Associations of lead exposure and dose measures with erythrocyte protein kinase C activity in 212 current Korean lead workers.

Lead can replace calcium in enzyme assays that measure protein kinase C activity and lead activates protein kinase C in human erythrocytes after exposure to lead in vitro. To examine the relevance of these observations to lead exposure in humans, we studied the associations of lead found in blood or tibia with activation of protein kinase C in erythrocytes isolated from workers in the lead industry. We examined erythrocytes among 212 lead workers, with a mean (+/-SD) age of 39.1 (10.0) years and exposure duration of 8.1 (6.5) years and measured protein kinase C activation by an in vitro back-phosphorylation assay. After adjustment for potential confounding factors (age and sex), tibia lead and exposure duration were significantly associated with erythrocyte protein kinase C activation (both p values < 0.05). No associations were observed between protein kinase C activation and blood-lead or zinc-protoporphyrin levels. These findings suggest that human exposure to lead results in activation of erythrocyte protein kinase C, which may be directly relevant to the neurotoxicity of lead.

Immediate early gene expression in PC12 cells exposed to lead: requirement for protein kinase C.

We previously demonstrated induction of c-fos mRNA in PC12 cells exposed to lead that was dependent on new transcription. In the current work, we examined two signal transduction mechanisms that are activated by lead and have been shown to mediate induction of c-fos mRNA. One mechanism involves protein kinase C, and the other requires calmodulin-dependent protein kinase II. Significant increases in the levels of c-fos, c-jun, and egr-1 but not NGFIB mRNA were observed in PC12 cells exposed to lead or phorbol 12-myristate 13-acetate. In contrast, PC12 cells depolarized with 56 mM K+ displayed an increase in c-fos, egr-1, and NGFIB but not c-jun mRNA. Similar to other activators of protein kinase C, lead increased AP-1 and Egr-1 DNA binding activity. Additionally, lead increased luciferase activity in cerebellar granule cells transfected with an AP-1 luciferase reporter construct. Lead did not increase c-fos mRNA in PC12 cells that were depleted of protein kinase C by a 24-h treatment with phorbol 12,13-dibutyrate or incubated with the protein kinase C inhibitor H-7. In contrast, an inhibitor of calmodulin-dependent protein kinase, KN-62, and an inhibitor of calmodulin, W-7, did not block the induction of c-fos mRNA by lead. An increase in serum-response element DNA-binding activity was observed in nuclear extracts from PC12 cells exposed to lead. It is interesting that lead activated protein kinase C isoforms delta and epsilon, but not isoforms alpha and beta. In conclusion, lead appears to induce the expression of immediate early genes by a mechanism that requires protein kinase C.

Increased AP-1 DNA binding activity in PC12 cells treated with lead.

The possibility that the mechanism of lead neurotoxicity may be at the level of transcription was investigated in PC12 cells. In electrophoretic mobility gel shift assays Pb2+ was found to increase activator protein-1 complex (AP-1) DNA binding activity in PC12 cells; the increase was time- and concentration-dependent. Exposure to Pb2+ also resulted in an increase in AP-1-driven transcription in cerebellar granule cells transfected with a luciferase gene reporter construct. The increase in AP-1 DNA binding activity by Pb2+ required protein synthesis. The increase was mediated by protein kinase C because depletion of protein kinase C and an inhibitor of protein kinase C prevented the increase in AP-1 DNA binding activity by Pb2+. Fra-2 and JunD were found in supershift assays to be the major components of the AP-1 that was increased by Pb2+. In summary, our studies indicate that Pb2+ increases AP-1 DNA binding activity in PC12 cells by a pathway that requires protein kinase C and new protein synthesis.

Induction of c-fos mRNA by lead in PC 12 cells.

Addition of lead acetate to PC 12 pheochromocytoma cells elicits induction of c-fos, an immediate early response gene. Induction of c-fos was concentration- and time-dependent: the lowest concentration of lead acetate tested that induced c-fos was 10 microM; induction was observed after a 30 min incubation and remained high after 90 min. Treatment with lead acetate and cycloheximide superinduced c-fos mRNA. Actinomycin D, an inhibitor of mRNA transcription, decreased the level of c-fos mRNA induced by lead acetate by almost 80%. Cadmium chloride and zinc chloride did not induce c-fos mRNA. Since the c-fos gene encodes a transcription factor, Pb2+ has the potential to deregulate the expression of other genes.

Distinct mechanisms of neurotransmitter release from PC 12 cells exposed to lead.

Two enzymes, protein kinase C and microsomal Ca(2+)-ATPase help regulate levels of Ca2+ in many types of cells. Since proteins that regulate Ca2+ often influence sensitivity to Pb2+, we determined the possible roles played by protein kinase C and microsomal Ca(2+)-ATPase for the Pb(2+)-evoked release of norepinephrine (NOR) in PC cells. NOR release was observed at 10 microM Pb2+ when PC 12 cells were stimulated with inhibitors of microsomal Ca(2+)-ATPase such as thapsigargin, cyclopiazonic acid, or 2,5-di-(t-butyl)-hydroquinone. At 5 microM, Pb2+ evoked the release of NOR in PC 12 cells stimulated with activators of protein kinase C such as phorbol 12-myristate 13-acetate (PMA) or (-)-7-octylindolactam. NOR release was observed at 1 microM Pb2+ in the presence of both PMA and thapsigargin. Ni2+ and Cd2+ blocked NOR release stimulated by Pb2+ in the presence of thapsigargin but not by PMA. NOR released by thapsigargin stimulation was not altered in PC 12 cells depleted of protein kinase C. Two proteins found in vesicles, chromogranin B and secretogranin-II were released with NOR. Our results indicate that in PC 12 cells, PB(2+)-evokes the release of neurotransmitters. Furthermore, thapsigargin and PMA increase the cell's sensitivity to Pb2+ by different pathways.

Role of anion exchange and thiol groups in the regulation of potassium efflux by lead in human erythrocytes.

Pb2+ is thought to enter erythrocytes through anion exchange (AE) and to remain in the cell by binding to thiol groups. To define the role of AE mechanisms and thiol groups in Pb2+ toxicity, we studied the effects of drugs and conditions that modify AE and that modify thiol groups on the ability of Pb2+ to stimulate potassium efflux as measured with 86Rb. The most potent stimulation of 86Rb efflux by Pb2+ occurred when conditions were optimal for the AE mechanism--that is, when bicarbonate was included in the buffer or a buffer made with Nal or NaCl rather than NaClO4 or NaNO3 was used. Furthermore, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid and 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfuonic acid, potent inhibitors of the AE mechanism, completely inhibited stimulation of the 86Rb efflux by Pb2+. These conditions or inhibitors did not affect stimulation of the 86Rb efflux by ionomycin plus Ca2+. A role for Ca2+ channels was dismissed because the inorganic Ca2+ channel blockers, Cd2+ or Mn2+, did not prevent stimulation of 86Rb efflux by Pb2+ but did inhibit stimulation by ionomycin plus Ca2+. 86Rb efflux was more sensitive to Pb2+ if erythrocytes were treated for 15 min with thiol-modifying reagents that enter cells, such as iodoacetamide, N-ethylmaleimide, or dithiothreitol, than to reduced glutathione, a thiol-modifying reagent that is not permeable to the cell. Thus, in erythrocytes the AE mechanism and internal thiol groups are critical factors that affect the stimulation of a Ca(2+)-dependent process by Pb2+.

Phosphorylation of membrane proteins in erythrocytes treated with lead.

In immature rat microvessels, endothelial cells and glioma cells, exposure to lead results in an increase in the level of protein kinase C in membranes. In this paper we have extended these studies to human erythrocytes and, in addition, studied the phosphorylation of membrane proteins. A significant increase in the phosphorylation of membrane cytoskeletal proteins of molecular mass 120, 80, 52 and 45 kDa was observed in human erythrocytes treated for 60 min with lead acetate at concentrations greater than 100 nM. These same proteins were phosphorylated when erythrocytes were treated for 10 min with 50 nM phorbol 12-myristate 13-acetate (PMA). Similarly, protein kinase C activity was elevated and an increase in the amount of protein kinase C-alpha was observed in membranes from erythrocytes exposed to concentrations of lead acetate above 100 nM. No changes, however, in the activities of cAMP-dependent protein kinase, protein phosphatases I and IIA or casein kinase were observed. Phosphorylation of these membrane proteins stimulated by lead acetate or by PMA was not observed in erythrocytes depleted of protein kinase C by a 72-h treatment with 500 nM phorbol 12,13-dibutyrate. Finally, no changes in the levels of calcium or diacylglycerol were observed in erythrocytes stimulated with 100 nM lead acetate. These results indicate that, in erythrocytes, lead acetate stimulates the phosphorylation of membrane cytoskeletal proteins by a mechanism dependent on protein kinase C. Since levels of calcium or diacylglycerols did not increase, it appears that lead may activate the enzyme by a direct interaction.

Characterization of synaptic vesicles and related neuronal features in nerve growth factor and ras oncogene differentiated PC12 cells.

PC12 cells can differentiate into neuron-like cells after treatment with either nerve growth factor (NGF) or transduction with a retrovirus which expresses the K-ras oncogene. The concomitant treatment of NGF plus ras differentiates PC12 cells further than either agent alone with respect to neurite outgrowth, acetylcholinesterase levels, and most strikingly, the number of synaptic vesicle (SV) clusters. These SV clusters in PC12 cell neurites closely resemble those in the presynaptic terminals of neurons. Such SV clusters have not been described in cell lines previously. The SV clusters from all three differentiated groups (NGF, ras, and NGF plus ras) were similar in size, shape, and configuration, except that the ones in the doubly treated group occur in higher frequency and have more vesicles. The synaptic nature of these vesicle clusters was demonstrated by their regulated depletion after potassium stimulation. Furthermore, these vesicle clusters stained positively for two SV-associated proteins, synapsin I and synaptophysin, by EM immunocytochemistry (ICC). Such SV clusters in a cell line are very useful for characterizing the regulated release of SVs and the distribution of SV-related antigens in intact cells. Analysis by SDS-gel electrophoresis and immunoblotting indicated that synapsin I levels are higher in all three differentiated groups compared to untreated cells; whereas synaptophysin levels are lower in cells exposed to NGF alone or with NGF and ras double treatment. Possible convergence and/or divergence on the mechanisms of NGF and ras differentiation in PC12 cells are discussed.

Effect of ganglioside GM1 on arachidonic acid release in bovine aortic endothelial cells.

A role for the ganglioside GM1 in arachidonic acid release in bovine aortic endothelial cells (BAEC) was investigated. [3H]Arachidonic acid-labeled BAEC were preincubated with GM1 and incubated with one of four different stimulators. GM1 inhibited arachidonic acid release when stimulated with maitotoxin or melittin but not with ionomycin or thapsigargin. A 10 microM GM1 concentration achieved a 50% and 100% inhibition of the maitotoxin and melittin responses, respectively. The selective inhibition displayed by GM1 on the maitotoxin and melittin responses was not due to its ability to bind calcium since all four drugs, maitotoxin, melittin, ionomycin, and thapsigargin, required extracellular calcium. The effect of GM1 was not specific to arachidonic acid release. GM1 at 50 microM inhibited phosphatidylinositol polyphosphate (PIP) hydrolysis mediated by melittin, but did not affect hydrolysis mediated by ionomycin. Moreover, the activity of GM1 was not restricted to phospholipid metabolism since it also inhibited calcium influx that was stimulated by maitotoxin or melittin but not by ionomycin. We conclude that GM1 is not a specific inhibitor of phospholipases in bovine aortic endothelial cells, but rather its activity is dependent on the type of stimulant used to activate the cell.

Inhibition of astroglia-induced endothelial differentiation by inorganic lead: a role for protein kinase C.

Microvascular endothelial function in developing brain is particularly sensitive to lead toxicity, and it has been hypothesized that this results from the modulation of protein kinase C (PKC) by lead. We examined the effects of inorganic lead on an in vitro model of central nervous system endothelial differentiation in which astroglial cells induce central nervous system endothelial cells to form capillary-like structures. Capillary-like structure formation within C6 astroglial-endothelial cocultures was inhibited by lead acetate with 50% maximal inhibition at 0.5 microM total lead. Inhibition was independent of effects on cell viability or growth. Under conditions that inhibited capillary-like structure formation, we found that lead increased membrane-associated PKC in both C6 astroglial and endothelial cells. Prolonged exposure of C6 cells to 5 microM lead for up to 16 h resulted in a time-dependent increase in membranous PKC as determined by immunoblot analysis. Membranous PKC increased after 5-h exposures to as little as 50 nM lead and was maximal at approximately 1 microM. Phorbol esters were used to determine whether PKC modulation was causally related to the inhibition of endothelial differentiation by lead. Phorbol 12-myristate 13-acetate (10 nM) inhibited capillary-like structure formation by 65 +/- 5%, whereas 4 alpha-phorbol 12,13-didecanoate was without effect. These findings suggest that inorganic lead induces cerebral microvessel dysfunction by interfering with PKC modulation in microvascular endothelial or perivascular astroglial cells.

Astroglial membrane structure is affected by agents that raise cyclic AMP and by phosphatidylcholine phospholipase C.

The role of signal transduction mechanisms in the production of the characteristic orthogonal arrays of particle assemblies in the astroglial plasma membrane was investigated in vitro by freeze-fracture electron microscopy. Agents which raise cellular cAMP levels and subsequently activate protein kinase A, such as forskolin (50 microM), isoproterenol (10 microM) and 8-bromo-cAMP (1 mM), increased the density, the number of assemblies per unit area of cleaved cell membrane, and the frequency of astrocytes with assemblies. Agents that lead to the activation of protein kinase C, such as phorbol 12,13-myristate acetate (at 50 nM) and choline-dependent phospholipase C (at 0.01-0.1 U ml-1), did not affect the assembly concentration. Thus, protein kinase A but not protein kinase C appears to be involved in the production of assemblies or their insertion into the astroglial plasma membrane. Although choline-dependent phospholipase C did not affect the astroglial assemblies, it caused the non-assembly, background particles to aggregate. A choline-dependent phospholipase C from a different source (B. cereus) was also active though at a higher concentration. Phospholipases of different specificities, such as phospholipase A2, phospholipase D or inositol-dependent phospholipase C were inactive over a wide range of concentrations. Two other astroglia derived cells, Müller cells and cells of the C6 glioma cell line, were also similarly affected by choline-dependent phospholipase C, while six other cells types including neurons, endothelial cells and fibroblasts were unaffected. It appears that phosphatidylcholine plays a significant role in determining the membrane structure of astrocytes. In a search for a means of isolating the assemblies, the binding of three lectins: ConA, WGA and PNA, conjugated to gold, was tested by label-fracture to ascertain whether the assemblies have an external oligosaccharide component. None of the lectins bound specifically to assemblies.

Retinal microvessels express less gamma-glutamyl transpeptidase than brain microvessels.

In this investigation we localized and compared the level of gamma-glutamyl transpeptidase (GGTP) activity in retinal and brain preparations using histochemical, enzymatic and in situ hybridization assays. We compared GGTP distribution to another microvessel specific enzyme, alkaline phosphatase (AP). In the rat brain, GGTP activity was observed in microvessels and choroid plexus by a histochemical method. Similar studies in the rat retina revealed activity in the pigment epithelium but only a very weak reaction in microvessels. Histochemical staining for alkaline phosphatase was observed in both retinal and brain microvessels choroid plexus and pigment epithelium. Biochemical analysis verified that GGTP activity was significantly lower in retinal than brain microvessels, while alkaline phosphatase activity was similar in both types of microvessels. GGTP specific activity of bovine brain and retinal microvessels was 185 +/- 39 mUnits and 8.5 +/- 1.5 mUnits (p less than 0.001), respectively. By contrast, alkaline phosphatase specific activity in brain and retinal microvessels was 732 +/- 139 and 471 +/- 114 (p greater than 0.1), respectively. Choroid plexus and retinal pigment epithelium exhibited similar levels of GGTP and alkaline phosphatase. Differences in GGTP expression between retinal and brain microvessels were also observed on the mRNA level. In situ hybridization studies revealed that brain microvessels expressed four times more GGTP specific mRNA than retinal microvessels. We conclude that retinal microvessels do not express high levels of GGTP which may make them more vulnerable than brain microvessels to injuries mediated by leukotrienes and oxidative stress.

Gamma-glutamyl transpeptidase activity in brain microvessels exhibits regional heterogeneity.

Brain microvessels form a tight blood-tissue permeability barrier and express high levels of specific enzymes, including gamma-glutamyl transpeptidase (GGTP). This differentiation is thought to be induced by perivascular astrocytes. By using histochemical methods, we found that the percentage of GGTP-positive vessels varied considerably in different areas of rat brain. Enzyme activity was not found in the pineal gland or the median eminence, where the blood-brain barrier is not expressed. In areas where the blood-brain barrier is expressed, the percentage of GGTP-positive vessels varied from 8% in the optic nerve to 100% in the anterior commissure. The neocortex showed a lower percentage of GGTP-positive vessels (2-15%) than anterior olfactory nucleus (42%), subiculum (70%), hippocampus (48%), and striatum (50-58%). Alkaline phosphatase, another brain microvessel-enriched enzyme, did not show these marked regional differences. The morphometric histochemical results were verified by enzymatic assays in homogenates of different regions from rat and bovine brain and in microvessel preparations of bovine putamen and neocortex. During the postnatal development of rat brain, the difference between neocortex and striatum appeared after day 20. The regional heterogeneity of brain microvessels may be caused by astrocytic heterogeneity and reflect regional heterogeneity in microvascular function.

Glial shape and cytoskeletal protein synthesis.

We investigated whether the shape of astroglial derived cells influences the expression of cytoskeletal proteins. In reaggregating cultures GFAP, vimentin and actin synthesis was approximately 52%, 50% and 37% the level found in monolayer cultures, respectively. Monolayer cultures consisted of polygonal shaped cells adhering to plastic, while reaggregating cultures were comprised of round cells growing in a suspension like culture. Additionally, human glioma cells induced to grow as round cells on poly-2-hydroxyethyl methacrylate (polyhema) coated plastic exhibited a level of GFAP synthesis that was approximately 20% the level displayed by polygonal shaped cells grown on uncoated plastic. Glioma cells initially grown on a polyhema surface and replated onto uncoated plastic were capable of reinitiating GFAP synthesis. Thus, alterations in the synthesis of GFAP and other cytoskeletal proteins can occur when astrocytes change their shape.

Mechanisms of lead neurotoxicity.

During the past several years, there has been a renewed interest in the mechanisms by which lead poisoning disrupts brain function. In part, this is related to clinical observations that imply an absence of threshold for toxicity in the immature brain. Many of the neurotoxic effects of lead appear related to the ability of lead to mimic or in some cases inhibit the action of calcium as a regulator of cell function. At a neuronal level, exposure to lead alters the release of neurotransmitter from presynaptic nerve endings. Spontaneous release is enhanced and evoked release is inhibited. The former may be due to activation of protein kinases in the nerve endings and the latter to blockade of voltage-dependent calcium channels. This disruption of neuronal activity may, in turn, alter the developmental processes of synapse formation and result in a less efficient brain with cognitive deficits. Brain homeostatic mechanisms are disrupted by exposure to higher levels of lead. The final pathway appears to be a breakdown in the blood-brain barrier. Again, the ability of lead to mimic or mobilize calcium and activate protein kinases may alter the behavior of endothelial cells in immature brain and disrupt the barrier. In addition to a direct toxic effect upon the endothelial cells, lead may alter indirectly the microvasculature by damaging the astrocytes that provide signals for the maintenance of blood-brain barrier integrity.

Some neuronal properties of PC12 cells differentiated by the K-ras oncogene.

When infected with a virus containing the Kirsten-ras oncogene, rat phaeochromocytoma or PC12 cells elaborated neurites and ceased mitosis, that is, they underwent neuronal differentiation. Such differentiated cells could be replaced and maintained up to 20 weeks in vitro without the need of an exogenous, continuous supply of nerve growth factor (NGF). The neurites of K-ras infected PC12 cells, filled with microtubules and actin which was concentrated within the growth cones, resembled those of primary neurons in vitro. As in the NGF-primed PC12 cells, two types of secretory vesicles were present in the K-ras-infected PC12 neurites: large (100 nm), dense core granules, and small (45 nm), clear vesicles. Compared to naive PC12 cells, K-ras infected PC12 cells had (a) higher activities of acetylcholinesterase and choline acetyltransferase, two enzymes involved in acetylcholine metabolism; (b) enhanced activity of tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis; (c) a higher, evoked norepinephrine release; and (d) similar levels of sodium-dependent uptake of both choline and norepinephrine. Although the total content of catecholamines in K-ras-differentiated PC12 cells was less than that of naïve cells, both norepinephrine and dopamine were present in substantial amounts and norepinephrine was released after stimulation. According to their enzymatic activity, these cells can also synthesize acetylcholine and thus have potential as donors for the intracerebral replacement of either catecholaminergic or cholinergic neurotransmitters.

Regulation of cAMP levels by protein kinase C in C6 rat glioma cells.

Cultures of rat C6 rat glioma cells exhibit a diminished response to isoproterenol and forskolin after being treated with phorbol 12,13-dibutyrate (PDbU). An IC50 for PDbU of 38 +/- 5 nM and 62 +/- 8 nM was observed in the isoproterenol and forskolin response, respectively. Similarly, C6 cultures exhibited a diminished response to isoproterenol and forskolin after an overnight incubation with phospholipase C. We previously demonstrated that this treatment will increase diacylglycerol levels in these cells (Bressler: J Neurochem 48:181-186, 1987). An IC50 for phospholipase C of 6.0 +/- 0.1 x 10(-1) and 7.0 +/- 0.1 x 10(-1) units/ml was observed for the isoproterenol and forskolin response, respectively. A kinetic analysis suggests that the site of PDbU-mediated inhibition to beta-adrenergic and forskolin stimulation was different. Degradation of cAMP was a contributory factor since elevated cAMP levels decreased faster in PDbU treated cells than in nontreated cells. In addition, PDbU treated cells exhibited a significantly higher level of phosphodiesterase activity. We conclude that activation of protein kinase C and subsequent stimulation of phosphodiesterase activity contributes to the inhibition of the beta-adrenergic and forskolin mediated increase in cAMP levels in intact C6 rat glioma cells. The consequences of lower cAMP levels in sustaining differentiated function in the C6 rat glioma cell line will be discussed.

Second stage tumor promoters: differences in biological potency and phorbol ester receptor affinity in C6 cells.

We have shown that the second stage tumor promoters mezerein (MEZ) and phorbol 12-retinoate 13-acetate (PRA) inhibit the gluccocorticoid-induced increase in glycerol phosphate dehydrogenase (GPDH) activity in C6 rat glioma cells with ED 50-values of 3.9 and 2.9 nM, respectively. Phorbol 12-myristate 13-acetate (PMA) was 10-fold less potent. MEZ was likewise more potent than PMA for inhibition of cAMP formation in response to isoproterenol. Binding competition studies using [3H]phorbol 12,13-dibutyrate ([3H]PDBu) yielded apparent Ki-values for MEZ and PRA of 50-70 nM. The large difference between the biological potencies of MEZ and PRA and their affinity for the major phorbol ester receptor suggest they may be acting through a more complicated mechanism in these cells.

Neoplastic transformation of newborn rat astrocytes in culture.

A subpopulation of rat glial cells, derived from the astroglial population of newborn cerebral cortex cell cultures, spontaneously transformed in culture. Unlike the pretransformed cells, the transformed cells formed pile-up colonies, exhibited anchorage-independent growth, and were tumorigenic in young rats. Both the pretransformed and transformed cells exhibited differentiated properties characteristic of glial cells. For example, the pretransformed cells possessed hydrocortisone-inducible glutamine synthetase (GS), a property restricted to astrocytes in the central nervous system. As was anticipated, these cells did not exhibit either of two oligodendroglial characteristics, hydrocortisone-inducible glycerol phosphate dehydrogenase (GPDH) or the induction of lactate dehydrogenase (LDH) by N6,O6-dibutyryl cyclic adenosine 3':5'monophosphate (Bt2 cAMP). Unexpectedly, the transformed cells expressed the induction of glycerol phosphate dehydrogenase and lactate dehydrogenase but lost the glutamine synthetase induction. Both the pretransformed and transformed cells were examined ultrastructurally. Neither cell type exhibited glial filaments (9-10 nm), a structure typical of astrocytes. Rather, the pretransformed cells were characterized by distinct longitudinal filaments near the cell surface and the absence of microtubules. On the other hand, the only cytoskeletal element visible in transformed cells were microtubules. Our work demonstrates that, like other rodent cell types, rat glial cells can spontaneously transform in culture. It also shows that the expression of differentiated properties are sensitive to the transformation process.

Chemotaxis of rat brain astrocytes to platelet derived growth factor.

Using a purified population of rat brain astrocytes prepared from neonatal cortex, we investigated the chemotaxis of astroglia to several well characterized growth factors. Chemotactic activity for astrocytes was found for the platelet derived growth factor with a half maximal response occurring at 0.5 ng/ml as compared with a value of 2-3 ng/ml obtained for NIH/3T3 fibroblasts in control experiments. Other growth factors including epidermal growth factor, fibroblast growth factor and insulin were inactive as chemoattractants. Affinity purified fibronectin was also found to stimulate the migration of astroglia, with half maximal doses of approximately 1 microgram/ml relative to maximal responses to platelet derived growth factor.