Endothelin-1 decreases glutamate uptake in primary cultured rat astrocytes

Abstract

Endothelin-1 (ET-1) is a potent vasoconstrictor peptide that is also known to induce a wide spectrum of biological responses in nonvascular tissue. In this study, we found that ET-1 (100 nM) inhibited the glutamate uptake in cultured astrocytes expressing the glutamate/aspartate transporter (GLAST); astrocytes did not express the glutamate transporter-1 (GLT-1). The V(max) and the K(m) of the glutamate uptake were reduced by 57% and 47%, respectively. Application of the ET(A) and ET(B) receptor antagonists BQ-123 and BQ-788 partly inhibited the ET-1-evoked decrease in the glutamate uptake, whereas the nonspecific ET receptor antagonist bosentan completely inhibited this decrease. Incubation of the cultures with pertussis toxin abolished the effect of ET-1 on the uptake. The ET-1-induced decrease in the glutamate uptake was independent of extracellular free Ca(2+) concentration, whereas the intracellular Ca(2+) antagonists thapsigargin and 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester abolished the effect of ET-1 on the glutamate uptake. Incubation with the protein kinase C (PKC) antagonist staurosporine, but not with the fatty acid-binding protein bovine serum albumin, prevented the ET-1-induced decrease in the glutamate uptake. These results suggest that ET-1 impairs the high-affinity glutamate uptake in cultured astrocytes through a G protein-coupled mechanism, involving PKC and changes in intracellular Ca(2+).

Glutamate released by Japanese encephalitis virus‐infected microglia involves TNF‐α signaling and contributes to neuronal death

“…Although microglia express EAATs, the activity of microglia-associated EAAT remains controversial. Microglia have less effect in the clearance of extracellular glutamate (Leonova et al, 2001;Liang et al, 2008). In response to lipopolysaccharide stimulation, no remarkable glutamate uptake activity can be detected in microglia.…”

Microglial GLT‐1 is upregulated in response to herpes simplex virus infection to provide an antiviral defence via glutathione

“…Immunocytochemistry was performed essentially as previously described (Leonova et al, 2001). The cells were stained with the following: rabbit polyclonal antibody against glial fibrillary acidic protein (GFAP; DAKO, Carpinteria, CA) diluted 1:100, goat monoclonal antibody against microtubuli associated protein 2 (MAP2; Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1:100, rabbit polyclonal antibody against GLT-1 (Covalab, Lyon, France) diluted 1:100, or the in-house produced and characterized (Liljeqvist et al, 1999) mouse monoclonal antibodies against glycoprotein C1 (gC1; B1.C1.B4; Olofsson et al, 1999) diluted 1:100, and glycoprotein G2 (gG2; O1.C5.B2; Liljeqvist et al, 1998) diluted 1:100.…”

Rapid increase of glial glutamate uptake via blockade of the protein kinase A pathway

“…In contrast to PKC, protein kinase A (PKA) does neither phosphorylate GLT-1 nor GLAST, although both of them contain consensus sequences for phosphorylation (Anderson and Swanson, 2000). However, there are indications that the PKA pathway as well might be involved in the regulation of glutamate transporter activity (Hannson and Ronnback, 1991;Leonova et al, 2001).…”

“…Since the glial glutamate transporters GLT-1 and GLAST are often regulated by a particular signaling pathway in a reciprocal manner, we used primary cultures of undifferentiated glial cells which almost exclusively express GLAST and which are accepted to represent a model system that closely reflects the intracellular milieu observed in vivo (Leonova et al, 2001;Robinson, 2002;Schlag et al, 1998;Zelenaia et al, 2000). Using these cells we performed experiments to show that GF 109203X, a broad-spectrum inhibitor of different protein kinases, increases glutamate uptake of cortical glial cells within seconds.…”