S100 beta stimulates calcium fluxes in glial and neuronal cells.

The glial-derived protein S100 beta can act as a mitogen or a neurotrophic factor, stimulating proliferation of glial cells or differentiation of immature neurons. We report here that dimeric S100 beta evokes increases in intracellular free calcium concentrations ([Ca2+]i) in both glial cells and neuronal cells. The [Ca2+]i increase exhibited a rapid transient component which was not affected by removal of extracellular calcium and a sustained component which appeared to require influx of extracellular calcium through Ni(2+)-sensitive channels. S100 beta also stimulated hydrolysis of phosphoinositides, suggesting a mobilization of calcium from intracellular stores. These data suggest that although the final biological responses of neuronal and glial cells to S100 beta are different, transduction of the S100 beta signal in both cell types involves changes in [Ca2+]i.     

Insulin rapidly stimulates L-arginine transport in human aortic endothelial cells via Akt

Insulin stimulates endothelial NO synthesis, at least in part mediated by phosphorylation and activation of endothelial NO synthase at Ser1177 and Ser615 by Akt. We have previously demonstrated that insulin-stimulated NO synthesis is inhibited under high culture glucose conditions, without altering Ca(2+)-stimulated NO synthesis or insulin-stimulated phosphorylation of eNOS. This indicates that stimulation of endothelial NO synthase phosphorylation may be required, yet not sufficient, for insulin-stimulated nitric oxide synthesis. In the current study we investigated the role of supply of the eNOS substrate, L-arginine as a candidate parallel mechanism underlying insulin-stimulated NO synthesis in cultured human aortic endothelial cells. Insulin rapidly stimulated L-arginine transport, an effect abrogated by incubation with inhibitors of phosphatidylinositol-3'-kinase or infection with adenoviruses expressing a dominant negative mutant Akt. Furthermore, supplementation of endothelial cells with extracellular L-arginine enhanced insulin-stimulated NO synthesis, an effect reversed by co-incubation with the L-arginine transport inhibitor, L-lysine. Basal L-arginine transport was significantly increased under high glucose culture conditions, yet insulin-stimulated L-arginine transport remained unaltered. The increase in L-arginine transport elicited by high glucose was independent of the expression of the cationic amino acid transporters, hCAT1 and hCAT2 and not associated with any changes in the activity of ERK1/2, Akt or protein kinase C (PKC). We propose that rapid stimulation of L-arginine transport contributes to insulin-stimulated NO synthesis in human endothelial cells, yet attenuation of this is unlikely to underlie the inhibition of insulin-stimulated NO synthesis under high glucose conditions.     

INFgamma stimulates arginine transport through system y+L in human monocytes

Freshly isolated human monocytes transport L-arginine mostly through a sodium independent, NEM insensitive pathway inhibited by L-leucine in the presence, but not in the absence of sodium. Interferon-gamma (IFNgamma) stimulates this pathway, identifiable with system y+L, and markedly enhances the expression of SLC7A7, the gene that encodes for system y+L subunit y+LAT1, but not of SLC7A6, that codes for the alternative subunit y+LAT2. System y+ plays a minor role in arginine uptake by monocytes and the expression of system y+-related genes, SLC7A1 and SLC7A2, is not changed by IFNgamma. These results demonstrate that system y+L is sensitive to IFNgamma.     

Uptake of biogenic amines by glial cells in culture I. A neuronal-like transport system for serotonin

Rat C6 astrocytoma cells take up serotonin (5HT) via a high affinity carrier mediated system with Km of 1 micromolar, and a second component of lower affinity. This high affinity 5HT transport system is rapid, concentrative, and highly sodium and temperature dependent. Chlorimipramine and Lilly 110140 preferentially block the glial 5HT but not NE uptake. This preferential inhibition has previously been shown for synaptosomes and brain slices. Norepinerphrine (NE) and to a lesser extent dopamine (DA) block the glial 5HT uptake, suggesting a partial overlap between the catecholamine and indoleamine glial carrier systems. 5-Hydroxy but not 6-hydroxy dopamine inhibits the high affinity 5HT transport in glia. A variety of ring hydroxylated indoleamine analogs block this glial 5HT transport; of the compounds tested, 5, 7 dihydroxytryptamine is the least effective inhibitor. Phenylethylamine (PEA) and its 0-methylated derivatives block synaptosomal and glial 5HT transport equally well. These observations suggest that cultured C6 cells used as models of glia possess a 5HT transport system which kinetically and pharmacologically resembles a neuronal 5HT transport system.     

L-arginine immunoreactive enteric glial cells in the enteric nervous system of rat ileum

L-arginine is a precursor of nitric oxide (NO) that may be involved in neuronal activity in the gastrointestinal tract. It is known that NO is formed from L-arginine by NO synthase which is localized in neurons in the enteric nervous system. The present study demonstrated that significant L-arginine immunoreactivity was present in the enteric ganglia. Ultrastructural examination showed that L-arginine immunoreactivity was present in the ganglionic glial cells but not in neurons. These findings suggest that enteric glial cells may represent the main reservoir of L-arginine, which may possibly be transferred to neurons when used.     

Arginine transport through system y(+)L in cultured human fibroblasts: normal phenotype of cells from LPI subjects

Inlysinuric protein intolerance (LPI), impaired transport of cationicamino acids in kidney and intestine is due to mutations of theSLC7A7 gene. To assess the functional consequences of the LPI defect in nonepithelial cells, we have characterized cationic aminoacid (CAA) transport in human fibroblasts obtained from LPI patientsand a normal subject. In both cell types the bidirectional fluxes ofarginine are due to the additive contributions of two Na⁺-independent, transstimulated transport systems. One ofthese mechanisms, inhibited by N-ethylmaleimide (NEM) andsensitive to the membrane potential, is identifiable with systemy⁺. The NEM- and potential-insensitive component,suppressed by L-leucine only in the presence ofNa⁺, is mostly due to the activity of systemy⁺L. The inward and outward activities of the two systemsare comparable in control and LPI fibroblasts. Both cell types expressSLC7A1 (CAT1) and SLC7A2 (CAT2B and CAT2A) aswell as SLC7A6 (y+LAT2) and SLC7A7 (y+LAT1). Weconclude that LPI fibroblasts exhibit normal CAA transport throughsystem y⁺L, probably referable to the activity ofSLC7A6/y+LAT2.

     

Arachidonic acid inhibits glycine transport in cultured glial cells.

The effects of arachidonic acid on glycine uptake, exchange and efflux in C6 glioma cells were investigated. Arachidonic acid produced a dose-dependent inhibition of high-affinity glycine uptake. This effect was not due to a simple detergent-like action on membranes, as the inhibition of glycine transport was most pronounced with cis-unsaturated long-chain fatty acids, whereas saturated and trans-unsaturated fatty acids had relatively little or no effect. Endogenous unsaturated non-esterified fatty acids may exert a similar inhibitory effect on the transport of glycine. The mechanism for this inhibitory effect has been examined in a plasma membrane vesicle preparation derived from C6 cells, which avoids metabolic or compartmentation interferences. The results suggest that part of the selective inhibition of glycine transport by arachidonic acid could be due to the effects of the arachidonic acid on the lipid domain surrounding the carrier.     

Mitochondrial carbonic anhydrase in the nervous system: expression in neuronal and glial cells

Carbonic anhydrase (CA) V is a mitochondrial enzyme that has been reported in several tissues of the gastrointestinal tract. In liver, it participates in ureagenesis and gluconeogenesis by providing bicarbonate ions for two other mitochondrial enzymes: carbamyl phosphate synthetase I and pyruvate carboxylase. This study presents evidence of immunohistochemical localization of CA V in the rodent nervous tissue. Polyclonal rabbit antisera against a polypeptide of 17 C-terminal amino acids of rat CA V and against purified recombinant mouse isozyme were used in western blotting and immunoperoxidase stainings. Immunohistochemistry showed that CA V is expressed in astrocytes and neurons but not in oligodendrocytes, which are rich in CA II, or capillary endothelial cells, which express CA IV on their plasma face. The specificity of the immunohistochemical results was confirmed by western blotting, which identified a major 30-kDa polypeptide band of CA V in mouse cerebral cortex, hippocampus, cerebellum, spinal cord, and sciatic nerve. The expression of CA V in astrocytes and neurons suggests that this isozyme has a cell-specific, physiological role in the nervous system. In astrocytes, CA V may play an important role in gluconeogenesis by providing bicarbonate ions for the pyruvate carboxylase. The neuronal CA V could be involved in the regulation of the intramitochondrial calcium level, thus contributing to the stability of the intracellular calcium concentration. CA V may also participate in bicarbonate ion-induced GABA responses by regulating the bicarbonate homeostasis in neurons, and its inhibition could be the basis of some neurotropic effects of carbonic anhydrase inhibitors.     

Base-exchange enzymic system for the synthesis of phospholipids in neuronal and glial cells and their subfractions: a possible marker for neuronal membranes

Abstract— The calcium-dependent incorporation of L-[3-¹⁴C]serine and [1,2^?14C]ethanolamine into the phospholipid of isolated neuronal and glial cells from rabbit brain was studied, and the distribution of the enzymic system among the correspondent subfractions was examined. The neuronal cell-enriched fraction was found to possess a much higher rate of exchange of both bases than the glial cell-enriched fraction. Among the sub-fractions isolated from the neuronal and glial cells, those corresponding to neuronal plasma membranes and microsomes showed a noticeably higher exchange of serine and ethanolamine compared to the corresponding subfractions from glia. Neuronal/glial ratios of about 6–8 were found for the exchange activity in both plasma membrane-enriched fraction and in microsomes. Synaptosomes and synaptosomal subfractions contained low activities. It is concluded that the calcium-dependent enzymic system for the exchange of serine, ethanolamine and other nitrogenous bases with endogenous phospholipid is concentrated mostly in the neuronal perikaryal membranes, and could be used as a neuronal marker.     

Accumulation of sulfatide in neuronal and glial cells of arylsulfatase A deficient mice

Arylsulfatase A (ASA) degrades sulfatide, seminolipid and lactosylceramide sulfate, glycolipids recognized by the Sulph I antibody although sulfatide is considered the main antigen. Sulfatide is myelin associated but studies have shown a minor distribution also in non-myelin forming cells. The aim of this work was to further study sulfatide in neurons and astrocytes by immunohistochemistry, facilitated by investigation of tissue from adult ASA deficient (ASA ?/?) mice. Cells with a low presence of sulfatide might be detected due to lack of ASA activity and accumulation of Sulph I antigens. Sulfatide positive astrocytes and neurons were more numerous and intensely stained in ASA ?/? mice, demonstrating a sulfatide accumulation compared to controls. Sulph I staining was especially increased in the molecular layer of cerebellum, in which Purkinje cell dendrites displayed an altered morphology, and in layer IV–VI of cerebral cortex. In hippocampus, immunostaining was found in neuronal cytoplasm in ASA ?/? but in nuclear membranes of control mice. We observed a gray matter astrogliosis, which appeared to be associated to sulfatide accumulation. In addition, the developmental change (<20 months) of Sulph I antigens, galactosylceramide, phospholipids and cholesterol were followed by lipid analyses which verified sulfatide and seminolipid accumulation in adult ASA ?/? mice, although no lactosylceramide sulfate could be detected. In addition to demonstrating sulfatide in neurons and astrocytes, this study supports the value of ASA ?/? mice as a model for metachromatic leukodystrophy and suggests that accumulation of sulfatide beyond myelin might contribute to the pathology of this disease.     

Glucagon stimulates the A system for neutral amino acid transport in isolated hepatocytes of adult rat.

It has been found that both the peroxidase and synthetase activity of sheep vesicular gland microsomes catalyze the oxygenation of singlet oxygen trapping or quenching agents. Furthermore the synthetase was also readily inactivated by these agents, particularly bilirubin, and suggests that singlet oxygen formed by the peroxidase activity may initiate prostaglandin biosynthesis. The singlet oxygen agents also protected the synthetase from self-catalyzed destruction or inactivation by peroxides and suggest that singlet oxygen may also be responsible for the inactivation.     

Beta-synuclein regulates Akt activity in neuronal cells. A possible mechanism for neuroprotection in Parkinson's disease

Recent studies have shown that the neurodegenerative process in disorders with Lewy body formation, such as Parkinson's disease and dementia with Lewy bodies, is associated with alpha-synuclein accumulation and that beta-synuclein might protect the central nervous system from the neurotoxic effects of alpha-synuclein. However, the mechanisms are unclear. The main objective of the present study was to investigate the potential involvement of the serine threonine kinase Akt (also known as protein kinase B) signaling pathway in the mechanisms of beta-synuclein neuroprotection. For this purpose, Akt activity and cell survival were analyzed in synuclein-transfected B103 neuroblastoma cells and primary cortical neurons. Beta-synuclein transfection resulted in increased Akt activity and conferred protection from the neurotoxic effects of rotenone. Down-regulation of Akt expression resulted in an increased susceptibility to rotenone toxicity, whereas transfection with a lentiviral vector encoding for beta-synuclein was protective. The effects of beta-synuclein on the Akt pathway appear to be by direct interaction between these molecules and were independent of upstream signaling molecules. Taken together, these results indicate that the mechanisms of beta-synuclein neuroprotection might involve direct interactions between beta-synuclein and Akt and suggest that this signaling pathway could be a potential therapeutic target for neurological conditions associated with parkinsonism and alpha-synuclein aggregation.     

Characteristics and adaptive regulation of glycine transport in cultured glial cells.

The transport of glycine in C6 glioma cells takes place mainly in a heterogeneous Na+-dependent manner which can be resolved into different components. A Na+- and Cl(-)-dependent component with high affinity for glycine is pH-sensitive and inhibited by sarcosine, all these characteristics corresponding to System Gly. The low-affinity component of the transport of glycine can be discriminated as two components, namely System A and System ASC. The main proportion of glycine transport through the low-affinity system is carried out by the ASC System, which appears to be constitutively expressed by the cells. The adaptive response of the low-affinity Na+-dependent transport of glycine to amino acid deprivation was identified with System A on the basis of its ion-dependency, pH-sensitivity and by inhibition analysis. The possible physiological role of the high- and low-affinity components of the transport system for glycine in glial cells is discussed.     

A glucose-activated electron transfer system in the plasma membrane stimulates the H(+)-ATPase in Penicillium cyclopium.

Hyphal cells of three fungal species of the genus Penicillium reduced the nonpermeable, external electron acceptor hexabromoiridate IV (HBI IV). In Penicillium cyclopium, the rate of HBI IV reduction by hyphal cells was drastically increased by the addition of beta-glucose. The stimulation showed high specificity for this sugar and did not require its uptake and cellular metabolism. Cell wall oxidases (e.g., glucose oxidase) did not seem to be involved in the reduction of HBI IV, as no measurable H2O2 was formed from added glucose and removal of oxygen had no effect. We propose that there is a glucose-binding component outside the plasma membrane which controls transmembrane electron fluxes in response to external glucose. Reduction of HBI IV was accompanied by rapid acidification of the cellular interior (measured by confocal pH topography). Subsequently, the outer medium was acidified of the cellular interior (measured by confocal pH topography). Subsequently, the outer medium was acidified with an e-/H+ stoichiometry of > 1. In plasma membrane vesicles containing endogenous electron donors, the membrane-residing fluoroprobe Di-8-ANEPPS reported a transient depolarization of the membrane potential triggered by the external electron acceptor. Inhibitors of ATP-dependent proton pumping enhanced the extent of this depolarization, inhibited the subsequent normalization of membrane potential, and, in whole cells, reduced the amount of redox-triggered proton extrusion. From these and other findings, it is concluded that the observed trans-plasma membrane redox process activates the H(+)-ATPase via membrane depolarization and cytosolic acidification.     

A mechanism of restricted human immunodeficiency virus type 1 expression in human glial cells.

We characterized in detail the life cycle of human immunodeficiency virus type 1 (HIV-1) in human glioma H4/CD4 cells which stably express transfected CD4 DNA (B. Volsky, K. Sakai, M. Reddy, and D. J. Volsky, Virology 186:303-308, 1992). Infection of cloned H4/CD4 cells with the N1T strain of cell-free HIV-1 (HIV-1/N1T) was rapid and highly productive as measured by the initial expression of viral DNA, RNA, and protein, but all viral products declined to low levels by 14 days after infection. Chronically infected, virus-producing H4/CD4 cells could be obtained by cell cloning, indicating that HIV-1 DNA can integrate and remain expressed in these cells. The HIV-1 produced in H4/CD4 cells was noninfectious to glial cells, but it could be transmitted with low efficiency to CEM cells. Examination of viral protein composition by immunoprecipitation with AIDS serum or anti-gp120 antibody revealed that HIV-1/N1T-infected H4/CD4 cells produced all major viral proteins including gp160, but not gp120. Deglycosylation experiments with three different glycosidases determined that the absence of gp120 was not due to aberrant glycosylation of gp160, indicating a defect in gp160 proteolytic processing. Similar results were obtained in acutely and chronically infected H4/CD4 cells. To determine the generality of this HIV-1 replication phenotype in H4/CD4 cells, nine different viral clones were tested for replication in H4/CD4 cells by transfection. Eight were transiently productive like N1T, but one clone, NL4-3, established a long-lived productive infection in H4/CD4 cells, produced infectious progeny virus, and produced both gp160 and gp120. We conclude that for most HIV-1 strains tested, HIV-1 infection of H4/CD4 is restricted to a single cycle because of the defective processing of gp160, resulting in the absence of gp120 on progeny virus.     

Fibroblasts, glial, and neuronal cells are involved in extravascular prothrombin activation.

A membrane-associated prothrombin activator (MAPA) was found on various cultured cells derived from non-hematopoietic cells [Sekiya, F. et al. (1994) J. Biol. Chem. 269, 32441-32445]. In this study, we investigated the enzymatic properties of this enzyme using protease inhibitors. While the metalloproteinase inhibitor, o-phenanthroline, had no effect, some Kunitz type serine protease inhibitors attenuated MAPA activity. Recombinant tissue factor pathway inhibitor (rTFPI) also markedly reduced the activity (IC(50), 1. 3+/-0.6 x 10(-10) M). MAPA activity is, therefore, most likely to be due to factor Xa. We evaluated the effect of exogenous factor Xa on MAPA activity. Factor Xa-dependent prothrombin activation was observed on fibroblast cells (apparent K(d), 1.47+/-0.72 nM). Activation was also observed on glial and neuronal cells, which expressed MAPA activity. These results imply that membrane-bound factor Xa results in MAPA activity on these cells. Therefore, we considered the involvement of factor Va, a component of prothrombinase, in this activity. We examined whether or not the prothrombinase complex is assembled on these cells. Prothrombin was activated in a manner dependent on both exogenous factor Xa and factor Va (apparent K(d) of 0.51-1.81 nM for factor Va). These results indicate that the prothrombinase complex forms specifically on various extravascular cells. Although the prothrombinase complex can be assembled on monocytes and lymphocytes, it is not known why these cells can activate prothrombin specifically. These cells which have the capacity for prothrombin activator activity could also activate factor X; i.e. cells with factor X activation activity were able to convert prothrombin. These observations suggest that thrombin was generated via two procoagulant activities; factor X activation and subsequent prothrombinase complex formation on the surface of these cells. This mechanism may explain the various pathological states involving or resulting from extravascular thrombin and fibrin formation.