Acidic Amino Acid Transport Characteristics of a Newly Developed Conditionally Immortalized Rat Type 2 Astrocyte Cell Line (TR-AST).

Abstract

ABSTRACT. To characterize acidic amino acid transport in type 2 astrocytes, we established conditionally immortalized rat astrocyte cell lines (TR-AST) from newly developed transgenic rats harboring temperature-sensitive SV40 large T-antigen gene. TR-AST exhibitedThis study demonstrated that GLT-1, system xC -, and GLAST were expressed in TR-AST, which has the characteristics of type 2 astrocytes and is able to transport acidic amino acids.Key words: acidic amino acid/GLT-1/GLAST/system xC -/transport/type 2 astrocyte Acidic amino acids have conflicting roles at the central nervous system (CNS) because they contribute both to excitatory neurotransmission and excitatory damage (Zorumski and Olney, 1993). Therefore, acidic amino acid transporters in the brain play an important role in maintaining the concentrations below neurotoxic levels (Kanai and Hediger, 1996). System X AG -and system x C -are acidic amino acid transporters, the mRNAs of which are referred to as EAAT (excitatory amino acid transporter) (Palacín et al., 1998), and xCT (Sato et al., 1999), respectively. System X AG -recognizes L-glutamic acid (L-Glu), L-aspartic acid (L-Asp), and D-aspartic acid (D-Asp) in an Na + -dependent manner, while system x C -functions as a complex of xCT and the heavy chain of 4F2 cell surface antigen (4F2hc) protein, accepting L-Glu in an Na + -independent manner, and exchanging L-cystine (Bannai and Kitamura, 1980;Sato et al., 1999).More than a decade ago, two types of astrocytes were found in the rat optic nerve by Raff et al. based on differences in their morphology, cell surface gangliosides, and growth characteristics (Raff et al., 1983). They further con-*To whom correspondence should be addressed: Department of Molecular Biopharmacy and Genetics, Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba, Aramaki, Aoba-ku, Sendai 980-8578, Japan.Tel: +81-22-217-6831, Fax: +81-22-217-6886 E-mail: terasaki@mail.pharm.tohoku.ac.jp Abbreviations: 4F2hc, heavy chain of 4F2 cell surface antigen; ALS, amyotrophic lateral sclerosis; CNS, central nervous system; EAAT, excitatory amino acid transporter; GFAP, glial fibrillary acidic protein; GLAST, L-glutamate/L-aspartate transporter; GLT-1, L-glutamate transporter; SV, simian virus; TR-AST, conditionally immortalized rat type 2 astrocyte cell line; tsA58 TG rat, transgenic rat harboring temperature-sensitive SV40 large T-antigen gene.

Distribution of the Cystine/Glutamate Antiporter System x−c in the Brain, Kidney, and Duodenum

“…System x c -in the Brain, Duodenum, and Kidney 2001), in mouse kidney and intestine by RT-PCR (Bassi et al 2001), in primary neuronal cultures (Shih et al 2003), immortalized (Tetsuka et al 2001) and primary (Gochenauer and Robinson 2001) astrocyte cultures, and in the HT22 neuronal cell line (Lewerenz et al 2003). One would expect that 4F2hc would be more widely distributed than xCT, because it is paired with numerous other amino acid light chain transporters and, in general, that is what we found.…”

“…One would expect that 4F2hc would be more widely distributed than xCT, because it is paired with numerous other amino acid light chain transporters and, in general, that is what we found. Although we did not include choroid plexus in our 4F2hc immunostaining panel, system x 2 c activity has been demonstrated in a conditionally immortalized mouse choroid plexus epithelial cell line (Tetsuka et al 2001). 4F2hc staining is somewhat weaker in the human brain leptomeninges than that for xCT ( Figure 2F vs. Figure 3F), although the qualitative nature of immunohistochemistry makes it difficult to make value comparisons.…”

Advances in the Cell Biology of Transport via the Inner Blood-Retinal Barrier: Establishment of Cell Lines and Transport Functions

“…This prompts the hypothesis that the inner BRB expresses different molecules than the BBB. In addition to TR-iBRB, TR-rPCT, and TR-MUL cells, we have established BBB cell lines, a conditionally immortalized rat brain capillary endothelial cell line (TR-BBB), 75) pericytes (TR-PCT), 76) and an astrocyte cell line (TR-AST) 77) from tsA58 Tg rats. The background of gene expression in these cell lines is similar, since these lines were established from the same rat strain using the same procedure.…”

Functional expression of rat ABCG2 on the luminal side of brain capillaries and its enhancement by astrocyte‐derived soluble factor(s)

“…Cell cultures TR-BBB13, TR-AST4 and TR-PCT1 cells are conditionally immortalized BCEC, astrocyte and pericyte cell lines (Hosoya et al 2000b;Tetsuka et al 2001;Asashima et al 2002) that have been used as an in vitro BBB model . The cells were grown in Dulbecco's modified Eagle's medium (DMEM, Nissui Pharmaceutical, Tokyo, Japan) supplemented with 20 mM sodium bicarbonate, 15 ng/mL ECGF, 100 U/mL benzylpenicillin potassium, 100 lg/mL streptomycin sulfate and 10% fetal bovine serum (Moregate, Bulimba, Australia) (culturemedium A).…”