Neonatal Rat Astrocytes Research Articles

Collagen containing neonatal astrocytes stimulates regrowth of injured fibers and promotes modest locomotor recovery after spinal cord injury

The use of collagen as a vehicle to transplant neonatal astroglial cells into the lesioned spinal cord of the adult rat allows a precise application of these cells into the lesion gap and minimizes the migration of the transplanted cells. This approach might lead to anatomical and functional recovery. In the present study, 20 adult female Wistar rats were subjected to a dorsal hemisection at thoracic spinal cord levels. Cultured cortical neonatal rat astrocytes were transplanted into the lesion with collagen as a vehicle (N = 10). Prior to transplantation, the cultured astroglial cells were labelled with fast blue. Control rats received collagen implants only (N = 10). During 1 month of survival time, functional recovery of all rats was continuously monitored. Histological data showed that the prelabelled astroglial cells survived transplantation and were localized predominantly in the collagen implant. Virtually no fast blue-labelled GFAP-positive astroglial cells migrated out of the implant into the adjacent host spinal cord. The presence of transplanted neonatal astroglial cells resulted in a significant increase in the number of ingrowing neurofilament-positive fibers (including anterogradely labeled corticospinal axons) into the implant. Ingrowing fibers were closely associated with the transplanted astroglial cells. The implantation of neonatal astroglial cells did result in modest temporary improvements of locomotor recovery as observed during open-field locomotion analysis (BBB subscore) or during crossing of a walkway (catwalk).

Differences in the regulation of BDNF and NGF synthesis in cultured neonatal rat astrocytes

Using a new brain-derived neurotrophic factor (BDNF) specific enzyme-immunoassay, we determined the basal cellular content of BDNF protein in neonatal rat astrocytes in primary culture, thus confirming the ability of astrocytes to synthesize BDNF in addition to nerve growth factor (NGF). We subsequently monitored the influence of different pharmacological agents: neurotransmitter receptor agonists, cytokines, and second messenger up-regulators, on the synthesis of BDNF and NGF. Marked differences in the regulation of their synthesis by the above pharmacological agents were observed in our study.The basal cellular levels of BDNF protein in cultured neonatal rat cortical and cerebellar astrocytes were 15.9±0.3 and 18.7±0.4pg BDNF/mg cell protein, respectively, and differ significantly between astrocytes from different brain regions, whereas NGF levels were the same (16.1±0.3 and 16.2±0.7pg NGF/mg cell protein, respectively). Screening different neurotransmitter systems for their influence on BDNF and NGF synthesis in cortical astrocytes revealed that dopamine (0.15mM) is a potent up-regulator of BDNF protein synthesis in astrocytes, while kainic acid (50μM) and histamine (1μM) did not raise the cellular level of BDNF protein. Dopamine had no influence on NGF synthesis, while kainic acid caused minor, and histamine marked, elevation of NGF cellular content. Tumor necrosis factor-α (30ng/ml) and interleukin-1β (10U/ml) treatments did not influence BDNF synthesis, whereas they markedly increased NGF protein cellular level. We also confirmed (using forskolin (20μM) and phorbol 12-myristate 13-acetate (TPA) (100nM)) that adenylate cyclase and protein kinase C participate in the downstream signaling responsible for the stimulation of BDNF synthesis, whereas in the regulation of NGF synthesis only the participation of protein kinase C was confirmed.Our results indicate that astrocyte-derived neurotrophins could play a role in distinct brain functions under physiological conditions and in the pathogenesis as well as possible treatment of different neurodegenerative disorders.

Morphine Prevents Glutamate‐Induced Death of Primary Rat Neonatal Astrocytes Through Modulation of Intracellular Redox

This study is designed to investigate the effect of morphine on glutamate‐induced toxicity of primary rat neonatal astrocytes. Glutamate decreases the intracellular GSH level, and thereby induces cytolysis of astrocytes and C6 glial cells accompanied by apoptotic features. Glutamate‐induced cytotoxicity is protected by morphine and antioxidants such as GSH and NAC, whereas MK‐801, an antagonist of glutamate receptor NMDA does not protect astrocytes against glutamate toxicity. Also, morphine antagonist, naloxone, as well as selective ligands for opioid receptor subtypes, including DAMGO, DPDPE, and U69593, do not inhibit the protective effect of morphine on glutamate‐induced cytotoxicity. Morphine significantly prevents the depletion of GSH by glutamate and thereby inhibits the generation of H2O2 in a dose‐dependent manner. Furthermore, morphine prevents the change of mitochondrial permeability transition by glutamate. Taken together, we suggest that morphine protects the primary rat neonatal astrocytes from glutamate toxicity via modulation of intracellular redox status.

Regional Variation in the Activation Threshold for 1,3-DNB-Induced Mitochondrial Permeability Transition in Brainstem and Cortical Astrocytes

1,3-Dinitrobenzene (DNB) produces edematous, glio-vascular lesions in brainstem nuclei with high energy demands. Astrocytes in vulnerable brainstem nuclei appear to be an early and selective target of DNB and other nitroaromatic compounds, though the molecular basis of this susceptibility is poorly understood. It has been postulated that mitochondria are a principal target of DNB in sensitive cell types [Neuropathol. Appl. Neurobiol. 13 (5) (1987) 371], where redox-cycling of DNB increases levels of reactive oxygen species and disrupts cellular energy metabolism. The present study investigates the role of regional differences in activation of the mitochondrial permeability transition pore (mtPTP) by DNB in brainstem and cortical astrocytes and examines the expression of Bcl-2 proteins as potential regulators of mtPTP function. Neonatal rat astrocytes were cultured from both DNB-sensitive (brainstem) and insensitive (cortex) brain regions and evaluated for DNB-induced alterations in cell morphology and mitochondrial function. Exposure to DNB resulted in rapid changes in the morphology of brainstem astrocytes consistent with loss of ion homeostasis and initiation of necrotic cell death. These changes were not observed in cortical astrocytes at corresponding concentrations of DNB and were prevented in brainstem astrocytes by the mtPTP inhibitor, bongkrekic acid, suggesting that mitochondrial dysfunction is involved in DNB-induced morphological changes in brainstem astrocytes. Mitochondrial depolarization in brainstem astrocytes was observed at DNB concentrations as low as 10 μM, whereas no loss of mitochondrial membrane potential (Δ Ψ mt) occurred in cortical astrocytes at less than 100 μM DNB. DNB-induced loss of Δ Ψ mt followed apparent first-order kinetics, with EC 50-values for half-maximal rates of mitochondrial depolarization of ∼23 and ∼290 μM in brainstem cortical astrocytes, respectively. DNB-induced mitochondrial depolarization was prevented by pretreatment with bongkrekic acid, indicating that loss of Δ Ψ mt was mediated by activation of the mtPTP. Inhibition of succinate dehydrogenase (SDH) activity occurred in astrocytes from both brain regions exposed to DNB and was blocked in brainstem, but not cortical, astrocytes by bongkrekic acid. Constitutive expression of Bcl-X L was high in cortical tissue and astrocytes, whereas Bax expression was low. However, Bax was highly expressed in brainstem tissue and astrocytes and Bcl-X L expression was markedly lower. The expression of Bcl-2 was similar in both brain regions. These data suggest that the selective vulnerability of brainstem astrocytes to DNB is due to a lower threshold for activation of the mtPTP that is be mediated, in part, by distinct expression patterns of Bcl-2 proteins rather than by intrinsic differences in susceptibility of the electron transport chain.

Therapeutic effects of astrocytes expressing both tyrosine hydroxylase and brain-derived neurotrophic factor on a rat model of Parkinson’s disease

Tyrosine hydroxylase (TH) and brain-derived neurotrophic factor (BDNF), expressed in normal astrocytes, were used in combination for the treatment of Parkinson’s disease (PD) symptoms in a rat model. Normal neonatal rat astrocytes were co-transfected with a vector expressing BDNF (AAVBDNF) and a retroviral vector expressing TH (termed TH-BDNF-DA + cells), and then implanted into the striatum of PD rats induced by 6-hydroxydopamine. TH-BDNF-DA + cells compensated for a severe insufficiency of endogenous dopaminergic neurons in the PD rats, resulting in a significant improvement of PD symptoms. The decrease in the rotational rate of PD rats implanted with TH-BDNF-DA + cells was more marked than that in PD rats implanted with normal astrocytes expressing either TH or BDNF alone (termed TH + and BDNF + cells, P<0.01 and 0.001, respectively), and suggested a synergistic effect between TH and BDNF. In contrast, the rotational rate was not altered from the baseline in PD rats without treatment or implanted with parental rat astrocytes alone ( P>0.05). BDNF protected the dopaminergic neurons from apoptosis induced by 6-hydroxydopamine, and significantly increased the long-term survival of TH-positive cells in the striatum. Our data indicate that the combined use of TH and BDNF has a synergistic therapeutic effect, and is more efficient for the treatment of PD than a single gene therapy using either TH or BDNF alone.

Methylmercury-mediated inhibition of 3H- d-aspartate transport in cultured astrocytes is reversed by the antioxidant catalase

Astrocytes are essential for removal of glutamate from the extracellular space in the central nervous system. The neurotoxic heavy metal methylmercury potently and specifically inhibits the transport of glutamate in cultured astrocytes by an unknown mechanism. Glutamate transport in astrocytes is also inhibited by reactive oxygen species. A glutamate-induced transporter current is inhibited both by reactive oxygen species and thiol oxidizing agents. These observations suggest that oxidation of the transporter might mediate methylmercury-induced inhibition of glutamate transport. In the present study, we examined the ability of thiol reducing or oxidizing agents to inhibit transport of 3H- d-aspartate, a glutamate analog, in primary cultures of neonatal rat astrocytes. To assess if methylmercury-mediated inhibition of 3H-aspartate transport was due to overproduction of reactive oxygen species, we tested the ability of Trolox, α-phenyl- tert-butyl nitrone (PBN), or catalase to attenuate the methylmercury-induced inhibition of aspartate uptake. Neither the thiol reducing agent dithiothreitol (DTT), nor the thiol oxidizing agent 5,5′-dithio-bis(2-nitrobenzoic) acid (DTNB) had any effect on 3H-aspartate transport suggesting that the thiol redox state does not alter transporter function. In contrast, the antioxidant catalase (1000 U/ml) significantly attenuated methylmercury-induced inhibition of 3H-aspartate uptake, suggesting that excess reactive oxygen species, specifically H 2O 2, inhibit the function of an astrocytic excitatory amino acid transporter (EAAT1). Prolonged exposure (6 h) to inhibitors of glutamate transport significantly decreased EAAT1 mRNA levels suggesting that transporter expression is related to function. This study suggests that methylmercury-induced overproduction of H 2O 2 is a mechanism for inhibition of glutamate transport and transporter expression in cultured astrocytes.

Protective effects of morphine in peroxynitrite-induced apoptosis of primary rat neonatal astrocytes: potential involvement of G protein and phosphatidylinositol 3-kinase (PI3 kinase)

Opiates, such as morphine, have been used extensively in the clinical management of pain due to their potent analgesic effect. Astrocytes, representing a major non-neuronal cell population in the CNS, contain opioid receptors that are actively involved in several brain functions. This study was designed to evaluate the effects by which morphine, a preferential mu-opioid receptor agonist, contributes to cytotoxicity of nitric oxide (NO) species, including NO and peroxynitrite (ONOO −), in primary rat neonatal astrocytes. Primary astrocytes isolated from the cerebral cortex of 1- to 2-day-old Sprague–Dawley rats were treated with morphine, naloxone, and 3-morpholinosydnonimine (SIN-1), a donor of peroxynitrite. Morphine significantly protected primary rat astrocytes from apoptosis mediated by sodium nitroprusside, an NO donor, and SIN-1 in a dose-dependent manner, whereas it did not in other types of cells including C6 glioma, RAW 264.7, and HL-60 cells. Moreover, naloxone antagonized the protective effects of morphine on SIN-1-induced apoptosis. Morphine also inhibited the nuclear condensation and fragmentation of SIN-1-treated cells that was antagonized by naloxone pretreatment. The protective role of morphine in SIN-1-induced apoptosis was dependent on an intracellular antioxidant system such as GSH. Furthermore, the effects of morphine on SIN-1-induced cytotoxicity were prohibited by pretreatment with the G i protein inhibitor, pertussis toxin, and the phosphatidylinositol 3-kinase (PI3 kinase) inhibitors, wortmannin and LY294002. Taken together, these results suggest that morphine may protect primary rat astrocytes from apoptosis by NO species via the signaling cascades that involve both G protein and PI3 kinase.

β-Amyloid (1–42) affects MTT reduction in astrocytes: implications for vesicular trafficking and cell functionality

Beta-amyloid (Aβ) peptide deposition in the brains of Alzheimer’s disease patients results in reactive astrogliosis which may enhance neuronal cell death. Aβ has also been reported to impair important supportive astrocyte functions, such as glutamate uptake in vitro. We studied the effect of amyloid β-peptide (Aβ) on 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction, cellular ATP content, lactate release, and proliferation using neonatal rat astrocyte cultures. Aβ(1–42) decreased MTT reduction potently in the absence of cell death, but did not affect cellular ATP levels or lactate release. Moreover, the cells displayed increased proliferation after incubation with Aβ(1–42), confirming that the decreased MTT reduction was not deleterious to cell viability. Aβ(1–42) enhanced transfer of MTT dye to the cell surface leading to cessation of MTT reduction and cell death. Bafilomycin A1, but not brefeldin A, prevented the enhanced trafficking of MTT, suggesting that lysosomes, but not Golgi apparatus, are involved. Our results show that the viability of astrocytes themselves is not diminished by β-amyloid peptide. However, Aβ alters vesicular trafficking in astrocytes, which may disturb the supportive function of astrocytes in the brains of patients with Alzheimer’s disease.

7β‐hydroxysterol is cytotoxic to neonatal rat astrocytes in primary culture when cAMP levels are increased

7β‐hydroxysterol is cytotoxic to neonatal rat astrocytes in primary culture when cAMP levels are increased

Attachment of primary neonatal rat astrocytes to vitronectin is mediated by integrins αvβ5 and α8β1: modulation by the type 1 plasminogen activator inhibitor

Vitronectin is expressed in a cell-specific manner in the developing brain and concentrated in the brain during disease processes, such as germinal matrix hemorrhage and infarction, in which there is breakdown of the blood-brain barrier. In this study, we identified the integrin receptors that mediate attachment of primary neonatal rat astrocytes to vitronectin. Using fluorescent activated cell sorter and immunoprecipitation analyses, we established that the vitronectin receptor integrins alphavbeta5 and alpha8beta1, but not alphavbeta3, are expressed on neonatal rat astrocytes. Attachment of the neonatal astrocytes to vitronectin was inhibited (85%) in an additive manner by neutralizing anti-alphavbeta5 and anti-beta1 antibodies. Attachment to vitronectin was also inhibited in a dose-dependent manner by the type I plasminogen activator inhibitor (PAI-1), a serine protease inhibitor. Our data demonstrate that unstimulated primary neonatal rat astrocytes attach to vitronectin, utilizing integrins alphavbeta5 and alpha8beta1, and that this attachment is regulated by PAI-1.

Cytokine-regulated secretion of nerve growth factor from cultured rat neonatal astrocytes.

Interactions involved in the regulation of nerve growth factor (NGF) release by inflammatory cytokines: interleukin-1β (IL-1β), interleukin-6 (IL-6), tumour necrosis factor-α (TNF-α) and transforming growth factor-β1 (TGF-β1) were examined in rat neonatal cortical astrocytes in primary culture. Abstract Exposure of cultured astrocytes to IL-1β, IL-6, and TGF-β1 resulted in the stimulation of NGF secretion. Treatment of cells for 24 h with IL-1β (10 U/ml), IL-6 (5 ng/ml) and TGF-β1 (5 ng/ml) caused 3-, 1.8-, and 2.8-fold increase in NGF secretion as compared to the control cells. In contrast, TNF-α (30 ng/ml) by itself had no stimulatory action on NGF release whereas co-stimulation of astrocytes with IL-1β and TNF-α showed a synergistic interaction. Co-treatment of astrocytes with IL-1β and TGF-β1 increased NGF secretion in an additive way, whereas simultaneous application of IL-1β and IL-6 resulted in the inhibitory effect on NGF secretion. Our results suggest that interactions between cytokines used cause the stimulation of NGF secretion through different regulatory mechanisms.

GSH transport in human cerebrovascular endothelial cells and human astrocytes: evidence for luminal localization of Na +-dependent GSH transport in HCEC

The purpose of the present study was to identify and localize glutathione (GSH) transport in an in vitro tissue culture model of blood–brain barrier (BBB). The localization of Na +-dependent GSH transport in an immortalized cell line of human cerebrovascular endothelial cells (HCEC) and asymmetry of transport in Transwell™ studies were investigated. Initial studies with cultured HCEC established a significant (45%) Na +-dependency for GSH uptake in cultured HCEC pretreated with acivicin, an inhibitor of γ-glutamyltranspeptidase (GGT). Transendothelial electrical resistance (TEER) and uptake of [ 35 S ]GSH from luminal and abluminal fluids of HCEC were measured in Na +-containing and Na +-free (choline chloride) buffers using cells grown on gelatin-coated membrane filters. TEER of HCEC monolayers in regular medium was 40.1±8.0 Ω cm 2. Human astrocyte-conditioned medium (ACM) caused no change in TEER, but increased GGT activity approximately threefold when measured in cell lysates. Luminal and abluminal GSH uptake increased in a time-dependent fashion and were not affected by inhibition of GGT activity with acivicin. Sodium dependency was only observed for luminal uptake (Na +-containing 2.41±0.15 vs. Na +-free 0.96±0.03 pmol/30 min/million cells, p<0.001) but not for abluminal uptake (1.02±0.13 vs. 1.11±09, p>0.05). Apparent efflux via the luminal membrane was lower in the presence of sodium as compared to that without sodium, further suggesting that a Na +-dependent uptake process for GSH is operative at this membrane. GSH uptake and efflux were also demonstrated in neonatal rat and fetal human astrocytes, both exhibiting partial Na +-dependency of uptake. In conclusion, our results show for the first time, that HCEC and astrocytes take up GSH by both Na +-dependent and -independent mechanisms. The Na +-dependent GSH transport process in HCEC appears to be localized to luminal plasma membranes of HCEC.

Morphine Protects Peroxynitrite-induced Cell Death in Primary Rat Neonatal Astrocytes

Morphine Protects Peroxynitrite-induced Cell Death in Primary Rat Neonatal Astrocytes

7?-hydroxysterol is cytotoxic to neonatal rat astrocytes in primary culture when cAMP levels are increased

We have shown previously that 7beta-hydroxycholesterol (7betaOHCH) and 7beta-hydroxycholesteryl-3-oleate (7betaOHCH- 3-OL) are potent inhibitors of lesion-induced astrogliosis in the rat cortex or spinal cord; these substances reduce reactive astrocyte proliferation and hypertrophy. In this study, we employed cultured newborn rat astrocytes with increased cAMP levels as an in vitro model of reactive astrocytes. Treatment with either dibutyryl-cAMP (dbcAMP) or isoproterenol resulted in morphologic differentiation of astrocytes which became fibrous. Concomitant incubation with 30 microM 7betaOHCH and dbcAMP (or isoproterenol) provoked the cells to retract and was cytotoxic. When the beta-adrenergic receptor-mediated cAMP increase was abolished by propranolol, the 7betaOHCH cytotoxicity was inhibited. Immunocytochemical labelling for glial fibrillary acidic protein (GFAP) and beta-tubulin and electron microscopy suggested that intermediate filament and microtubular organizations were modified by 7betaOHCH. Analysis of the activity of cAMP-dependent protein kinase (PKA) in astrocytes treated with dbcAMP and 7betaOHCH showed a rapid and marked inhibition of the phosphotransferase activity which lasted for 24 hr. We suggest that this culture system provides an experimental system to study the molecular mechanisms involved in the effect of oxysterols on astrocytic hypertrophy. The cytotoxicity of 7betaOHCH seems to be mediated by inhibition of PKA, which phosphorylates intermediate filaments and the transcription factor cyclic AMP responsive element binding.

Effects of epidermal growth factor and dibutyryl cyclic adenosine monophosphate on the migration pattern of astrocytes grafted into adult rat brain.

Neonatal rat astrocytes transplanted into host brains migrate in specific patterns, which are determined by the developmental stage of the host brain and the region of implantation. We hypothesized that the differentiation state of the implanted astrocytes could also affect astrocyte migration. Astrocytes derived from neonatal rats (1-4 d) were placed in culture and exposed to growth- or differentiation-promoting agents (e.g., epidermal growth factor or dibutyryl cyclic adenosine monophosphate). Treated cells were then injected into different regions of the adult rat brain. At 3, 6, and 9 days after implantation, the extent and pattern of astrocyte migration after injection into the cortex, hippocampus, and corpus callosum were assessed. Astrocytes pretreated with either factor did not migrate during the first 3 days after implantation into the host cortex and hippocampus, whereas untreated cells migrated extensively by Day 3. After 9 days, implanted cells that had been pretreated with dibutyryl cyclic adenosine monophosphate began to demonstrate migratory activity, whereas those exposed to epidermal growth factor remained at the site of implantation. These findings corresponded to the effects of these agents in culture. On the other hand, cells implanted into the corpus callosum migrated in spite of pretreatment. Epidermal growth factor and dibutyryl cyclic adenosine monophosphate each altered the cells in culture such that they were inhibited from migrating after transplantation into the host cortex and hippocampus. This finding suggests that the activation of either growth or differentiation cascades partially inhibits the migratory ability in these cells either through effects on their internal migratory potentials or their responsiveness to external migratory signals. In contrast, cells implanted into the corpus callosum migrated in spite of pretreatment, suggesting that this structure may present migratory cues sufficient to override the effects of treatment.

Neuregulins and ErbB receptors in cultured neonatal astrocytes

Neuregulins (NRGs) are a family of growth factors involved in signaling between neurons and glial cells of the peripheral and central nervous system. NRGs are synthesized and secreted by a number of cell types including Schwann cells, neurons, and oligodendrocytes. NRG transduction signals are mediated by the erbB family of receptor tyrosine kinases. These NRGs may be important for paracrine or autocrine signaling during development, injury, and the normal functioning of the central nervous system. In this study, we characterize the NRGs and erbB receptors expressed by cultured neonatal rat astrocytes. Using immunoblotting protocols with pan-specific antibodies, we identified eleven NRG molecular weight isoforms from approximately 16 kDa to 105 kDa in cultured neonatal rat astrocytes. Immunocyotchemistry with isoform-specific antibodies revealed the expression of both major isoform families (NRGalpha, NRGbeta). Additionally, astrocyte-conditioned media contained two molecular weight isoforms of NRGs. We detected mRNA expression of NRGalpha and NRGbeta in astrocytes by amplifying mRNA transcripts with reverse transcription polymerase chain reaction. Furthermore, we confirm that cultured astrocytes express all four erbB receptors as detected by immunocytochemical and immunoblotting techniques. These data indicate that astrocytes contain and secrete NRGs.

Regulation of type 3 iodothyronine deiodinase expression in cultured rat astrocytes: role of the Erk cascade.

The type 3 iodothyronine deiodinase (D3) metabolizes thyroid hormones to inactive metabolites in many tissues, including the brain. In the present studies, we have examined the mechanisms by which T3 (T3), retinoic acid, 12-O-tetradecanoyl phorbol 13-acetate (TPA), and basic fibroblast growth factor (bFGF) induce D3 expression in primary cultures of neonatal rat astrocytes. In untreated cells, D3 messenger RNA (mRNA) was essentially undetectable by Northern analysis and RT-PCR. However, all four agents induced expression of a 2.4-kb D3 transcript as well as D3 activity. Induction of D3 by TPA and bFGF was more rapid than that by T3 and retinoic acid, and T3 potentiated the stimulatory effects of TPA and bFGF. D3 induction by TPA was blocked by GF 109203X, an inhibitor of protein kinase C. In addition, the effects of TPA and bFGF were partially prevented by PD 98059, a specific inhibitor of MEK and the Erk signaling cascade. These studies demonstrate that multiple growth factors and hormones regulate D3 activity in cultured astrocytes by inducing D3 mRNA expression. In addition, the stimulatory effects of TPA and bFGF on D3 mRNA and activity appear to be mediated at least in part by activation of the MEK/Erk signaling cascade.

Regulation of cytosol–nucleus pH gradients by K +/H + exchange mechanism in the nuclear envelope of neonatal rat astrocytes

In order to study the subcellular heterogeneity of intracellular H + concentration in reactive astrocytes, the pH in the nucleus and cytosol of cultured astrocytes was measured using a confocal laser scanning microscope (CLSM) and pH indicator dye, 5′(and 6′)-carboxyseminaphthofluorescein (carboxy SNAFL-1). The change in intracellular pH was indexed by the fluorescence ratio ( F 535/ F 610) at an excitation wavelength of 514.5 nm. The in vitro fluorescence ratio increased as pH decreased. This ratio in the nucleus was significantly lower than that in the cytosol of astrocytes when perfused by HEPES-buffered Hanks' balanced salt solution (HHBSS) at pH 7.4. Acid stimulations of cells (pH 5.0) raised the fluorescence ratio in both nucleus and cytosol. However, the increase in the fluorescence ratio of the nucleus was less than that of cytosol. Treatment with a K +/H + ionophore, nigericin (20 μM), reversibly nullified this cytosol–nucleus pH gradient. These findings suggest that a buffering mechanism(s) for maintaining of intracellular pH exists between the nucleus and cytosol, and a K +/H + exchanger may act on the nuclear envelope to eventuate intranuclear pH maintenance in the living cells.

Transforming growth factor‐β mediates the neurotrophic effect of fibroblast growth factor‐2 on midbrain dopaminergic neurons

Fibroblast growth factor (FGF)-2 is an established neurotrophic factor for dopaminergic (DAergic) neurons in the ventral midbrain. Its survival and differentiation-promoting effects on DAergic neurons in vitro and in vivo are crucially dependent on the presence, numerical expansion and maturation of astroglial cells. We show now that transforming growth factor (TGF)-beta, an established trophic factor for DAergic neurons and product of astroglial cells, mediates the trophic effect of FGF-2 on DAergic neurons cultured from the embryonic rat midbrain floor. Antibodies to TGF-beta that neutralize the isoforms -beta1, -beta2 and -beta3 abolish the trophic effect of FGF-2. FGF-2 increases TGF-beta3 mRNA and amounts of biologically active TGF-beta determined in a mink lung epithelial cell assay in a time-dependent manner. FGF-2 also induces levels of active TGF-beta in neonatal rat astrocytes cultured from midbrain, striatum and cortex. We conclude that TGF-beta is required for mediating the survival promoting effect of FGF-2 on DAergic and, possibly, cortical and striatal neurons grown in the presence of glial cells.

Transforming growth factor-beta mediates the neurotrophic effect of fibroblast growth factor-2 on midbrain dopaminergic neurons

Fibroblast growth factor (FGF)-2 is an established neurotrophic factor for dopaminergic (DAergic) neurons in the ventral midbrain. Its survival and differentiation-promoting effects on DAergic neurons in vitro and in vivo are crucially dependent on the presence, numerical expansion and maturation of astroglial cells. We show now that transforming growth factor (TGF)-beta, an established trophic factor for DAergic neurons and product of astroglial cells, mediates the trophic effect of FGF-2 on DAergic neurons cultured from the embryonic rat midbrain floor. Antibodies to TGF-beta that neutralize the isoforms -beta1, -beta2 and -beta3 abolish the trophic effect of FGF-2. FGF-2 increases TGF-beta3 mRNA and amounts of biologically active TGF-beta determined in a mink lung epithelial cell assay in a time-dependent manner. FGF-2 also induces levels of active TGF-beta in neonatal rat astrocytes cultured from midbrain, striatum and cortex. We conclude that TGF-beta is required for mediating the survival promoting effect of FGF-2 on DAergic and, possibly, cortical and striatal neurons grown in the presence of glial cells.