Endothelin (ET) is a putative pathogenetic mediator associated with brain trauma and ischemia. Because a link between neuronal damage after these injuries and glial Na(+)-dependent L-glutamate transporter activity has been suggested, we investigated the effect of ET on the glutamate clearance ability of astrocytes. Dibutyryl cyclic adenosine monophosphate (dBcAMP), which is widely used to induce differentiation of cultured astrocytes, markedly increased [(3)H]glutamate transport activity in a concentration- and time-dependent manner. In the presence of ET, however, dBcAMP decreased the glutamate uptake. This effect was efficiently prevented by an antagonist of ET(B) receptor, but not of ET(A) receptor. ET per se was virtually ineffective. Eadie-Hofstee analysis demonstrated that dBcAMP increased the V(max) value of glutamate uptake activity by 43.4% in the absence of ET, but decreased it by 41.4% in the presence of ET, without apparent changes in the K(m) value. Accordingly, Western blot analysis indicated that the change in transport activity correlated closely with that in expression level of the glial glutamate transporter GLAST. These results may represent the mechanisms by which ET aggravates trauma- and ischemia-elicited neuronal damage.

Although amyloid ␤-protein (A␤) has long been implicated in the pathogenesis of Alzheimer's disease, little is known about the mechanism by which A␤ causes dementia. A␤ leads to neuronal cell death in vivo and in vitro, but recent evidence suggests that the property of the amnesic characteristic of Alzheimer's disease can be explained by a malfunction of synapses rather than a loss of neurons. Here we show that prolonged treatment with A␤ augments the glutamate clearance ability of cultured astrocytes and induces a dramatic decrease in glutamatergic synaptic activity of neurons cocultured with the astrocytes. Biotinylation assay revealed that the enhancement of glutamate uptake activity was associated with an increase in cell-surface expression of GLAST, a subtype of glial glutamate transporters, without apparent changes in the total amount of GLAST. This phenomenon was blocked efficiently by actindisrupting agents. Thus, A␤-induced actin-dependent GLAST redistribution and relevant synaptic malfunction may be a cellular basis for the amnesia of Alzheimer's disease.Amyloid ␤-protein (A␤), 1 a peptide with 40 -42 residues, is a main element of senile plaque, a hallmark of Alzheimer's disease (AD) (1, 2), and is accumulated highly in the forebrain of AD patients, as well as transgenic mice overexpressing mutant ␤-amyloid precursor protein (␤APP), which develop AD-like pathology (3, 4). Although numerous studies showed that exogenously applied or endogenously produced A␤ leads to neuronal cell death, the amnesic feature of AD cannot be explained by the neuronal loss alone (5). Indeed, accumulating evidence indicates that A␤ induces severe impairment of excitatory neurotransmission in the hippocampus (6 -8) and thereby may cause memory deficits (9). In mutant ␤APP transgenic mice, such synaptic malfunction often appears in advance of A␤ plaque formation (10, 11), and cognitive deterioration is also observed without apparent neurodegeneration (4, 12). A␤-induced synaptic deterioration rather than neuronal loss is, therefore, likely to be a main cause of early AD dementia (5, 13). However, the mechanisms by which A␤ causes such synaptic malfunction remain to be elucidated.Excitatory neurotransmission is tightly regulated by a rapid clearance of the neurotransmitter glutamate from the extracellular milieu through Na ϩ -dependent L-glutamate transporters that are expressed on astrocytes, i.e. GLAST and GLT-1 (14, 15). We therefore investigated the effect of A␤ on glutamate uptake activity in cultured cortical astrocytes. Here we show for the first time that A␤ ending at 42 residues (A␤ (1-42)) induces an increase in the activity of GLAST. This work further demonstrates that A␤ (1-42) stimulates actin-dependent GLAST redistribution from subcellular compartment to the cell surface. Such up-regulation of GLAST function may attenuate glutamatergic synaptic efficacy. EXPERIMENTAL PROCEDURESMaterials-Chemically synthesized A␤ (1-40) and A␤ (1-42) were gifts from Dr. T. Shirasawa (Department of Molecular Genetics, Tokyo Metropo...

Although expression of the glial glutamate transporter GLAST is tightly regulated during development and under pathophysiological conditions, little is known about endogenous modulators of GLAST expression. Because growth factors are generally believed to regulate glial functions, we addressed their possible contribution to GLAST regulation in cultured rat astrocytes. Of the six growth factors tested (basic fibroblast growth factor (bFGF), insulin-like growth factor-1 (IGF-1), epidermal growth factor (EGF), insulin, platelet-derived growth factor, and hepatocyte growth factor), bFGF, IGF-1 and EGF enhanced [3H]glutamate transport activity in a concentration-dependent manner. These effects were accompanied by an increase in the Vmax value for transport activity and in GLAST protein and mRNA levels, which suggests that GLAST expression is transcriptionally regulated by the growth factors. Interestingly, the effects reached a peak after 36 hours of exposure to growth factors, and rapidly returned to baseline by 48 hours. A combination of IGF-1 with either bFGF or EGF showed an additive effect on the glutamate uptake activity, but a combination of bFGF and EGF did not. Pharmacological blockade of protein kinase C inhibited the effects of IGF-1 and EGF, but not bFGF. By contrast, genistein, an inhibitor of tyrosine kinases, blocked the effects of bFGF and EGF without affecting the effect of IGF-1. These results suggest that the growth factors activate different signaling pathways for GLAST upregulation. The present study may indicate a novel regulatory system of glial glutamate transporters.