Lysette Mutkus

The uptake of cysteine in cultured primary astrocytes and neurons

One of the vitally important functions of glutathione (GSH) is to adequately protect cells against toxic chemicals, reactive oxygen metabolites and free radical species. The amino acid, cysteine, is the key rate-limiting substrate for the biosynthesis of GSH, and the maintenance of adequate intracellular GSH levels is dependent upon the extracellular availability and transport of cysteine into cells. In the present study, primary cultures of astrocytes and neurons were employed to characterize cysteine transport systems. Both astrocytes and neurons used Na(+)-dependent systems as the major route for cysteine uptake (80-90% of total), while Na(+)-independent uptake represented a minor component of total transport (10-20% of total). Among the Na(+)-dependent systems, X(AG(-)) was the major contributor (approx. 80-90%) for cysteine uptake in both neurons and astrocytes, with a minor contribution from the ASC transport system (Na(+)-dependent neutral amino acid transport system for alanine, serine, and cysteine). In the Na(+)-independent transport systems (10-20% of total cysteine transport), multifunctional ectoenzyme/amino acid transporter gamma-glutamyltranspeptidase (GGT), and the neutral amino acid L-system contributed approximately equally towards cysteine uptake, in both neurons and astrocytes. The present studies demonstrate that astrocytes and neurons accumulate cysteine by both Na(+)-dependent and Na(+)-independent uptake systems, with major uptake occurring through the X(AG(-)) system and minor uptake via the ASC, GGT and L-systems.

Tumor-associated stromal cells as key contributors to the tumor microenvironment.

The tumor microenvironment is a heterogeneous population of cells consisting of the tumor bulk plus supporting cells. It is becoming increasingly evident that these supporting cells are recruited by cancer cells from nearby endogenous host stroma and promote events such as tumor angiogenesis, proliferation, invasion, and metastasis, as well as mediate mechanisms of therapeutic resistance. In addition, recruited stromal cells range in type and include vascular endothelial cells, pericytes, adipocytes, fibroblasts, and bone-marrow mesenchymal stromal cells. During normal wound healing and inflammatory processes, local stromal cells change their phenotype to become that of reactive stroma. Under certain conditions, however, tumor cells can co-opt these reactive stromal cells and further transition them into tumor-associated stromal cells (TASCs). These TASCs express higher levels of proteins, including alpha-smooth muscle actin, fibroblast activating protein, and matrix metalloproteinases, compared with their normal, non-reactive counterparts. TASCs are also known to secrete many pro-tumorigenic factors, including IL-6, IL-8, stromal-derived factor-1 alpha, vascular endothelial growth factor, tenascin-C, and matrix metalloproteinases, among others, which recruit additional tumor and pro-tumorigenic cells to the developing microenvironment. Here, we review the current literature pertaining to the origins of recruited host stroma, contributions toward tumor progression, tumor-associated stromal cells, and mechanisms of crosstalk between endogenous host stroma and tumor cells.

Methylmercury enhances arachidonic acid release and cytosolic phospholipase A2 expression in primary cultures of neonatal astrocytes

Cytosolic phospholipase A(2) (cPLA(2)) stimulates the hydrolysis of sn-2 ester bond in membrane phospholipids releasing arachidonic acid (AA) and lysophospholipids. The present study examined the effect of methylmercury (MeHg) on cPLA(2) activation and AA release in primary cultures of neonatal rat cerebral astrocytes. Astrocytes were preloaded overnight at 37 degrees C with 3H-AA to metabolically label phospholipids. The effect of MeHg on the activation of cPLA(2) was measured by the release of 3H-AA from astrocytes over 120 min. MeHg (5 microM) caused a significant increase in AA release at 10, 30, 60, and 120 min, whereas 2.5 microM MeHg significantly increased AA release only at 120 min. MeHg-induced increase in 3H-AA release was due to cPLA(2) activation, since arachidonyl trifluoromethyl ketone (AACOCF(3)), a selective inhibitor of cPLA(2), completely abolished MeHgs effect. Consistent with these observations, MeHg (5.0 and 10.0 microM) increased cPLA(2) mRNA (6 h) and cPLA(2) protein expression (5.0 and 10.0 microM; 24 h). The time-course of these effects suggests an immediate direct or indirect effect of MeHg on cPLA(2) activation and 3H-AA release as well as a long-term effect involving the induction of cPLA(2). Thin layer chromatographic analysis of 3H-AA-labeled phospholipids showed that MeHg-stimulated astrocyte 3H-AA release was not due to increased incorporation of 3H-AA into the putative substrates of cPLA(2). These results invoke cPLA(2) as a putative target for MeHg toxicity, and support the notion that cPLA(2)-stimulated hydrolysis and release of AA play a critical role in MeHg-induced neurotoxicity.

Mercuric chloride, but not methylmercury, inhibits glutamine synthetase activity in primary cultures of cortical astrocytes

Methylmercury (MeHg) is highly neurotoxic with an apparent dose-related latency period between time of exposure and the appearance of symptoms. Astrocytes are known targets for MeHg toxicity and a site of mercury localization within the central nervous system (CNS). Glutamine synthetase (GS) is an enzyme localized predominately within astrocytes. GS converts two potentially toxic molecules, glutamate and ammonia, to the relatively non-toxic amino acid, glutamine. During prolonged exposure to MeHg, inorganic mercury (I-Hg) accumulates within the brain, suggesting in situ demethylation of MeHg to I-Hg. To determine if speciation of mercurials would differentially alter GS activity and expression, neonatal rat primary astrocyte cultures were exposed to MeHg or mercuric chloride (HgCl2) for 1 or 6 h. MeHg produced no changes in GS activity, protein, or mRNA at any time or dose tested. In contrast, HgCl2 produced a dose dependent decrease in astrocytic GS activity at both 1 and 6 h. There were no changes in GS protein or mRNA levels following HgCl2 exposure. Additional studies were carried out to determine GS activity in cell lysates incubated with HgCl2 or MeHg. In cell lysates, HgCl2 was three-times more potent than MeHg in inhibiting GS activity. The inhibition of GS activity in cell lysates by HgCl2 was reversed by the addition of dithiothreitol (DTT), while DTT did not restore GS activity following MeHg. These data suggest that astrocytic GS activity is not inhibited by physiologically relevant concentrations of MeHg, but is inhibited by I-Hg, which is present in CNS following chronic MeHg exposure.

Methylmercury inhibits cysteine uptake in cultured primary astrocytes, but not in neurons

The maintenance of adequate intracellular glutathione (GSH) concentrations is dependent on the availability and transport of the rate-limiting substrate, cysteine. A suggested mechanism of methylmercury (MeHg) neurotoxicity in brain involves the formation of oxygen radicals, and a decrease in intracellular levels of GSH. Recently, we have characterized various cysteine transport systems (both Na(+)-dependent and -independent) in cerebrocortical astrocytes and hippocampal neurons. The present study was carried out to investigate the effect of MeHg on cysteine uptake in both astrocytes and neurons, and to determine whether cysteine transport is differentially affected in the two cell types by MeHg treatment. Sixty-minute pretreatment with MeHg caused significant concentration-dependent inhibition in cysteine uptake in astrocytes, but not in neurons. As most of the cysteine transport is Na(+)-dependent (80-90% of total), additional studies focused on MeHgs effect on the Na(+)-dependent cysteine transporters X(AG(-)) and ASC. An additive inhibitory effect on cysteine uptake was observed in astrocytes treated with MeHg (5 microM) plus sub-maximal inhibitory concentrations (0.1 and 0.5 mM) of threo-beta-hydroxy-aspartate (THA), a specific inhibitor of the Na(+)-dependent transporter, X(AG(-)), compared to astrocytes treated with MeHg (P<0.001) or THA alone (P<0.05). There was no additive effect of MeHg and maximal inhibitory concentrations of THA (1.0 and 5.0 mM) on astrocytic cysteine uptake inhibition. Additional studies examined the sensitivity of the Na(+)-dependent ASC transport system to MeHg treatment. Maximal inhibitory concentration of L-serine (10 mM) alone had a rather modest inhibitory effect on cysteine uptake, and when applied in the presence of MeHg there was no additive effect. These results suggest that the inhibition of cysteine uptake by MeHg in astrocytes occurs through specific inhibition of both the X(AG(-)) as well as the ASC transport system.

Foreign metallothionein-I expression by transient transfection in MT-I and MT-II null astrocytes confers increased protection against acute methylmercury cytotoxicity

The mechanisms associated with metallothionein (MT) gene regulation are complex and poorly understood. Only a modest increase in brain MT expression levels is attained by exposure to metals, MT gene transfection, and MT gene knock-in techniques. Accordingly, in the present study, MT null astrocytes isolated from transgenic mice deficient in MT-I and MT-II genes were introduced as a zero background model of MT expression. MT protein levels were determined by western blot analysis. MT proteins in MT-I and MT-II null astrocytes were undetectable. Transient MT-I gene transfection increased the levels of foreign MT expression in MT-I and MT-II null astrocytes by 2.3-fold above basal levels in wild-type astrocytes. Intracellular Na(2)51CrO(4) efflux and D-[2,3-3H]aspartate uptake were studied as indices of acute methylmercury (MeHg) (5 microM) cytotoxicity. In MT-I and MT-II knockout astrocytes MeHg led to significant (p<0.01) increase in Na(2)51CrO(4) efflux and a significant (p<0.05) decrease in the initial rate (1 min) of D-[2, 3-3H]aspartate uptake compared to MT-I and MT-II knockout controls. Transfection of the MT-I gene in MT-I and MT-II null mice significantly (p<0.01) decreased the effect of MeHg on Na(2)51CrO(4) efflux in MT null, as well as wild-type astrocytes. MT-I gene transfection in MT-I and MT-II null astrocytes reversed the inhibitory effect of MeHg on D-[2,3-3H]aspartate uptake, such that initial rates of uptake in MT-I transfected cells in the presence and absence of MeHg (5 microM) were indistinguishable. These results demonstrate that: (1) astrocytes lacking MTs are more sensitive to MeHg than those with basal MT protein levels, (2) the MT-I gene can be overexpressed in MT-I and MT-II null astrocytes by transient MT-I gene transfection, and (3) that foreign MT expression endows astrocytes with increased resistance to MeHg.

Isolation of Neonatal Rat Cortical Astrocytes for Primary Cultures

There is increasing interest in the role of astrocytes as mediators of neurotoxicity. This unit describes a method for preparing astrocyte cultures of greater than 95% purity by enzymatic dissociation from neonatal rat brain. These preparations have both high yield and high viability.

The uptake of manganese in brain endothelial cultures

The present study focused on central nervous system (CNS) transport kinetics of manganese phosphate and manganese sulfate; these findings were correlated with the transport kinetics of manganese chloride (MnCl2), a model Mn compound that has been previously studied. A series of studies was performed to address the transport of Mn salts in confluent cultured endothelial cells. The initial rate of uptake (5 min) of Mn salts (chloride, sulfate, and phosphate) in rat brain endothelial (RBE4) cell cultures is salt-dependent, with the highest rates of uptake for Mn chloride and Mn sulfate (as reflected by the greatest displacement of 54Mn compared with control). Mn phosphate had a lower rate of uptake than the other two Mn salts. These data show that brain endothelial cells efficiently transport Mn sulfate.

Transendothelial permeability of chlorpyrifos in RBE4 monolayers is modulated by astrocyte-conditioned medium

The immortalized rat brain endothelium 4 (RBE4) cell line preserves many features of the in vivo brain endothelium. It has been used as an in vitro model of the blood-brain barrier (BBB). Astrocyte-endothelial cell interactions are crucial for maintenance of BBB characteristics. The present study investigated morphological and permeability properties of the RBE4 cell line. Immunohistochemical studies showed positive staining in RBE4 cells for E-cadherin, a Ca(2+)-dependent cell-cell adhesion molecule. Western blot immunoassay showed that RBE4 cells consistently express E-cadherin and that its expression significantly increased (P<0.001) in the presence of astrocyte-conditioned medium (ACM). The transendothelial permeability of chlorpyrifos, an organophosphorus insecticide, was significantly decreased (P<0.001) when the RBE4 cells were grown in ACM compared with control medium. Additional studies were carried out to determine whether chlorpyrifos is a substrate for the multidrug resistance protein, P-glycoprotein (P-gp). No significant change in chlorpyrifos transendothelial permeability was noted in the presence of verapamil, a P-gp blocker. Thus, in this system, chlorpyrifos is not a substrate for P-gp. This work demonstrates that with additional refinements the RBE4 monolayers might serve as a useful in vitro model for the study of BBB permeability and modulation by astrocyte-derived soluble factors.

Aspartate and Glutamate Transport in Acutely and Chronically Ethanol Exposed Neonatal Rat Primary Astrocyte Cultures

Maintenance of the ionic and osmotic composition of the extracellular fluid (ECF) is essential for the optimal functioning of the central nervous system (CNS). Changes in ion and neurotransmitter levels in the cerebrospinal fluid (CSF) can have profound effects on the processing and transmission of neuronal signals. Cell swelling during correction of isotonic imbalances can produce a series of events leading to inappropriate release of excitatory amino acids (EAA). Given the osmoregulatory demands of the CNS, it is not surprising that it possesses well-developed osmoregulatory mechanisms capable of maintaining both extracellular and intracellular ionic composition and volume within narrow limits, despite large fluctuations in the ionic composition and osmolarity of the plasma. We have undertaken a series of studies to test the hypothesis that ethanol (EtOH) acts as an osmotic stressor and stimulates osmoregulatory processes in astrocytes. In the course of these studies, we have investigated the effects of acute and chronic exposure to EtOH on cell volume, as well as uptake and release of amino acids in neonatal rat primary astrocyte cultures.