Yoshinori Moriyama

Metabotropic Glutamate Receptors Negatively Regulate Melatonin Synthesis in Rat Pinealocytes

Rat pinealocytes receive noradrenergic innervation that stimulates melatonin synthesis in a cAMP-mediated manner. In addition to melatonin, we showed previously that pinealocytes secretel-glutamate through an exocytic mechanism. The released glutamate inhibits norepinephrine (NE)-dependent melatonin synthesis. Consistent with this observation, specific agonists of class II metabotropic glutamate receptors (mGluRs), including 1-(1S,3R)-aminocyclopentane-1,3-dicarboxylic acid (tACPD), inhibited NE-dependent melatonin synthesis, whereas agonists for other types of glutamate receptors did not. Furthermore, reverse transcription-PCR, Northern blotting, and immunohistochemistry analyses indicated expression of class II mGluR3 in pinealocytes. Inhibitory guanine nucleotide-binding protein (Gi) was also detected in pinealocytes. l-Glutamate or agonists of class II receptors decreased NE- or forskolin-dependent increase of cAMP and serotonin-N-acetyltransferase activities to similar extents. These effects were blocked by pertussis toxin or dibutyryl cAMP. These results indicate that the inhibitory cAMP cascade is involved in the glutamate-evoked inhibition of melatonin synthesis. We propose that the glutaminergic system negatively regulates NE-dependent melatonin synthesis in rat pinealocytes.

l‐Aspartate but Not the d Form Is Secreted Through Microvesicle‐Mediated Exocytosis and Is Sequestered Through Na+‐Dependent Transporter in Rat Pinealocytes

Abstract: 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.

Lack of vacuolar proton ATPase association with the cytoskeleton in osteoclasts of osteosclerotic (oc/oc) mice

We examined the pathogenetic mechanism underlying the lack of bone resorption in osteosclerotic oc/oc mice. An immunoelectron microscopic analysis revealed that in the osteoclasts of these mice, no ruffled borders formed, and that vacuolar H+‐ATPase (V‐ATPase) was present throughout the cytoplasm but not on the apical membranes. The activity of V‐ATPase in oc/oc mice was similar to that in normal mice. In normal spleen cell‐derived osteoclast‐like cells (OCLs), immunoreactivity for V‐ATPase was detected in association with Triton X‐100‐insoluble cellular structure, but not in oc/oc spleen cell‐derived OCLs. Moreover, in renal proximal convoluted tubules of oc/oc mice, the basal striation did not form. These results suggest that osteosclerosis in oc/oc mice is possibly due to the dissociation of V‐ATPase and cytoskeleton in osteoclasts.

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 KC1 in the presence of Ca2+ at 37°C. The ability of glutamate secretion disappeared when the cells were incubated at below 20°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 ω‐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.

Synaptic Vesicle Protein SV2B, but Not SV2A, Is Predominantly Expressed and Associated with Microvesicles in Rat Pinealocytes

Abstract: Microvesicles are endocrine counterparts of neuronal synaptic vesicles, and accumulate and secrete classic neurotransmitters. In mammalian pinealocytes, microvesicles accumulate l‐glutamate through a vesicular glutamate transporter and secrete it through exocytosis. To characterize the molecular organization of microvesicles in more detail, we investigated in this study the expression and localization of synaptic vesicle protein 2 (SV2) in rat pinealocytes. RT‐PCR analysis indicated that transcripts specific for two isoforms, SV2A, a ubiquitous form present in neuronal and endocrine cells, and SV2B, a neuron‐specific form, are amplified in pineal RNAs. Northern blotting with specific transcripts indicated that the mRNA for SV2B is predominantly expressed, whereas that for SV2A is below the detection limit. Site‐specific antibodies against SV2B recognized a single 72‐kDa polypeptide in the pineal membrane fraction, whereas anti‐SV2A antibodies did not recognize any polypeptides. Immunohistochemical analysis of cultured cells indicated that SV2B is expressed in pinealocytes but not in other types of cells. SV2B is present in somata and is especially rich in processes, which are filled with microvesicles. SV2B is colocalized with synaptophysin and synaptotagmin, markers for microvesicles. Immunoelectron microscopy indicated that SV2B is associated with microvesicles. These results indicated that SV2B, but not SV2A, is expressed in rat pinealocytes and associated with microvesicles. As SV2B is also expressed in cultured αTC6 clonal pancreatic α cells, SV2B is not a protein specific for neurons.

The L-type Ca2+ channel is involved in microvesicle-mediated glutamate exocytosis from rat pinealocytes

Abstract: Pinealocytes, parenchymal cells of the pineal gland, secrete glutamate through microvesicle‐mediated exocytosis upon depolarization by KC1 in the presence of Ca2+, which is involved in a novel paracrine‐like intercellular signal transduction mechanism in neuroendocrine organs. In the present study, we investigated whether or not the L‐type Ca2+ channel is involved in the microvesicle‐mediated glutamate secretion from cultured rat pinealocytes. Nifedipine, a specific antagonist of the L‐type Ca2+ channel, inhibited the Ca2+‐dependent glutamate exocytosis by 48% at 20 uM. Other L‐type Ca2+ channel antagonists, such as nitrendipine, showed similar effects. 1,4‐Dihydro‐2,6‐dimethyl‐5‐nitro‐4[2‐(trifluoromethyl)‐phenyl]‐3‐pyridinecarboxylic acid methyl ester (BAY K8644), an agonist of the L‐type Ca2+ channel, at 1 uM, on the other hand, stimulated the glutamate exocytosis about 1.6‐fold. Consistently, these Ca2+ channel antagonists inhibited about 50% of the Ca2+ uptake, whereas BAY K8644 increased the uptake 5.3‐fold. An antibody against the carboxyl‐terminal region of the rabbit L‐type Ca2+ channel recognized polypeptides of pinealocytes with apparent molecular masses of 250 and 270 kDa, respectively, and immunostained the plasma membrane region of the pinealocytes. These results strongly suggested that the entry of Ca2+ through L‐type Ca2+ channel(s), at least in part, triggers microvesicle‐mediated glutamate exocytosis in pinealocytes.

Localization of N-ethylmaleimide-sensitive fusion protein in pinealocytes

N-ETHYLMALEIMIDE-sensitive fusion protein (NSF), a protein necessary for vesicular docking and/or fusion, was detected immunohistochemically in pinealocytes. NSF was distributed similarly to synaptophysin and vacuolar-type H+-ATPase (V-ATPase), marker proteins for synaptic-like microvesicles (MVs) abundantly present in pinealocytes. A subcellular fractionation study indicated that >95% of NSF was present as a membrane- bound form and that some NSF was associated with MVs. Like neuronal NSF, the protein was not solubilized from membranes with either 2mM Mg-ATP or 2% sodium carbonate, suggesting that NSF was tightly bound to the membranes. NSF was also detected in purified MVs from bovine posterior pituitaries. Since MVs are the organelles in which transmitters are stored, these results suggest that NSF is involved in the MV- mediated exocytosis of transmitters from endocrine cells.

Identification of D-aspartate in rat pheochromocytoma PC12 cells.

D-Aspartate is now known to be present in mammalian neuronal and endocrine cells in vivo, and may play some role(s) in neurocrine and endocrine functions. However, origin of D-aspartate is unknown. Here, we report that free D-aspartate (108 pmoles/3 x 10(7) cells) is present in the cultured PC12 cells, a rat pheochromocytoma cell line, as determined with immunohistochemical techniques as well as high performance liquid chromatography (HPLC) on a Pirkle-type chiral column. The amount of D-aspartate does not change with the passage. The culture medium does not contain D-aspartate. These results strongly suggest the presence of a de novo biosynthetic pathway for D-aspartate in the endocrine cells.

L-Aspartate-evoked inhibition of melatonin production in rat pineal glands

Our previous studies in rat indicated that pinealocytes secrete L-glutamate through microvesicle-mediated exocytosis to regulate negatively melatonin production. Recently, we further found that pinealocytes secrete L-aspartate through microvesicle-mediated exocytosis. In the present study, we investigated the role of L-aspartate in the melatonin production in isolated rat pineal glands. It was found that L-aspartate inhibits norepinephrine-stimulated melatonin production as well as serotonin N-acetyltransferase activity reversibly and dose-dependently, the concentrations required for 50% inhibition being 150 and 175 microM, respectively. L-Asparagine and oxaloacetate, metabolites of L-aspartate, had no effect on the melatonin production. These results suggest that pinealocytes use L-aspartate, as well as L-glutamate, as a negative regulator for melatonin production.