Functional and molecular identification of sodium-coupled dicarboxylate transporters in rat primary cultured cerebrocortical astrocytes and neurons.
Na+-coupled carboxylate transporters (NaCs) mediate the uptake of tricarboxylic acid cycle intermediates in mammalian tissues. Of these transporters, NaC3 (formerly known as Na+-coupled dicarboxylate transporter 3, NaDC3/SDCT2) and NaC2 (formerly known as Na+-coupled citrate transporter, NaCT) have been shown to be expressed in brain. There is, however, little information available on the precise distribution and function of both transporters in the CNS. In the present study, we investigated the functional characteristics of Na+-dependent succinate and citrate transport in primary cultures of astrocytes and neurons from rat cerebral cortex. Uptake of succinate was Na+ dependent, Li+ sensitive and saturable with a Michaelis constant (Kt) value of 28.4 microM in rat astrocytes. Na+ activation kinetics revealed that the Na+ to succinate stoichiometry was 3:1 and the concentration of Na+ necessary for half-maximal transport was 53 mM. Although uptake of citrate in astrocytes was also Na+ dependent and saturable, its Kt value was significantly higher (approximately 1.2 mM) than that of succinate. Unlabeled succinate (2 mM) inhibited Na+-dependent [14C]succinate (18 microM) and [14C]citrate (4.5 microM) transport completely, whereas unlabeled citrate inhibited Na+-dependent [14C]succinate uptake more weakly. Interestingly, N-acetyl-L-aspartate, which is the second most abundant amino acid in the nervous system, also completely inhibited Na+-dependent succinate transport in rat astrocytes. The inhibition constant (Ki) for the inhibition of [14C]succinate uptake by unlabeled succinate, N-acetyl-L-aspartate and citrate was 15.9, 155 and 764 microM respectively. In primary cultures of neurons, uptake of citrate was also Na+ dependent and saturable with a Kt value of 16.2 microM, which was different from that observed in astrocytes, suggesting that different Na+-dependent citrate transport systems are expressed in neurons and astrocytes. RT-PCR and immunocytochemistry revealed that NaC3 and NaC2 are expressed in cerebrocortical astrocytes and neurons respectively. These results are in good agreement with our previous reports on the brain distribution pattern of NaC2 and NaC3 mRNA using in situ hybridization. This is the first report of the differential expression of different NaCs in astrocytes and neurons. These transporters might play important roles in the trafficking of tricarboxylic acid cycle intermediates and related metabolites between glia and neurons.
Yodoya
Etsuo
E
Department of Biopharmaceutics, Kyoto Pharmaceutical University, Yamashina-ku, Kyoto, Japan.
Wada
Miyuki
M
Shimada
Ayumi
A
Katsukawa
Hiromi
H
Okada
Naoki
N
Yamamoto
Akira
A
Ganapathy
Vadivel
V
Fujita
Takuya
T
D000818:Animals; D000831:Animals, Newborn; D001224:Aspartic Acid / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D001253:Astrocytes / Q000187:drug effects / Q000378:metabolism*; D001693:Biological Transport, Active / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000378:metabolism*; D019343:Citric Acid / Q000378:metabolism; D002952:Citric Acid Cycle / Q000187:drug effects / Q000502:physiology; D029382:Dicarboxylic Acid Transporters / Q000187:drug effects / Q000378:metabolism*; D008094:Lithium / Q000494:pharmacology; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D029363:Organic Anion Transporters, Sodium-Dependent / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D012964:Sodium / Q000378:metabolism* / Q000494:pharmacology; D019802:Succinic Acid / Q000378:metabolism; D027981:Symporters / Q000187:drug effects / Q000378:metabolism*
D029382:Dicarboxylic Acid Transporters; D029363:Organic Anion Transporters, Sodium-Dependent; C490478:Slc13a2 protein, rat; D027981:Symporters; D019343:Citric Acid; D001224:Aspartic Acid; C000179:N-acetylaspartate; D008094:Lithium; D012964:Sodium; D019802:Succinic Acid