Fibroblast growth factors-5 and -9 distinctly regulate expression and function of the gap junction protein connexin43 in cultured astroglial cells from different brain regions

Astroglial cells contribute to neuronal maintenance and function in the normal and diseased brain. Gap junctions formed predominantly by connexin43 (cx43) provide important pathways to coordinate astroglial responses. We have previously shown that fibroblast growth factor (FGF)-2, which occurs ubiquitously in the CNS, downregulates gap junction communication in cortical and striatal, but not in mesencephalic astroglial cells in vitro (Reuss et al. Glia 22:19-30, 1998). Other members of the FGF family expressed in the CNS include FGF-5 and FGF-9. We show that both FGF-5 and FGF-9, like FGF-2, downregulate astroglial gap junctions and functional coupling. However, their effects are strikingly different from different brain regions, with regard to astroglial cells. FGF-5 specifically affects mesencephalic astroglial cells without changing coupling of cortical and striatal astroglia, while FGF-9 reduces gap junctional coupling in astroglia from all three brain regions. Both cx43 mRNA and protein levels as well as functional coupling assessed by dye spreading are affected. To clarify whether brain region-specific effects of FGFs on astroglial coupling are due to differential expression of FGF receptors (FGFR), we monitored expression of the four known FGFR mRNAs in astroglial cultures by RT-PCR. Irrespective of their regional origin, astroglial cells express mRNAs for FGFR-2 and FGFR-3. In summary, our results provide evidence for an important role of FGF-2, -5, and -9 in a distinct, CNS region-specific regulation mechanism of astroglial gap junction communication. The molecular basis underlying the regionally distinct responsiveness of astrocytes to different FGFs may be sought beyond distinct FGFR expression.     

TNF alpha increases activity of gamma-glutamyl transpeptidase in cultured rat astroglial cells

To investigate the presence of gamma-glutamyl transpeptidase (gammaGT) in brain cells, cultures enriched for astroglial cells, neurons, oligodendroglial cells, and microglial cells were studied. Astroglial cultures contained a specific gammaGT activity of 2.3 +/- 0.9 nmol/min/mg protein. A similar specific gammaGT activity was measured for oligodendroglial cultures, whereas microglial cells and neurons contained less than 30% of the specific gammaGT activity of astroglial cultures. The activity of gammaGT in astroglial cultures was elevated strongly by the presence of tumor necrosis factor-alpha (TNFalpha) in a time- and concentration-dependent manner. Maximal activity of gammaGT was observed after incubation of astroglial cultures for 3 days with 30 ng/mL TNFalpha. Under these conditions the specific gammaGT activity was increased by threefold compared to controls. Presence of the gammaGT-inhibitor acivicin completely inhibited gammaGT activity both in TNFalpha-treated and in control cells. In addition, the increase in astroglial gammaGT activity after application of TNFalpha was prevented completely by the presence of the protein synthesis inhibitor cycloheximide. gammaGT is involved in extracellular processing of glutathione (GSH) that is exported by astroglial cells. After TNFalpha-treatment the concentration of GSH in the medium of astroglial cells was reduced significantly compared to control cells. In conclusion, the data presented demonstrate that TNFalpha stimulates gammaGT synthesis in astroglial cells and thereby improves the capacity to process GSH exported by these cells.     

Attenuated transcriptional responses to oxidative stress in the aged rat brain

To investigate the release of glutathione (GSH) from brain cells, cultures enriched for astroglial cells, neurons, oligodendroglial cells, and microglial cells derived from rat brain were studied. During incubation of astroglial cultures, GSH accumulated in the medium with a rate of 3.1 +/- 0.6 nmol x h(-1) x mg protein(-1). In contrast, only marginal amounts of extracellular GSH were detectable in the media of the other brain cell cultures investigated. The mechanism of GSH release from astroglial cells, as yet, has not been reported. Multidrug resistance protein 1 (Mrp1), a transport protein known to mediate cellular export of glutathione disulfide and glutathione conjugates, is expressed in astroglial cultures. Inhibitors of Mrp1 were used to test for a function of this transporter in mediating GSH release from astroglial cells. The presence of the competitive Mrp1 inhibitor MK571 at a concentration of 50 microM inhibited the rate of GSH release by 63%. In contrast, the low concentration of 1 microM of MK571 increased the rate of GSH release by 83%. This bimodal concentration-dependent effect of MK571 is in accord with literature data for the effects of Mrp1 substrates on GSH release from cells. In addition, the presence of cyclosporin A (10 microM) reduced the GSH release rate significantly and completely blocked the stimulating effect of 1 microM MK571 on the release of GSH from astroglial cells. In conclusion, the data presented are a strong indication that Mrp1 participates in the release of GSH from astroglial cells.     

Microglial cells in culture express a prominent glutathione system for the defense against reactive oxygen species

To obtain information on the glutathione metabolism of microglial cells, the content of glutathione and activities of enzymes involved in the defense against peroxides were determined for microglia-rich cultures from rat brain. These cultures contain approximately 90% microglia cells as determined by immunocytochemical staining for glial markers, by the phagocytosis activity of the cells and by the production of superoxide after stimulation of the cells with phorbolester. For these cultures, a glutathione content of 41.2 +/- 11.2 nmol/mg protein and a specific activity of glutathione reductase of 15.2 +/- 3.2 nmol/(min x mg protein) were determined. These values are significantly higher than those found for astroglial or neuronal cultures. In addition, with 68.7 +/- 23.5 nmol/(min x mg protein), the specific activity of glutathione peroxidase in microglial cultures was 78% higher than in cultured neurons. The specific catalase activity of microglial cultures was less than 40% that of astroglial or neuronal cultures. Microglial cultures contain only marginal amounts of oxidized glutathione. However, on application of oxidative stress by incubation of microglial cultures with hydrogen peroxide or with the superoxide-producing hypoxanthine/xanthine oxidase system, cellular glutathione was rapidly oxidized. These results demonstrate that microglial cells have a prominent glutathione system, which is likely to reflect the necessity for self-protection against reactive oxygen species when produced by these or surrounding brain cells.     

AMPA-Selective glutamate receptor subunits in astroglial cultures

We analysed AMPA ionotropic receptor subunits at the mRNA level (GluR-1 to -4) and at the protein level (GluR-1 and GluR-2/3/4C) in “primary astroglial cultures” (non-neuronal cell cultures highly enriched in glial fibrillary acidic protein [GFAP] positive cells) prepared from newborn rat cerebral hemispheres, cerebral cortex, hippocamps, and striatum and in “brain non-neuronal cell cultures” (low percentage of GFAP positive cells) prepared from cerebellum, brainstem, mesencephalon, and hypothalamus. For comparison, we also determined ampa subunit mRNA and protein levels in different brain regions. By Northern blot analysis mRNAs for the AMPA receptor subunits (glur-1,-2,-3,-4) were detected in primary rat cerebral hemispheres astroglial cultures. Immunoblotting analysis with anti-GluR-1 and anti GluR-2/3/4C polyclonal antibodies confirmed the presence of low leve of immunoreactive proteins of the same size of those identifice in vivo as GluR subunits. Expression of GluR genes varied depending on the brain area used as starting material for the preparation of the cultures: GluR-1, -2, and -3 were mailly expressed in cortical cultures, while GluR-4 expression predominated in brainstem derived cultures. Interestingly this pattern of expression correlates with that observed in the intact brain, where high levels of GluR-4 mRNA and low levels of the other GluR subunits were found in the brainstem. In conclusion our results confirm the existence of glutmate ionotropic receptors of the AMPA type in primary astroglial cultrues and suggest that GluR-4 is the main AMPA receptor subunit expressed in non neuronal cells of the central nervous system. © 1993 Wiley-Liss, Inc.     

Regionally distinct regulation of astroglial neurotransmitter receptors by fibroblast growth factor-2

Fibroblast growth factor (FGF)-2 is an abundant astroglial cytokine. We have previously shown that FGF-2 downregulates gap junctions in primary astroglial cultures (B. Reuss et al., 1998, Glia 22, 19-30). We demonstrate now that FGF-2 induces astroglial dopamine (DA) sensitivity and D1 dopamine-receptor (D1DR) antigen and message in cortical and striatal astroglial cultures. On the functional level 10 micromol/L DA triggered transient increases in astroglial [Ca(2+)](i). In gap-junction-coupled cells, no FGF-2-dependent changes in proportions of DA-responsive cells were observable. However, uncoupling with octanol or 18alpha-glycirrhetinic acid isolated the smaller population of astrocytes intrinsically sensitive to DA which was significantly increased by FGF-2 in cortical and striatal cultures. Administration of DR-specific substances revealed that FGF-2 upregulated D1DR. These results indicate that downregulation of astroglial gap junctions by FGF-2 is accompanied by an upregulation of D1DR and DA sensitivity, adding a new aspect to the role of FGF-2 in the regulation of brain functions.     

Induction of type III-deiodinase activity in astroglial cells by retinoids

Thyroid hormones and retinoic acid (RA) are important modulators of growth, development, and differentiation. Type III deiodinase (D-III), which catalyzes thyroid hormones degradation in the brain and in cultured astroglial cells, is induced in astroglial cells by multiple pathways, including cAMP, 12.0-tetradecanoylphorbol-13-acetate (TPA), fibroblast growth factors, and thyroid hormones themselves. In the present study, the effects of retinoids on D-III activity were examined in astroglial cells cultures in a chemically defined medium devoid of hormones and growth factors. Incubation of astroglial cells with 5 μM all-trans-RA caused up to 200-fold increase in D-III activity, which reached a plateau after 48 h. The retinoid-induced increase in D-III activity was concentration dependent (0.5 μM all-trans-RA and 9-cis-RA producing half-maximal effect). Retinol was effective at physiological concentrations (1 and μM). The 48 h effects of 5 μM all-trans-RA and 10 nM thyroid hormones on D-III activity were at least additive. Addition of 2 nM acidic fibroblast growth factor or 1 mM 8-bromo-cAMP for the last 8 h of a 48 h incubation with 5 μM all-trans-RA did not alter the induction by all-trans-RA, whereas 0.1 μM TPA in the same conditions produced an additive effect with all-trans-RA. All-trans-RA (5 μM) had little or no effect on type II deiodinase, the enzyme which catalyzes the activation of thyroxine to 3,5,3′-triiodothyronine. The potent action of retinoids on the enzyme responsible for thyroid hormones degradation in the brain may protect the brain from the effects of 3,5,3′-triiodothyronine in regions influenced by retinoids. © 1994 Wiley-Liss, Inc.     

Replacement of glucose by sorbitol in growth medium causes selection of astroglial cells from heterogeneous primary cultures derived from newborn mouse brain

Primary cultures derived from the brains of newborn mice are quantitatively dominated by astroglial cells, but contain also oligodendroglial, phagocytic and ependymal cells. When confluent cultures are fed with glucose-free growth medium containing 25 mM sorbitol for 14 days, oligodendroglial, phagocytic and ependymal cells are eliminated from the culture, as judged by morphological and immunocytochemical criteria. The remaining cells stain positively for vimentin and glial fibrillary acidic protein and, therefore, can be considered as astroglial cells. Inoculation of freshly dissociated mouse brain cells in the absence of glucose in a sorbitol-containing medium is not possible; however, feeding of the cultures from day 2 on with sorbitol instead of glucose results in a pure astroglial culture at confluency. Therefore glucose-free growth medium supplemented with sorbitol can be considered a selective medium for astroglial cells in primary mouse glial cultures.     

Astroglial cells: glucocorticoid target cells in the brain

Glutamine synthetase (GS), an enzyme localized in astroglial cells in the brain, is directly implicated in brain detoxification. An ontogenic study of GS activity was performed in homogenates from four distinct brain areas in comparison with the respective astrocytes obtained in primary cultures. GS was induced by hydrocortisone in the astrocytes of all brain areas studied; only cerebellum and cerebral hemisphere astroglial cells had a higher specific activity when compared with the corresponding homogenates. N6O2-Dibutyryl adenosine 3',5'-cyclic monophosphate (dBc AMP), insulin, soluble brain factors, and noradrenaline (NA) were also able to modulate GS activity. Brain factors as well as dBc AMP interfered with hydrocortisone induction of GS. Regulation by hydrocortisone paralleled the variation in its concentration in brain during development. We conclude that astroglial cells are target cells for glucocorticoids, which may modulate ammonia detoxification in these cells.     

Alpha isoform of smooth muscle actin is expressed in astrocytes in vitro and in vivo

We have previously reported that astroglial cell lines derived from spontaneously immortalized mouse cerebellar cultures as well as primary astrocyte cultures express the mRNA of the alpha isoform of smooth muscle actin. In this report, we have used an antiserum specific for the alpha smooth muscle actin protein to investigate the presence and the pattern of expression of alpha smooth muscle actin protein at the cellular level with immunocytochemical methods. The results show that an anti-smooth muscle vessels alpha actin antiserum labels a typical actin network in the D19 astroglial cell clone and in flat astrocytes of primary cultures derived from various CNS regions of embryonic and postnatal mice. Furthermore, this antiserum labels distinct populations of astrocytes in the adult mouse brain, in particular in the corpus callosum and the fornix. However, in the corpus callosum, astrocytic processes are strongly labeled by anti-SMV alpha actin antibodies only in parasagittal planes. Thus, alpha smooth muscle actin represents a new marker for subsets of astrocytes.     

Thyroid hormone and conditioned medium effects on astroglial cells from hypothyroid and normal rat brain: Factor secretion,cell differentiation,and proliferation

The effects of triiodothyronine (T₃)on cell morphology were examined in cerebral hemisphere and cerebellar astrocyte cultures obtained from normal and hypothyroid neonatal rats. T₃-treatment induced morphological changes in astrocytes from cerebral hemispheres. This morphological effect was produced earlier if astrocytes were treated with conditioned medium obtained from cerebral hemisphere astrocyte cultures previously exposed to 50 nM T₃. T₃ or conditioned medium-treatment produced faster morphological changes in hypothyroid rat cerebral hemisphere astrocyte monolayers. Cerebellar astrocytes from normal brain did not respond to thyroid hormone with morphological changes, but proliferated after T₃-treatment. However, hypothyroid cerebellar astrocyte cultures exhibited morphological changes, differently than normal cells. We verified that T₃ may induce astrocyte secretion of factor(s) that promotes morphological differentiation in cerebral hemisphere astroglial cultures and stimulates the proliferation of cerebellar astrocytes. Astrocytes obtained from hypothyroid animals were more sensitive to secreted factors than normal cells. These results emphasize the heterogeneity and the importance of glial cells to normal brain development and open new questions about thyroid hormone therapy in hypothyroidism. © 1995 Wiley-Liss, Inc.     

Astrocyte cultures from adult rat brain. Derivation, characterization and neurotrophic properties of pure astroglial cells from corpus callosum

It has not as yet been routinely possible to derive primary cultures of glial cells from adult rat brain tissue even when adopting strategies that have proven successful with perinatal tissue. We now report that in response to a surgical lesion and a period of postoperative ‘priming’ in vivo, proliferating cultures of astroglial cells can be derived from the normally quiescent glia of the corpus callosum region of the adult rat brain. In such cultures the predominance of astroglia and the virtual absence of oligodendroglia and neurons has been established by the use of a variety of cell-type specific antisera. Fibroblasts, the only other cell type identified, when not numerous could be succesfully eliminated by treatment of the cultures with anti-Thy-1 antibodies and guinea pig compliment. Pure astroglial cells from adult brain have been sub-cultured and maintained for up to 4 months in vitro, providing suitable quantities of cells for studies on the trophic interaction between glia and neurons. In long-term culture the adult astrocytes maintain a flattened undiffirentiated morphology but readily assume a stellate shape with long branching processes upon the addition of a crude homogenate from bovine pituitary.     

The output of neuronotrophic and neurite-promoting agents from rat brain astroglial cells: a microculture method for screening potential regulatory molecules

Throughout embryonic development, as well as in response to injury of the central nervous system, astroglial cells may present neurons with a critical supply of neuronotrophic and neurite-promoting factors which control, respectively, neuronal survival and axonal growth. The identification of such astroglial cell-derived factors, as well as of specific extrinsic agents regulating their production, will require the use of in vitro techniques. We define here a new microculture system in which added agents can be screened for their ability to enhance or inhibit the output of trophic and neurite-promoting factors from purified neonatal rat brain astroglial cells. With such a procedure, thousands of replicate secondary astroglial cultures can be set-up and maintained in chemically defined medium, on a defined substratum and in a viable, low proliferative stable state. These cultured astroglial cells release into their medium at least three distinct and separable types of agents addressing nerve cells in vitro: (i) high molecular weight trophic factors (Mr greater than 10,000) which support the survival of embryonic peripheral neurons; (ii) low molecular weight trophic agents (Mr less than 10,000) supporting embryonic central neurons; and (iii) polyornithine-binding neurite-promoting factors which enhance neuritic regeneration for both peripheral and central neurons. The temporal release patterns of these three agents from astroglial cultures are quite distinct suggesting that their output is independently regulated.     

Primary cultures from defined brain areas; effects of seeding time on cell growth, astroglial content and protein synthesis

The influence of seeding time on cell growth, astroglial content and on protein synthesis during cultivation was determined in primary cultures from 3 phylogenetically different brain areas from rat cerebral cortex, striatum and brainstem. Brainstem cultivated from 17-day-old embryos and all the cultures studied from the 3 brain areas of newborn and 7-day-old rat showed a similar increase in total and water-soluble protein during cultivation. Glial fibrillary acidic protein (GFAp, alpha-albumin) levels increased with age in all cultures studied. There was a rapid increase in GFAp (alpha-albumin) between 1 and 2 weeks in cultures from newborn and between 2 and 3 weeks in brainstem cultures from 17-day-old embryos, these increases being slower thereafter. Incorporation of [3H]valine into soluble protein was lower in 3-week-old cultures than in 1- and 2-week-old cultures derived from newborn and 7-day-old rat brain. The incorporation rates were similar in comparisons of the various cultures. Similar results were obtained from embryonic cultures, although the decrease in incorporation rate was between 3 and 4 weeks. The efficiency of incorporation (% TCA-precipitated material/total [3H]activity) was higher in 2- and 3-week-old than in 1-week-old cultures from newborn and 7-day-old rats and in 3- and 4-week-old cultures of brainstem from 17-day-old rat embryos. These findings suggest a cell differentiation during cultivation. The results show that seeding time has a variable influence on cultures from the different brain areas studied concerning cell growth, astroglial content and probably differentiation during cultivation. Embryonic cell cultures seem, in general, to develop one week later than neonatal and postnatal ones. Cultures of newborn rat cells from cerebral cortex, striatum and brainstem show many similarities in the above parameters during cultivation. This is also the case for brainstem cultures from embryonic rat.     

Inhibition of endotoxin-induced nitric oxide synthase production in microglial cells by the presence of astroglial cells: A role for transforming growth factor β

In mixed glial cell cultures from cerebral cortices of newborn rats, endotoxin induces inducible nitric oxide (iNOS), nitric oxide (NO), and interleukin-1β (IL-1β) production in microglial cells. Earlier we demonstrated that endotoxin induced iNOS but not IL-1β expression in microglial cells is inhibited by the presence of astroglial cells. In the present paper we describe studies on the mechanism by which astroglial cells exert selective suppressive action on iNOS expression by microglial cells. Expression of iNOS and IL-1β was studied by single or double label immunocytochemical techniques and cell identification was performed with GSA-I-B₄-isolectin and an antibody against GFAP. Production of IL-1β and NO was determined by measurement of IL-1β and nitrite concentrations in cell lysates and the culture medium, respectively. TGFβ, a cytokine known to inhibit NO production by endotoxin challenged macrophages, was measured in culture medium of mixed glial cell cultures using a bioassay. Microglial, astroglial, and mixed glial cell cultures produced similar concentrations of TGFβ. The potential effect of TGFβ was studied by using immunoneutralizing antibodies against TGFβ1 and TGFβ2 on the induction of iNOS in microglial cells in the presence of astroglial cells. Incubation of the mixed glial cell culture with these TGFβ antibodies (3 μg/ml) markedly increased endotoxin-induced NO production and iNOS expression in microglial cells, whereas the production of IL-1β was not affected. The antibodies against TGFβ1 and TGFβ2 marginally increased NO production in pure microglial cell cultures, nonetheless in cultures of purified microglial cells recombinant TGFβ1 and TGFβ2 together with endotoxin inhibited NO production. We conclude that the presence of astroglial cells is essential for the inhibitory effect of TGFβ on NO production by microglial cells (possibly) by activation of TGFβ or by increasing the sensitivity of microglial cells for TGFβ. GLIA 19:190–198, 1997. © 1997 Wiley-Liss, Inc.     

Differential erbB signaling in astrocytes from the cerebral cortex and the hypothalamus of the human brain

Studies in rodents have shown that astroglial erbB tyrosine kinase receptors are key regulatory elements in neuron-glia communication. Although both astrocytes and deregulation of erbB functions have been implicated in the pathogenesis of many common human brain disorders, erbB signaling in native human brain astrocytes has never been explored. Taking advantage of our ability to perform primary cultures from the cortex and the hypothalamus of human fetuses, we conducted a thorough analysis of erbB signaling in human astrocytes. We showed that human cortical astrocytes express erbB1, erbB2, and erbB3, whereas human hypothalamic astrocytes express erbB1, erbB2, and erbB4 receptors. Ligand-dependent activation of different erbB receptor heterodimeric complexes in these two populations of astrocytes translated into different morphological and proliferative responses. Although morphological plasticity was more pronounced in hypothalamic astrocytes than in cortical astrocytes, the former showed a lower mitogenic potential. Decreasing erbB4 expression via siRNA-mediated gene knockdown revealed that erbB4 constitutively restrains basal proliferative activity in hypothalamic astrocytes. We further show that treatment of human astrocytes with a protein kinase C activator results in rapid tyrosine phosphorylation of erbB receptors that involves cleavage of endogenous membrane bound erbB ligands by metalloproteinases. Together, these results indicate that erbB signaling in primary human brain astrocytes is functional, region-specific, and can be activated in a paracrine and/or autocrine manner. In addition, by revealing that some aspects of astroglial erbB signaling are different between human and rodents, our results provide a molecular framework to explore the potential involvement of astroglial erbB signaling deregulation in human brain disorders.     

Colocalization of fructose-1,6-bisphosphatase and glial fibrillary acidic protein in rat brain

Immunofluorescence studies of rat brain sections demonstrated an exclusive colocalization of the gluconeogenic key enzyme fructose-1,6-bisphosphatase (FBPase) with the astroglial marker glial fibrillary acid protein, indicating FBPase in brain as an astrocyte-specific enzyme. This conclusion was supported by the presence of FBPase activity in astroglia-rich but not neuron-rich primary cultures derived from rat brain.     

A Neurotoxic Prion Protein Fragment Induces Rat Astroglial Proliferation and Hypertrophy

Prion-related encephalopathies are characterized by the accumulation of an abnormal prion protein isoform (PrP^Sc) and the deposition of PrP amyloid in the brain. This process is accompanied by neuronal loss and astrogliosis. We recently showed that a synthetic peptide corresponding to residues 106–126 of human PrP is amyloidogenic and causes neuronal death by apoptosis in vitro. In the present study we investigated the effects of 1- and 14-day exposures of rat astroglial cultures to mtcromolar concentrations of this peptide as well as peptides homologous to other portions of PrP, a peptide corresponding to residues 25–35 of amyloid-β protein, and a scrambled sequence of PrP 106–126. No significant changes were observed after 1-day exposure of cultures to any peptide. Conversely, 14-day treatment with PrP 106–126 (50 μM) resulted in a 5-fold increase in glial fibrillary acidic protein (GFAP) expression, as evaluated by Northern and Western blot analyses, and a 1.5-fold increment in cell number. Light and electron microscopy immunohistochemistry showed an enlargement in size and density of astroglial processes, and an increase in GFAP-immunoreactive intermediate filaments. These changes were not observed after 14-day treatment of cultures with the other peptides, including PrP 106–126 scrambled. The increase in GFAP expression of astroglial cultures exposed to PrP 106–126 was quantitatively similar to that found in scrapie-infected hamster brains. These results suggest that the PrP region corresponding to residues 106–126 is biologically active, and that cerebral accumulation of peptides including this sequence might be responsible for both the neuronal degeneration and the astrogliosis that occur in prion-related encephalopathies.     

SITS-inhibitable Cl- transport and Na+-dependent H+ production in primary astroglial cultures.

The uptake and efflux of Cl- were measured in primary astroglial cultures from neonatal rat brain using 36Cl- as a tracer. Both uptake and efflux were found to be inhibited by the specific anion inhibitor SITS. The rate of Cl- efflux showed a broad optimum at pH values greater than 7.5, and both this pH dependence and the effect of SITS suggests that these cells contain a Cl- in equilibrium Cl- or Cl- in equilibrium HCO3- exchange carrier similar to that described in erythrocytes. In addition, the cells rapidly lost Cl- when placed in media of decreasing Cl- concentrations, and ploting the initial rate of uptake of 36Cl- as a function of external Cl- concen-ration gave an apparent Km for Cl- uptake of 56 mM. Pretreatment of these cultures with DBcAMP is known to cause the cells to form numerous processes, resulting in their morphology more closely resembling that of astroglia in brain. Treatment with DBcAMP resulted in decreased equilibrium levels of 36Cl- and a small decrease in the initial rate of uptake of 36Cl-, but did not affect inhibition by SITS. Addition of Na+ to the cells suspended in Na+-free media specifically increased the rate of acidification of the medium. These observations suggest that these cells have both Cl- in equilibrium HCO3- and Na+ in equilibrium H+ exchange processes which, if these cultures can be considered to be representative of cells in vivo, may also occur in astroglial cells in the central nervous system. Based on these results and other work, a model is proposed by which these processes would lead to the astroglial swelling which is often observed in vivo in pathological conditions.