Primary Astroglial Cell Cultures Research Articles

Interactions between valproate, glutamate, aspartate, and GABA with respect to uptake in astroglial primary cultures.

Astrocytes have been proposed to regulate the extracellular space in the brain, even if rather little is known about their specific functions. One possibility for obtaining more knowledge on the functions of astroglial cells is to examine how they respond on exposure to pharmacological agents. Na(+)-valproate is an anticonvulsive drug which is used in the treatment of several types of epilepsy. The mechanisms of action of the drug are not fully understood, but the GABA-ergic system, both in neurons and astrocytes, has been shown to be affected. In the present study, the effects of valproate were investigated on astroglial cells in primary cultures from newborn rat cerebral cortex. The transport of the drug itself and its effects on the transport of the amino acid transmitters glutamate, aspartate and gamma-aminobutyric acid (GABA) into astrocytes were examined. The [3H]valproate transport into the astrocytes was increased after exposure to L-glutamate but not L-aspartate. On the other hand, after acute exposure for the drug, the transport of [3H]L-glutamate and [3H]L-aspartate decreased, as also did the affinity but not the transport capacity for the [3H]GABA uptake. However, after 5 days chronic valproate exposure, no effects could be seen on the uptake kinetics of L-glutamate or L-aspartate. For GABA, the affinity decreased, while the transport capacity remained unchanged compared with controls. The results showed that valproate, glutamate, aspartate and GABA were capable of interacting significantly with each others transport into the astrocytes.

Agonist-evoked Ca2+ transients in primary astroglial cultures--modulatory effects of valproic acid.

Cytosolic Ca2+ ([Ca2+]i) activity was measured in individual type 1 astroglial cells in primary culture after exposure to glutamate (Glu), quisqualate (QA), gamma-aminobutyric acid (GABA), 5-hydroxytryptamine (5HT), and noradrenaline (NA) by using the Ca2+ indicator dye fura-2/AM in a computerized microspectrofluorimetric system. Various patterns of Ca2+ transients were observed, but the most common was biphasic, having an initial sharp peak, rising immediately after stimulation, and then declining to a lower but sustained Ca2+ level. The only substance that diverged from this pattern was GABA, which induced a Ca2+ response with longer latency and a single-phase curve. The effects of the anticonvulsive drug Na(+)-valproate (VPA) were also investigated. After both acute and chronic (5-7 days) exposure to 10(-4) M VPA, the GABA-evoked rises in [Ca2+]i were completely inhibited. VPA also had acute effects on the 5HT- and Glu-evoked Ca2+ spikes. The Ca2+ responses after 5HT stimulation were greatly reduced after exposure to 10(-4) M VPA. The responses after glutamate stimulation were, on the contrary, increased after a similar exposure. No VPA effects were seen on the curve patterns of QA and NA stimulations. The most frequent agonist-evoked responses were seen after stimulation with 5HT and NA, where over 80% of the tested cells responded. For QA and Glu, the response frequencies were about 40% each, while for GABA it was 20%. The responses after 5HT and NA stimulation were blocked to baseline levels after exposure to ketanserin (5HT2 receptor antagonist) and a combination of prazosin, yohimbine, and propranolol (alpha 1, alpha 2, and beta adrenoceptor antagonists, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Arachidonic acid cycloxygenase and lipoxygenase pathways are differently activated by platelet activating factor and the calcium-ionophore A23187 in a primary culture of astroglial cells

The aim of our study has been to investigate the metabolism of endogenous arachidonic acid or that of radiolabeled arachidonate in astroglial cells, stimulated with platelet activating factor (PAF) and with the calcium-ionphore A23187. Primary cultures of astroglial cells were obtained from brain cortex of one-day-old rats and were characterized by immunofluorescent staining vs glial fibrillary acidic protein. In labeled cells, diacylglycerol was formed after stimulation with platelet activating factor, whereas mainly the release of labeled arachidonic acid from phospholipids was observed after stimulation with calcium-ionophore. Both PAF and the calcium-ionophore A23187 actively stimulated the formation of the cycloxygenase products PGD 2, TXB 2 and 6-keto-PGF 1α measured by radio- or enzyme-immunoassay. Differences were observed, instead, in the formation of the lipoxygenase metabolites, the hydroxyeicosatetraenoic acids, which were measured by high pressure liquid chromatography (HPLC) with on line radiodetection for the labeled products, and Leukotriene C 4, measured by radioimmunoassay. The formation of hydroxyacids by stimulated cells was confirmed by gas chromatography-mass spectrometry (GC-MS). In labeled cells, both agonists induced the formation of 12- and 15-hydroxyeicosatetraenoic acids, whereas stimulation of unlabeled cells with calcium ionophore resulted in formation of 12-hydroxyeicosatetraeonic acid and Leukotriene C 4. Our results suggest that in astroglial cells, PAF, a compound which is produced in several tissues including brain, mobilizes a selected arachidonic acid pool, possibly associated with diacylglycerol production, from phospholipids, thus activating the conversion of the released fatty acid via the cyclo and the 12-lipoxygenase pathways.

Alpha 1-adrenergic receptor coupling with phospholipase-C is negatively regulated by protein kinase-C in primary cultures of hypothalamic neurons and glial cells.

In the present work we evaluated the interactions of adrenergic receptors with phospholipase-C (PLC) and protein kinase-C (PKC), using an in vitro system of hypothalamic neurons and astroglial cells in primary cultures. The study was performed on immature neurons after 7 days in vitro (7 Div), that is before synaptogenesis, as well as on mature cells (14 Div). Comparisons were made between neurons and glial cells at the corresponding developmental stages. Norepinephrine (NE) increased inositol phosphates (IPs) formation in a dose- and time-dependent manner. The NE effect was mediated by alpha 1-receptor (alpha 1R) and was observed in young cells before synaptogenesis as well as in mature neuronal cultures; its amplitude was enhanced during the latter stage of the neuronal development. The coupling of alpha 1R with PLC was partially sensitive to pertussis toxin treatment and did not implicate the activation of calcium voltage-dependent channels. Activation of PKC by 12-O-Tetradecanoylphorbol 13-acetate (TPA) inhibited in a time-dependent manner the NE-stimulated production of IPs in young and mature hypothalamic neurons; however, in PKC depleted cells NE-induced IPs formation remained unchanged. In hypothalamic astroglial cell cultures the adrenergic stimulus of IPs generation was also mediated by alpha 1R. The effect was observed at both developmental stages, with a greater response in 14 Div cultures, and was insensitive to pertussis toxin treatment. As in neurons, activation of PKC resulted in inhibition of NE-induced IPs formation. These data indicate that functional interrelation between alpha 1R, PLC, and PKC is already present in immature neurons and glial cells and progressively develops in culture.

A double staining technique for simultaneous demonstration of astrocytes and microglia in brain sections and astroglial cell cultures.

We developed a double staining technique for simultaneous demonstration of astrocytes and microglial cells in histological brain sections and cell cultures. The procedure included a histochemical stain specific for microglial cells and an immunocytochemical stain specific for astroglial cells, with postponement of the final visualization of the staining products until both reactions had been performed. First, microglial cells were specifically but invisibly labeled by histochemical reaction for nucleoside diphosphatase (NDPase). Then the astroglial cells were labeled by performing the first parts of the immunocytochemical reaction for glial fibrillary acidic protein (GFAP). Finally, in a series of intervening steps, the NDPase reaction product was visualized and stabilized by treatment with ammonium sulfide and silver nitrate, while the 1-naphthol basic dye method was used to visualize the GFAP immunoreactive product. As an end product, the NDPase-positive microglial cells were brown and the GFAP-reactive astroglial cells blue. The two types of glial cells were clearly distinguishable in vibratome sections of rat brain tissue and in primary astroglial cell cultures, and we never observed cells that stained for both NDPase and GFAP. When the GFAP antibody was replaced by the OX-42 antibody, which recognizes microglial cells and macrophages, double staining of microglial cells was observed. The staining protocol has wide applications in studies of the functional interactions between microglial and astroglial cells in the normal brain and in different pathological states with neuronal or axonal degeneration, just as it can be used for experimental studies in cell cultures.

Mercuric Chloride Affects Morphology and Thymidine Incorporation into DNA in Astroglial Cells in Culture

Primary astroglial cell cultures from neonatal rat cerebral cortex revealed morphological changes when incubated in HgCl2 at various concentrations. After incubation for 30 minutes with 5 x 10-6 HgCl2, most cells changed their form from a flat to a more rounded shape and extended processes. After longer incubations (>60 minutes) or at higher concentrations (≥10-5M), vacuoles appeared in many cells. [3H]-Thymidine incorporation into DNA was slightly increased after acute incubation in 10-9M HgCl2, while decreases were obtained after incubation in the μM range. The results are important due to the functions of astroglia during development and in the mature nervous system.

The presence of glutathione in primary neuronal and astroglial cultures from rat cerebral cortex and brain stem

The concentration of the tripeptide glutathione (GSH) was measured in primary cultures of neurons and astroglial cells from rat cerebral cortex and brain stem. The concentration of GSH was found to be approximately 20 nmol/mg protein in the neuronal culture from the cerebral cortex and ca. 40 nmol/mg protein in the neuronal brain stem cultures. A GSH concentration of approximately 20 nmol/mg was observed in the astrocyte cultures from both brain regions. The possibility to increase the GSH concentration was tested by incubating the cultures in the presence of the GSH precursor gamma-glutamylcysteine (gamma-GC). In the cultured astrocytes gamma-GC produced a dose-dependent increase in GSH. A similar increase was observed in the neuronal cultures, but this effect failed to reach statistical significance.

Ganglioside characterization of rat astroglial cells in primary culture: Detection of the ganglio and lacto series gangliosides

The ganglioside composition in primary astroglial cell cultures, plated from newborn Sprague Dawley rat neocortex has been characterized. The ganglioside sialic acid content in these cultures reached a value of ca 6 nmol/mg protein after 7 days in culture and remained constant to day 15. The major gangliosides at day 15 were GM3, constituting ca 80%, and GD3, constituting 10% of the total ganglioside sialic acid. Gangliosides of the gangliotetraose series, GM1, GD1a, GD1b and GT1b, were determined by immunostaining on TLC plates using cholera toxin B-subunit as lectin. Taken together they comprised 6% of total ganglioside sialic acid at day 15. Using specific anti-ganglioside monoclonal antibodies, gangliosides of the lacto series have, for the first time, been detected in primary astroglial cultures, by immunological staining of TLC plates. 3′LM1 was found throughout the culture period while 3′-isoLM1 was found only days 0–6, and Fuc-3′-isoLM1 days 6–16 in culture.

Development and regulation of excitatory amino acid receptors involved in the release of arachidonic acid in cultured hippocampal neural cells

Release of [ 3H]arachidonic acid mediated by excitatory amino acid (EAA) receptors was investigated from prelabelled primary cultures of hippocampal neurons and astroglial cells. Treatment with N- methyl- d-aspartate (NMDA), quisqualate (QA) and kainate resulted in age- and dose-dependent stimulation of [ 3H]arachidonic acid release. During development, the maximum response for NMDA was observed relatively earlier (at 7 days) than those for QA and kainate (at 14 days) in the hippocampal neuronal cultures. The half maximal effects were obtained at about 15 μM NMDA at all ages studied and about 0.5 μM QA at 14 and 20 days. At optimum concentrations NMDA- and QA-induced releases were additive. Unlike with neurons, treatment with all the 3 EAA receptor agonists, NMDA, QA and kainate, had no significant effect on [ 3H]arachidonate release in hippocampal astroglial cells. In cultured 14-day-old neurons, the increases in NMDA- and QA-mediated [ 3H]arachidonic acid release were completely blocked by the NMDA receptor antagonist, 2-amino-5-phosphonovaleric acid, and the ionotropic QA receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione, respectively. But the ionotropic QA receptor agonist α-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) had no significant effect on [ 3H]arachidonate release, indicating that interaction between ionotropic QA and metabolotropic QA receptors may be essential for optimal QA-mediated arachidonic acid release. At physiological concentrations of Mg 2+ (1.2 mM), AMPA was found to potentiate NMDA-induced release of [ 3H]arachidonic acid; the effect appeared to be related to a removal of Mg 2+ blockade mediated by mild depolarisation. Our findings would suggest a role for associative stimulation of different types of EAA receptors in the effector mechanisms responsible for signal transduction and synaptic plasticity in the hippocampus.

Exposure toN-methyl- d-aspartate increases release of arachidonic acid in primary cultures of rat hippocampal neurons and not in astrocytes

The release of [ 3H]arachidonic acid (ARA) was investigated from prelabelled primary cultures of hippocampal neurons and astroglial cells. The activation ofN-methyl- d-aspartate (NMDA) subtype of glutamate receptors resulted in a dose-dependent stimulation of [ 3H]ARA release. The half maximal effect was obtained at about 15 μM NMDA, whereas the maximum concentration (50 μM NMDA) produced about a 2-fold increase in 7-day-old cultures. This elevation in [ 3H]ARA release was blocked in a dose-related manner by the NMDA receptor antagonist, 2-amino-5-phosphonovaleric acid (APV), and by Mg 2+ which blocks NMDA receptor-linked Ca 2+ ion channels. The removal of external Ca 2+ inhibited NMDA-induced release, whereas treatment with calcimycin (A 23187, a Ca 2+ ionophore) greatly increased the [ 3H]ARA release. The inhibitors of phospholipase A 2, nordihydroguaiaretic acid and mepacrine, decreased the NMDA-dependent [ 3H]ARA release in a dose-related manner, maximum inhibition reaching to about 90% at high doses. Entry of Ca 2+ brought about by opening the voltage-sensitive channels by high K + had no effect on the release of [ 3H]ARA, indicating that NMDA gated channels are situated in a part of the neuron where Ca 2+ entry through this route is more efficiently coupled to the activation of phospholipase A 2. Treatment with NMDA had no significant effect on [ 3H]ARA release in hippocampal astroglial cells as opposed to neurons. This was not due to inability of astrocytes to release ARA, for ATP still evoked [ 3H]ARA release, and this was markedly inhibited by mepacrine. It is suggested that ARA act as both intracellular and intercellular messengers in the functioning of NMDA receptors in synaptic transmission and plasticity in the hippocampus.

Transport of valproate and its effects on GABA uptake in astroglial primary culture

The antiepileptic drug Na(+)-valproate (VPA) is a broadspectrum anticonvulsant. It has been proposed to be involved in the inhibitory mechanisms of GABA-ergic systems. In this study, transport of the drug and possible influence on the GABA uptake were investigated in primary astroglial cell cultures from newborn rat cerebral cortex. The results show a Na+ and K+ independent high affinity uptake for VPA, with km and Vmax not significantly different from those observed for the GABA uptake. In the presence of the drug, the Km-value of the GABA uptake increased. The GABA uptake inhibitors guvacine, (RS)-Cis-4-OH-nipecotic acid and 4,5,6,7-tetrahydroisoxazolo (4,5- c) pyridin-3-ol (THPO) did not influence upon the uptake of VPA, suggesting a transport mechanism for the drug, separated from the GABA uptake carrier.

Interactions between cyclic AMP and inositol phosphate transduction systems in astrocytes in primary culture

Astroglial cells in primary culture possess receptors with cyclic AMP and inositol phosphates (IP) as second messengers. The beta-receptor agonist, isoproterenol induces an increase in the accumulation of cyclic AMP, the alpha 2-receptor agonist clonidine inhibits the isoproterenol-induced accumulation of cyclic AMP, while the alpha 1-receptor agonist phenylephrine acts only on the inositol phosphate system. 5-Hydroxytryptamine (5-HT) stimulates, the formation of inositol phosphate, while isoproterenol and clonidine per se do not affect the inositol phosphate system. In the present paper the possibility of interactions between the cyclic AMP and the inositol phosphate transduction systems were investigated. In the presence of 10(-5) M 5-HT, in itself ineffective on the formation of cyclic AMP, isoproterenol stimulated the accumulation of cyclic AMP far more than in the absence of 5-HT. The potentiation was blocked by the 5-HT2 receptor antagonist ketanserin. On the other hand, there were no indications for a beta-receptor influence on the 5-HT-induced inositol phosphate formation. Stimulation of the alpha 2-receptor did not induce accumulation of inositol phosphate but significantly potentiated 5-HT2-receptor transduction, as measured by hydrolysis of phosphoinositide and formation of inositol phosphate. Stimulation by 5-HT also increased the formation of inositol phosphate after adrenergic stimulation and this effect was found to be synergistic at certain concentrations of adrenergic agonists. In addition, there was a statistically significant accumulation of cyclic AMP in the presence of both 5-HT and phenylephrine, none of which stimulated cyclic AMP alone. The results suggest specific interactions between the cyclic AMP and inositol phosphate systems on cultured astroglial cells.

Characterization of cysteinyl-leukotriene formation in primary astroglial cell cultures

The formation and composition of cysteinyl-leukotrienes (LT) in primary astroglial cell cultures prepared from newborn rat brain has been studied. Small amounts of cysteinyl-LT determined in terms of LTC4-like material in the supernatants of the cultures, became detectable after stimulation of the cells with 10(-5) M ionophore A23187. Cysteinyl-LT formation increased with time, reaching about 600 pg (mg protein)-1 after 60 min incubation. In contrast, considerable thromboxane (TX) B2 synthesis was found at 5 min following A23187-stimulation (about 30 ng TXB2 (mg protein)-1). The synthesis of cysteinyl-LT was abolished by 5 x 10(-5) M nordihydroguaiaretic acid (NDGA). Irrespective of the duration of incubation, blockage of prostanoid synthesis by 10(-6) M indomethacin did not result in increased cysteinyl-LT production. Reversed phase HPLC combined with radioimmunological detection showed that, after 60 min incubation in the presence of A23187, LTC4 and LTD4 accounted for practically all the LTC4-like immunoreactive material in the supernatants of cell cultures. No significant amounts of LTE4 could be detected. The results show that astrocytes may contribute to brain LTC4 and LTD4 synthesis. However, the cellular site of cerebral LTE4 formation seems to be other than the astroglia.

Sodium and potassium uptake in primary cultures of rat astroglial cells induced by long-term exposure to the basic astroglial growth factor (AGF2).

Astroglial cell cultures were derived from newborn rat forebrain and cultured for 5 days in serum containing-, and for an additional 4 days in a serum-free, defined medium. At the end of this 9-day-long period, basic astroglial growth factor (AGF2) was administered to the culture medium (10 ng per ml). Cells were subsequently cultured in AGF2 containing serum-free, defined medium for further two weeks. At definite intervals of culturing, unidirectional influx of both Na+ and K+ (INa and IK, respectively) was determined by applying 22Na and 42K. The AGF2-treated cultures showed highly increased, amiloride-sensitive INa at the early exposure period (2-8 hours), similar to that we have reported about cultured astroglia exposed to AGF2 for minutes. They also exhibited significant furosemide-sensitive-, while relatively poor ouabain-sensitive component of INa. However, at later periods of exposure to AGF2, INa was significantly reduced, particularly due to the decrease of its amiloride-sensitive component, while its furosemide-sensitive component further increased with the time of AGF2 treatment. In contrast to INa, the IK in the cultures exposed to AGF2 increased significantly in the course of the long-term exposure period, particularly the ouabain-, and furosemide-sensitive-components, while its amiloride-sensitive component, similarly to that of INa, decreased.(ABSTRACT TRUNCATED AT 250 WORDS)

Ethanol potentiates serotonin stimulated inositol lipid metabolism in primary astroglial cell cultures

Serotonin-stimulated activation of phospholipase C in primary astroglial cell cultures was studied as a mean of evaluating the effect of acute ethanol exposition on this signal transduction system. The addition of 50–150 mM ethanol prior to stimulation with 10 −5M serotonin led to a potentiation of the serotonin-induced [ 3H]-inositol phosphate formation and an increased incorporation of [ 3H]-inositol into the three phosphoinositides studied. This potentiating effect of ethanol was observed only when ethanol was added together with serotonin. No stimulatory effect ofethanol perse was found. Furthermore, ethanol had no effect on arginin-vasopressin, bradykinin or phenylephrine stimulated inositol lipid metabolism.

Stimulation of astroglial serotonin receptors produces culture media which regulates growth of serotonergic neurons

Our work has been concerned with the role of high affinity serotonin receptors in regulating the development of the serotonergic system. In previous studies, we have found evidence that these receptors occur on astroglial cells and that their number is developmentally linked. The current work is aimed at investigating the mechanism by which these receptors may regulate serotonin neuronal growth. Primary cultures of astroglial cells were exposed to serotonin (5-HT) or the selective receptor agonists 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-D-PAT, for 5-HT 1a receptors) or trifluoro-methyl-phenyl-piperazine (TFMPP) and m-chlorophenylpiperazine (mCPP) (for 5-HT 1b receptors). Media was collected after 4 or 24 h, and added to primary cultures of serotonergic neurons. Growth was determined by specific uptake of radiolabeled serotonin into the cultures. Our results show the presence of a factor(s) in the glial-conditioned media which can be stimulatory or toxic to serotonin neurons, depending on the neuronal plating density. This factor is significantly present after 24 h, is found in both brainstem and cortical astroglial-conditioned media and appears to be linked to the 5-HT 1a receptor. Thus, it appears possible that the serotonergic neuronal system can regulate its own development through an action on astroglial cells.

Arachidonic acid metabolism in cultured astrocytes from rat embryo and in C 6 glioma cells

Arachidonic acid metabolism in primary cultures of astroglial cells prepared from cerebra of rat embryos was examined. Arachidonic acid was mainly metabolized through the lipoxygenase pathway and the major metabolites formed were 12-hydroxyeicosatetraenoic acid (12-HETE), 11-HETE and 15-HETE. By contrast, in C 6 cells, which are considered to be of astroglial origin, arachidonic acid was mainly metabolized through the cyclooxygenase pathway and the major metabolites formed were prostaglandin (PG)E 2, PGF 2α and throm☐ane B 2.

Effect of potassium depolarization on phosphate-activated glutaminase activity in primary cultures of cerebellar granule neurons and astroglial cells during development

The cerebellar granule cells are believed to be glutamatergic neurons. During the normal development of granule cells grown in a chemically defined medium, the specific activity of phosphate-activated glutaminase increased from 60 at 3 days to 150 (nmol/h/mg protein) at 15 days in vitro. Treatment with 25 mM K + for the last 2 days elevated glutaminase activity in an age-dependent manner: about 100% at 3 and 6 days, 75% at 10 days, and 40% at 15 days in vitro. The enhancement of glutaminase in granule cells was dose-dependent. The half-maximal effect was obtained at about 20 mM K +, whereas the maximum concentration, which produced about a 2.5-fold increase in 3-day-old cultures was about 40 mM K +. The voltage-sensitive Na + channel inhibitor tetrodotoxin had no effect on the depolarization-induced activity in granule cells. However, the increase in glutaminase by 25 mM K + was significantly blocked by both organic (nifedipine) and inorganic (Ni 2+ and Mg 2+) calcium antagonists, indicating that elevation in activity may be mediated through transmembrane Ca 2+ entry into granule cells. In contrast to neurons, in cultured cerebellar astrocytes, the activity of glutaminase slightly decreased during development, and treatment with 25 mM K + had no significant effect on this enzyme activity. The present findings, together with previous observations, would indicate that depolarization with K +, which is believed to mimic in vivo presynaptic stimulation, could be one of the mechanisms that selectively controls the development and function of neurons, when measured in terms of the activity of the enzymes involved in the synthesis of cell-specific neurotransmitters.

Regulation of glutamate and GABA transport by adrenoceptors in primary astroglial cell cultures

Un astrocytes grown in primary cultures from cerebral cortex of neonatal rats, α1-adrenoceptors regulate the active uptake of glutamate followed by an activation of glutamic oxaloacetate transaminase (GOT; EC 2.6.1.1) and a slight activation of glutamine synthetase (GS; EC 6.3.1.2) activity. The β-adrenoceptors regulate the active uptake of GABA, and this is followed by an activation of γ-aminobutyric acid α-ketoglutarate transaminase (GABA-T; EC 2.6.1.19.). The data suggest that astrocyte adrenoceptors may modulate neurotransmitter induced neuronal excitability.

Toluene treatment in vitro and calcium-regulated protein phosphorylation in primary astroglial cell cultures from the rat striatum

Primary astroglial cell cultures from striata of newborn rats were exposed to toluene in vitro at doses of between 4.7 and 150 μmol/ml. The protein content of the cell cultures, about 1 mg/ml, was not affected by the toluene treatment. Calcium-induced back-phosphorylation was used to detect changes in the phosphorylation state of calcium-regulated glial phosphoproteins. The 11 most back-phosphorylated protein bands were separated by gel electrophoresis and analysed by autoradiography and computer-assisted image analysis. The back-phosphorylation of the 59-kD protein band was increased by 150% at 9.4 μmol/ml toluene, but no further increase was observed with increasing concentrations. This increase is believed to correspond to a toluene-induced decrease in calcium-regulated protein phosphorylation. Furthermore, there was a trend for decreased back-phosphorylation of the 140-kD band and for increased back-phosphorylation of the 110-kD band in response to the higher concentrations of toluene used. The back-phosphorylation of the 42-, 44-, 48-, 51-, 54-, 63-, 74- and >200-kD protein bands showed no dose-related changes in response to the toluene treatment. Taken together, the data indicate that the 59-kD protein in astrocytes from the rat striatum is specifically sensitive to toluene treatment in vitro.