Gouri Shanker

Identification and characterization of uptake systems for cystine and cysteine in cultured astrocytes and neurons: evidence for methylmercury-targeted disruption of astrocyte transport.

Maintenance of appropriate intracellular glutathione (GSH) levels is crucial for cellular defense against oxidative damage. A suggested mechanism of methylmercury (MeHg) neurotoxicity implicates the involvement of oxygen radical formation and a decrease in cellular levels of GSH. Astrocytes play an important role in providing GSH precursors to neurons, and as will be discussed in this review, altered GSH homeostasis likely leads to impairment of astrocytic handling of glutamate, and neuronal energy metabolism. The review summarizes recent observations on transport systems for cysteine and cystine, precursors of GSH, in primary cultures of astrocytes and neurons, and their sensitivity to MeHg treatment.

Methylmercury inhibits the in vitro uptake of the glutathione precursor, cystine, in astrocytes, but not in neurons

Maintenance of adequate intracellular glutathione (GSH) levels is vital for intracellular defense against oxidative damage. The toxic effects of methylmercury (MeHg) are attributable, at least in part, to elevated levels of reactive oxygen species, and thus decreases in GSH synthesis may increase methylmercury toxicity. Astrocytes have recently been proposed to play an essential role in providing GSH precursors to neurons. Therefore, cystine transport, a prerequisite to GSH production, was characterized in cultured astrocytes and neurons, and the effects of methylmercury on this transport were assessed. Astrocytes and neurons both possessed temperature dependent transport systems for cystine. Astrocytes accumulated cystine by Na+-independent (X(C)-) and -dependent (X(AG)-) systems while neurons used exclusively Na+-independent systems. Inhibition of the X(AG)- transport system decreased cystine transport in astrocytes to levels equivalent to those in sodium-depleted conditions, suggesting that cystine is carried by a glutamate/aspartate transporter in astrocytes. Inhibition of the multifunction ectoenzyme/amino acid transporter gamma-glutamyltranspeptidase (GGT) decreased cystine transport in both neurons and astrocytes. Inhibition of System X(C)- with quisqualate also decreased cystine uptake in both astrocytes and neurons. These data demonstrate that cultured astrocytes accumulate cystine via three independent mechanisms, System X(AG)-, System X(C)-, and GGT, while cultured hippocampal neurons use System X(C)- and GGT exclusively. Inhibition of cystine uptake in astrocytes by methylmercury appears to be due to actions on the System X(AG)- transporter.

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.

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.

The Consequences of Methylmercury Exposure on Interactive Functions between Astrocytes and Neurons

Methylmercury (MeHg) is a highly neurotoxic, environmentally ubiquitous chemical that exerts its toxic effects by largely unknown mechanisms. Maintenance of optimal intracellular concentrations of glutathione (GSH) is vital for cellular defenses against damage from free radicals. Since astrocytes play an essential role in providing GSH precursors to neurons, studies were directed at the effect of MeHg on cystine transport in both cell types. Astrocytes accumulated cystine via three independent transporters, referred to as system XAG-, system XC-, and gamma-glutamyltranspeptidase (GGT). In contrast, neurons accumulated cystine exclusively via system XC- and GGT. MeHg potently inhibited cystine uptake in astrocytes (but not in neurons), and this effect could be fully accounted for by inhibition of the system XAG- transporter. The transport of glutamate in astrocytes is also inhibited by reactive oxygen species (ROS). Accordingly, additional studies examined the ability of thiol reducing or oxidizing agents to inhibit the astrocytic transport of 3H-D-aspartate, a glutamate analog. The antioxidant catalase significantly attenuated MeHg-induced inhibition of astrocytic 3H-aspartate uptake. Combinedly, these studies suggest that inhibition of cystine uptake and decreased astrocytic GSH levels and efflux reduce the availability of precursors for GSH synthesis in neurons. In addition, MeHg-induced generation of H2O2 plays a role in the inhibition of astrocytic glutamate transport. These effects likely increase neuronal vulnerability to MeHg-induced oxidative stress, and excess N-methyl D-aspartate (NMDA) receptor activation leading to neuronal demise.

Astrocyte-mediated methylmercury neurotoxicity.

Methylmercury (MeHg) is a potent neurotoxicant. Any source of environmental mercury represents a potential risk for human MeHg poisoning, because the methylation of inorganic mercury to MeHg in waterways results ultimately in its accumulation in the sea food chain, which represents the most prevalent source for human consumption. A small amount of MeHg accumulates in the central nervous system (CNS), particularly in astrocytes. Astrocytic swelling, excitatory amino acid (EAA) release and uptake inhibition, as well as EAA transporter expression inhibition are known sequelae of MeHg exposure. Herein, we review the effect of MeHg on additional transport systems (for cystine and cysteine) as well as arachidonic acid (AA) release and cytosolic phospholipase A2 (cPLA2) regulation and attempt to integrate the effects of MeHg in astrocytes within a mechanistic hypothesis that explains the inability of these cells to maintain control of the proper milieu of the extracellular fluid and, in turn, leads to neuronal demise.

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.

Estrogen modulates the inducible expression of platelet-derived growth factor mrna by monocyte/macrophages

We examined the effects of estrogen, 12-O-tetradecanoylphorbol 13 acetate (TPA), and lipopolysaccharide (LPS) on the gene expression of platelet-derived growth factor (PDGF) by the monocyte/macrophage cell line, THP-1. THP-1 cells were exposed to TPA for 48 or 96 hours to induce differentiation. Some were treated with LPS in the last 3 hours and/or ethinyl estradiol (estrogen) (10(-9) M) in the last 20 hours. Total cellular RNA was isolated and cDNA was synthesized and then coamplified (with an internal control, beta-actin, product size 1126 bp) using polymerase chain reaction (PCR) and a set of primers for PDGF-A (product size 225 bp), PDGF-B (217 bp), or PDGF beta-receptor (PDGF-R) (228 bp). The products were separated on an agarose gel and the ratios of radioactivity incorporated into PDGF PCR products to beta-actin products were used to assess the relative changes in the levels of PDGF mRNA abundance in response to various inducers. TPA induced the expression of PDGF-A mRNA, whereas LPS had no effect. Treatment of TPA-stimulated cells with estrogen caused a 61% and 190% increase in PDGF-A mRNA (p < 0.05) at 48 and 96 hours, respectively. Addition of estrogen to cells treated with both TPA and LPS did not cause any significant change in the amounts of the transcripts. In contrast to PDGF-A mRNA, attempts to visualize and estimate PDGF-B and PDGF-R mRNA were unsuccessful. This was probably due to low levels of these transcripts in THP-1 cells. The results indicate that estrogen modulates PDGF-A gene expression by monocyte/macrophages and suggest that estrogen may influence atherogenesis at the vascular level.

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.

Dibutyryl cyclic AMP-induced changes in neuron-astroglia interactions and fibronectin immunocytochemistry in dissociated rat cerebellar cultures

In mixed primary embryonic CNS cultures flat astroglia grow exclusively underneath the initially formed neuronal network. This invasive under-growth results in neuronal detachment and degeneration. The present study sought to find out whether or not morphological differentiation of astroglia, from flat to process-bearing cells, could alter astroglial-neuronal growth relationships in rat cerebellar cultures. Morphological differentiation of astroglia was induced by treatment with dibutyryl cyclic AMP. The results demonstrate that in contrast to flat astroglia, large stellate astroglia can grow over the neurite bundles, and that in these dibutyryl cyclic AMP-treated cultures neurons can persist. Immunocytochemical studies show that the extracellular matrix protein fibronectin is present in these cultures and appears to be associated with flat astroglia rather than with stellate astroglia. The study indicates that in the presence of dibutyryl cyclic AMP transformed stellate astroglia interact differently with neurons and with the growth substratum as compared with flat astroglia.