Rat Astroglial Cells Research Articles

Ethanol-induced injury in rat primary cortical astrocytes involves oxidative stress: effect of idebenone

Ethanol-induced neurological disorders have recently been characterised. Indeed, evidence has been collected indicating that chronic ethanol consumption leads to direct or indirect changes in the viability of central nervous system cells. Here we investigated the role of free radical overproduction in primary cortical rat astroglial cells undergoing chronic treatment with ethanol (100 μM). In particular, exposure of astroglial cell cultures to ethanol for 12 consecutive days produced an increased release of lactic dehydrogenase, a decrease on glutamine synthase activity being both effects accompanied by decrease in astroglial viability as detected by MTT (Thiazolyl Blue) test. These effects were accompanied by an increased formation of malondialdehyde (a marker of lipid peroxidation) and by abnormal formation of heat shock protein, being both effects antagonised by liposomally entrapped idebenone, a non-peptidyl free radical scavenger. Taken together, these results suggest that ethanol-induced injury on astroglial cells are mediated by abnormal formation of free radical species and this may represent a useful approach in the treatment of ethanol-related brain disorders.

Cellular and molecular pharmacological studies on membrane receptor-signaling and stress-responses in the brain

Studies on the cellular and molecular mechanism of neurotransmitter receptor-signaling and of neuronal and glial cell responses to stresses seem to be important to elucidate the action mechanism of centrally-acting drugs and to develop novel therapeutics against several diseases in the brain. The present review shows our findings with regard to the membrane receptor-signaling mechanism including serotonin, noradrenaline, glutamate receptors, ion channels, G-proteins, protein kinases and drug actions in Xenopus oocytes injected with rat brain mRNA, NG108-15 cells and brain membranes. Regarding the results of studies on the inter- and intra-cellular mechanism of neurons and glial cells against cerebral ischemia/hypoxia, we review the involvement of a transcription factor NF-kappa B in LPS-elicited inducible NO synthase (iNOS) expression in rat astroglial cells. Then we describe possible involvement of: 1) ADP-ribosylation/nitrosylation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and 2) decrease in mitochondrial membrane potential, release of caspase-3 from mitochondria and degradation of the inhibitor of caspase-activated DNase by activated caspase in NO-induced neuronal apoptosis. We observed that hypoxia results in expression of a molecular chaperon such as protein disulfide isomerase (PDI) and HSP70 in astroglial cells. Our recent findings indicate that overexpression of PDI in the rat hippocampus (in vivo) and in neuroblastoma SK-N-MC cells (in vitro) significantly suppress the hypoxia-induced neuronal death. From physiological/pathophysiological and pharmacological aspects, we review the importance of studies on the cellular and molecular mechanism of membrane receptor-signaling and of stress-responses in the brain to identify functional roles of neuro-glial- as well as neuro-neuronal interaction in the brain.

Changes in antioxidant enzyme expression in response to hydrogen peroxide in rat astroglial cells.

Oxidative stress has been causally linked to a variety of neurodegenerative diseases. To clarify the role of the antioxidant enzyme (AOE) system in oxidative brain damage primary cultures of rat astroglial cells were exposed to hydrogen peroxide (H2O2). Expression of AOEs and several parameters for cell viability and functionality were measured. In our experiments astrocytes responded to low concentrations of H2O2 exposure with a pronounced generation of ROS which ran parallel with induction of lipid peroxidation. This distinct oxidative stress was not reflected in cell viability or functionality parameters measured. Cytotoxicity, a decrease in glutathione content of astrocytes, and impairment of mitochondrial functions became obvious only for higher concentrations of H2O2. After H2O2 exposure catalase, manganese superoxide dismutase, and glutathione peroxidase expression levels were found to be increased, whereas copper/zinc superoxide dismutase mRNA expression was not affected. These data indicate that the AOE system of astrocytes can be directly regulated by oxidative stress and may thus contribute to protection of cells against oxidative insults.

Apoptosis induced by 2-chloro-adenosine and 2-chloro-2?-deoxy-adenosine in a human astrocytoma cell line: Differential mechanisms and possible clinical relevance

We have previously demonstrated that 2-chloro-adenosine (2-CA) can induce apoptosis of rat astroglial cells (Abbracchio et al. [1995] Biochem. Biophys. Res. Commun. 213:908-915). In the present study, we have characterized, for the first time, the effects induced on a human astrocytoma cell line (ADF cells) by both 2-CA and its related analog 2-chloro-2'-deoxy-adenosine (2-CdA, that is employed as anti-cancer agent in chronic lymphoid malignancies). Exposure of these cells to either adenosine analog resulted in time- and concentration-dependent apoptosis. Experiments with pharmacological agents known to interfere with adenosine receptors, its membrane transporter, and intracellular nucleoside kinases showed that: (i) cell death induced by either adenosine analog did not depend on extracellular adenosine receptors, but on a direct intracellular action; however, only in the case of 2-CA, was entry into cells mediated by the specific nitrobenzyl-tioinosine-sensitive transporter; (ii) for both adenosine analogs, induction of apoptosis required the phosphorylation/activation by specific intracellular nucleoside kinases, i.e., adenosine kinase for 2-CA, and deoxycytidine kinase for 2-CdA. In addition, only in the case of 2-CdA, was induction of apoptosis preceded by a block of cells at the G2/M phase of the cell cycle. Finally, at concentrations of either analog that killed about 80-90% of astrocytoma cells, a significantly lower effect on the viability of primary cortical neurons was observed. In conclusion, both adenosine analogs can trigger apoptosis of human astrocytoma cells, albeit with different mechanisms. This effect together with the relative sparing of neuronal cells, may have potential clinical implications for the therapy of tumors of glial origin.

Interleukin-10, interleukin-4, and transforming growth factor-beta differentially regulate lipopolysaccharide-induced production of pro-inflammatory cytokines and nitric oxide in co-cultures of rat astroglial and microglial cells.

The pro-inflammatory cytokines interleukin-1beta (IL-1beta), IL-6, tumor necrosis factor-alpha (TNF-alpha), and nitric oxide (NO) can be produced by activated glial cells and play a critical role in various neurological diseases. Using primary co-cultures of rat microglial and astroglial cells, we investigated the effects of the anti-inflammatory cytokines transforming growth factor-beta1 (TGF-beta1)/beta2, IL-4, and IL-10 on the production of (pro-) inflammatory mediators after stimulation of the cells with lipopolysaccharide (LPS; 0.1 micrograms/ml, 24 h). IL-10 (10 and 100 ng/ml) and IL-4 (5 and 50 U/ml) suppressed the LPS-induced production of NO, IL-6, and TNF-alpha in a dose-dependent manner, whereas TGF-beta1/beta2 (2 and 20 ng/ml) only suppressed NO production. LPS-induced levels of IL-1beta were suppressed by IL-10, but not by IL-4 and TGF-beta1/beta2. Conversely, co-incubation of the glial cells with LPS and antibodies to TGF-beta1/beta2 selectively enhanced LPS-induced NO production, whereas co-incubation with antibody to IL-10 enhanced LPS-induced production of all pro-inflammatory cytokines and NO. This finding strongly suggests that effective concentrations of TGF-beta1/beta2 and IL-10 are produced by LPS-stimulated glial cell co-cultures. Production of IL-10 in these co-cultures was confirmed by measurement of rat IL-10 by radioimmunoassay. We conclude that anti-inflammatory cytokines affect the production of inflammatory mediators in LPS-activated co-cultures of microglial and astroglial cells differentially.

Stimulation of endothelin B receptor modulates the inflammatory activation of rat astrocytes.

Inside the brain tissue, endothelins play numerous important biological roles. One of the targets, astrocytes, predominantly display endothelin receptor subtype B (ET(B)). On cultured primary rat astroglial cells, we analyzed the effect of IRL1620, a selective ET(B) receptor agonist, on the production of nitric oxide (NO) and the synthesis of interleukin (IL)-6 and tumor necrosis factor (TNF)-alpha. We performed these experiments in the presence or absence of interferon-gamma (IFN-gamma) and/or lipopolysaccharide (LPS). IRL1620 decreases NO production under basal conditions and after IFN-gamma stimulation. However, during LPS-induced NO production, IRL1620 enhances this release. The basal IL-6 secretion and especially the LPS-induced synthesis are enhanced by the IRL1620 stimulation. The LPS-dependent TNF-alpha production is increased by the ET(B) stimulation. The IRL1620-induced decrease of basal NO production is not dependent on Ca2+ entry or on phospholipase C (PLC) activation, as shown by the use of LaCl3 and U73122, respectively. In the presence of LPS, the IRL1620 potentiation of NO production is inhibited by LaCl3 and U73122. The IRL1620-induced increase of IL-6 is dependent on PLC activation. These results suggest that endothelins can have dual effects depending on the costimulatory factors present. Endothelins thus have important immunomodulatory functions in the brain.

Cyclic AMP regulates G oα protein and mRNA levels by modulating the transcriptional rate of G oα gene

In rat astroglial cells, four G oα transcripts were found: G o2α mRNA (5.7 kb) and three G o1α mRNAs (4.0, 3.0 and 2.3 kb). However, G o2α but little G oα1 proteins were present in membrane-enriched fractions. Culturing astroglial cells with forskolin (10 μM) or isoproterenol (10 μM) a β-adrenergic agonist increased transiently in a time-dependent manner the levels of G oα proteins. The degradation rate of G oα proteins was slightly decreased by the cAMP treatment. In parallel, forskolin (10 μM) treatment increased transiently the amounts of both G o1α and G o2α mRNAs. The relative transcription rate of G oα gene was increased by 1.7-fold in forskolin-treated cells whereas the half-lives of G o1α and G o2α mRNAs were not significantly changed. These results suggest that cAMP regulates the transcription rate of G oα gene and this is compatible with the existence of a cAMP responsive element in the promoter of the G oα gene.

Sodium salicylate and 17beta-estradiol attenuate nuclear transcription factor NF-kappaB translocation in cultured rat astroglial cultures following exposure to amyloid A beta(1-40) and lipopolysaccharides.

In recent years inflammatory mechanisms have become increasingly appreciated as important steps in the Alzheimer's pathogenic pathway. There is accumulating evidence that amyloid beta-peptide (A beta), the peptide product of the cleavage of amyloid precursor protein, may promote or exacerbate local inflammation by stimulating glial cells to release immune mediators. In addition, clinical studies using nonsteroidal antiinflammatory drugs have found a reduced risk for Alzheimer's disease with their use. Here we show that the neurotoxic A beta, a major plaque component, and lipopolysaccharides (LPS), an immune reaction-triggering portion of bacterial membranes, are both potent activators of the nuclear transcription factor NF-kappaB in primary rat astroglial cells. The activation was found to be concentration- and time-dependent and could be attenuated in the presence of NF-kappaB decoy nucleotides. The pretreatment by either 17beta-estradiol (1-10 microg) or sodium salicylate (3-30 mM) reduced the A beta (LPS)-induced activation of NF-kappaB by 48 (50%) and 60% (50%) of activated levels, respectively. In addition, 17beta-estradiol (10 microM) and sodium salicylate (10 mM) were able to attenuate the increase in interleukin-1beta levels following exposure to 25 microM A beta. Our data suggest that the aberrant gene expression is at least in part due to A beta-induced activation of NF-kappaB, a potent immediate-early transcriptional regulator of numerous proinflammatory genes; this event takes place in astroglial cells. The results of our experiments provide a further understanding of the effects of estrogen and aspirin on astroglial cells exposed to A beta and LPS.

Neosynthesis and Activation of Rho by Escherichia coli Cytotoxic Necrotizing Factor (CNF1) Reverse Cytopathic Effects of ADP-ribosylated Rho

Clostridium botulinum exoenzyme C3 inactivates the small GTPase Rho by ADP-ribosylation. We used a C3 fusion toxin (C2IN-C3) with high cell accessibility to study the kinetics of Rho inactivation by ADP-ribosylation. In primary cultures of rat astroglial cells and Chinese hamster ovary cells, C2IN-C3 induced the complete ADP-ribosylation of RhoA and concomitantly the disassembly of stress fibers within 3 h. Removal of C2IN-C3 from the medium caused the recovery of stress fibers and normal cell morphology within 4 h. The regeneration was preceded by the appearance of non-ADP-ribosylated RhoA. Recovery of cell morphology was blocked by the proteasome inhibitor lactacystin and by the translation inhibitors cycloheximide and puromycin, indicating that intracellular degradation of the C3 fusion toxin and the neosynthesis of Rho were required for reversal of cell morphology. Escherichia coli cytotoxic necrotizing factor CNF1, which activates Rho by deamidation of Gln(63), caused reconstitution of stress fibers and cell morphology in C2IN-C3-treated cells within 30-60 min. The effect of CNF1 was independent of RhoA neosynthesis and occurred in the presence of completely ADP-ribosylated RhoA. The data show three novel findings; 1) the cytopathic effects of ADP-ribosylation of Rho are rapidly reversed by neosynthesis of Rho, 2) CNF1-induced deamidation activates ADP-ribosylated Rho, and 3) inhibition of Rho activation but not inhibition of Rho-effector interaction is a major mechanism underlying inhibition of cellular functions of Rho by ADP-ribosylation.

Protective properties of tin- and manganese-centered porphyrins against hydrogen peroxide-mediated injury in rat astroglial cells.

Tin-mesoporphyrin (tin-mp), a potent inhibitor of heme oxygenase, and manganese (III) tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP), a potent superoxide dismutase mimetic, reduced H2O2 toxicity in cultures of transformed rat astroglial cells if added 30 min before, or at the same time as, H2O2. Reduced toxicity was not observed if treatment was delayed for 60 min, the time by which H2O2 was essentially eliminated from cultures. Coadministration of tin-mp and MnTMPyP did not increase protection over either compound administered individually. Tin-mp, but not MnTMPyP, was stable in culture. MnCl2 was not protective, suggesting that protection by MnTMPyP was not dependent on manganous ion, a by-product of MnTMPyP breakdown. Protection by tin-mp and MnTMPyP was not associated with metalloporphyrin-mediated induction of heme oxygenase-1 or with changes in heme oxygenase-2 on western blots. Whereas protective concentrations of tin-mp did not have superoxide dismutase-mimetic properties in vitro, protective concentrations of MnTMPyP partially inhibited heme oxygenase. The data support the hypothesis that heme oxygenase inhibition is protective against acute oxidative injury.

Structure-activity relationships of a series of analogues of the octadecaneuropeptide ODN on calcium mobilization in rat astrocytes.

The octadecaneuropeptide ODN (QATVGDVNTDRPGLLDLK), originally characterized as an endogenous ligand for central-type benzodiazepine receptors, increases intracellular calcium concentration ([Ca2+]i) in rat astroglial cells. A series of ODN analogues was synthesized, and each compound was studied for its ability to induce Ca2+ mobilization in cultured rat astrocytes. Replacement of each amino acid by an L-alanine residue (AlaScan) showed that the N-terminal region of the molecule was relatively tolerant to alanine substitution (2-8, 10), except for the Ala9-substituted analogue (9) which was totally devoid of activity. Pyroglutamization (21) and acetylation (22) of the Gln1 residue reduced the Ca2+ response suggesting that a free N-terminal amine function is required for full activity of ODN. Alanine substitution of the residues in the C-terminal region of the molecule (11-14, 16-18) significantly reduced the biological activity of ODN. In particular, modifications of the Leu15 residue (15, 20) abolished the Ca2+-mobilizing activity. The analogues [Ala9]ODN (9), [Ala15]ODN (15), [D-Thr9]ODN (19), and [D-Leu15]ODN (20) partially antagonized the Ca2+ response evoked by ODN. Most importantly, the octapeptide ODN11-18 (OP, 24) produced a dose-response curve that was superimposable to that obtained with ODN, indicating that the C-terminal region of the molecule possesses full biological activity. Finally, the AlaScan of OP revealed that replacement of the Leu5 residue by Ala (29) or D-Leu (33) totally suppressed the calcium response, confirming the crucial contribution of the Leu15 residue of ODN to the biological activity of the neuropeptide.

The effect of ethanol and acetaldehyde on microsomal and mitochondrial membrane fatty acid profiles in cultured rat astroglia.

It has been shown that free radical damage may be involved in ethanol-induced cytotoxicity in cultured neural cells. Since changes in oxidative metabolism and the resulting lipid peroxidation readily modify biological membranes and alter cell functions we studied the effect of ethanol and its metabolite acetaldehyde on rat astroglial fatty acids profiles in the most common lipid classes of mitochondrial and microsomal membranes, i.e. phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Rat astroglial cells were grown for 1 week in the presence of 50 m M or 100 m M ethanol. To examine acetaldehyde effects we used a 4-day co-culture model consisting of astroglial cells and alcohol dehydrogenase-transfected Chinese hamster ovary (CHO) cells. Acetaldehyde produced by these cells reached 172 mu M and 265 mu M, respectively, for ethanol concentrations of 10 and 20 m M. Mitochondrial and microsomal membranes were prepared by differential centrifugation, phosphatidylcholine and phosphatidylethanolamine were separated using thin layer chromatography and fatty acid quantitation was performed by GLC. Neither ethanol nor acetaldehyde changed the mitochondrial phosphatidylcholine or phosphatidylethanolamine profiles of total saturated, mono-unsaturated or polyunsaturated fatty acids. However, some significant alterations in particular fatty acids appeared especially after acetaldehyde but also after the highest ethanol dose. In microsomal phosphatidylcholine monounsaturated fatty acids were significantly increased after both, ethanol and acetaldehyde exposure. Among polyunsaturated fatty acids, arachidonic acid was found to be especially affected by both ethanol and acetaldehyde. Similar decreases were observed in adrenic, docosapentaenoic and docosahexaenoic acids in the groups treated with ethanol. In microsomal phosphatidylethanolamine, ethanol and acetaldehyde decreased monounsaturated and some polyunsaturated fatty acids. These data support the role of peroxidative processes in cultured rat astroglia exposed to ethanol and point to the role of acetaldehyde in this mechanism.

Regulation of nerve growth factor secretion and mRNA expression by bacterial lipopolysaccharide in primary cultures of rat astrocytes.

The present work was undertaken to study the effect of bacterial lipopolysaccharide (LPS), a potent activator of the host inflammatory response, on the synthesis of nerve growth factor (NGF) by newborn rat brain astrocytes. Treatment of primary rat astroglial cells cultured in chemically defined medium with LPS resulted in a dose-dependent accumulation of NGF mRNA, and an increased release of NGF protein in the cell medium. NGF mRNA levels were maximal after 24 hr of stimulation (8-fold increase), whereas extracellular NGF peaked after 72 hours of treatment (17-fold increase). This dramatic increase of extracellular NGF was abrogated if cells were treated with actinomycin D or cycloheximide, a fact which implies that the accumulation of extracellular NGF by LPS-treated cells requires DNA transcription and RNA translation. Stimulation of NGF synthesis and secretion was: (i) unaffected by treatment with the protein kinase C inhibitor bisindolylmaleimide, and (ii) prevented by forskolin and 3-isobutyl-1-methylxanthine, two agents which increase cAMP levels. Inhibition of LPS effect was also obtained with apigenin, a proposed inhibitor of the mitogen-activated protein kinase pathway. Results thus show that LPS stimulates NGF synthesis by astroglial cells through a mechanism that is independent of protein kinase C (PKC), antagonized by cAMP-elevating agents, and probably mediated by the mitogen-activated protein kinase cascade. The data raise the possibility that LPS exerts stimulatory effects on NGF synthesis that are independent of those elicited by astrocyte-derived inflammatory lymphokines such as IL-1beta, TNF alpha or TGF beta1.

A Putative Tetrapeptide Antagonist Prevents β-Amyloid-Induced Long-Term Elevation of [Ca2+]iin Rat Astrocytes

Comparative fluorimetric studies on the long-term (8-hour) action of β[1-42]amyloid and its shorter fragments β[1-40], β[25-35] and β[31-35] on the steady-state intracellular Ca2+concentration in primary cultures of rat astroglial cells using the Ca2+-sensitive fluorescent probe Fura-2 AM revealed higher 340/380 fluorescence excitation ratios in the treated cells as compared to the untreated controls. All the peptides were found to induce similar cellular effects, suggesting the [31-35] region as the putative active centre of the molecule. No significant alteration was detectable in Fura-2 fluorescence using the Ca2+-insensitive excitation wavelength of 367 nm, indicating that the observed changes reflect a real alteration in the Ca2+concentration of the cells. Moreover, no considerable difference was observed in the total protein content of treated and untreated cells. Co-treatment of the cells with Pr-Ile-Ile-Gly-Leu-NH2(Pr-IIGL) peptide, an analogue of the [31-34] region of β[1-42]-amyloid, was found to effectively antagonize the β[1-42]-amyloid-induced elevation of the fluorescence excitation ratio, leaving the 367-nm fluorescence unaffected. To the best of the authors’ knowledge, this is the first report on an analogue of β-amyloid peptide capable of blocking one of its physiological effects, thereby raising the possibility that this sequence could prove to be a lead compound for designing effective β-amyloid antagonists.

Metabotropic glutamate receptor expression in cultured rat astrocytes and human gliomas.

In order to confirm the existence of metabotropic glutamate receptors in astroglial cultures and to provide information on different receptor subtypes, the expression of different mGluRs was analysed in cultures highly enriched in rat astroglial cells. mRNA levels for mGluR1, 2, 3, 4, 7 were undetectable by Northern blot analysis in primary type-1 astroglial cultures derived from total cerebral hemispheres, cerebral cortex and striatum. Interestingly, these cultures expressed a low, but detectable, level of mGluR5 mRNA. The more sensitive technique Reverse Transcription-Polymerase Chain Reaction (RT-PCR) confirmed the presence of mGluR5 transcript in cultured astrocytes and, in addition, revealed the presence of mGluR3 mRNA. The lack of expression of mGluR5 in CG-4 cells, a rat cell line able to differentiate in type-2 astrocytes or oligodendrocytes depending on the culture conditions, suggested that the presence of mGluR5 was not a general feature of cells of glial origin. Moreover, all the examined mGluR transcript were undetectable by RT-PCR in CG4 cells. In order to confirm the possible expression of mGluR5 in cell of glial origin we examined the mRNA levels for this receptor in tissue samples from human gliomas obtained after surgical resection of the tumors: only 1 sample (grade II astrocytoma), out of 8 examined, showed the presence of mGluR5 mRNA. In conclusion our data show that the only cloned metabotropic receptor linked to phosphoinositide hydrolysis, whose expression is detectable in cultured type-1 astrocytes, in mGluR5. It remains to be established if the low level of expression of mGluR3 could be responsible for the group II metabotropic glutamate receptor activity previously observed in cultured astroglial cells.

Insulin promotes the hydrolysis of a glycosyl phosphatidylinositol in cultured rat astroglial cells.

Glycosyl phosphatidylinositols have been implicated in insulin signaling through their action as precursors of second messenger molecules in peripheral tissues. In the present study, cultured rat astrocytes were used to investigate whether glycosyl phosphatidylinositol might be involved in the mechanism of insulin signal transduction in neural cells. A glycosyl phosphatidylinositol sensitive to hydrolysis by both phosphatidylinositol-specific phospholipase C and glycosyl phosphatidylinositol-specific phospholipase D and to nitrous acid deamination was purified. When astrocytes were exposed to 10 nM insulin, a rapid and significant reduction in the content of glycosyl phosphatidylinositol was observed within 1-2 min. In addition, an inverse concentration-dependent relationship between glycosyl phosphatidylinositol and diacylglycerol levels was found, suggesting a phospholipase C-mediated hydrolysis of glycosyl phosphatidylinositol in response to insulin. The effects of insulin were mediated through its own receptors and not through insulin-like growth factor (IGF)-I and/or IGF-II receptors, as demonstrated by affinity cross-linking studies. Also, the effects of 5 nM IGF-1 or 5 nM IGF-II on glycosyl phosphatidylinositol and diacylglycerol levels were different from those caused by insulin and were not essentially modified by pretreatment of the cells with either platelet-derived growth factor (PDGF) or epidermal growth factor (EGF). When cells were sequentially incubated with PDGF and EGF, a reduction in both glycosyl phosphatidylinositol and diacylglycerol contents was observed; the diacylglycerol but not the glycosyl phosphatidyl content was reversed after incubation with IGF-I, and especially with IGF-II, for 10 min. Despite the remarkable homology among insulin, IGF-I, and IGF-II, our results indicate that in astrocytes these compounds probably use different signal transduction pathways.

Long-term effect of forskolin on the activation of adenylyl cyclase in astrocytes.

Long-term (48-h) forskolin treatment of rat astroglial cells led to a slight decrease (30-40%) in the response to isoproterenol, vasoactive-intestinal peptide, guanyl 5'-(beta gamma-imido)diphosphate, guanosine 5'-O-(3-thiotriphosphate) [GTP(S)], and AIF4- in crude membrane fractions. In contrast, the acute stimulatory effect of forskolin was increased by 1.25-1.5-fold. These two opposite effects of forskolin were mediated by a cyclic AMP-dependent mechanism. No changes in Gs alpha, Gi alpha, or G beta protein levels could be determined by immunoblotting using specific antisera. No significant differences were observed in the ability of G proteins extracted from control and forskolin-treated cells to reconstitute a full adenylyl cyclase activity in membranes from S49 cyc- cells, lacking Gs alpha protein. Gs alpha proteins were detected in two pools of membranes, one in the heavy sucrose fractions and the other in light sucrose fractions. Forskolin treatment of the cells shifted Gs alpha protein toward the light-density membranes. We did not find any significant change in the distribution of adenylyl cyclase. In contrast to the decreased stimulation of adenylyl cyclase activity by agonists acting via Gs alpha, observed in the crude membrane fraction, the responses of adenylyl cyclase to forskolin as well as to GTP(S) were increased in the purified plasma membrane fractions. These results may indicate that sensitization of the catalyst appears to be the dominant component in the astroglial cell response to long-term treatment by forskolin.

Cell cycle kinetics and commitment in newborn, adult, and tumoral astrocytes

In terms of cell cycle phases, mammalian astrocytes maintain the capacity to leave G0G1 and enter S phase in response to brain injury or due to neoplastic transformation. This report compares proliferative behavior in vitro, particularly departure from G0, in three types of rat astroglial cells — newborn astrocytes, astrocytes from gelatin implants into the traumatized striata of adults, and astrocytoma cells (C6 glioma). Newborn and adult astrocytes demonstrated nearly identical proliferation kinetics as determined by peaks in cell number and rates of DNA synthesis. C6 glioma (C6G) proliferated more rapidly. Exit from G0 was examined by shift-down of serum from 10 to 0.1% for 48 h, followed by return to 10% at time 0. Synchronization of newborn and adult astrocytes in this way resulted in a 12 h lag phase (G0G1) followed by a 6–10-fold surge in DNA synthesis and a corresponding increase in S-phase nuclei from < 15% to > 70%. Timing of S-phase commitment was established in late G1 by resistance to the inhibitors cycloheximide and mevinolin. Decay of commitment was assessed by addition of hydroxyurea (HU) at 10 h to cause accumulation at the G1S boundary. Removal of HU after an additional 14, 16, and 20 h resulted respectively in these percentages of maximal S-phase DNA synthesis in newborn and adult astrocytes: 75 ± 9, 60 ± 8, 23 ± 3, and 87 ± 20, 62 ± 7, 34 ± 5. In contrast, synchronization of C6G resulted in a 6 h lag before a surge in DNA synthesis and an increase in S-phase nuclei from < 20% to 100%. Cell cycle commitment occurred earlier with C6G, and decay of commitment was not observed, even after 20 h of HU treatment. Thus, these in vitro techniques for cell cycle analysis are applicable to astrocytes obtained from developing and adult brain, and to at least some astroglioma cells. Furthermore, this comparative study showed that important cell cycle parameters differ markedly in the non-tumoral astrocytes and glioma cells. These differences could lead to strategies for selective targeting of the proliferation of neoplastic astroglia.

Cell cycle kinetics and commitment in newborn, adult, and tumoral astrocytes

In terms of cell cycle phases, mammalian astrocytes maintain the capacity to leave G 0 G 1 and enter S phase in response to brain injury or due to neoplastic transformation. This report compares proliferative behavior in vitro, particularly departure from G 0, in three types of rat astroglial cells — newborn astrocytes, astrocytes from gelatin implants into the traumatized striata of adults, and astrocytoma cells (C6 glioma). Newborn and adult astrocytes demonstrated nearly identical proliferation kinetics as determined by peaks in cell number and rates of DNA synthesis. C6 glioma (C6G) proliferated more rapidly. Exit from G 0 was examined by shift-down of serum from 10 to 0.1% for 48 h, followed by return to 10% at time 0. Synchronization of newborn and adult astrocytes in this way resulted in a 12 h lag phase ( G 0 G 1 ) followed by a 6–10-fold surge in DNA synthesis and a corresponding increase in S-phase nuclei from < 15% to > 70%. Timing of S-phase commitment was established in late G 1 by resistance to the inhibitors cycloheximide and mevinolin. Decay of commitment was assessed by addition of hydroxyurea (HU) at 10 h to cause accumulation at the G 1 S boundary. Removal of HU after an additional 14, 16, and 20 h resulted respectively in these percentages of maximal S-phase DNA synthesis in newborn and adult astrocytes: 75 ± 9, 60 ± 8, 23 ± 3, and 87 ± 20, 62 ± 7, 34 ± 5. In contrast, synchronization of C6G resulted in a 6 h lag before a surge in DNA synthesis and an increase in S-phase nuclei from < 20% to 100%. Cell cycle commitment occurred earlier with C6G, and decay of commitment was not observed, even after 20 h of HU treatment. Thus, these in vitro techniques for cell cycle analysis are applicable to astrocytes obtained from developing and adult brain, and to at least some astroglioma cells. Furthermore, this comparative study showed that important cell cycle parameters differ markedly in the non-tumoral astrocytes and glioma cells. These differences could lead to strategies for selective targeting of the proliferation of neoplastic astroglia.

Clusterin (SGP-2) induction in rat astroglial cells exposed to prion protein fragment 106-126.

Prion-related encephalopathies are characterized by the accumulation of an abnormal prion protein isoform (PrPSc) associated with neuronal degeneration and astrogliosis. The synthetic peptide homologous to PrP fragment 106-126 (PrP 106-126) induced in vitro neuronal apoptosis and glial proliferation. We used Northern blot analysis and the RNA polymerase chain reaction to assess the expression of several genes associated with programmed cell death and proliferation. Blots of total RNA extracted from neuronal and astroglial cells exposed to PrP 106-126 for between 1 h and 7 days were hybridized with probes recognizing c-fos, c-jun, c-myc, p53, hsp-70 and bcl-2 mRNA. Except for a slight decrease in bcl-2 mRNA in neuronal cells, no change in other transcripts was evident. Since clusterin (apolipoprotein J) mRNA levels are increased in prion-related encephalopathies and clusterin immunoreactivity has been located in association with PrPSc in Gerstmann-Sträussler-Scheinker brain, the expression of clusterin was determined in neuronal and astroglial cells chronically exposed to PrP 106-126. Although the induction of clusterin has been involved in the apoptotic mechanism in other experimental conditions, its expression was unchanged in PrP 106-126-treated neurons, while a three-fold induction of clusterin mRNA was observed in astrocytes exposed to PrP 106-126. To investigate whether the clusterin up-regulation was simply associated with the astroglial proliferative stimulus of PrP 106-126 or was specifically induced by the peptide, we measured clusterin expression in astrocytes cultured in fetal calf serum-free medium and exposed to PrP 106-126 or fetal calf serum restoration. In this condition the PrP peptide, like fetal calf serum, increased the glial proliferation rate, but only PrP 106-126 doubled clusterin mRNA. The selectivity of this effect indicates that PrPSc is directly involved in the clusterin up-regulation seen in prion-related encephalopathies and is associated with astroglial cells.