Myelin Gene Expression in Immortalized Schwann Cells: Relationship to Cell Density and Proliferation

Abstract: Myelin gene expression was investigated in the immortalized S16 Schwann cell line grown in the presence and absence of serum and at different densities. Protein expression was monitored by western blotting, and message levels were determined by RNase protection assays. To study cell proliferation rates at different cell densities and serum conditions. [³H]thymidine uptake assays and cell counts were performed. Although serum deprivation decreased cell proliferation as expected, the proliferation of S16 cells was unchanged or slightly increased at high density under the conditions of our experiments in either serum-containing or serum-free medium. This increased cell division at high density appeared to be due to greater release of an autocrine growth factor to the medium by dense cell populations. For both sparse and dense cells, substantially more P₀ glycoprotein (P₀) and myelin-associated glycoprotein (MAG) per milligram of total cellular protein were expressed when the cells were proliferating slowly in defined medium in comparison with more rapidly proliferating cells in serum-containing medium. Furthermore, in both serum-containing and defined media, dense cell populations expressed more MAG and P₀ than sparse ones. P₀ mRNA and MAG mRNA levels generally paralleled protein levels. The level of mRNA for peripheral myelin protein-22 (PMP-22) was also increased at high cell density but did not change much when proliferation was decreased by serum deprivation. PMP-22 protein was not detected under any of the growth conditions. The changes in expression of these genes with growth conditions may be specific for myelin proteins, because the expression of a nonmyelin glycoprotein, L1, remained constant. The level of cyclic AMP in the cells did not change with the different growth conditions tested. The results indicate that the S16 Schwann cell line mimics primary or secondary Schwann cells by down-regulating myelin gene expression when it proliferates more rapidly in the presence of serum. Furthermore, in both the presence and absence of serum, there was greater expression of myelin genes at high cell density that was not associated with a decreased proliferative rate. Because evidence for a role of secretory factors in affecting myelin gene expression was not obtained by treating sparse S16 cells with medium conditioned by dense S16 cells, the results suggest that the higher expression of myelin genes at high density may be mediated by cell-to-cell contact.     

Regulation of Myelination: Biosynthesis of the Major Myelin Glycoprotein by Schwann Cells in the Presence and Absence of Myelin Assembly

Schwann cell biosynthesis of the major myelin glycoprotein, P0, was investigated in the crush-injured adult rat sciatic nerve, where there is myelin assembly, and in the permanently transected nerve, where there is no myelin assembly. Endoneurial fractions from desheathed rat sciatic nerves distal to the crush were compared with similar fractions from the permanently transected nerves at 7, 14, 21, 28, and 35 days after injury. The Schwann cell expression of this asparagine-linked glycoprotein was evaluated after sodium dodecyl sulfate-pore gradient electrophoresis by Coomassie Blue and silver stain and by autoradiography after direct overlay of radioiodinated lectins [wheat germ agglutinin, gorse agglutinin, and concanavalin A (Con A)]. As evaluated by these parameters, the concentration of P0 after crush decreased and subsequently increased as a function of time after injury, corresponding to the events of demyelination and remyelination. After permanent transection, the P0 concentration decreased following the same time course found after crush. At subsequent time points, P0 could not be detected with Coomassie Blue stain, silver stain, or wheat germ agglutinin. Both gorse agglutinin and Con A, however, showed binding to P0. Radioactive precursor incorporation studies with [3H]fucose or [3H]-mannose into endoneurial slices at 35 days posttransection revealed active oligosaccharide processing of P0 glycoprotein by Schwann cells in this permanent transection model. Compared with other Schwann cell glycoproteins in the transected nerve, the highest level of incorporation of [3H]mannose was found in P0 which accounted for 42.7% of the incorporated label. In contrast, incorporation of [3H]mannose into endoneurial slices at 35 days after crush accounted for only 13.3% in P0. In addition, higher levels of Con A binding were observed in P0 in the transected nerve compared with the contralateral control or the crushed nerve. Both the [3H]fucose incorporation and gorse agglutinin binding to P0 in the transected nerve suggest posttranslational processing of this glycoprotein in the Golgi apparatus; however, the absence of wheat germ agglutinin binding, the high level of mannose incorporation, and the high level of binding by Con A imply that additional processing steps are required prior to its assembly into myelin.     

Posttranslational Protein Modification: Biosynthetic Control Mechanisms in the Glycosylation of the Major Myelin Glycoprotein by Schwann Cells

The posttranslational processing of the asparagine-linked oligosaccharide chain of the major myelin glycoprotein (P0) by Schwann cells was evaluated in the permanently transected, adult rat sciatic nerve, where there is no myelin assembly, and in the crush injured nerve, where there is myelin assembly. Pronase digestion of acrylamide gel slices containing the in vitro labeled [3H]mannose and [3H]fucose P0 after electrophoresis permitted analysis of the glycopeptides by lectin affinity and gel filtration chromatography. The concanavalin A-Separose profile of the [3H]mannose P0 glycopeptides from the transected nerve revealed the high-mannose-type oligosaccharide as the predominant species (72.9%), whereas the normally expressed P0 glycoprotein that is assembled into the myelin membrane in the crushed nerve contains 82.9-91.9% of the [3H]mannose radioactivity as the complex-type oligosaccharide chain. Electrophoretic analysis of immune precipitates verified the [3H]mannose as being incorporated into P0 for both the transected and crushed nerve. The high-mannose-type glycopeptides of the transected nerve isolated from the concanavalin A-Sepharose column were hydrolyzed by endo-beta-N-acetylglucosaminidase H, and the oligosaccharides were separated on Biogel P4. Man8GlcNAc and Man7GlcNAc were the predominant species with radioactivity ratios of 12.5/7.2/1.4/1.0 for the Man8, Man7, Man6, and Man5 oligosaccharides, respectively. Jack bean alpha-D-mannosidase gave the expected yields of free Man and ManGlcNAc from these high-mannose-type oligosaccharides. The data support the notion that at least two alpha-1,2-mannosidases are responsible for converting Man9GlcNAc2 to Man5GlcNAc2. The present experiments suggest distinct roles for each mannosidase and that the second mannosidase (I-B) may be an important rate-limiting step in the processing of this glycoprotein with the resulting accumulation of Man8GlcNAc2 and Man7GlcNAc2 intermediates. Pulse chase experiments, however, demonstrated further processing of this high-mannose-type oligosaccharide in the transected nerve. The [3H]mannose P0 glycoprotein with Mr of 27,700 having the predominant high-mannose-type oligosaccharide shifted its Mr to 28,500 with subsequent chase. This band at 28,500 was shown to have the complex-type oligosaccharide chain and to contain fucose attached to the core asparagine-linked GlcNAc residue. The extent of oligosaccharide processing of this down-regulated glycoprotein remains to be determined.     

Regulation of Myelin P0 Glycoprotein Synthesis in Cultured Rat Schwann Cells and Continuous Rat PNS Cell Lines

We studied the effects of agents that raise intracellular cyclic AMP on synthesis of myelin components by cultured neonatal rat sciatic nerve Schwann cells and by continuous PNS cell lines derived from the fusion of neonatal rat sciatic nerve Schwann cells with rat RN22 Schwannoma. Treatment with N6,2'-O-dibutyryl cyclic AMP (dibutyryl cyclic AMP) caused a fourfold increase in Schwann cell incorporation of 35SO4 into sulfogalactosylceramide (sulfatide), and elicited a 10- to 20-fold increase in such incorporation by the continuous PNS cell lines; a similar effect on PNS cell line sulfatide radiolabelling was obtained with forskolin. Cultured Schwann cells expressed barely detectable levels of myelin P0 glycoprotein (P0) mRNA and myelin basic protein (MBP) mRNA. Treatment of the Schwann cells with axolemmal fragments or with dibutyryl cyclic AMP did not elicit a detectable increase in the levels of these mRNAs. The PNS cell lines constitutively expressed much higher levels of P0 mRNA than did the Schwann cells, and synthesized immunochemically demonstrable P0 glycoprotein, but did not express MBP. Treatment of the PNS cell lines with dibutyryl cyclic AMP markedly reduced expression of P0 mRNA and also diminished immunoreactive P0 glycoprotein. These PNS cell lines should prove useful for further studies of the control of Schwann cell differentiation.     

Biosynthesis of Neutral Glucocerebroside Homologues in the Absence of Myelin Assembly After Nerve Transection

The biosynthesis of myelin-associated glycolipids during various stages of myelination was studied by in vitro incorporation of [3H]Gal, [3H]Glc, or [35S]sulfate into the endoneurium of rat sciatic nerve. In the normal adult nerve, where the level of myelin assembly is substantially reduced and Schwann cells are principally involved in maintaining the existing myelin membrane, [3H]Gal was primarily incorporated into monogalactosyl diacylglycerol (MGDG) and the galactocerebrosides (GalCe) with lower levels of incorporation into the sulfatides. Such incorporation was enhanced 35 days after crush injury of the adult rat sciatic nerve, which is characterized by active myelin assembly. In contrast, at 35 days after permanent nerve transection where there is no axonal regeneration or myelin assembly, the incorporation of [3H]Gal or [3H]Glc into GalCe was nearly undetected whereas the incorporation of [3H]Gal into MGDG was completely inhibited. Instead, the 3H-labeled glycolipids in transected nerve were identified as the glucocerebrosides (GlcCe) and oligohexosylceramide derivatives with tetrahexosylceramide being a major product. In contrast, [35S]sulfate was incorporated into endoneurial sulfatides in the transected nerve, which suggests that endogenous GalCe rather than newly synthesized GalCe served as the substrate for the sulfotransferase reaction. The GlcCe homologues are not considered as constituents of the myelin membrane but are likely plasma membrane components synthesized in the absence of myelin assembly. It is likely that the cells responsible for GlcCe biosynthesis are Schwann cells, since they comprise 90% of the total endoneurial cell area in the distal nerve segment at 35 days after transection.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of G Protein Subtypes in Peripheral Nerve and Cultured Schwann Cells

In this study, we investigated the expression of various G proteins in whole sciatic nerves, in myelin and nonmyelin fractions from these nerves, and in membranes of immortalized Schwann cells. In myelin, nonmyelin, and Schwann cell membranes we detected two 39-40-kDa pertussis toxin substrates that were resolved on separation on urea-gradient gels. Two cholera toxin substrates with apparent molecular masses of 42 and 47 kDa were present in nerve and brain myelin and in Schwann cell membranes. In these membranes, a third 45-kDa cholera toxin substrate, which displayed the highest labeling, was also present. Immunoblotting with specific antisera allowed the identification of G(o) alpha, Gi1 alpha, Gi2 alpha, Gi3 alpha, Gq/G11 alpha, and the two isoforms of Gs alpha in nerve homogenates, nerve, and brain myelin fractions. In Schwann cell membranes we identified G(o) alpha, Gi2 alpha, Gi3 alpha, and proteins from the Gq family, but no immunoreactivity toward anti-Gi1 alpha antiserum was detected. In these membranes, anti-Gs alpha antibody recognized the three cholera toxin substrates mentioned above, with the 45-kDa band displaying the highest immunoreactivity. Relative to sciatic nerve myelin, the Schwann cell membranes revealed a significantly higher expression of Gi3 alpha and the absence of Gi1 alpha. The different distribution of G proteins among the different nerve compartments might reflect the very specialized function of Schwann cells and myelin within the nerve.     

Progesterone Stimulates the Activity of the Promoters of Peripheral Myelin Protein-22 and Protein Zero Genes in Schwann Cells

Abstract: To understand better the mechanisms by which progesterone (PROG) promotes myelination in the PNS, cultured rat Schwann cells were transiently transfected with reporter constructs in which luciferase expression was controlled by the promoter region of either the peripheral myelin protein-22 (PMP22) or the protein zero (P₀) genes. PROG stimulated the P₀ promoter and promoter 1, but not promoter 2, of PMP22. The effect of PROG was specific, as estradiol and testosterone only weakly activated promoters. Dose-response curves for stimulation of both promoter constructs by PROG were biphasic. RU486, a PROG antagonist, did not abolish the effect of PROG, but stimulated promoter activities by itself. In the human carcinoma cell line T47D expressing high levels of PROG receptor, PROG did not stimulate the P₀ and PMP22 promoters, whereas the promoter region of the mouse mammary tumor virus was fully activated. Thus, the activation by PROG of promoter activity of two peripheral myelin protein genes is Schwann-cell specific.     

Isolation and Partial Characterization of Plasmalemma from Quiescent Schwann Cells in Denervated Cat Sciatic Nerve

Fractions enriched in plasma membranes have been obtained from peripheral nerves enriched 89% in quiescent Schwann cells. Fractions were prepared from the intrafascicular tissue of desheathed distal stumps of cat sciatic nerve 8-10 weeks after transection and suture in the upper thigh. Tissue enriched in Schwann cells was minced, homogenized, and centrifuged to remove nuclei and undispersed tissue. Centrifugation of the resulting supernatant produced a pellet that was osmotically shocked, layered over a discontinuous sucrose gradient, and recentrifuged. Fractions enriched in plasma membrane (PM) markers were pooled, osmotically shocked for 16 h, layered over a second discontinuous sucrose density gradient, and recentrifuged. Membrane fractions (0.6 M:0.85 M and 0.85 M:1.0 M interfaces) contained a homogeneous population of unilamellar vesicles free of myelin. The 0.85 M fraction was enriched in 5'-nucleotidase, 2',3'-cyclic nucleotide 3'-phosphohydrolase. and specific [3H]ouabain binding, 4.8-, 3.0-, and 5.7-fold over the crude homogenate, respectively. These fractions also demonstrated low enzyme activities for succinate dehydrogenase, lactate dehydrogenase, and glucose-6-phosphatase (9, 13, and 15% of control values, respectively). Protein yield of the PM fraction (0.85 M) was approximately 0.6 mg/g of denervated nerve. This preparation should be suitable to characterize the surface properties of Schwann cells free of neuronal regulation.     

Insulin Is Released from Rat Brain Neuronal Cells in Culture

Depolarization of neuronal cells in primary culture from the rat brain by potassium ions in the presence of calcium or by veratridine caused a greater than three-fold stimulation of release of immunoreactive insulin. HPLC of the released insulin immunoreactivity from the neuronal cultures comigrated with the two rat insulins. The depolarization-induced release of insulin was inhibited by cycloheximide and was specific for neuronal cultures since potassium ions failed to cause the release in comparably prepared astrocytic glial cells from the rat brain. Prelabelling of neuronal cultures with [3H]leucine followed by depolarization resulted in the release of radioactivity that immunoprecipitated with insulin antibody. The release of [3H]insulin was biphasic. These observations suggest that neuronal cells from the brain have the capacity to synthesize insulin that could be released under depolarization conditions.     

Role of Intracellular pH in the Axolemma- and Myelin-Induced Proliferation of Schwann Cells

In order to provide additional information on the biochemical events that interact to cause Schwann cells to proliferate, we have monitored the intracellular pH of Schwann cells that have been stimulated to divide with myelin-enriched fractions (MEF) or axolemma-enriched fractions (AEF). The intracellular pH of Schwann cells was monitored using 2',7'-bis(carboxymethyl)-5(6)-carboxyfluorescein (BCECF), which displays an increase in fluorescence upon alkalinization. Both AEF and MEF caused dose-dependent increases in the intracellular fluorescence of the Schwann cell cultures. At their maximum doses, AEF and MEF stimulation resulted in a 260 and 300% increase in intracellular fluorescence, respectively. The increase in intracellular fluorescence was abolished when cells were stimulated in Na+-free media, suggesting a role for the Na+/H+ exchanger. Mitotic stimulation required integrity of the Na+/H+ exchanger, as inhibition of the Na+/H+ exchanger for periods up to 1 h after addition of mitogen caused a significant inhibition of subsequent mitosis. Phorbol esters, which can potentiate AEF- and MEF-induced Schwann cell proliferation, increased intracellular fluorescence fivefold, an effect which was also dependent upon the presence of Na+ in the culture media. The specificity of the increase in intracellular pH for AEF and MEF was tested by incubating Schwann cells with liver microsomes and a biologically inactive phorbol alcohol, neither of which is significantly mitogenic for Schwann cells. Neither liver microsomes nor phorbol alcohol had a significant effect on intracellular pH. The implications of the increase in intracellular pH in Schwann cells with respect to inositol phospholipid metabolism, protein kinase C activation, and cellular proliferation are discussed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein Carboxylmethyltransferase Activity in Intact, Differentiated Neuroblastoma Cells: Quantitation by S[3H]Adlenosylmethionine Prelabeling

Abstract: Protein carboxylmethyltransferase has been proposed to play a role in the regulation of neuroblastoma differentiation (Kloog et al., 1983). When we investigated this hypothesis further, different results for methyl ester formation were obtained when measured in acid-precipitated proteins and in proteins separated by acidic polyacrylamide gel electrophoresis, following the incubation of intact neuroblastoma cells with [³H]methionine. These unexpected findings led to the development of a modified assay using S -[³H]-adenosylmethionine as the radiolabeled precursor for quan-titating carboxyl methylation in intact cells. Data obtained from either acid-precipitated proteins or those separated on an electrophoresis gel following S -[³H]adenosylmethionine incubation directly correlated with data obtained from proteins separated by electrophoresis following [³H]methionine incubation. Using each of the three methods, an approxi-mately twofold increase in the carboxyl methylation of cellular proteins was detected in neuroblastoma cells differentiated by reducing the serum concentration from 10 to 0.5%, but not in those cells differentiated with either 5 m M hexa-methylene bisacetamide or 2% dimethyl sulfoxide. The finding that all detectable methyl acceptor proteins are increasingly methylated following 0.5% serum treatment and that this modification is substoichiometric suggests that protein carboxyl methylation is not an essential component of the differentiation process in neuroblastoma cells.     

Chronic Ethanol Treatment Decreases [3H]Epibatidine and [3H]Nicotine Binding and Differentially Regulates mRNA Levels of Nicotinic Acetylcholine Receptor Subunits Expressed in M10 and SH-SY5Y Neuroblastoma Cells

Abstract: For a study of the underlying mechanisms of a possible interaction between ethanol and nicotinic receptors during ethanol dependence, the aim of this work was to investigate the effect of chronic ethanol exposure on nicotinic receptor subtypes in a transfected fibroblast cell line (M10 cells) stably expressing α4β2 nicotinic receptor subtype and an SH-SY5Y neuroblastoma cell line expressing α3, α5, α7, β2, and β4 nicotinic acetylcholine receptor (nAChR) subunits. A significant dose-related decrease (−30–80%) in number of [³H]nicotine binding sites was observed in ethanol-treated (25–240 m M ) compared with untreated M10 cells. Similarly, 4-day treatment with ethanol in concentrations relevant to chronic alcoholism (100 m M ) decreased the number of nicotinic receptor binding sites in the SH-SY5Y cells when measured using [³H]epibatidine. When M10 cells were chronically treated with nicotine, ethanol partly inhibited the up-regulation of nicotinic receptors when present in the cells together with nicotine. Chronic treatment for 4 days with 100 m M ethanol significantly decreased the mRNA level for the α3 nAChR subunit (−39%), while the mRNA levels for the α7 (+30%) and α4 (+22%) subunits were significantly increased. Chronic ethanol treatment did not affect the mRNA levels for the β2 nAChR subunit. Changes in the levels of nAChR protein and mRNA may have adaptive significance and be involved in the development of dependence, tolerance, and addiction to chronic ethanol and nicotine exposure. They also may be targets for therapeutic strategies in the treatment of ethanol and nicotine dependence.     

In Vivo Regulation of [3H]Acetylcholine Recognition Sites in Brain by Nicotinic Cholinergic Drugs

The in vivo regulation of [3H]acetylcholine [( 3H]ACh) recognition sites on nicotinic receptors in rat brain was examined by administering drugs that increase stimulation of nicotinic cholinergic receptors, either directly or indirectly. After 10 days of treatment with the cholinesterase inhibitor diisopropyl fluorophosphate, [3H]ACh binding in the cortex, thalamus, striatum, and hypothalamus was decreased. Scatchard analyses indicated that the decrease in binding in the cortex was due to a reduction in the apparent density of [3H]ACh recognition sites. In contrast, after repeated administration of nicotine (5-21 days), the number of [3H]ACh recognition sites was increased in the cortex, thalamus, striatum, and hypothalamus. Similar effects were observed in the cortex and thalamus following repeated administration of the nicotinic agonist cytisin. The nicotinic antagonists mecamylamine and dihydro-beta-erythroidine did not alter [3H]ACh binding following 10-14 days of administration. Further, concurrent treatment with these antagonists and nicotine did not prevent the nicotine-induced increase in these binding sites. The data indicate that [3H]ACh recognition sites on nicotinic receptors are subject to up- and down-regulation, and that repeated administration of nicotine results in a signal for up-regulation, probably through protracted desensitization at the recognition site.     

Preferential Induction by Stress of the N-Methyl-d-Aspartate Recognition Domain in Discrete Structures of Rat Brain

Abstract: Immobilization stress in water for 3 h was effective in inducing significant potentiation of [³H](+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d] cyclohepten-5,10-imine ([³H]MK-801) binding 5 days after the stressful manipulation in rat hypothalamus and cerebellum when determined before equilibrium in the absence of any added agonists, in addition to resulting in marked reduction of rearing behaviors of animals. However, the stressful manipulation failed to modulate the [³H]MK-801 binding in other central regions examined, and binding of either [³H]dl -α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid or [³H]kainic acid was not significantly affected in all brain structures studied 5 days after the stress application. In contrast, the stressful procedures potentiated binding of both l -[³H]glutamic ([³H]Glu) and [³H]dl -(E)-2-amino-4-propyl-5-phosphono-3-pentenoic ([³H]CGP-39653) acids in the hypothalamus and cerebellum 5 days later, without affecting binding of [³H]-glycine and 5,7-dichloro[³H]kynurenic acid. The systemic administration of corticosterone mimicked the stress manipulation at doses of 5–50 mg/kg in terms of inducing significant enhancement of binding of both [³H]Glu and [³H]CGP-39653 in the hypothalamus and cerebellum when determined 5 days after the single administration. The translation inhibitor cycloheximide was effective in preventing the stress-induced potentiation of [³H]Glu binding in the cerebellum, without altering that in the hypothalamus. Furthermore, the stressful handling significantly increased the densities of [³H]Glu binding sites in the hypothalamus and cerebellum, with the affinities being unchanged. These results suggest that stress may preferentially potentiate binding of radioligands to the N-methyl-d -aspartate recognition domain through facilitation of de novo biosynthesis in rat hypothalamus and cerebellum.     

脂肪干细胞上清液培养雪旺细胞的实验研究

目的:研究应用脂肪干细胞上清液培养新生小鼠雪旺细胞的可行性。方法:取新生(出生5-7天)C57BL/6小鼠的坐骨神经,采用0.2%的复合胶原酶NB4消化法分离获取细胞,然后应用雪旺细胞条件培养基(SCCM)和C57BL/6小鼠的脂肪干细胞上清液(ADSC-CM)分别培养雪旺细胞。用0.2%复合胶原酶NB4差速分离纯化这两种方法培养的雪旺细胞,每48 h纯化1次,共进行2次纯化。应用P75免疫荧光染色方法鉴别两组P2代雪旺细胞并比较两组雪旺细胞的纯度和生长情况。结果:脂肪干细胞上清液培养的雪旺细胞纯化两次后,数量明显增多,其纯度与雪旺细胞条件培养基相比没有明显差异(P0.05)。结论:脂肪干细胞上清液可以较好的培养雪旺细胞,可以作为一种新的廉价方便的培养基代替雪旺细胞条件培养基来培养许雪旺细胞。     

Down-Regulation of the Noradrenaline Transporter by Interferon-α in Cultured Bovine Adrenal Medullary Cells

Abstract: The aim of this study was to investigate the effect of long-term treatment with interferon (IFN)-α on the noradrenaline transporter of bovine adrenal medullary cells. Treatment of cultured adrenal medullary cells with IFN-α caused a decrease in uptake of [³H]noradrenaline by the cells in time (4–48 h)- and concentration (300–1,000 U/ml)-dependent manners. IFN-β also inhibited [³H]noradrenaline uptake to a lesser extent than did IFN-α, whereas IFN-γ had little effect. An anti-IFN-α antibody reduced the effect of IFN-α on [³H]noradrenaline uptake. Saturation analysis of [³H]noradrenaline uptake showed that the inhibitory effect of IFN-α was due to a reduction in the maximal uptake velocity ( V _max) values without altering apparent Michaelis constant ( K _m) values. Incubation of cells with IFN-α caused a translocation of protein kinase C from the soluble to the particulate fraction in the cells. The effect of IFN-α on [³H]noradrenaline uptake was diminished in protein kinase C-down-regulated cells. Incubation of cells with IFN-α for 48 h significantly reduced the specific binding of [³H]desipramine to crude plasma membranes isolated from cells. Scatchard analysis of [³H]desipramine binding revealed that IFN-α decreased the maximal binding ( B _max) values without any change in the dissociation constant ( K _D) values. These findings suggest that IFN-α suppresses the function of noradrenaline transporter by reducing the density of the transporter in cell membranes through, at least in part, a protein kinase C pathway.     

Expression of Excitatory Amino Acid Receptors by Cerebellar Cells of the Type-2 Astrocyte Cell Lineage

Abstract: We have used postnatal rat cerebellar astrocyte-enriched cultures to study the excitatory amino acid receptors present on these cells. In the cultures used, type-2 astrocytes (recognized by the monoclonal antibodies A2B5 and LB1) selectively took up γ-[³H]aminobutyric acid ([³H]GABA) and released it when incubated in the presence of micromolar concentrations of kainic and quisqualic acids. The releasing effect of kainic acid was concentration dependent in the range of 5–100 μ M . Quisqualate was more effective than kainate in the lower concentration range but less effective at concentrations at which its releasing activity was maximal (∼50 μ M ). N -Methyl- d -aspartic acid and dihydrokainate (100 μ M ) did not stimulate [³H]GABA release from cultured astrocytes. l -Glutamic acid (20–100 μ M ) stimulated [³H]GABA release as effectively as kainate. The stimulatory effects of kainate and quisqualate on [³H]GABA release were completely Na⁺ dependent; that of kainate was also partially Ca²⁺ dependent. Kynurenic acid (50–200 μ M ) selectively antagonized the releasing effects of kainic acid and also that of l -glutamate; quisqualate was unaffected. Quisqualic acid inhibited the releasing effects of kainic acid when both agonists were used at equimolar concentrations (50 μ M ). d -[³H]aspartate was taken up by both type-1 and type-2 astrocytes, but only type-2 astrocytes released it in the presence of kainic acid. Excitatory amino acid receptors with a pharmacology similar to that of the receptors present in type-2 astrocytes were also expressed by the immature, bipotential progenitors of type-2 astrocytes and oligodendrocytes.     

Proliferative and morphological effects of endothelins in Schwann cells: roles of p38 mitogen-activated protein kinase and Ca(2+)-independent phospholipase A2.

The regulation of Schwann cell (SC) proliferation and morphology is critical to nerve homeostasis. We have previously reported that endothelins (ETs) regulate the activity of different effectors in SC including adenylyl cyclase, phospholipases C and A2 and mitogen-activated protein kinases (MAPKs). These effects imply a possible participation of ETs in the regulation of SC phenotype. We have now investigated the effects of endothelins on the proliferation and morphology of SC, and compared them with the responses to platelet-derived growth factor (PDGF), a known mitogen in these cells. Both endothelin-1 (ET-1) and PDGF increased the incorporation of [3H]thymidine and the proportion of SC in S and G2/M, with a concomitant decrease in the G0/G1 stage cells. Treatment with ET-1 produced rapid changes in the morphology of the SC, characterized by the appearance of cell spreading with shorter processes. The response to ET-1 was considered to represent a proliferative phenotype, in contrast to the effects of forskolin, which decreased [3H]thymidine incorporation in immortalized SC (iSC) and lead to a differentiated morphology with longer extensions. While both ET-1 and PDGF displayed a proliferative effect on SC, treatment with PDGF did not affect the morphology of these cells to a significant extent. A role for p38 MAPK and Ca(2+)-independent phospholipase A2 in the changes in morphology and proliferation of iSC driven by ET-1 was suggested by the effects of selective inhibitors of these pathways [SB202190 and HELSS, respectively]. The unique pattern of signaling pathways recruited by ET-1 and its combined effects on regulation of phenotype and proliferation of SC suggest an important role for this peptide during nerve degeneration/regeneration.