Functional expression of a GLT-1 type Na+-dependent glutamate transporter in rat pinealocytes.
Pinealocytes, the neuroendocrine cells that produce melatonin, accumulate glutamate in microvesicles through a specific vesicular transporter energetically coupled with vacuolar-type proton ATPase. The glutamate is secreted into the extracellular space through microvesicle-mediated exocytosis and then stimulates neighboring pinealocytes, resulting in inhibition of norepinephrine-dependent melatonin synthesis. In this study, we identified and characterized the plasma membrane-type glutamate transporter in rat pinealocytes. The [3H]glutamate uptake by cultured pinealocytes was driven by extracellular Na+, saturated with the [3H]glutamate concentration used, and significantly inhibited by L-glutamate, L-aspartate, beta-threo-hydroxyaspartate, pyrrolidine dicarboxylate, and L-cysteine sulfinate, substrates or inhibitors of the plasma membrane glutamate transporter. Consistently, the clearance of extracellular glutamate, as measured by HPLC, was also dependent on Na+ and inhibited by beta-threo-hydroxyaspartate and L-cysteine sulfinate. Immunological studies with site-specific antibodies against three isoforms of the Na+-dependent glutamate transporter (GLT-1, GLAST, and EAAC1) revealed the expression of only the GLT-1 type transporter in pineal glands. Expression of the GLT-1 type transporter in pineal glands was further demonstrated by means of reverse transcription-polymerase chain reaction with specific DNA probes. Immunohistochemical analysis indicated that the immunological counterpart(s) of the GLT-1 is localized in pinealocytes. These results suggested that the GLT-1-type Na+-dependent transporter is expressed and functions as a reuptake system for glutamate in rat pinealocytes. The physiological role of the transporter in the termination of the glutamate signal in the pineal gland is discussed.
Yamada
H
H
Department of Cell Membrane Biology, Institute of Scientific and Industrial Research, Osaka University, Japan.
Yatsushiro
S
S
Yamamoto
A
A
Hayashi
M
M
Nishi
T
T
Futai
M
M
Yamaguchi
A
A
Moriyama
Y
Y
D018528:ATP-Binding Cassette Transporters / Q000378:metabolism*; D027322:Amino Acid Transport System X-AG; D000818:Animals; D002478:Cells, Cultured; D018698:Glutamic Acid / Q000493:pharmacokinetics; D007150:Immunohistochemistry; D010870:Pineal Gland / Q000166:cytology / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D012964:Sodium / Q000502:physiology*
D018528:ATP-Binding Cassette Transporters; D027322:Amino Acid Transport System X-AG; D018698:Glutamic Acid; D012964:Sodium