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γ-Aminobutyric acid (GABA) is an inhibitory amino acid and acts as an intercellular transmitter in the central nervous system and peripheral tissues. In pineal glands, GABA is supposed to be a paracrine-like modulator of secretion of melatonin, although its mode of action, especially the sites of GABA signal appearance, is unknown. Vesicular inhibitory amino acid transporter (VIAAT) is a potential marker for the GABAergic phenotype. Here we presented evidence that VIAAT is expressed in GFAP-expressing astrocytes and a subpopulation of OX42-expressing microglia, but not in pinealocytes in cultured cells of rat pineal glands. The VIAAT-expressing cells also exhibit GABA immunoreactivity. Essentially the same results were obtained for pineal glands. These results suggest that GABA is stored and secreted from astrocytes and a subpopulation of microglia in pineal glands. [Copyright &y& Elsevier]

Presents information on a study which examined the structure and function of microvesicles in mammalian pinealocytes and the role of acidic organelles in a novel regulatory mechanism for hormonal synthesis and secretion. Features of the microvesicle-mediated signal-transduction on the pineal gland; Information on vacuolar-adenosine triphospatase in microvesicles; Proposed mechanism for melatonin synthesis through a paracrine network.

The solute carrier 18B1 (SLC18B1) is the most recently identified gene of the vesicular amine transporter family and is conserved in the animal kingdom from insects to humans. Proteoliposomes containing the purified human SLC18B1 protein transport not only monoamines, but also polyamines, such as spermidine (Spd) and spermine (Spm), using an electrochemical gradient of H+ established by vacuolar H+-ATPase (V-ATPase) as the driving force. SLC18B1 gene knockdown abolished the exocytosis of polyamines from mast cells, which affected the secretion of histamine. SLC18B1 gene knockout decreased polyamine levels by ~20% in the brain, and impaired short- and long-term memory. Thus, the SLC18B1 protein is responsible for the vesicular storage and release of polyamines, and functions as a vesicular polyamine transporter (VPAT). VPAT may define when, where, and how polyamine-mediated chemical transmission occurs, providing insights into the more versatile and complex features of amine-mediated chemical transmission than currently considered. • Vesicular polyamine transporter (VPAT) was identified as a new SLC18 family member. • VPAT is responsible for vesicular storage and release of polyamines. • VPAT may define when, where, and how polyamine-mediated chemical transmission occurs. • VPAT is involved in metabolism of polyamine in brain. • Amine-mediated chemical transmission seems to be more complex than considered. [ABSTRACT FROM AUTHOR]

Glutamate transporters are expressed throughout the CNS where their major role is to clear released glutamate from presynaptic terminals. Here, we report a novel function of the transporter in rat pinealocytes. This electrogenic transporter conducted inward current in response to L-glutamate and L- or D-aspartate and depolarized the membrane in patch-clamp experiments. Ca2+ imaging demonstrated that the transporter-mediated depolarization induced a significant Ca2+ influx through voltage-gated Ca2+ channels. The Ca2+ rise finally evoked glutamate exocytosis as detected by carbon-fiber amperometry and by HPLC. In pineal slices with densely packed pinealocytes, glutamate released from the cells effectively activated glutamate transporters in neighboring cells. The Ca2+ signal generated by KCl depolarization or acetylcholine propagated through several cell layers by virtue of the regenerative "glutamate-induced glutamate release." Therefore, we suggest that glutamate transporters mediate synchronized elevation of L-glutamate and thereby efficiently downregulate melatonin secretion via previously identified inhibitory metabotropic glutamate receptors in the pineal gland. [ABSTRACT FROM AUTHOR]

5-Hydroxytryptamine (5-HT) is a precursor and a putative modulator for melatonin synthesis in mammalian pinealocytes. 5-HT is present in organelles distinct from L-glutamate-containing synaptic-like microvesicles as well as in the cytoplasm of pinealocytes, and is secreted upon stimulation by norepinephrine (NE) to enhance serotonin N-acetyltransferase activity via the 5-HT[sub 2] receptor. However, the mechanism underlying the secretion of 5-HT from pinealocytes is unknown. In this study, we show that NE-evoked release of 5-HT is largely dependent on Ca[sup 2+] in rat pinealocytes in culture. Omission of Ca[sup 2+] from the medium and incubation of pineal cells with EGTA-tetraacetoxymethyl-ester inhibited by 59 and 97% the NE-evoked 5-HT release, respectively. Phenylephrine also triggered the Ca[sup 2+]-dependent release of 5-HT, which was blocked by phentolamine, an α antagonist, but not by propranolol, a β antagonist. Botulinum neurotoxin type E cleaved 25 kDa synaptosomal-associated protein and inhibited by 50% of the NE-evoked 5-HT release. Bafilomycin A1, an inhibitor of vacuolar H[sup +]-ATPase, and reserpine and tetrabenazine, inhibitors of vesicular monoamine transporter, all decreased the storage of vesicular 5-HT followed by inhibition of the NE-evoked 5-HT release. Agents that trigger L-glutamte exocytosis such as acetylcholine did not trigger any Ca[sup 2+]-dependent 5-HT release. Vice versa neither NE nor phenylephrine caused synaptic-like microvesicle-mediated L-glutamate exocytosis. These results indicated that upon stimulation of a adrenoceptors pinealocytes secrete 5-HT through a Ca[sup 2+]-dependent exocytotic mechanism, which is distinct from the exocytosis of synaptic-like microvesicles. [ABSTRACT FROM AUTHOR]

Abstract: The mammalian pineal gland uses L-glutamate as an intercellular chemical transmitter to regulate negatively melatonin synthesis. To receive glutamate signals, pinealocytes express at least three kinds of glutamate receptors: metabotropic receptor types 3 and 5 and an ionotropic receptor, GluR1. In this study, we examined whether or not the fourth class of ionotropic receptor, δ, which is known for its nondefinitive molecular function and its unique expression pattern in brain, is expressed in pineal gland. RT-PCR analyses with specific probes indicated the expression of mRNA of δ2 but not that of δ1 in pineal gland and cultured pineal cells. Western blotting analysis with polyclonal antibodies specific to the carboxyl-terminal region of the δ2 receptor recognized a single 110-kDa polypeptide of cerebellar membranes and specifically immunostained Purkinje cells. The δ2 antibodies recognized a 110-kDa polypeptide of pineal membranes and specifically immunostained huge glial-like cells with the occasional presence of several long, branching processes in a pineal cell culture. δ2 is not uniformly distributed throughout the cells and is relatively abundant at the periphery of the cell bodies and long processes, where the terminals of synaptophysin-positive processes of pinealocytes, a site for glutamate secretion, are frequently present. The δ2-positive cells constitute a very minor population among total pineal cells (∼0.03%). Double immunolabeling with δ2 antibodies and antibodies against marker proteins for pineal interstitial cells clearly distinguishes δ2-positive pineal cells and other known interstitial cells, including glial fibrillary acidic protein- or vimentin-positive glial-like cells. These results indicated that the δ2 glutamate receptor is expressed in a novel subpopulation of pineal glial-like cells in culture and suggest the presence of a glutamate-mediated intercellular... [ABSTRACT FROM AUTHOR]

Abstract: Vesicular monoamine transporters (VMATs) are involved in chemical transduction in monoaminergic neurons and various endocrine cells through the storage of monoamines in secretory vesicles. Mammalian pinealocytes contain more 5-hydroxytryptamine (5-HT) than any other cells and are expected on contain VMAT, although no information is available so far. Upon the addition of ATP, radiolabeled 5-HT was taken up by a particulate fraction prepared from cultured rat pinealocytes. The 5-HT uptake was inhibited significantly by bafilomycin A1 (an inhibitor of vacuolar H+-ATPase), 3,5-di-tert-butyl-4-hydroxybenzyli-denemalononitrile (a proton conductor), or reserpine (an inhibitor of VMAT). RT-PCR analysis suggested that VMAT type 1 (VMAT1), but not type 2, is expressed. Antibodies against VMAT1 recognized a single polypeptide with an apparent molecular mass of ~55 kDa, and specifically immunostained pinealocytes. VMAT1 immunoreactivity was high in the vesicular structures in the varicosities of long branching processes and was associated with 5-HT, but not with synaptophysin, a marker protein for microvesicles. The 5-HT immunoreactivity in the long branching processes disappeared upon incubation with reserpine. These results indicate that 5-HT, at least in part, is stored in vesicles other than microvesicles in pinealocytes through a mechanism similar to that of various secretory vesicles. [ABSTRACT FROM AUTHOR]