Effects of dexamethasone on the differentiation of membrane structure in cultured astrocytes.

Astrocytic processes investing vascular structures or forming the surface of mammalian brain have large numbers of orthogonally packed aggregates of intramembrane particles, termed "assemblies." Similar particle aggregates are expressed by astrocytes derived from neonatal rat forebrain in secondary culture, but they are much more uniformly distributed across the membranes of the cultured cells. Dexamethasone, a potent glucocorticoid, affects the differentiation of astrocyte membrane structure in two patterns, depending on the rate of proliferation in the culture. When confluent secondary cultures of astrocytes are exposed to 5 microM dexamethasone, the densities of assemblies increase, and in some cells approach the values present in the glial limitans in vivo. However, when rapidly proliferating astrocytes are exposed to dexamethasone during the first week of secondary culture, most of the astrocytes fail to express any assemblies. The rate of astrocyte proliferation is slowed, and a lower cell density is reached during the first 2 weeks of secondary culture in dexamethasone. The suppression of assemblies is transient: as the cultures approach confluence, the proportion of cells expressing assemblies increases to nearly control levels, and the density of assemblies increases to greater than control values in some astrocytes. Certain of the effects of dexamethasone on cultured astrocytes may have relevance for understanding the mechanism(s) of its action in treating cerebral edema.     

Membrane structure in cultured astrocytes

Astrocytes cultured from the brains of neonatal rat pups acquire at least two specializations of intramembrane particle distribution: 'assemblies' and gap junctions. The number and appearance of assemblies in the cultured astrocytes is not markedly influenced by the presence or absence of a collagen substrate, and the range of concentrations of assemblies in astrocytic membranes is fairly stable from 7 through 28 days in culture. The assemblies are not concentrated in apposition to the substrate, even though the astrocytic membranes containing the highest concentration of assemblies in vivo are apposed to basal lamina. Quantitative analysis shows that assemblies are not uniformly distributed over the plasmalemma of a single cell, raising the possibility that the nature of cells around an astrocytic process may influence its membrane composition in vitro.     

Serum modulates cyclic AMP-dependent morphological changes in cultured neurohypophysial astrocytes

The effects of serum on the morphological plasticity exhibited by pituicytes in explant cultures of the neurohypophysis of adult rats have been examined. Cultured pituicytes are normally nonstellate, protoplasmic, amorphous cells (< 25% are stellate with a distinct cell body and phase bright processes). After incubation (90 min) of pituicyte cultures in a HEPES buffered salt solution (HBSS) supplemented with isoproterenol or forskolin, the fraction of stellate pituicytes significantly increased. The increase in the fraction of stellate cells induced by isoproterenol was not reversed by subsequent incubation in isoproterenol-free HBSS for 90 min. In contrast, after stellation was induced in cultures by exposure to forskolin (90 min), the fraction of stellate cells was significantly reduced if these cultures were incubated in forskolin-free, serum (0.5%) supplemented HBSS for the same duration. Serum also blocked the increase in the fraction of stellate pituicytes induced by forskolin. These experiments suggest that serum components may have a significant role in controlling the plasticity of neuroglial relations in the neurohypophysis priviously demonstrated in vivo.     

Morphological differentiation of cultured astrocytes is blocked by cadmium or cobalt

The morphological transformation of astrocytes in primary culture from flat to process bearing is induced by cyclic adenosine monophosphate (cAMP). Calcium currents in cultured astrocytes are also enhanced by cAMP. This correlation suggests the hypothesis that cAMP-induced calcium entry causes the morphological differentiation. The experiments reported here show that cadmium or cobalt, which block astrocyte calcium channels, also block the cAMP-induced morphological differentiation. These results support the hypothesized link between calcium entry and astrocyte differentiation.     

Activation of substance P receptors leads to membrane potential responses in cultured astrocytes

Cultured astrocytes from rat cortex and spinal cord responded with different types of membrane potential changes upon brief (10 seconds) applications of the natural neurokinin agonists substance P and neurokinin A. The most prominent type of response was a long-lasting membrane depolarization. In some cells, an initial rapid depolarization followed by a partial repolarization preceded the slow depolarizing event. Few astrocytes responded with a hyperpolarization of the membrane. Selective agonists at the NK-1 receptive site, substance P-methyl ester (SP-OME) and septide, mimicked the response to the natural neurokinins as did DiMe-C7, a selective NK-3 receptor agonist. A putative neurokinin antagonist, (D-Arg1,D-Pro2,D-Trp7,9,Leu11)SP (DADPDT) partially blocked membrane potential responses induced by substance P, SP-OME, septide, DiMe-C7, and NKA. The authors conclude that astrocytes express NK-1 and NK-3 receptors, which upon activation affect the electrical properties of these cells.     

Endothelins decrease the expression of aquaporins and plasma membrane water permeability in cultured rat astrocytes

Some of the aquaporins (AQPs), a family of water channel proteins, are expressed in astrocytes. The expression of astrocytic AQPs is altered after brain insults, such as ischemia and head trauma. However, little is known about the regulation of AQP expressions. Endothelins (ETs), which are vasoconstrictor peptides, regulate several pathophysiological responses of astrocytes. In this study, the effects of ETs on AQP expressions and plasma membrane water permeability were examined in cultured rat astrocytes. Determination of AQP mRNA copy numbers revealed that AQP1 and AQP4 were expressed prominently in cultured astrocytes. ET-1 (100 nM) and Ala1,3,11,1?-ET-1 (an ETB receptor agonist, 50 nM) decreased the AQP4 and AQP9 mRNA levels in cultured astrocytes, but the AQP1, -3, -5, and -8 mRNA levels remained unchanged. BQ788, an ETB receptor antagonist, reduced the effects of ET-1 on astrocytic AQP mRNAs, whereas FR139317, an ETA antagonist, had no effect. Immunoblot analyses revealed that treatment with ET-1 decreased the protein contents of AQP4 and AQP9 in both total cell lysates and plasma membrane fractions of cultured astrocytes. ET-1 and Ala1,3,11,1?-ET-1 decreased hypoosmolarity-induced water influxes into cultured astrocytes. In the presence of 30 μM Hg2+, which inhibits water movement through AQP1 and AQP9, the hypoosmolarity-induced water influxes were reduced. Phloretin, an inhibitor of AQP9, did not greatly affect the water influxes. The ET-induced decreases in water influxes were obtained in the presence of Hg2+ and phloretin. These results suggest that ET is a factor that regulates AQP expressions and water permeability in astrocytes.     

Effects of volatile anaesthetics on the membrane potential and ion channels of cultured neocortical astrocytes

Volatile anaesthetics cause changes in the membrane resting potential of central neurons. This effect probably arises from actions on neuronal ion channels, but may also involve alterations in the ion composition of the extracellular space. Since glial cells play a key role in regulating the extracellular ion composition in the brains of mammals, we analyzed the effects of halothane, isoflurane and enflurane on the membrane conductances and ion channels of cultured cortical astrocytes. Astrocytes were dissociated from the neocortex of 0–2-day old rats and grown in culture for 3–4 weeks. Anaesthetic-induced changes in the membrane potential were recorded in the whole cell current-clamp configuration of the patch-clamp technique. We further studied the effects of halothane and enflurane on single ion channels in excised membrane patches. At concentrations corresponding to 1–2 MAC (1 MAC induces general anaesthesia in 50% of the patients and rats), membrane potentials recorded in the presence of enflurane, isoflurane and halothane did not differ significantly from the control values. At higher concentrations, effects of enflurane and halothane, but not of isoflurane, were statistically significant. Single-channel recordings revealed that halothane and enflurane activated a high conductance anion channel, which possibly mediated the effects observed during whole cell recordings. In less than 10% of the membrane patches, volatile anaesthetics either increased or decreased the mean open time of K⁺-selective ion channels without altering single-channel conductances. In summary, it seems unlikely that the actions of volatile anaesthetics described here are involved in the state of general anaesthesia. Statistically significant effects occurred at concentrations ten times higher than those required to cause half-maximal depression of action potential firing of neocortical neurons in cultured brain slices. However, it cannot be excluded that the changes observed in the membrane conductance of cortical astrocytes disturb the physiological function of these cells, thereby influencing the membrane resting potential of neurons.     

Acetaldehyde cytotoxicity in cultured rat astrocytes

The effect of acetaldehyde on astrocytes have been investigated because not only do they play an important role in brain maturation but also recent reports have shown their delayed proliferation following both 'in vivo' and 'in vitro' ethanol exposure. Biochemical parameters related to apoptotic and necrotic processes were examined in primary cultures of rat astrocytes exposed for 4 days to acetaldehyde generated from ethanol by co-cultured alcohol dehydrogenase-transfected Chinese hamster ovary cells. Acetaldehyde levels in the culture media attained concentrations of approximately 450 microM. To study ethanol effects, alcohol oxidation was inhibited by 4-methylpyrazole (an inhibitor of alcohol dehydrogenase). Acetaldehyde but not ethanol increased intracellular calcium levels by 155%. Moreover, significant DNA fragmentation was detected using a random oligonucleotide primed synthesis assay, by flow cytometry and when using agar gel electrophoresis. Transglutaminase activity was elevated in the cells treated with acetaldehyde but when acetaldehyde formation was inhibited by 4-methylpyrazole the enzyme activity was unaffected. Nitrate levels in the culture media were unchanged. Additionally, microscopic examination of cell nuclei revealed chromatin condensation in astrocytes exposed to acetaldehyde. It can be concluded, that in 'in vitro' acetaldehyde exposed rat astrocytes apoptotic pathways are activated.     

Trauma-induced cell swelling in cultured astrocytes

Brain edema and associated increased intracranial pressure are major consequences of traumatic brain injury that account for most early deaths after traumatic brain injury. An important component of brain edema after traumatic brain injury is astrocyte swelling (cytotoxic edema). To examine the pathophysiologic mechanisms of trauma-induced astrocyte swelling, we used an in vitro fluid percussion trauma model. Exposure of cultured rat astrocytes to 5 atm of pressure resulted in significant cell swelling at 1 to 24 hours posttrauma that was maximal at 3 hours. Because oxidative/nitrosative stress, mitochondrial permeability transition (mPT), and mitogen-activated protein kinases (MAPKs) have been implicated in astrocyte swelling in other neurologic conditions, we examined their potential roles in this model. We previously showed increased free radical generation after in vitro trauma and show here that trauma to astrocytes increased the production of nitric oxide. Trauma also induced mPT and increased phosphorylation (activation) of MAPKs (extracellular signal-regulated kinase 1/2, c-Jun-N-terminal kinase, and p38-MAPK); these changes were diminished by antioxidants and the nitric oxide synthase inhibitor N-nitro-l-arginine methyl ester. Antioxidants, N-nitro-l-arginine methyl ester, the mPT inhibitor cyclosporin A, and inhibitors of MAPKs all significantly diminished trauma-induced astrocyte swelling. These findings demonstrate that direct mechanical injury to cultured astrocytes brings about cell swelling, and that blockade of oxidative/nitrosative stress, mPT, and MAPKs significantly reduce such swelling.     

Pharmacological characterization of the glutamate receptor in cultured astrocytes

Cultured astrocytes from neonatal rat cerebral hemispheres are depolarized by the excitatory neurotransmitter glutamate. In this study we have used selective agonists of different neuronal glutamate receptor subtypes, namely, the N-methyl-D-aspartate (NMDA), kainate, and quisqualate type, to characterize pharmacologically the glutamate receptor in astrocytes. The agonists of the neuronal quisqualate receptor, alpha-amino-3-hydroxy-5-methyl-4-isoxazole-4-propionic acid (AMPA) and quisqualate, depolarized the membrane. Kainate, an agonist of the neuronal kainate receptor, depolarized astrocytes more effectively than quisqualate. Combined application of kainate and quisqualate depolarized astrocytes to a level which was intermediate to that evoked by quisqualate and kainate individually. Agonists activating the neuronal NMDA receptor, namely NMDA and quinolinate, were ineffective. Application of NMDA did not alter the membrane potential even in combination with glycine or in Mg2+-free solution, conditions under which neuronal NMDA receptor activation is facilitated. The nonselective agonists L-cysteate, L-homocysteate, and beta-N-oxalylamino-L-alanine (BOAA) mimicked the effect of glutamate. Dihydrokainate, a blocker of glutamate uptake, did not, and several antagonists of neuronal glutamate receptors only slightly affect the glutamate response. These findings suggest that astrocytes express one type of glutamate receptor which is activated by both kainate and quisqualate, lending further support to the notion that cultured astrocytes express excitatory amino acid receptors which have some pharmacological similarities to their neuronal counterparts.     

Ryanodine-induced intracellular calcium mobilisation in cultured astrocytes

Changes in intracellular Ca²⁺ concentration were monitored in cultured cortical astrocytes challenged with ryanodine and ATP. Ryanodine elicited a modest, in comparison to ATP, increase in cytosolic Ca²⁺ concentration in approximately 60% of the cell fields examined. This effect was evident and, in fact, was augmented when incubations were performed in Ca²⁺ concentration were dose dependent, desensitised rapidly, and could not be mimicked by caffeine. Exposure to ryanodine was without effect on eicosanoid release from these cells nor did it influence Ca²⁺ mobilisation and eicosanoid release in response to ATP. In contrast, caffeine attenuated part of the ATP-evoked increase in intracellular Ca²⁺ concentration in the majority of cells tested and abolished its effect on eicosanoid release.     

Glutamine-induced free radical production in cultured astrocytes

Ammonia is a neurotoxin implicated in the pathogenesis of hepatic encephalopathy, Reye's syndrome, inborn errors of the urea cycle, glutaric aciduria, and other metabolic encephalopathies. Brain ammonia is predominantly metabolized to glutamine in astrocytes by glutamine synthetase. While the synthesis of glutamine has generally been viewed as the principal means of ammonia detoxification, this presumed beneficial effect has been questioned as growing evidence suggest that some of the deleterious effects of ammonia may be mediated by glutamine rather than ammonia per se. Since ammonia is known to induce the production of free radicals in cultured astrocytes, we investigated whether such production might be mediated by glutamine. Treatment of astrocytes with glutamine (4.5 mM) increased free radical production at 2-3 min (95%; P < 0.05), as well as at 1 and 3 h (42% and 49%, respectively; P < 0.05). Similarly treated cultured neurons failed to generate free radicals. Free radical production by glutamine was blocked by the antioxidants deferoxamine (40 microM) and alpha-phenyl-N-tert-butyl-nitrone (250 microM), as well as by the nitric oxide synthase inhibitor N(omega)-nitro-L-arginine methyl ester (500 microM). Free radical production was also blocked by 6-diazo-5-oxo-L-norleucine (1 mM), an inhibitor of glutaminase, suggesting that ammonia released by glutamine hydrolysis may be responsible for the generation of free radicals. Additionally, the mitochondrial permeability transition inhibitor, cyclosporin A, blocked free radical production by glutamine. The results indicate that astrocytes, but not neurons, generate free radicals following glutamine exposure. Glutamine-induced oxidative and/or nitrosative stress may represent a key mechanism in ammonia neurotoxicity.     

Manganese induces cell swelling in cultured astrocytes

Manganese in excess is neurotoxic and causes a CNS disorder that resembles Parkinson's disease (manganism). Manganese highly accumulates in astrocytes, which renders these cells more vulnerable to its toxicity. Consistent with this vulnerability, manganese has been shown to cause histopathological changes in astrocytes (Alzheimer type II change), generates oxidative stress and bring about mitochondrial dysfunction, including the induction of the mitochondrial permeability transition (mPT) in astrocytes. In addition to manganism, increased brain levels of manganese have been found in hepatic encephalopathy, a chronic neurological condition associated with liver dysfunction, wherein Alzheimer type II astrocytic changes are also observed. As low-grade brain edema, possibly secondary to astrocyte swelling, has been reported in hepatic encephalopathy, we hypothesized that manganese may contribute to such edema. We therefore exposed cultured astrocytes to manganese (Mn(3+)) acetate (25 and 50microM) for different time periods and examined for changes in cell volume. Manganese dose-dependently induced astrocyte swelling; such swelling was first observed at 12h (28%), which further increased (54%) at later time points (24-48h). Pretreatment of astrocyte cultures with antioxidants, including vitamin E, the spin trapping agent PBN, and the iron-chelating agent desferroximine, as well as the nitric oxide synthase inhibitor l-NAME, all significantly blocked (50-80%) astrocyte swelling caused by manganese, suggesting that oxidative/nitrosative stress is involved in the mechanism of such swelling. Cyclosporin A, an inhibitor of mPT also blocked (90%) manganese-induced astrocyte swelling. The data indicate that manganese exposure results in astrocyte swelling and such swelling, at least in part, may be caused by oxidative stress and/or mPT. Astrocyte swelling by manganese may represent an important aspect of manganese neurotoxicity, and may be a factor in low-grade brain edema associated with chronic hepatic encephalopathy.     

Characterization of stretch-activated ion channels in cultured astrocytes

The presence of a stretch-activated channel in rat cerebellar astrocytes in culture is described. This stretch-sensitive channel is K⁺ -selective and its open probability increases with suction following a Boltzmann-like distribution with half activation at 45 mm Hg. Kinetic analysis of the single-channel data indicated that there are two open and two closed states and that the shortest time constants of both open and closed states are the most sensitive to suction. A symmetrical two-barrier-one-site permeation model can quantitatively describe the inward rectification of the single-channel current to voltage relations. It is suggested that this stretch-activated channel plays a role in the regulatory volume response of astrocytes to hyposmotic conditions. © 1993 Wiley-Liss, Inc.     

Neurons modulate oxytocin receptor expression in rat cultured astrocytes: involvement of TGF-beta and membrane components.

We examined the effect of neurons on oxytocin (OT) receptors (OTR) and OTR gene expression in cultured astrocytes. The addition of neuron-conditioned medium induced an increase of both OTR binding and OTR mRNA level. This effect was enhanced after the medium was boiled or acidified. As it is known that transforming growth factor-beta (TGF-beta) can be released from carrier proteins by acid or heat, TGF-beta1 and 2 were tested and found to induce an increase of OTR binding. Furthermore, TGF-beta antibody abolished the stimulatory effect of normal or acidified neuron-conditioned medium. Neurons added to cultured astrocytes without contact mimicked the stimulatory effect of the conditioned medium. In contrast, neurons added with contact, induced a decrease in OTR binding and an increase of mRNA level, whereas neuronal membranes induced a decrease of both OTR binding and mRNA levels. In conclusion, the present data demonstrate that in vitro, neurons are able to modulate astrocytic OTR expression at the level of both protein and mRNA. They stimulate this expression through their release of TGF-beta and inhibit it by the action of unknown membrane components.