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. Glutamate is involved in the negative regulation of norepinephrine-stimulated melatonin production. In this study, we found that aspartate is also released from cultured rat pinealocytes during the exocytosis of glutamate. The release of aspartate was triggered by addition of KCI or A23187 (a Ca²⁺ ionophore) in the presence of Ca²⁺ and was proportional to the amount of L-glutamate released. Furthermore, the release of aspartate 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. Immunohistochemical analyses with antibodies against aspartate and synaptophysin 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 contain the D form in their cytoplasm, corresponding to ~30% of the total free aspartate. Radiolabeled L-aspartate was taken up by the microsomal fraction from bovine pineal glands in a Na⁺- dependent manner. The Na⁺-dependent uptake of L-aspartate was strongly inhibited by L-cysteine sulfinate, β-hydroxyaspartate, and L-serine-O- sulfate, inhibitors for the Na⁺-dependent glutamate/aspartate transporter on the plasma membrane. Na⁺-dependent sequestration of L-aspartate was also 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 transporter at the plasma membrane. The possible role of L-aspartate as an intercellular chemical transmitter in the pineal gland is discussed.