Characterization of rapid and high-affinity uptake of L-serine in neurons and astrocytes in primary culture.
The non-essential amino acid L-serine was shown to be required to support the survival of rat cerebellar Purkinje neurons because of lack of the expression of the L-serine biosynthesis enzyme 3-phosphoglycerate dehydrogenase in them. In the present study, we investigated L-[(3)H]serine uptake in primary cultures of neurons and astrocytes from the rat telencephalon. In both neurons and astrocytes, L-[(3)H]serine uptake was dependent on temperature and Na(+) ions, and exhibited a single component of high-affinity uptake sites (K(m)=15.0 and 17.2 micro M for neurons and astrocytes, respectively). Kinetic analysis of L-[(3)H]serine uptake also revealed that the uptake into neurons was faster than that into astrocytes. The selectivity of inhibition by amino acids of the L-[(3)H]serine uptake resembled that of the system ASC transporters ASCT1 and ASCT2. Neutral amino acids L-alanine, L-serine, L-cysteine, and L-threonine strongly inhibited the uptake by both cell types. Furthermore, in astrocytes, but not in neurons, L-valine and L-proline also inhibited L-[(3)H]serine uptake. Neither alpha-methyl aminoisobutyric acid (a system A-specific substrate) nor 2-aminobicyclo(2,2,1)heptane-2-carboxylic acid (a system L-specific substrate) inhibited the uptake of L-[(3)H]serine in both neurons and astrocytes. Expression of ASCT transporters in both neurons and astrocytes was examined by use of reverse transcriptase polymerase chain reaction and immunoblot analysis. Whereas transcripts (mRNAs) of both ASCT1 and ASCT2 transporters were detected in astrocytes, only the mRNA of the former subtype was detected in neurons. Immunoblot analysis confirmed the presence of ASCT1 in both neurons and astrocytes. These findings indicate that neurons accumulate a high level of L-serine by using a Na(+)-dependent, high-affinity transport system, operating predominantly through the ASCT1 transporter subtype.
Yamamoto
Toshifumi
T
Laboratory of Molecular Recognition, Graduate School of Integrated Science, Yokohama City University, 22-2 Seto, Kanazawa-ku, Yokohama, 236-0027, Japan. yamamoto@yokohama-cu.ac.jp
Nishizaki
Itone
I
Furuya
Shigeki
S
Hirabayashi
Yoshio
Y
Takahashi
Kenzo
K
Okuyama
Shigeru
S
Yamamoto
Hideko
H
D026962:Amino Acid Transport System ASC / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D021542:Amino Acids, Neutral / Q000494:pharmacology; D000818:Animals; D001253:Astrocytes / Q000737:chemistry / Q000166:cytology / Q000378:metabolism*; D018929:Cell Culture Techniques; D005333:Fetus; D007700:Kinetics; D015778:Minor Histocompatibility Antigens; D009474:Neurons / Q000737:chemistry / Q000166:cytology / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D012694:Serine / Q000378:metabolism*; D012964:Sodium / Q000494:pharmacology; D013696:Temperature
D026962:Amino Acid Transport System ASC; D021542:Amino Acids, Neutral; D015778:Minor Histocompatibility Antigens; D012333:RNA, Messenger; C420504:Slc1a4 protein, rat; C498849:Slc1a5 protein, rat; D012694:Serine; D012964:Sodium