Microvesicle‐mediated exocytosis of glutamate is a novel paracrine‐like chemical transduction mechanism and inhibits melatonin secretion in rat pinealocytes

Abstract

Mammalian pinealocytes are neuroendocrine cells that synthesize and secrete melatonin, these processes being positively controlled by norepinephrine derived from innervating sympathetic neurons. Previously, we showed that pinealocytes contain a large number of microvesicles (MVs) that specifically accumulate L-glutamate through a vesicular glutamate transporter and contain proteins for exocytosis such as synaptobrevin 2 (VAMP2). These findings suggested that the MVs are counterparts of synaptic vesicles and are involved in paracrine-like chemical transduction in the pineal gland. Here, we show that pinealocytes actually secrete glutamate upon stimulation by KCl in the presence of Ca2+ at 37 degrees C. The ability of glutamate secretion disappeared when the cells were incubated at below 20 degrees C. Loss of the activity was also observed on successive stimulation, but it was recovered after 12 hr incubation. A low concentration of cadmium chloride or omega-conotoxin GVIA inhibited the secretion. Botulinum neurotoxin E cleaved synaptic vesicle-associated protein 25 (SNAP-25) and thus inhibited the secretion. The released L-glutamate stimulated pinealocytes themselves via glutamate receptor(s) and inhibited norepinephrine-stimulated melatonin secretion. These results strongly suggest that pinealocytes are glutaminergic paraneurons, and that the glutaminergic system regulates negatively the synthesis and secretion of melatonin. The MV-mediated paracrine-like chemical transduction seems to be a novel mechanism that regulates hormonal secretion by neuroendocrine cells.

d-Aspartate Is Stored in Secretory Granules and Released through a Ca2+-dependent Pathway in a Subset of Rat Pheochromocytoma PC12 Cells

“…These properties may reflect complex membrane dynamics including the charging and discharging of neurotransmitters. Similar phenomena were observed for Ca 2ϩ -dependent exocytosis found in various endocrine cells and neuronal cells (15,(42)(43)(44). The relatively slow rate of D-aspartate release (minute order as shown in Fig.…”

Ca2+-Dependent Exocytosis of l-Glutamate by αTC6, Clonal Mouse Pancreatic α-Cells

“…Its efficiency was gradually restored with incubation and had recovered completely after 12 h, suggesting that charged and discharged processes are involved in the KCl-induced Ca 2ϩ -dependent glutamate release. These properties are similar to those of exocytosis of glutamate by synaptic vesicles and synaptic-like microvesicles (17,30,31), supporting the idea that glutamate is secreted through exocytosis.…”

“…As the first step of the study, we examined whether ␣TC6 cells release Lglutamate through an exocytic mechanism. In analogy to the glutamate exocytosis by neurons and pinealocytes (17,30,31), the glutamate concentration in the medium of cultured ␣TC6 cells was measured by HPLC after stimulation of the cells with KCl. An appreciable amount of L-glutamate (2.52 Ϯ 0.4 nmol per 10 6 cells, which corresponds to 23% total free glutamate, 50 determinations) has been released by ␣TC6 cells at 10 min after the addition of KCl in the presence of Ca 2ϩ .…”

“…The sensitivity to BoNT/E constitutes evidence of Ca 2ϩ -dependent regulated exocytosis because this neurotoxin is a protease that is specific to SNAP25, splitting the SNAP25 and resulting in inhibition of exocytosis via secretory granules, synaptic vesicles, and synaptic-like microvesicles (17,19,(37)(38)(39). BoNT/E cleaved SNAP25, yielding a lowmolecular fragment that migrated faster on a polyacrylamide gel during electrophoresis (Fig.…”

Differentiation-associated Na+-dependent Inorganic Phosphate Cotransporter (DNPI) Is a Vesicular Glutamate Transporter in Endocrine Glutamatergic Systems

“…DNPI Is Co-localized with SLMVs in Pinealocytes-Besides the central nervous system, peripheral endocrine tissues possess glutamatergic systems (3,4). Mammalian pinealocytes accumulate L-glutamate in SLMVs, counterparts of synaptic vesicles in endocrine cells, and secrete it to the extracellular space through exocytosis (28,29). Vesicular glutamate transporter is responsible for the storage of L-glutamate in pineal SLMVs (27,30).…”