Functional studies in cultured astrocytes

Studies using primary cultures of astrocytes have made essential contributions to the understanding of astrocytic functions and neuronal-astrocytic interactions. The purposes of this article are to (i) outline principles and methodologies used in the preparation of such cultures and caveats for the interpretation of the observations made; (ii) summarize astrocytic functions in turnover of the amino acid transmitters glutamate and gamma-aminobutyric acid (GABA), in energy metabolism and in Na+,K+-ATPase-catalyzed processes and emphasize the degree to which the observations have been confirmed in intact tissue; (iii) describe regulations of astrocytic functions by transmitters and by calcium channel activity; and (iv) indicate suggestions for future functional studies using astrocytes in primary cultures and emphasize that some of the conclusions about neuronal-astrocytic interactions reached on the basis of studies in cultured cells and confirmed in intact tissue may not yet have been completely integrated into general neuroscience knowledge.     

Lysosomal cysteine cathepsins: signaling pathways in apoptosis

Apoptosis is the major mechanism by which eukaryotic organisms eliminate potentially dangerous, superfluous and damaged cells. Initially, nuclei and mitochondria were found to be the key organelles involved in the process. However, recent data suggest that lysosomes and the endoplasmic reticulum also play important roles in the process. A number of different stimuli were found to directly or indirectly target the lysosomal membrane, thereby inducing lysosomal permeabilization and the release of cysteine cathepsins and the aspartic protease cathepsin D into the cytosol. Once in the cytosol, cathepsins can trigger cell death by different mechanisms. Here we discuss the different signaling pathways used by lysosomal proteases to trigger apoptosis and their potential role in physiological processes.     

Vesicle mobility studied in cultured astrocytes

Astrocytes release many neuroactive substances, which are stored in membrane bound vesicles and may play a role in synapse modulation and in the coupling between neuronal activity and the local blood flow. However, the mobility of these vesicles in astrocytes has not been studied yet. We here used a fluorescently tagged proatrial natriuretic peptide to label single vesicles and dynamic microscopy to monitor their mobility. To track and analyze labeled vesicles, we employed a computer software. We found two modes of vesicle mobility, directional and non-directional. The mobility of non-directional vesicles is likely determined mainly by free diffusion. Only directional vesicles displayed a straight-line motion. The relationship of mean square displacement with time in directional vesicles resembled a quadratic function, indicating that in addition to free diffusion other mechanisms may contribute to vesicle movements in astrocytes, the biophysical properties of which are similar to those of neurons.     

Biosynthesis of lysosomal cathepsins B and H in cultured rat hepatocytes

The biosynthesis of lysosomal cysteine proteases, cathepsins B and H, was investigated by using pulse-chase experiments in vivo in primary cultures of rat hepatocytes. Cathepsins B and H were isolated from either total cell extracts or culture medium labeled with [35S]methionine by immunoprecipitation and analyzed for their molecular forms. Within 60 min of chase, cellular proforms of cathepsins B of 39 kDa and H of 41 kDa were converted to single-chain form cathepsins B of 29 kDa and H of 28 kDa, respectively, and persisted as these forms even after 12-h chase periods. The proforms of cathepsins B and H derived from pulse-labeling experiments showed complete susceptibility to endoglycosidase H treatment, indicating that these proenzymes bear high-mannose-type oligosaccharides at the stage of initial events of biosynthesis. In the presence of tunicamycin, unglycosylated proenzymes of cathepsins B of 35 kDa and H of 34 kDa were found to be secreted into the extracellular medium without undergoing proteolytic processing. Furthermore, in the presence of swainsonine, a potent inhibitor of Golgi mannosidase II, considerable amounts of the proenzymes were secreted and accumulated in the medium during chasing periods. These results suggest that the oligosaccharide moiety of these enzymes would be necessary for the intracellular sorting mechanism. In monensin-treated cells, the conversion of intracellular proenzymes to mature enzymes was significantly inhibited and the proenzymes were secreted into the medium. In the presence of chloroquine or ammonium chloride, proteolytic processing of the proenzymes was completely prevented and the enhanced secretion of proenzymes was observed. These results suggest that in the presence of lysosomotropic amines the intracellular sorting of proenzymes might not occur properly during biosynthesis.     

Characterization of cystine uptake in cultured astrocytes

Glutathione is involved in the maintenance of the structural and functional integrity of membrane proteins, in protection against free radicals and oxidative stress, and in the detoxification of xenobiotics. The cellular uptake of cystine is the rate limiting step in the biosynthesis of glutathione. The precise mechanism for such uptake is not clear as some reports indicate that the uptake occurs through a glutamate-cystine antiporter (system X(c)(-)), whereas, others suggest that it is taken up by the glutamate transporter (system X(AG)). Our studies in cultured astrocytes derived from neonatal rats showed that glutamate, D- and L-aspartate inhibited cystine uptake; that factors that increased intracellular glutamate levels, which would have enhanced the activity of the antiporter, did not stimulate cystine uptake; that the uptake was sodium dependent and partially chloride dependent; that the b(o,+) and ASC systems, which have been shown to carry cystine in some cells, did not mediate cystine uptake in astrocytes; that glutamate uptake blockers such as L-aspartate-beta-hydroxamate (AbetaH) and L-trans-pyrrolidine-2,4-dicarboxylate (PDC), as well as cystine uptake inhibitor L-alpha-aminoadipate (AAA) potently reduced cystine uptake. Additionally, deferoxamine (100 microM) as well as ammonium chloride (5 mM), both of which inhibit glutamate uptake, also inhibited cystine uptake. Taken together, our findings indicate that astrocytes take up cystine through a similar, if not identical, system used to take up glutamate. Interference of cystine uptake by astrocytes through the glutamate transport system may have profound effects on the redox state and the structural and functional integrity of the CNS.     

Copper export from cultured astrocytes

Copper is an essential trace metal that is required as a catalytic co-factor or a structural component of several important enzymes. However, since excess of copper can also harm cells due to its potential to catalyse the generation of toxic reactive oxygen species, transport of copper and the cellular copper content are tightly regulated. Astrocytes are known to efficiently take up copper ions, but it was not known whether these cells are also able to export copper. Treatment of astrocyte-rich primary cultures for 24 h with copper chloride caused a concentration-dependent increase in the specific cellular copper content. During further 24 h incubation in the absence of copper chloride, the copper-loaded astrocytes remained viable and released up to 45% of the accumulated copper. The rate of copper export was proportional to the amount of cellular copper, was almost completely prevented by lowering the incubation temperature to 4 °C and was partly prevented by the endocytosis inhibitor amiloride. Copper export is most likely mediated by the copper ATPase ATP7A, since this transporter is expressed in astrocyte cultures and its cellular location is strongly affected by the absence or the presence of extracellular copper. The potential of cultured astrocytes to export copper suggests that astrocytes provide neighbouring cells in brain with this essential trace element.     

Swelling and potassium uptake in cultured astrocytes

The intracellular water content of astrocytes in primary cultures shows a biphasic swelling pattern on exposure to various increased external K+ concentrations over the range of 1.5-100 mM. The two phases (physiological, 1.5-12 mM K+; pathological, 25-100 mM K+) are based on two different mechanisms. Both can be blocked by low Cl- solutions and involve intensive net uptake of K+. However, the physiological phase consists of the activation of a KCl + NaCl carrier, while the Na+ in turn is pumped out by Na+-K+ ATPase, with a resultant net accumulation of KCl. At pathological K+ concentrations the KCl + NaCl carrier is less active because the Na+ driving force, its energy source, is reduced (owing to depolarization by K+). However, the Donnan equilibrium across the cell membrane is heavily disturbed, which leads to passive KCl accumulation. The results suggest that volume changes in cultured glial cells during exposure to high K+ should be taken into consideration since they disguise K+ accumulation when only ion activity is measured.     

Comparative analysis of optogenetic actuators in cultured astrocytes

Astrocytes modulate synaptic transmission via release of gliotransmitters such as ATP, glutamate, d-serine and l-lactate. One of the main problems when studying the role of astrocytes in vitro and in vivo is the lack of suitable tools for their selective activation. Optogenetic actuators can be used to manipulate astrocytic activity by expression of variants of channelrhodopsin-2 (ChR2) or other optogenetic actuators with the aim to initiate intracellular events such as intracellular Ca²⁺ ([Ca²⁺]_i) and/or cAMP increases. We have developed an array of adenoviral vectors (AVV) with ChR2-like actuators, including an enhanced ChR2 mutant (H134R), and a mutant with improved Ca²⁺ permeability (Ca²⁺ translocating channelrhodopsin, CatCh). We show here that [Ca²⁺]_i elevations evoked by ChR2(H134R) and CatCh in astrocytes are largely due to release of Ca²⁺ from the intracellular stores. The autocrine action of ATP which is released under these conditions and acts on the P2Y receptors also contributes to the [Ca²⁺]_i elevations. We also studied effects evoked using light-sensitive G-protein coupled receptors (opto-adrenoceptors). Activation of optoα1AR (Gq-coupled) and optoβ2AR (G_s-coupled) resulted in astrocytic [Ca²⁺]_i increases which were suppressed by blocking the corresponding intracellular signalling cascade (phospholipase C and adenylate cyclase, respectively). Interestingly, the bulk of [Ca²⁺]_i responses evoked using either optoAR was blocked by an ATP degrading enzyme, apyrase, or a P2Y1 receptor blocker, MRS 2179, indicating that they are to a large extent triggered by the autocrine action of ATP. We conclude that, whilst optimal tools for control of astrocytes are yet to be generated, the currently available optogenetic actuators successfully initiate biologically relevant signalling events in astrocytes.     

Effects of ammonia onl-glutamate uptake in cultured astrocytes

The effect of ammonia onl-glutamate (L-GLU) uptake was examined in cultured astrocytes. Acute ammonia treatment (5–10 mM) enhanced L-[³H]GLU uptake by 20–42% by increasing the V_max; this persisted for 2 days and then started to decline. Ammonia, however, did not affect the uptake ofd-[³H]aspartate (D-ASP), a non-metabolizable analog of L-GLU, that uses the same transport carrier as L-GLU. Also, L-GLU uptake was not affected during the first 2 min of the assay. Thus, ammonia did not have an acute effect on L-GLU transport (translocation); rather, ammonia enhanced the accumulation or “trapping” of L-GLU or its by-products. Chronic ammonia treatment, on the other hand, inhibited L-GLU transport in astrocytes by ∼30–45% and this was due to a decrease in V_max, suggesting that the number of L-GLU transporters was decreased. This inhibitory effect was observed after 1 day of treatment and persisted for at least 7 days. The inhibition of L-GLU transport was partially reversible following removal of ammonia. The effects of ammonia on L-GLU transport and uptake may explain the abnormal L-GLU neurotransmission observed in hyperammonemia/hepatic encephalopathy, and the brain swelling associated with fulminant hepatic failure.     

Exocytotic release of ATP from cultured astrocytes

Astrocytes appear to communicate with each other as well as with neurons via ATP. However, the mechanisms of ATP release are controversial. To explore whether stimuli that increase [Ca(2+)](i) also trigger vesicular ATP release from astrocytes, we labeled ATP-containing vesicles with the fluorescent dye quinacrine, which exhibited a significant co-localization with atrial natriuretic peptide. The confocal microscopy study revealed that quinacrine-loaded vesicles displayed mainly non-directional spontaneous mobility with relatively short track lengths and small maximal displacements, whereas 4% of vesicles exhibited directional mobility. After ionomycin stimulation only non-directional vesicle mobility could be observed, indicating that an increase in [Ca(2+)](i) attenuated vesicle mobility. Total internal reflection fluorescence (TIRF) imaging in combination with epifluorescence showed that a high percentage of fluorescently labeled vesicles underwent fusion with the plasma membrane after stimulation with glutamate or ionomycin and that this event was Ca(2+)-dependent. This was confirmed by patch-clamp studies on HEK-293T cells transfected with P2X(3) receptor, used as sniffers for ATP release from astrocytes. Glutamate stimulation of astrocytes was followed by an increase in the incidence of small transient inward currents in sniffers, reminiscent of postsynaptic quantal events observed at synapses. Their incidence was highly dependent on extracellular Ca(2+). Collectively, these findings indicate that glutamate-stimulated ATP release from astrocytes was most likely exocytotic and that after stimulation the fraction of quinacrine-loaded vesicles, spontaneously exhibiting directional mobility, disappeared.     

Gliotoxic action of glutamate on cultured astrocytes

Because of the well-documented importance of glutamate clearance by astrocytes in protecting neurons against excitotoxicity, it was interesting to examine whether L-glutamate exerts a toxic action on cultured astrocytes. Cell damage was evaluated by measuring activity of lactate dehydrogenase (LDH) released into the culture medium. Exposure of astrocyte cultures of the neonatal rat cerebral cortex to L-glutamate resulted in a concentration- and time-dependent increase in the release of LDH. L-Glutamate-induced gliotoxicity appeared to be mediated predominantly by the increase of oxidative stress because the reduced glutathione content and its effects were almost completely blocked by vitamin E and pyrrolidinedithiocarbamate. To support this notion further, the supplementation or depletion of intracellular reduced glutathione content attenuated or worsened L-glutamate toxicity, respectively. Activation of the glutamate transporter mimicked the action of L-glutamate on astrocytes. In addition, degrees of cell damage were not directly correlated to the levels of glutamate uptake. Moreover, the mechanism of this toxicity required energy and macromolecular synthesis. Taken together, brief exposure to L-glutamate resulted in glutamate uptake and cell swelling, whereas sustained exposure injured astrocytes via oxidative stress instead of the excitatory mechanism.     

Inositol phospholipid hydrolysis in cultured astrocytes and oligodendrocytes

Cultures of astrocytes and oligodendrocytes were prelabeled with 3H-inositol and the accumulation of 3H-inositol phosphates was determined following stimulation with a number of neuroactive substances. In astrocytes, norepinephrine (NE) produced the greatest stimulation with significant increase also observed with bradykinin. In oligodendrocytes, the greatest stimulation was produced by carbachol with significant increase also produced by bradykinin, histamine and NE. Carbachol was found to be ineffective in producing stimulation in astrocytes. The accumulation of 3H-inositol phosphates in astrocytes in response to NE was found to be dependent on the presence of Li+. The NE stimulation in astrocytes was dose-dependent and had an EC50 of 1.2 microM. This stimulation was blocked by the low concentration of the alpha 1-adrenergic antagonist prazosin but not by the alpha 2-adrenergic antagonist yohimbine. The NE-stimulated accumulation of 3H-inositol phosphates in astrocytes was inhibited by the cyclic nucleotide phosphodiesterase inhibitor isobutylmethylxanthine as well as by the cAMP analog dibutyryl cAMP.     

NMDA-responsible, APV-insensitive receptor in cultured human astrocytes

The present study was conducted to assess N-methyl-D-aspartate (NMDA)-responsible receptors in cultured human astrocytes by monitoring whole-cell membrane currents. NMDA generated currents, that are potentiated by glycine and blocked by Mg2+, with the current/voltage relation showing a reversal potential of +/- 0 mV. The currents were not inhibited by either the selective NMDA receptor antagonist, D-2-amino-5-phosphonovaleric acid (APV), or the non-selective ionotropic glutamate receptor antagonist, kynurenic acid. The currents were inhibited only by 19% in Ca2+-free extracellular solution. Furthermore, GDPbetaS, a broad G-protein inhibitor, inhibited NMDA-induced currents to 82% of original levels. The results of the present study thus suggest that an NMDA-responsible, APV-insensitive receptor with low Ca2+ permeability, distinct from the neuronal NMDA receptors, is expressed in human astrocytes and that the receptor is regulated in part by an unknown G-protein-linked receptor.     

A voltage-gated chloride conductance in rat cultured astrocytes

Large voltage-dependent outward currents are recorded with the whole-cell patch-clamp technique from rat cultured astrocytes under conditions where an outward movement of potassium ions is excluded (either by blockage of the potassium channels pharmacologically or by replacement of the internal potassium by the impermeant large organic cation N-methyl-(+)-glucamine). The current, which is activated at potentials more positive than -40 to -50 mV, is normally carried by an inward movement of chloride ions. Its reversal potential is the same as the chloride equilibrium potential. With depolarization to +60 mV (for 225 ms) little or no inactivation of the current occurs: with depolarizations to +90 to +110 mV a time-dependent decay is seen. The current, which is often not marked immediately after formation of the whole-cell clamp, generally increases over a period of a few minutes to a maximum (after which it usually declines), as if some as yet unknown intracellular factor keeping the channels closed were being washed away from the membrane. The time course of this phenomenon is not affected by changing of the internal free calcium concentration (from 10(-8)M to 10(-6)M) or by an intracellular mixture of cyclic AMP (1 mM), ATP (4 mM) and Mg+ (2 mM). The conductance is slightly increased when the chloride of the bathing medium is replaced by bromide; is much reduced on replacement by methylsulphate, sulphate, isethionate, or acetate; and is virtually abolished on replacement by the large anion gluconate. The outward current is inhibited by the disulphonate stilbenes DIDS and SITS; this blocking action was initially partly reversible, although never completely so. It is suggested that the chloride conductance plays a role in the spatial buffering of potassium by astrocytes.     

Energy-dependent volume regulation in primary cultured cerebral astrocytes

Cell volume regulation and energy metabolism were studied in primary cultured cerebral astrocytes during exposure to media of altered osmolarity. Cells suspended in medium containing 1/2 the normal concentration of NaCl (hypoosmotic) swell immediately to a volume 40-50% larger than cells suspended in isoosmotic medium. The cell volume in hypoosmotic medium then decreases over 30 min to a volume approximately 25% larger than cells in isoosmotic medium. In hyperosmotic medium (containing twice the normal concentration of NaCl), astrocytes shrink by 29%. Little volume change occurs following this initial shrinkage. Cells resuspended in isoosmotic medium after a 30 min incubation in hypoosmotic medium shrink immediately to a volume 10% less than the volume of cells incubated continuously in isoosmotic medium. Thus, the regulatory volume decrease (RVD) in hypoosmotic medium involves a net reduction of intracellular osmoles. The RVD is partially blocked by inhibitors of mitochondrial electron transport but is unaffected by an inhibitor of glycolysis or by an uncoupler of oxidative phosphorylation. Inhibition of RVD by these metabolic agents is correlated with decreased cellular ATP levels. Ouabain, added immediately after hypoosmotic induced swelling, completely inhibits RVD, but does not alter cell volume if added after RVD has taken place. Ouabain also inhibits cell respiration 27% more in hypoosmotic medium than in isoosmotic medium indicating that the (Na,K)-ATPase-coupled ion pump is more active in the hypoosmotic medium. These data suggest that the cell volume response of astrocytes in hypoosmotic medium involves the net movement of osmoles by a mechanism dependent on cellular energy and tightly coupled to the (Na,K)-ATPase ion pump. This process may be important in the energy-dependent osmoregulation in the brain, a critical role attributed to the astrocyte in vivo.     

Involvement of calcium-sensing receptor in ischemia/reperfusion-induced apoptosis in rat cardiomyocytes

The calcium-sensing receptor (CaR) is a seven-transmembrane G-protein coupled receptor, which activates intracellular effectors, for example, it causes inositol phosphate (IP) accumulation to increase the release of intracellular calcium. Although intracellular calcium overload has been implicated in the cardiac ischemia/reperfusion (I/R)-induced apoptosis, the role of CaR in the induction of apoptosis has not been fully understood. This study tested the hypothesis that CaR is involved in I/R cardiomyocyte apoptosis by increasing [Ca2+]i. The isolated rat hearts were subjected to 40-min ischemia followed by 2 h of reperfusion, meanwhile GdCl3 was added to reperfusion solution. The expression of CaR increased at the exposure to GdCl3 during I/R. By laser confocal microscopy, it was observed that the intracellular calcium was significantly increased and exhibited a Deltapsim, as monitored by 5,5',6,6'-tetrachloro-1,1',3,3'- tetraethylbenzimidazolcarbocyanine iodide (JC-1) during reperfusion with GdCl3. Furthermore, the number of apoptotic cells was significantly increased as shown by TUNEL assay. Typical apoptotic cells were observed with transmission electron microscopy in I/R with GdCl3 but not in the control group. The expression of cytosolic cytochrome c and activated caspase-9 and caspase-3 was significantly increased whereas the expression of mitochondrial cytochrome c significantly decreased in I/R with GdCl3 in comparison to the control. In conclusion, these results suggest that CaR is involved in the induction of cardiomyocyte apoptosis during ischemia/reperfusion through activation of cytochrome c-caspase-3 signaling pathway.     

Osmolyte contents of cultured astrocytes grown in hypoosmotic medium

Primary rat cerebral astrocyte cultures were grown for 2 weeks in isoosmotic medium (305 mosmol) and then placed in similar medium with a reduced NaCl concentration. During the first hour of growth in this moderately hypoosmotic medium (240 mosmol), the cells lose 88% of their taurine contents, 62% of their alanine contents, and 54% of their aspartate contents while regaining normal volume. Loss of these amino acids accounts for 43% of observed volume regulation. Contents of these amino acids remain decreased during 24 h of growth in hypoosmotic medium. In contrast, potassium, glutamate, glutamine, and asparagine contents are not changed, relative to cells in isoosmotic medium, at time points between 1 h and 24 h of hypoosmotic exposure. The data suggest astrocytes contribute to net loss of amino acids, but not potassium, from brains exposed to hypoosmotic conditions in situ.     

Expression of phosphoinositide-specific phospholipase C isoenzymes in cultured astrocytes

Signal transduction from plasma membrane to cell nucleus is a complex process depending on various components including lipid signaling molecules, in particular phosphoinositides and their related enzymes, which act at cell periphery and/or plasma membrane as well as at nuclear level. As far as the nervous system may concern the inositol lipid cycle has been hypothesized to be involved in numerous neural as well as glial functions. In this context, however, a precise panel of glial PLC isoforms has not been determined yet. In the present experiments we investigated astrocytic PLC isoforms in astrocytes obtained from foetal primary cultures of rat brain and from an established cultured (C6) rat astrocytoma cell line, two well known cell models for experimental studies on glia. Identification of PLC isoforms was achieved by using a combination of RT-PCR and immunocytochemistry experiments. While in both cell models the most represented PI-PLC isoforms were beta4, gamma1, delta4, and epsilon, isoforms PI-PLC beta2 and delta3 were not detected. Moreover, in primary astrocyte cultures PI-PLC delta3 resulted well expressed in C6 cells but was absent in astrocytes. Immunocytochemistry performed with antibodies against specific PLC isoforms substantially confirmed this pattern of expression both in astrocytes and C6 glioma cells. In particular while some isoenzymes (namely isoforms beta3 and beta4) resulted mainly nuclear, others (isoforms delta4 and epsilon) were preferentially localized at cytoplasmic and plasma membrane level.     

Effects of homocysteine on metabolic pathways in cultured astrocytes

Homocysteine is an amino acid that is an important risk factor for several neurodegenerative diseases such as Alzheimer's and Parkinson's disease. Increased homocysteine levels induce neuronal cell death in a variety of neuronal types. However, very few studies have probed the effects of homocysteine in astrocytes. The present study investigated the effects of homocysteine on primary cultures of astrocytes by exposing astrocytes to 400 microM homocysteine for 20 h. Metabolic extracts of cells were prepared following a 4-h incubation in minimum medium with 5.5 mM [U-(13)C]glucose in the presence or absence of homocysteine and analysed using (13)C NMR. The expression level of pyruvate dehydrogenase kinase isoform 2 (PDK-2), NAD(P)H levels and mitochondrial membrane potential responses were investigated following culture with homocysteine. Metabolomic analysis was performed using (1)H NMR spectroscopy and pattern recognition analysis. Following incubation with homocysteine there was a significant decrease (48%) in the ratio of flux through pyruvate carboxylase (PC) and pyruvate dehydrogenase (PDH) which was due to an increased flux through PDH. In addition, homocysteine culture resulted in a significant reduction in PDK-2 protein expression. Following stimulation with glucose there was a significant increase in NAD(P)H levels and an impaired hyperpolarisation of the mitochondrial membrane in homocysteine-treated cells. Metabolomic analysis showed that the most discriminating metabolites following homocysteine treatment were choline and hypotaurine. In summary, the results demonstrated that sub-lethal concentrations of homocysteine caused significant metabolic changes and altered mitochondrial function in primary cultures of astrocytes.