystine-glutamate antiporter (System xc-) to normal and pathological glutamatergic signaling.

System x(c)(-) represents an intriguing target in attempts to understand the pathological states of the central nervous system. Also called a cystine-glutamate antiporter, system x(c)(-) typically functions by exchanging one molecule of extracellular cystine for one molecule of intracellular glutamate. Nonvesicular glutamate released during cystine-glutamate exchange activates extrasynaptic glutamate receptors in a manner that shapes synaptic activity and plasticity. These findings contribute to the intriguing possibility that extracellular glutamate is regulated by a complex network of release and reuptake mechanisms, many of which are unique to glutamate and rarely depicted in models of excitatory signaling. Because system x(c)(-) is often expressed on non-neuronal cells, the study of cystine-glutamate exchange may advance the emerging viewpoint that glia are active contributors to information processing in the brain. It is noteworthy that system x(c)(-) is at the interface between excitatory signaling and oxidative stress, because the uptake of cystine that results from cystine-glutamate exchange is critical in maintaining the levels of glutathione, a critical antioxidant. As a result of these dual functions, system x(c)(-) has been implicated in a wide array of central nervous system diseases ranging from addiction to neurodegenerative disorders to schizophrenia. In the current review, we briefly discuss the major cellular components that regulate glutamate homeostasis, including glutamate release by system x(c)(-). This is followed by an in-depth discussion of system x(c)(-) as it relates to glutamate release, cystine transport, and glutathione synthesis. Finally, the role of system x(c)(-) is surveyed across a number of psychiatric and neurodegenerative disorders.

Augmented cystine-glutamate exchange by pituitary adenylate cyclase-activating polypeptide signaling via the VPAC1 receptor.

In the central nervous system, cystine import in exchange for glutamate through system xc- is critical for the production of the antioxidant glutathione by astrocytes, as well as the maintenance of extracellular glutamate. Therefore, regulation of system xc- activity affects multiple aspects of cellular physiology and may contribute to disease states. Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuronally derived peptide that has already been demonstrated to modulate multiple aspects of glutamate signaling suggesting PACAP may also target activity of cystine-glutamate exchange via system xc-. In this study, 24-h treatment of primary cortical cultures containing neurons and glia with PACAP concentration-dependently increased system xc- function as measured by radiolabeled cystine uptake. Furthermore, the increase in cystine uptake was completely abolished by the system xc- inhibitor, (S)-4-carboxyphenylglycine (CPG), attributing increases in cystine uptake specifically to system xc- activity. Time course and quantitative PCR results indicate that PACAP signaling may increase cystine-glutamate exchange by increasing expression of xCT, the catalytic subunit of system xc-. Furthermore, the potentiation of system xc- activity by PACAP occurs via a PKA-dependent pathway that is not mediated by the PAC1R, but rather the shared vasoactive intestinal polypeptide receptor VPAC1R. Finally, assessment of neuronal, astrocytic, and microglial-enriched cultures demonstrated that only astrocyte-enriched cultures exhibit enhanced cystine uptake following both PACAP and VIP treatment. These data introduce a novel mechanism by which both PACAP and VIP regulate system xc- activity. Synapse 68:604-612, 2014. © 2014 Wiley Periodicals, Inc.

Drug-induced plasticity contributing to heightened relapse susceptibility: neurochemical changes and augmented reinstatement in high-intake rats.

A key in understanding the neurobiology of addiction and developing effective pharmacotherapies is revealing drug-induced plasticity that results in heightened relapse susceptibility. Previous studies have demonstrated that increased extracellular glutamate, but not dopamine, in the nucleus accumbens core (NAcc) is necessary for cocaine-induced reinstatement. In this report, we examined whether drug-induced adaptations that are necessary to generate cocaine-induced reinstatement also determine relapse vulnerability. To do this, rats were assigned to self-administer cocaine under conditions resulting in low (2 h/d; 0.5 mg/kg/infusion, i.v.) or high (6 h/d; 1.0 mg/kg/infusion, i.v.) levels of drug intake since these manipulations produce groups of rats exhibiting differences in the magnitude of cocaine-induced reinstatement. Approximately 19 d after the last session, cocaine-induced drug seeking and extracellular levels of glutamate and dopamine in the NAcc were measured. Contrary to our hypothesis, high-intake rats exhibited a more robust cocaine-induced increase in extracellular levels of dopamine but not glutamate. Further, increased reinstatement in high-intake rats was no longer observed when the D(1) receptor antagonist SCH-23390 was infused into the NAcc. The sensitized dopamine response to cocaine in high-intake rats may involve blunted cystine-glutamate exchange by system x(c(-)). Reduced (14)C-cystine uptake through system x(c(-)) was evident in NAcc tissue slices obtained from high-intake rats, and the augmented dopamine response in these rats was no longer observed when subjects received the cysteine prodrug N-acetyl cysteine. These data reveal a role for drug-induced NAcc dopamine in heightened relapse vulnerability observed in rats with a history of high levels of drug intake.

Neurotoxicity of isomers of the environmental toxin L-BMAA.

There is evidence that the environmental toxin ß-N-methylamino-L-alanine (L-BMAA) may be involved in neurodegenerative diseases. However, a number of controversies exist regarding L-BMAA, one of which is the possibility that when assaying for L-BMAA, its isomers are being detected instead. There are at least four isomers of BMAA that are known to occur: L-BMAA, ß-N-methylamino-D-alanine (D-BMAA), 2,4-diaminobutyric acid (DAB), and N-(2-aminoethyl)glycine (AEG). The fact that isomers of BMAA exist in nature also leads to the possibility that they are involved in toxicity. We set out to determine both the potency and the mechanism of toxicity of L-BMAA, D-BMAA, DAB, asnd AEG using primary cortical cultures. The results were surprising with the following order of potency of toxicity: AEG > DAB > D-BMAA > L-BMAA. These results suggest that AEG may be an overlooked neurotoxin. We found that AEG induced toxicity through mGluR5 receptors and induction of oxidative stress. While the potential role of L-BMAA in neurodegenerative diseases has been emphasized, other isomers of L-BMAA, particularly AEG, are actually more potent toxins, and could therefore potentially contribute to neurodegenerative diseases.

Mechanisms of beta-N-methylamino-L-alanine induced neurotoxicity.

Since the initial discovery that the amino acid beta-N-methylamino-L-alanine (BMAA) was a neurotoxin, a great deal has been learned about its mechanism of action. However, exactly how it causes death of motor neurons, and how its actions may interact with other neurotoxins or pathological conditions, is not well understood. The focus of study on the mechanism of BMAA toxicity has been on its action as a glutamate receptor agonist. There is evidence that BMAA has effects on all of the main types of glutamate receptors: NMDA, AMPA/kainate, and metabotropic receptors. However, recent results suggest that BMAA may also act through other mechanisms to induce neuronal death. One such action is on the cystine/glutamate antiporter (system xc(-)). Through its effect of system xc(-), BMAA can induce oxidative stress and increase extracellular glutamate. This action of BMAA provides an attractive mechanism for the multiple neurological deficits that BMAA has been implicated in inducing.

Effects of chelators on mercury, iron, and lead neurotoxicity in cortical culture.

Chelation therapy for the treatment of acute, high dose exposure to heavy metals is accepted medical practice. However, a much wider use of metal chelators is by alternative health practitioners for so called "chelation therapy". Given this widespread and largely unregulated use of metal chelators it is important to understand the actions of these compounds. We tested the effects of four commonly used metal chelators, calcium disodium ethylenediaminetetraacetate (CaNa2EDTA), D-penicillamine (DPA), 2,3 dimercaptopropane-1-sulfonate (DMPS), and dimercaptosuccinic acid (DMSA) for their effects on heavy metal neurotoxicity in primary cortical cultures. We studied the toxicity of three forms of mercury, inorganic mercury (HgCl2), methyl mercury (MeHg), and ethyl mercury (thimerosal), as well as lead (PbCl2) and iron (Fe-citrate). DPA had the worst profile of effects, providing no protection while potentiating HgCl2, thimerosal, and Fe-citrate toxicity. DMPS and DMSA both attenuated HgCl2 toxicity and potentiated thimerosal and Fe toxicity, while DMPS also potentiated PbCl2 toxicity. CaNa2EDTA attenuated HgCl2 toxicity, but caused a severe potentiation of Fe-citrate toxicity. The ability of these chelators to attenuate the toxicity of various metals is quite restricted, and potentiation of toxicity is a serious concern. Specifically, protection is provided only against inorganic mercury, while it is lacking against the common form of mercury found in food, MeHg, and the form found in vaccines, thimerosal. The potentiation of Fe-citrate toxicity is of concern because of iron's role in oxidative stress in the body. Potentiation of iron toxicity could have serious health consequences when using chelation therapy.

Effects of dental composite resin monomers on dental pulp cells.

Methacrylate monomers found in many dental materials cause toxicity to dental pulp cells but the mechanism of the toxicity is poorly understood. We used cultured human dental pulp cells to test the effects of three commonly used monomers; bisphenol-A-glycidyl methacrylate (Bis-GMA), urethane dimethacrylate (UDMA), and triethyleneglycol dimethacrylate (TEGDMA). The order of toxicity was Bis-GMA>UDMA>TEGDMA. The toxicity correlated inversely with cystine uptake, with TEGDMA stimulating uptake and BisGMA and UDMA inhibiting uptake. Bis-GMA and UDMA induced oxidative stress, while TEGDMA did not. Toxicity correlated poorly with glutathione levels, as all compounds decreased cellular glutathione. TEGDMA is less toxic than Bis-GMA and UDMA likely because it stimulates cystine uptake and does not induce oxidative stress, the enhanced uptake of cystine appears to compensate for TEGDMA's direct interaction with glutathione. Bis-GMA and UDMA both deplete glutathione and inhibit cystine uptake leading to oxidative stress and cell death.

System x(c)- activity and astrocytes are necessary for interleukin-1 beta-mediated hypoxic neuronal injury.

The purpose of this study was to elucidate the cellular/biochemical pathway(s) by which interleukin-1beta (IL-1beta) contributes to the pathogenesis of hypoxic-ischemic brain damage. In vivo, IL-1 receptor type I (IL-1RI)-deficient mice showed smaller infarcts and less neurological deficits than wild-type animals after a 90 min reversible middle cerebral artery occlusion. In vitro, IL-1beta mediated an enhancement of hypoxic neuronal injury in murine cortical cultures that was lacking in cultures derived from IL-1RI null mutant animals and was blocked by the IL-1 receptor antagonist or an IL-1RI blocking antibody. This IL-1beta-mediated potentiation of hypoxic neuronal injury was associated with an increase in both cellular cystine uptake ([cystine]i) and extracellular glutamate levels ([glutamate]e) and was prevented by either ionotropic glutamate receptor antagonism or removal of L-cystine, suggesting a role for the cystine/glutamate antiporter (System x(c)-). Indeed, dual System x(c)-/metabotropic glutamate receptor subunit 1 (mGluR1) antagonism but not selective mGluR1 antagonism prevented neuronal injury. Additionally, cultures derived from mGluR1-deficient mice exhibited the same potentiation in injury after treatment with IL-1beta as wild-type cultures, an effect prevented by System x(c)-/mGluR1 antagonism. Finally, assessment of System x(c)- function and kinetics in IL-1beta-treated cultures revealed an increase in velocity of cystine transport (Vmax), in the absence of a change in affinity (Km). Neither the enhancement in [cystine]i, [glutamate]e, or neuronal injury were observed in chimeric cultures consisting of IL-1RI(+/+) neurons plated on top of IL-1RI(-/-) astrocytes, highlighting the importance of astrocyte-mediated alterations in System x(c)- as a novel contributor to the development and progression of hypoxic neuronal injury.

Repeated N-acetylcysteine administration alters plasticity-dependent effects of cocaine.

Cocaine produces a persistent reduction in cystine-glutamate exchange via system x(c)- in the nucleus accumbens that may contribute to pathological glutamate signaling linked to addiction. System x(c)- influences glutamate neurotransmission by maintaining basal, extracellular glutamate in the nucleus accumbens, which, in turn, shapes synaptic activity by stimulating group II metabotropic glutamate autoreceptors. In the present study, we tested the hypothesis that a long-term reduction in system x(c)- activity is part of the plasticity produced by repeated cocaine that results in the establishment of compulsive drug seeking. To test this, the cysteine prodrug N-acetylcysteine was administered before daily cocaine to determine the impact of increased cystine-glutamate exchange on the development of plasticity-dependent cocaine seeking. Although N-acetylcysteine administered before cocaine did not alter the acute effects of cocaine on self-administration or locomotor activity, it prevented behaviors produced by repeated cocaine including escalation of drug intake, behavioral sensitization, and cocaine-primed reinstatement. Because sensitization or reinstatement was not evident even 2-3 weeks after the last injection of N-acetylcysteine, we examined whether N-acetylcysteine administered before daily cocaine also prevented the persistent reduction in system x(c)- activity produced by repeated cocaine. Interestingly, N-acetylcysteine pretreatment prevented cocaine-induced changes in [35S]cystine transport via system x(c)-, basal glutamate, and cocaine-evoked glutamate in the nucleus accumbens when assessed at least 3 weeks after the last N-acetylcysteine pretreatment. These findings indicate that N-acetylcysteine selectively alters plasticity-dependent behaviors and that normal system x(c)- activity prevents pathological changes in extracellular glutamate that may be necessary for compulsive drug seeking.

Transport of BMAA into Neurons and Astrocytes by System xc.

The study of the mechanism of ß-N-methylamino-L-alanine (BMAA) neurotoxicity originally focused on its effects at the N-methyl-D-aspartate (NMDA) receptor. In recent years, it has become clear that its mechanism of action is more complicated. First, there are certain cell types, such as motor neurons and cholinergic neurons, where the dominate mechanism of toxicity is through action at AMPA receptors. Second, even in cortical neurons where the primary mechanism of toxicity appears to be activation of NMDA receptors, there are other mechanisms involved. We found that along with NMDA receptors, activation of mGLuR5 receptors and effects on the cystine/glutamate antiporter (system xc-) were involved in the toxicity. The effects on system xc- are of particular interest. System xc- mediates the transport of cystine into the cell in exchange for releasing glutamate into the extracellular fluid. By releasing glutamate, system xc- can potentially cause excitotoxicity. However, through providing cystine to the cell, it regulates the levels of cellular glutathione (GSH), the main endogenous intracellular antioxidant, and in this way may protect cells against oxidative stress. We have previously published that BMAA inhibits cystine uptake leading to GSH depletion and had indirect evidence that BMAA is transported into the cells by system xc-. We now present direct evidence that BMAA is transported into both astrocytes and neurons through system xc-. The fact that BMAA is transported by system xc- also provides a mechanism for BMAA to enter brain cells potentially leading to misincorporation into proteins and protein misfolding.

Effects of growth factors on dental pulp cell sensitivity to amalgam toxicity.

OBJECTIVES: The objective was to determine the effects of growth factor treatment on dental pulp cell sensitivity to toxicity. METHODS: The toxicity of zinc-containing and zinc-free dental amalgam was tested on four types of cells; glia, neurons, embryonic stem cells, and dental pulp cells. The effects of six different growth factors were tested on dental pulp cell sensitivity to amalgam toxicity. RESULTS: Zinc-containing amalgam was highly toxic to all cell types tested. Zinc-free amalgam was less toxic, but it was most toxic to dental pulp cells. Exposure of dental pulp cells to the growth factors IGF-I or BMP-7 had no effect on their morphology or rate of cell division, and did not alter their sensitivity to zinc-free amalgam toxicity. Exposure to EGF, bFGF, or TGF-beta altered the morphology and decreased the rate of cell division, and the cells were no longer sensitive to zinc-free amalgam toxicity. Exposure to BMP-2 also altered the morphology and decreased the rate of cell division, but the cells remained sensitive to zinc-free amalgam toxicity. SIGNIFICANCE: The results indicate that untreated dental pulp cells are highly sensitive to amalgam toxicity, but that sensitivity can be decreased by exposure to certain growth factors. Therefore, for treatment of conditions in which pulp cells may come in contact with substances released from dental materials, such as pulp capping, the use of amalgam should be avoided unless the pulp cells are first treated with the proper growth factors.

Pulp Capping Materials Alter the Toxicity and Oxidative Stress Induced by Composite Resins in Dental Pulp Culture.

OBJECTIVE: Direct pulp capping involves covering exposed pulp to preserve its viability. Calcium hydroxide materials have traditionally been the most commonly used pulp capping compounds; however, they can be toxic, and their success rate in pulp capping is variable. Recently, the compound mineral trioxide aggregate (MTA) has gained wide use for pulp capping. One advantage of MTA is its low toxicity. However, the effects of MTA and calcium hydroxide compounds on the toxicities of other dental materials have not been tested. The aim of this study is to determine whether different pulp capping materials can alter the toxicity of composite restoration materials. METHODS: We used cultured human dental pulp cells to test the toxicities of the calcium hydroxide pulp capping material Dycal and MTA. We then tested the abilities of these compounds to alter the toxicity of the composite materials Durafill and Flow Line and to induce oxidative stress. RESULTS: As expected, Dycal demonstrated toxicity, while MTA did not. However, when cells were exposed to subtoxic amounts of Dycal or MTA, then exposed to Durafill or Flow Line, changes in the composite materials induced toxicity. Treatment with Dycal had no effect on the toxicity of Durafill, but significantly attenuated the toxicity of Flow Line; meanwhile, MTA significantly enhanced the toxicity of Durafill but had no effect on the toxicity of Flow Line. Early changes in oxidative stress were correlated with later changes in cell death. Statistical calculations were performed using one-way ANOVA followed by the Bonferroni t-test. P-values <0.05 were considered to indicate significant differences. CONCLUSION: The results suggest that when choosing a pulp capping material, one factor that should be considered is the impact of that compound on the toxicity of the composite material used for restoration.

Regulation of System xc(-) by Pharmacological Manipulation of Cellular Thiols.

The cystine/glutamate exchanger (system xc (-)) mediates the transport of cystine into the cell in exchange for glutamate. By releasing glutamate, system xc (-) can potentially cause excitotoxicity. However, through providing cystine to the cell, it regulates the levels of cellular glutathione (GSH), the main endogenous intracellular antioxidant, and may protect cells against oxidative stress. We tested two different compounds that deplete primary cortical cultures containing both neurons and astrocytes of intracellular GSH, L-buthionine-sulfoximine (L-BSO), and diethyl maleate (DEM). Both compounds caused significant concentration and time dependent decreases in intracellular GSH levels. However; DEM caused an increase in radiolabeled cystine uptake through system xc (-), while unexpectedly BSO caused a decrease in uptake. The compounds caused similar low levels of neurotoxicity, while only BSO caused an increase in oxidative stress. The mechanism of GSH depletion by these two compounds is different, DEM directly conjugates to GSH, while BSO inhibits γ-glutamylcysteine synthetase, a key enzyme in GSH synthesis. As would be expected from these mechanisms of action, DEM caused a decrease in intracellular cysteine, while BSO increased cysteine levels. The results suggest that negative feedback by intracellular cysteine is an important regulator of system xc (-) in this culture system.

A Cytotoxic, Co-operative Interaction Between Energy Deprivation and Glutamate Release From System xc- Mediates Aglycemic Neuronal Cell Death.

The astrocyte cystine/glutamate antiporter (system xc(-)) contributes substantially to the excitotoxic neuronal cell death facilitated by glucose deprivation. The purpose of this study was to determine the mechanism by which this occurred. Using pure astrocyte cultures, as well as, mixed cortical cell cultures containing both neurons and astrocytes, we found that neither an enhancement in system xc(-) expression nor activity underlies the excitotoxic effects of aglycemia. In addition, using three separate bioassays, we demonstrate no change in the ability of glucose-deprived astrocytes--either cultured alone or with neurons--to remove glutamate from the extracellular space. Instead, we demonstrate that glucose-deprived cultures are 2 to 3 times more sensitive to the killing effects of glutamate or N-methyl-D-aspartate when compared with their glucose-containing controls. Hence, our results are consistent with the weak excitotoxic hypothesis such that a bioenergetic deficiency, which is measureable in our mixed but not astrocyte cultures, allows normally innocuous concentrations of glutamate to become excitotoxic. Adding to the burgeoning literature detailing the contribution of astrocytes to neuronal injury, we conclude that under our experimental paradigm, a cytotoxic, co-operative interaction between energy deprivation and glutamate release from astrocyte system xc(-) mediates aglycemic neuronal cell death.

Saturation of neuroprotective effects of adenosine in cortical culture.

Adenosine and adenosine A1 receptor agonists are often, but not always, protective against metabolic insults. The effects of an A1 agonist and antagonist on neuronal death were determined in cortical cell cultures. The A1 agonist cyclohexyladenosine did not attenuate neuronal death induced by oxygen-glucose deprivation, but did attenuate death caused by glucose deprivation or NMDA. Extracellular adenosine levels during oxygen-glucose deprivation were significantly higher than those during glucose deprivation or NMDA exposure. The A1 antagonist 8-cyclopentyltheophylline increased death induced by oxygen-glucose deprivation, but not that caused by glucose deprivation or NMDA exposure. Thus, while activation of A1 receptors can provide neuroprotection, the protective effect appears to become saturated by high levels of endogenous extracellular adenosine during oxygen-glucose deprivation.

Mechanisms of bFGF and NT-4 potentiation of necrotic neuronal death.

The effects of neurotrophic factors on necrotic neuronal death are controversial. In this study we found that both neurotrophin-4 (NT-4) and basic fibroblast growth factor (bFGF) potentiated necrotic neuronal death caused by exposure to oxygen-glucose deprivation or iron-citrate (Fe) in cortical cultures. However, there were significant differences in the actions of the two neurotrophic factors. Neurotrophin-4 protected against apoptotic neuronal death, while bFGF had no effect on apoptotic death in these cultures. Furthermore, potentiation of oxygen-glucose deprivation induced necrotic death by NT-4 required pretreatment (24 h), while pretreatment with bFGF had no effect. However, acute treatment with bFGF during oxygen-glucose deprivation did potentiate neuronal death. Both neurotrophic factors potentiated free radical mediated necrotic neuronal death induced by exposure to Fe. However, the RNA synthesis inhibitor, actinomycin-D, blocked the injury potentiation by NT-4, but not that caused by bFGF. Also, NT-4, but not bFGF, potentiated Fe induced necrotic death in pure neuronal cultures. Expression of mRNA for FGF receptors FGFR1 and FGFR2 was observed at high levels in astrocytes. The results indicate that the injury enhancing effects of bFGF are acute, while those of NT-4 require prolonged exposure and new protein synthesis. Furthermore, the effects of bFGF appear to be mediated through actions on astrocytes, while NT-4 appears to act directly on neurons. The fact that neurotrophic factors from two distinct families can potentiate neuronal death by two different mechanisms suggests that such injury potentiation may be a common concern regarding the use of neurotrophic factors.

In vitro neurotoxic evaluation of root-end-filling materials.

Root-end-filling materials have been tested for toxicity on several cell types, but their toxicity has not been tested on neurons. In this study we evaluated the neurotoxicity in murine cerebral cortical cell cultures of four commonly used root-end-filling materials: mineral trioxide aggregate, amalgam, Super EBA, and Diaket. Standardized amounts of each material were placed on culture-well inserts, allowing the material to be exposed to the culture bathing media without causing physical disruption of the cells. Cell death was quantified by assaying release of the cytosolic enzyme lactate dehydrogenase. Exposure of cortical cultures to freshly mixed or 7-day-old MTA did not cause significant neuronal death, whereas exposure to freshly mixed or 7-day-old amalgam, Super EBA, and Diaket resulted in significant neuronal death (p < .05). Thus, each material, except for mineral trioxide aggregate, can induce neurotoxicity, even when allowed to set thoroughly.