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 MeHg's 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 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.

Chronic ethanol produces increased taurine transport and efflux in cultured astrocytes.

Due to ethanol's low potency and low level of toxicity, high amounts of ethanol are consumed to achieve pharmacological effects. Blood levels of ethanol in chronic alcoholics may reach as high as 80-100 mM. We undertook a series of studies to determine if these high levels of ethanol stimulated osmoregulatory processes in cultured astrocytes. The uptake and efflux of taurine, the major osmoregulatory amino acid with potentially neuroprotective actions, was assessed. In addition, uptake and efflux of the excitatory amino acid aspartate was studied since astrocytes are vital in maintaining proper synaptic excitatory amino levels through uptake, metabolism, and efflux. Ethanol exposure for 96 h resulted in increased uptake of both 3H-taurine and 3H-D-asparate. There were no significant changes in transporter function at 24 h consistent with the delayed time course of transporter up-regulation seen during chronic hyperosmotic stress. Following EtOH withdrawal, efflux of preloaded 3H-taurine was significantly increased as compared to controls for up to 1 h. In contrast to the efflux profile seen during hypotonic induced swelling and regulatory volume decrease (RVD), no increased 3H-D-asparate efflux was demonstrated. Cell volume measurements suggest that inhibition of the normal RVD response be involved in the increased taurine release.