DI TNC1 Cells Research Articles

A Pro-Inflammatory Stimulus versus Extensive Passaging of DITNC1 Astrocyte Cultures as Models to Study Astrogliosis.

Astrogliosis is a process by which astrocytes, when exposed to inflammation, exhibit hypertrophy, motility, and elevated expression of reactivity markers such as Glial Fibrillar Acidic Protein, Vimentin, and Connexin43. Since 1999, our laboratory in Chile has been studying molecular signaling pathways associated with "gliosis" and has reported that reactive astrocytes upregulate Syndecan 4 and αVβ3 Integrin, which are receptors for the neuronal glycoprotein Thy-1. Thy-1 engagement stimulates adhesion and migration of reactive astrocytes and induces neurons to retract neurites, thus hindering neuronal network repair. Reportedly, we have used DITNC1 astrocytes and neuron-like CAD cells to study signaling mechanisms activated by the Syndecan 4-αVβ3 Integrin/Thy-1 interaction. Importantly, the sole overexpression of β3 Integrin in non-reactive astrocytes turns them into reactive cells. In vitro, extensive passaging is a simile for "aging", and aged fibroblasts have shown β3 Integrin upregulation. However, it is not known if astrocytes upregulate β3 Integrin after successive cell passages. Here, we hypothesized that astrocytes undergoing long-term passaging increase β3 Integrin expression levels and behave as reactive astrocytes without needing pro-inflammatory stimuli. We used DITNC1 cells with different passage numbers to study reactivity markers using immunoblots, immunofluorescence, and astrocyte adhesion/migration assays. We also evaluated β3 Integrin levels by immunoblot and flow cytometry, as well as the neurotoxic effects of reactive astrocytes. Serial cell passaging mimicked the effects of inflammatory stimuli, inducing astrocyte reactivity. Indeed, in response to Thy-1, β3 Integrin levels, as well as cell adhesion and migration, gradually increased with multiple passages. Importantly, these long-lived astrocytes expressed and secreted factors that inhibited neurite outgrowth and caused neuronal death, just like reactive astrocytes in culture. Therefore, we describe two DITNC1 cell types: a non-reactive type that can be activated with Tumor Necrosis Factor (TNF) and another one that exhibits reactive astrocyte features even in the absence of TNF treatment. Our results emphasize the importance of passage numbers in cell behavior. Likewise, we compare the pro-inflammatory stimulus versus long-term in-plate passaging of cell cultures and introduce them as astrocyte models to study the reactivity process.

Antioxidant Activity of Polysaccharides from the Edible Mushroom Pleurotus eryngii.

In this study the antioxidant and neuroprotective activity of an enriched polysaccharide fraction (EPF) obtained from the fruiting body of cultivated P. eryngii was evaluated. Proximate composition (moisture, proteins, fat, carbohydrates and ash) was determined using the AOAC procedures. The EPF was extracted by using, in sequence, hot water and alkaline extractions followed by deproteinization and precipitation with cold ethanol. Total α- and β-glucans were quantified using the Megazyme International Kit. The results showed that this procedure allows a high yield of polysaccharides with a higher content of (1-3; 1-6)-β-D-glucans. The antioxidant activity of EPF was detected from the total reducing power, DPPH, superoxide, hydroxyl and nitric oxide radical scavenging activities. The EPF was found to scavenge DPPH, superoxide, hydroxyl and nitric oxide radicals with a IC50 values of 0.52 ± 0.02, 1.15 ± 0.09, 0.89 ± 0.04 and 2.83 ± 0.16 mg/mL, respectively. As assessed by the MTT assay, the EPF was biocompatible for DI-TNC1 cells in the range of 0.006-1 mg/mL and, at concentrations ranging from 0.05 to 0.2 mg/mL, significantly counteracted H2O2-induced reactive oxygen species production. This study demonstrated that polysaccharides extracted from P. eryngii might be used as functional food to potentiate the antioxidant defenses and to reduce oxidative stress.

Ca2+ signaling as a mechanism of haloperidol-induced cytotoxicity in human astrocytes and assessing the protective role of a Ca2+ chelator.

Haloperidol, a typical antipsychotic medication, has been shown to possess various biological effects in different brain models. However, the impact of haloperidol on Ca2+ signaling in astrocytes is elusive. This study explored the effect of haloperidol on cytosolic free Ca2+ levels ([Ca2+]i) and viability, and established these two connections in Gibco® Human Astrocytes (GHAs) and DI TNC1 rat astrocytes. Haloperidol (5-20μM) caused [Ca2+]i rises in a concentration-dependent manner in GHAs but not in DI TNC1 cells. Furthermore, removal of extracellular Ca2+ reduced haloperidol's effect by approximately 30% in GHAs. Haloperidol (20-40μM) evoked concentration-dependent cytotoxicity in GHAs and DI TNC1 cells. However, chelating cytosolic Ca2+ with the Ca2+ chelator BAPTA/AM significantly reversed haloperidol's cytotoxicity only in GHAs. In GHAs, haloperidol-induced Ca2+ entry was inhibited by store-operated Ca2+ modulators (2-APB and SKF96365) and the protein kinase C (PKC) inhibitor GF109203X. This Ca2+ entry induced by haloperidol was confirmed by Mn2+ entry-induced quench of fura-2 fluorescence. In Ca2+-free medium, treatment with the endoplasmic reticulum Ca2+ pump inhibitor 2,5-di-tert-butylhydroquinone (BHQ) abolished haloperidol-induced [Ca2+]i rises. Conversely, treatment with haloperidol inhibited 45% of BHQ-evoked [Ca2+]i rises. Moreover, haloperidol-induced Ca2+ release from the endoplasmic reticulum was abolished by inhibition of phospholipase C (PLC) by U73122. Together, in GHAs but not in DI TNC1 cells, haloperidol caused Ca2+-associated cell death, induced Ca2+ entry via PKC-sensitive store-operated Ca2+ channels, and evoked PLC-dependent Ca2+ release from the endoplasmic reticulum. The protective effect of Ca2+ chelating on haloperidol-induced cytotoxicity in human astrocytes was also demonstrated.

Neuroprotection against Amyloid-β-Induced DNA Double-Strand Breaks Is Mediated by Multiple Retinoic Acid-Dependent Pathways.

In this study, we have investigated the role of all-trans-retinoic acid (RA) as a neuroprotective agent against Aβ1-42-induced DNA double-strand breaks (DSBs) in neuronal SH-SY5Y and astrocytic DI TNC1 cell lines and in murine brain tissues, by single-cell gel electrophoresis. We showed that RA does not only repair Aβ1-42-induced DSBs, as already known, but also prevents their occurrence. This effect is independent of that of other antioxidants studied, such as vitamin C, and appears to be mediated, at least in part, by changes in expression, not of the RARα, but of the PPARβ/δ and of antiamyloidogenic proteins, such as ADAM10, implying a decreased production of endogenous Aβ. Whereas Aβ1-42 needs transcription and translation for DSB production, RA protects against Aβ1-42-induced DSBs at the posttranslational level through both the RARα/β/γ and PPARβ/δ receptors as demonstrated by using specific antagonists. Furthermore, it could be shown by a proximity ligation assay that the PPARβ/δ-RXR interactions, not the RARα/β/γ-RXR interactions, increased in the cells when a 10 min RA treatment was followed by a 20 min Aβ1-42 treatment. Thus, the PPARβ/δ receptor, known for its antiapoptotic function, might for these short-time treatments play a role in neuroprotection via PPARβ/δ-RXR heterodimerization and possibly expression of antiamyloidogenic genes. Overall, this study shows that RA can not only repair Aβ1-42-induced DSBs but also prevent them via the RARα/β/γ and PPARβ/δ receptors. It suggests that the RA-dependent pathways belong to an anti-DSB Adaptative Gene Expression (DSB-AGE) system that can be targeted by prevention strategies to preserve memory in Alzheimer's disease and aging.

Uncovering malathion (an organophosphate insecticide) action on Ca2+ signal transduction and investigating the effects of BAPTA-AM (a cell-permeant Ca2+ chelator) on protective responses in glial cells

Malathion, one of commonly used organophosphate insecticides, has a wide range of toxic actions in different models. However, the effect of this compound on Ca2+ homeostasis and its related cytotoxicity in glial cells is elusive. This study examined whether malathion evoked intracellular Ca2+ concentration ([Ca2+]i) rises and established the relationship between Ca2+ signaling and cytotoxicity in normal human astrocytes, rat astrocytes and human glioblastoma cells. The data show that malathion induced concentration-dependent [Ca2+]i rises in Gibco® Human Astrocytes (GHA cells), but not in DI TNC1 normal rat astrocytes and DBTRG-05MG human glioblastoma cells. In GHA cells, this Ca2+ signal response was reduced by removing extracellular Ca2+. In Ca2+-free medium, pretreatment with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin abolished malathion-induced [Ca2+]i rises. Conversely, incubation with malathion abolished thapsigargin-induced [Ca2+]i rises. Inhibition of phospholipase C (PLC) with U73122 also blocked malathion-induced [Ca2+]i rises. In Ca2+-containing medium, malathion-induced [Ca2+]i rises was inhibited by store-operated Ca2+ channel blockers (2-APB, econazole or SKF96365) and the protein kinase C (PKC) inhibitor GF109203X. Malathion (5–25 μM) concentration-dependently caused cytotoxicity in GHA, DI TNC1 and DBTRG-05MG cells. This cytotoxic effect was partially prevented by prechelating cytosolic Ca2+ with BAPTA-AM (a selective Ca2+ chelator) only in GHA cells. Together, in GHA but not in DI TNC1 and DBTRG-05MG cells, malathion induced [Ca2+]i rises by inducing PLC-dependent Ca2+ release from the endoplasmic reticulum and Ca2+ entry via PKC-sensitive store-operated Ca2+ channels. Furthermore, malathion induced Ca2+-associated cytotoxicity, suggesting that Ca2+ chelating may have a protective effect on malathion-induced cytotoxicity in normal human astrocytes.

Inhibition of the PERK-Dependent Unfolded Protein Response Signaling Pathway Involved in the Pathogenesis of Alzheimer's Disease.

There is a body of evidence that neurodegenerative disease entities are directly correlated with the perturbations on the molecular level. Hence, the ER stress-mediated Unfolded Protein Response (UPR) is activated resulting in PERK-dependent phosphorylation of the Eukaryotic initiation factor 2 (eIF2α). Thus, the levels of ATF4 and CHOP proteins are significantly increased, which subsequently switches the pro-adaptive branch of the UPR into the pro-apoptotic directly leading to neuronal loss and initiation of the neurodegenerative process. The aim of the presented study was the evaluation of the biological activity of highly specific, small-molecule inhibitors of the PERKdependent UPR signaling pathway. The study was conducted on rat astrocytic DI TNC1 cell line. The level of p-eIF2α was measured by Western blot technique, the cytotoxicity of the investigated compound was assessed by the MTT assay and using the FITC-conjugated Annexin V (Annexin V-FITC) to indicate apoptosis and propidium iodide (PI) to indicate necrosis. The effect of tested compound on cell cycle progression was measured by flow cytometry, where the PI-labelled nuclei were analysed for DNA content. As a result one of the investigated compound LDN-0060609 triggers a significant inhibition of the eIF2α phosphorylation in DI TNC1 cell line. Moreover, we showed that compound LDN-0060609 is non-cytotoxic and has no effect on cell cycle progression. In conclusion, LDN-0060609 may constitute a novel, targeted treatment approach against neurodegenerative diseases, including Alzheimer's disease (AD), where pathogenesis and progression are closely associated with the overactivation of the PERK-dependent UPR signaling pathway.

Evaluation of cytotoxicity of propofol and its related mechanism in glioblastoma cells and astrocytes.

Propofol (2,6-diisopropylphenol), one of the extensively and commonly used anesthetic agents, has been shown to affect the biological behavior of various models. Previous researches have shown that propofol-induced cytotoxicity might cause anticancer effect in different cells. However, the mechanisms underlying the effect of propofol on cytotoxicity is still elusive in human glioblastoma cells. The aims of this study were to evaluate effects of propofol on cytotoxicity, cell cycle distribution and ROS production, and establish the relationship between oxidative stress and cytotoxicity in GBM 8401 human glioblastoma cells and DI TNC1 rat astrocytes. Propofol (20-30 μM) concentration-dependently induced cytotoxicity, cell cycle arrest, and increased ROS production in GBM 8401 cells but not in DI TNC1 cells. In GBM 8401 cells, propofol induced G2/M phase cell arrest, which affected the CDK1, cyclin B1, p53, and p21 protein expression levels. Furthermore, propofol induced oxygen stresses by increasing O2- and H2 O2 levels but treatment with the antioxidant N-acetylcysteine (NAC) partially reversed propofol-regulated antioxidative enzyme levels (superoxide dismutase, catalase, and glutathione peroxidase). Most significantly, propofol induced apoptotic effects by decreasing Bcl-2 but increasing Bax, cleaved caspase-9/caspase-3 levels, which were partially reversed by NAC. Moreover, the pancaspase inhibitor Z-VAD-FMK also partially prevented propofol-induced apoptosis. Together, in GBM 8401 cells but not in DI TNC1 cells, propofol activated ROS-associated apoptosis that involved cell cycle arrest and caspase activation. These findings indicate that propofol not only can be an anesthetic agent which reduces pain but also has the potential to be used for the treatment of human glioblastoma.

1,3-Dinitrobenzene neurotoxicity – Passage effect in immortalized astrocytes

Age-related disturbances in astrocytic mitochondrial function are linked to loss of neuroprotection and decrements in neurological function. The immortalized rat neocortical astrocyte-derived cell line, DI-TNC1, provides a convenient model for the examination of cellular aging processes that are difficult to study in primary cell isolates from aged brain. Successive passages in culture may serve as a surrogate of aging in which time-dependent adaptation to culture conditions may result in altered responses to xenobiotic challenge. To investigate the hypothesis that astrocytic mitochondrial homeostatic function is decreased with time in culture, low passage DI-TNC1 astrocytes (LP; #2–8) and high passage DI-TNC1 astrocytes (HP; #17–28) were exposed to the mitochondrial neurotoxicant 1,3-dinitrobenzene (DNB). Cells were exposed in either monoculture or in co-culture with primary cortical neurons. Astrocyte mitochondrial membrane potential, morphology, ATP production and proliferation were monitored in monoculture, and the ability of DI-TNC1 cells to buffer K+-induced neuronal depolarization was examined in co-cultures. In HP DI-TNC1 cells, DNB exposure decreased proliferation, reduced mitochondrial membrane potential and significantly decreased mitochondrial form factor. Low passage DI-TNC1 cells effectively attenuated K+-induced neuronal depolarization in the presence of DNB whereas HP counterparts were unable to buffer K+ in DNB challenge. Following DNB challenge, LP DI-TNC1 cells exhibited greater viability in co-culture than HP. The data provide compelling evidence that there is an abrupt phenotypic change in DI-TNC1 cells between passage #9–16 that significantly diminishes the ability of DI-TNC1 cells to compensate for neurotoxic challenge and provide neuroprotective spatial buffering. Whether or not these functional changes have an in vivo analog in aging brain remains to be determined.

TRPA1 Is Expressed in Central But Not in Peripheral Glia

TRPA1 are cation channels expressed in sensory neurons and in several other cell types. This channel is specifically activated by ally isothiocyanate (AITC), the pungent component of mustard oil, as well as by other electrophilic compounds. Although TRPA1 expression in central glia has been reported, its subcellular localization and its expression in peripheral glia have not been investigated before. In this paper we report the molecular and functional expression of TRPA1 in rat cortical astrocytes. Real-time RT-PCR identified low but significant amounts of TRPA1 mRNA in cortical astrocytes while no signal was seen in peripheral glia isolated from dorsal root ganglia (DRG) or in a glial cell line (DITNC-1). Calcium imaging showed AITC-induced signals in astro-cytes while no response in peripheral glia. AITC induced calcium signals in astrocytes in the presence and in the absence of extracellular calcium, suggesting an intracellular localization of TRPA1 channels. Whole cell electrophysiological recordings were performed in astrocytes, in peripheral glia and in DITNC-1 cells transfected with TRPA1 during AITC application. In TRPA1-transfected DITNC-1 cells typical TRPA1 currents were recorded with a reversal potential near 0 mV, consistent with the opening of a non-selective cation channel. No such currents were recorded in untransfected DITNC-1 cells, in astrocytes and in peripheral glial cells, where even high concentrations of AITC (up to 10 mM) induced no significant outward current. In astrocytes AITC transiently induced an outward rectifying current with the reversal potential near ?90 mV, consistent with K channel activation, likely activated by intracellular release of calcium. Our results suggest that TRPA1 channels are molecularly and functionally expressed in calcium-containing organelles of rat cortical astrocytes, with no expression in the plasma membrane.

A comparative study of protein carbonylation and mitochondrial dysfunction using the neurotoxicants 1,3-dinitrobenzene, 3-nitropropionic acid, and 3-chloropropanediol

This comparative evaluation of neurotoxicants previously identified as models of chemical-induced mitochondrial dysfunction and energy deprivation demonstrated that subtoxic concentrations of 1,3-dinitrobenzene (1,3-DNB), 3-nitropropionic acid (3-NPA), and 3-chloropropanediol (3-CPD) each led to concentration-dependent loss of the mitochondrial membrane potential (ΔΨm) associated with similar patterns of protein carbonylation. Subtoxic concentrations of each neurotoxicant were determined by measuring DI TNC1 cell viability using the MTS cell proliferation assay. Although exposure 1μM, 10μM, and 100μM concentrations of each toxicant did not result in loss of cell viability after 48h, exposure to each toxicant at these concentrations led to concentration-dependent loss of tetramethyl rhodamine methyl ester (TMRM) fluorescence over the same exposure period. Preincubation with the antioxidant, deferoxamine, was effective in preventing loss of TMRM flurorescence. Through the combined use of two-dimensional polyacrylamide gel electrophoresis (2D PAGE) and Oxyblot analysis, this study demonstrated that exposure to each toxicant resulted in the formation of distinctly similar patterns of protein carbonylation comprised of specific proteins identified with tandem MS/MS. Our results provide insight as to how exposure to different neurotoxicants that enhance oxidative stress may, in fact, lead to mitochondrial injury and subsequent toxicity through selective, yet shared, pathways of protein modification by oxidative carbonylation.

IL-10 overexpression attenuated expression of TNF-α and IL-1β activated by lipopolysaccharide in astrocytes.

To investigate the intervention effect of lentivirus expressing human IL-10 (LV-hIL-10) on activated astrocytes. DI TNC1 cell line was treated with different concentrations of lipopolysaccharide (LPS) and time points. The expression of proinflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukinb-1β (IL-1β) was detected by RT-PCR and ELISA. Moreover, the effect on the expression of proinflammatory cytokines TNF-α and IL-1β was analyzed in DI TNC1 cell lines infected with and without LV-hIL-10. The expression of TNF-α and IL-1β was increased in DI TNC1 induced by LPS. The expression of IL-10 was upregulated in DI TNC1 infected with LV-hIL-10. TNF-α and IL-1β were inhibited by IL-10 overexpression in DI TNC1 actived by LPS. DI TNC1 is activated by LPS and secretes proinflammatory cytokines TNF-α and IL-1β as immune-like cells, and these activation is inhibited by hIL-10 overexpression.

Proteomic identification of carbonylated proteins in 1,3-dinitrobenzene neurotoxicity

This study demonstrated that 1,3-dinitrobenzene-induced (1,3-DNB) oxidative stress led to the oxidative carbonlyation of specific protein targets in DI TNC1 cells. 1,3-DNB-induced mitochondrial dysfunction, as indicated by loss of tetramethyl rhodamine methyl ester (TMRM) fluorescence, was initially observed at 5h and coincided with peak reactive oxygen species (ROS) production. ROS production was inhibited in cells pre-treated with the mitochondrial permeability transition (MPT) inhibitor, bonkrekic acid (BkA). Pre-incubation with the antioxidant deferoxamine inhibited loss of TMRM fluorescence until 24h after initial exposure to 1,3-DNB. Two-dimensional polyacrylamide gel electrophoresis (2D PAGE) and subsequent Oxyblot analysis were used to determine if 1,3-DNB exposure led to the formation of protein carbonyls. Exposing DI TNC1 cells to 1,3-DNB led to marked protein carbonylation 45min following initial exposure. Pre-treatment with deferoxamine or Trolox reduced the intensity of protein carbonylation in DI TNC1 cells exposed to 1mM 1,3-DNB. Tandem MS/MS performed on protein samples isolated from 1,3-DNB-treated cells revealed that specific proteins within the mitochondria, endoplasmic reticulum (ER), and cytosol are targets of protein carbonylation. The results presented in this study are the first to suggest that the molecular mechanism of 1,3-DNB neurotoxicity may occur through selective carbonylation of protein targets found within specific intracellular compartments of susceptible cells.

GABA-B1 Receptors are Coupled to the ERK1/2 MAP Kinase Pathway in the Absence of GABA-B2 Subunits

In the current model of gamma-aminobutyric acid (GABA) B receptor function, there is a requirement for GABA-B(1/2) heterodimerisation for targetting to the cell surface. However, different lines of evidence suggest that the GABA-B(1) subunit can form a functional receptor in the absence of GABA-B(2). We observed coupling of endogenous GABA-B(1) receptors in the DI-TNC1 glial cell line to the ERK pathway in response to baclofen even though these cells do not express GABA-B(2). GABA-B(1A) receptors were also able to mediate a rapid, transient, and dose-dependent activation of the ERK1/2 MAP kinase pathway when transfected alone into HEK 293 cells. The response was abolished by G(i/o) and MEK inhibition, potentiated by inhibitors of phospholipase C and protein kinase C and did not involve PI-3-kinase activity. Finally, using bioluminescence resonance energy transfer and co-immunoprecipitation, we show the existence of homodimeric GABA-B(1A) receptors in transfected HEK293 cells. Altogether, our observations show that GABA-B(1A) receptors are able to activate the ERK1/2 pathway despite the absence of surface targetting partner GABA-B(2) in both HEK 293 cells and the DI-TNC1 cell line.

High-throughput RNAi screening in vitro: From cell lines to primary cells

Small interfering RNAs (siRNAs) are being used to induce sequence-specific gene silencing in cultured cells to study mammalian gene function. Libraries of siRNAs targeting entire human gene classes can be used to identify genes with specific cellular functions. Here we describe high-throughput siRNA delivery methods to facilitate siRNA library screening experiments with both immortalized and primary cells. We adapted chemical reverse transfection for immortalized adherent cell lines in a 96-well format. The method is fast, robust, and exceptionally effective for many cell types. For primary cells and immortalized cells that are recalcitrant to lipofection-based methods, we developed electropermeabilization (electroporation) conditions that facilitate siRNA delivery to a broad range of cell types, including primary human T-cells, hMSC, NHA, NDHF-Neo, HUVEC, DI TNC1, RPTEC, PC12, and K562 cells. To enable high-throughput electropermeabilization of primary cells, we developed a novel 96-well electroporation device that provides highly efficient and reproducible delivery of siRNAs. The combination of high-throughput chemical reverse transfection and electroporation makes it possible to deliver libraries of siRNAs to virtually any cell type, enabling gene function analysis and discovery on a genome scale.

Benzo[a]pyrene diol epoxide up-regulates COX-2 expression through NF-κB in rat astrocytes

Cyclooxygenase may be important in the pathogenesis of smoking-related cancer because it activates carcinogens and is highly inducible in inflammation. Benzo[a]pyrene (B[a]P) is one of the most common ingredients of cigarette smoke and benzo[a]pyrene diol epoxide (BPDE) is a metabolic product of B[a]P. Cigarette smoking-induced inflammation has been found in several tissues and in association with cyclooxygenase-2 (COX-2) expression. The contribution of COX-2 to peripheral inflammation is well documented, however, little is known about its role in brain inflammation. We studied COX-2 expression following treatment with BPDE in the cortical cells of Sprague–Dawley rats in vivo, as well as in DI TNC1 rat astrocytes and rat pheochromocytoma PC-12 cells (neurons) cultured in vitro. Our data showed that BPDE increases levels of COX-2 mRNA and protein in cortical cells of Sprague–Dawley rats. BPDE also increases levels of COX-2 mRNA in PC-12 and DI TNC1 cells. Induction of COX-2 protein was only found in DI TNC1 cells. Gel shift assay and western blot revealed increased NF-κB binding activity and protein level after treatment with BPDE. Experiments were performed to define the signaling mechanism by which BPDE induces COX-2, and suggested that BPDE-mediated COX-2 induction increases the risk of brain inflammation.

Regulatory pathways and uptake of L-DOPA by capillary cerebral endothelial cells, astrocytes, and neuronal cells.

We examined the nature and regulation of the inward L-3,4-dihydroxyphenylalanine (L-DOPA) transporter in rat capillary cerebral endothelial (RBE4) cells, type 1 astrocytes (DI TNC1), and Neuro-2a neuroblastoma cells. In all three cell types, the inward transfer of L-DOPA was largely promoted through the 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid-sensitive and sodium-independent L-type amino acid transporter. Only in DI TNC1 cells was the effect of maneuvers that increase intracellular cAMP levels accompanied by increases in L-DOPA uptake. Also, only in DI TNC1 cells was the effect of the guanylyl cyclase inhibitor LY-83583 accompanied by a 65% increase in L-DOPA accumulation, whereas the nitric oxide donor sodium nitroprusside produced a 25% decrease in L-DOPA accumulation. In all three cell types, the Ca2+/calmodulin inhibitors calmidazolium and trifluoperazine inhibited L-DOPA uptake in a noncompetitive manner. Thapsigargin (1 and 3 microM) and A-23187 (1 and 3 microM) failed to alter L-DOPA accumulation in RBE4 and Neuro-2a cells but markedly increased L-DOPA uptake in DI TNC1 cells. We concluded that L-DOPA in RBE4, DI TNC1, and Neuro-2a cells is transported through the L-type amino acid transporter and appears to be under the control of Ca2+/calmodulin-mediated pathways. Astrocytes, however, are endowed with other processes that appear to regulate the accumulation of L-DOPA, responding positively to increases in intracellular Ca2+ and cAMP and to decreases in cGMP.

The effect of acrylonitrile on gap junctional intercellular communication in rat astrocytes.

Rats chronically exposed to acrylonitrile (ACN) have shown a dose-dependent increase in the incidence of astrocytomas in the brain. The mechanism(s) by which ACN induces cancer in rodents has not been established. ACN does not appear to be directly genotoxic in the brain and thus a nongenotoxic mode of action has been proposed. Inhibition of gap junctional intercellular communication (GJIC) has been shown to be a property of many nongenotoxic carcinogens. The present study examined the effects of ACN on GJIC in a rat astrocyte transformed cell line, DI TNC1 cells (a target cell for ACN carcinogenicity) and primary cultured hepatocytes (a nontarget cell for ACN carcinogenicity). ACN inhibited GJIC in rat astrocytes in a dose-dependent manner. Inhibition of GJIC was observed following 2 h treatment with 0.10 mmol/L and 1.00 mmol/L ACN. However, in primary cultured hepatocytes, ACN exposed did not result in inhibition of GJIC even after 48 h of continued treatment. In the astrocytes, GJIC inhibition plateaued after 4 h of treatment and remained blocked throughout the entire experimental period examined. Inhibition of GJIC in DI TNC1 cells was reversed by removal of ACN from the culture medium after 4 or 24 h of treatment. Cotreatment of astrocytes with vitamin E reduced the effect of ACN-induced inhibition of GJIC. Similarly, inhibition of GJIC was prevented by treatment with 2-oxothiazolidine-4-carboxylic acid (OTC), a precursor of glutathione synthesis. Decreasing cellular glutathione by treatment with buthionine sulfoxamine alone (without ACN) did not affect GJIC in astrocytes. Collectively, these results demonstrate that treatment with ACN caused a selective inhibition of GJIC in rat DI TNC1 astrocytes (the target cell type), but not in rat hepatocytes (a nontarget tissue). Inhibition of GJIC in astrocytes was reversed by treatment with antioxidants and suggests a potential role for oxidative stress in ACN-induced carcinogenesis.

Regulation of energy metabolism by neurotransmitters in astrocytes in primary culture and in an immortalized cell line.

Evidence suggests that astrocytes might play an important role in cerebral energy metabolism. A recently developed cell line, called DI TNC1, displays several characteristic features of astrocytes. Thus, we have investigated in these cells a number of parameters related to energy metabolism. First, glycogen, the major energy reserve in the brain, is present in these cells and its levels are influenced by the glucose content of the growth medium and the presence of serum. Second, several neurotransmitters including noradrenaline and vasoactive intestinal peptide (VIP) induce a glycogenolytic response. Their effect on glycogen is paralleled by a similar effect on the formation of cyclic AMP, which is presumably the second messenger involved. Third, noradrenaline stimulates glucose utilization (as reflected by 2-deoxyglucose uptake) in DI TNC1 cells, an effect which is mimicked by the second messenger arachidonate. Interestingly, two actions of neurotransmitters, which are well characterized in primary astrocytes, are absent in DI TNC1 cells. These are the noradrenaline- and VIP-induced resynthesis of glycogen and the glutamate-stimulated glycolysis. In summary, the observations reported here lend further support to the concept that astrocytes are important for the control of brain energy metabolism. In addition, DI TNC1 cells might represent an interesting preparation to help decipher some of the astrocytic functions related to energy metabolism.