L-ASPARTATE BUT NOT THE D-FORM IS SECRETED THROUGH MICROVESICLE-MEDIATED EXOCYTOSIS AND IS SEQUESTERED THROUGH NA-DEPENDENT TRANSPORTER IN RAT PINEALOCYTES()

Rat pinealocytes accumulate glutamate in microvesicles and secrete it through exocytosis so as to transmit signals intercellularly. Glutamat e is involved in the negative regulation of norepinephrine-stimulated melatonin production. In this study, we found that aspartate is also r eleased from cultured rat pinealocytes during the exocytosis of glutam ate. The release of aspartate was triggered by addition of KCl or A231 87 (a Ca2+ ionophore) in the presence of Ca2+ and was proportional to the amount of L-glutamate released. Furthermore, the release of aspart ate was inhibited by both botulinum neurotoxin type E and L- or N-type voltage-gated Ca2+ channel blockers. Bay K 8644, an agonist for the L -type Ca2+ channel, stimulated the release of aspartate 2.1-fold. Immu nohistochemical analyses with antibodies against aspartate and synapto physin revealed that aspartate is colocalized with synaptophysin in a cultured pinealocyte. HPLC with fluorometric detection indicated that the released aspartate is of the L form, although pinealocytes also co ntain the D form in their cytoplasm, corresponding to similar to 30% o f the total free aspartate, Radiolabeled L-aspartate was taken up by t he microsomal fraction from bovine pineal glands in a Na+-dependent ma nner. The Na+-dependent uptake of L-aspartate was strongly inhibited b y L-cysteine sulfinate, beta-hydroxyaspartate, and L-serine-O-sulfate, inhibitors for the Na+-dependent glutamate/aspartate transporter on t he plasma membrane, Na+-dependent sequestration of L-aspartate was als o observed in cultured rat pinealocytes, which was inhibited similarly by these transporter inhibitors. These results strongly suggest that L-aspartate is released through microvesicle-mediated exocytosis from pinealocytes and is taken up again through the Na+-dependent transport er at the plasma membrane. The possible role of L-aspartate as an inte rcellular chemical transmitter in the pineal gland is discussed.