Microvesicles isolated from bovine pineal gland specifically accumulate <scp>l</scp>‐glutamate

Moriyama, Yoshinori; Yamamoto, Akira

doi:10.1016/0014-5793(95)00559-r

Cited by 55 publications

(52 citation statements)

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“…Furthermore, the MVs specifically accumulated L-glutamate in an energy-dependent manner (6). These results suggested that MVs are the organelles in pinealocytes that store and secrete L-glutamate.…”

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confidence: 77%

“…6. In brief, bovine pineal glands (about 50 glands for one preparation) obtained from a local slaughterhouse within a few hours after killing were washed, cut into small pieces with scissors, suspended in 80 ml of SME buffer (20 mM MOPS-Tris (pH 7.0) containing 0.3 M sucrose, 5 mM EDTA, 5 g/ml pepstatin A, and 5 g/ml leupeptin), and then homogenized in a Dounce homogenizer.…”

Section: Methodsmentioning

confidence: 99%

“…Very recently, we established a procedure for isolating MVs from bovine pineal glands and found that these vesicles were actually devoid of synapsin I but possess synaptotagmin and synaptobrevin 2, proteins necessary for vesicular transport (6). Furthermore, the MVs specifically accumulated L-glutamate in an energy-dependent manner (6).…”

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confidence: 99%

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Vesicular L-Glutamate Transporter in Microvesicles from Bovine Pineal Glands

Moriyama

Yamamoto

1995

Journal of Biological Chemistry

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Pinealocytes, endocrine cells that synthesize and secrete melatonin, possess a large number of synaptic-like microvesicles (MVs) containing the L-glutamate transporter (Moriyama, Y., and Yamamoto, A. (1995) FEBS Lett., 367, 233-236). In this study, the L-glutamate transporter in MVs isolated from bovine pineal glands was characterized as to its driving force, requirement of anions, and substrate specificity. Upon the addition of ATP, the MVs accumulated L-glutamate. The uptake was significantly dependent on the extravesicular Cl ؊ concentration, being negligible in the absence of Cl ؊ and maximum at 2-5 mM and decreasing gradually at 20 -100 mM. The membrane potential (inside positive) was maximum at 0 -10 mM Cl ؊ and then decreased gradually depending on the Cl ؊ concentration, whereas a pH gradient was practically absent without Cl ؊ and increased gradually up to 100 mM Cl ؊ . Ammonium acetate or nigericin plus K ؉ , a dissipator of a pH gradient, had little effect on or was slightly stimulatory toward the uptake, whereas valinomycin plus K ؉ inhibited both formation of the membrane potential and the glutamate uptake to similar extents. The ATP-and Cl ؊ -dependent glutamate uptake was inhibited by fluoride, iodide, or thiocyanate, without vacuolar H ؉ -ATPase being affected. An anion channel blocker, 4,4-diisothiocyanatostilbene-2,2-disulfonic acid, similarly inhibited the glutamate uptake in a Cl ؊ protectable manner. Furthermore, ATP-and glutamate-dependent acidification of MVs was observed when 4 mM Cl ؊ was present. Among more than 50 kinds of glutamate analogues tested, only a few compounds, including 1-aminocyclohexane-trans-1,3-dicarboxylic acid, caused similar acidification. A good correlation was observed between the acidification and the inhibition of glutamate uptake by glutamate analogues. These results indicated that 1) the major driving force of the glutamate uptake is the membrane potential, 2) Cl ؊ regulates the glutamate uptake, probably via anion-binding site(s) on the transporter, and 3) the transporter shows strict substrate specificity. Hence, the overall properties of the vesicular glutamate transporter in the MVs well matched those of the synaptic vesicle glutamate transporter. We concluded that the vesicular glutamate transporter, being similar if not identical to the neuronal counterpart, operates in endocrine cells.Pinealocytes are parenchymal endocrine cells of pineal glands that synthesize and secrete melatonin into the blood (1-3). At least two kinds of secretory vesicle-like organelles have been identified in pinealocytes: dense core granules, which are speculated to be involved in the storage and secretion of melatonin and some neuropeptides such as arginine vasotocin (3, 4), and a large number of synaptic-like microvesicles (MVs) 1 containing synaptophysin (5). Although histochemical evidence indicated that MVs are distinct from neuronal synaptic vesicles in their lack of synapsin I, the possible participation of MVs in some secretory pathways in pinealocytes was also speculated...

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confidence: 99%

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Vesicular L-Glutamate Transporter in Microvesicles from Bovine Pineal Glands

Moriyama

Yamamoto

1995

Journal of Biological Chemistry

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“…Pinealocytes also contain microvesicles and are capable of secreting glutamate (Moriyama and Yamamoto, 1995a;Yamada et al, 1996a,b). Thus, like glutaminergic neurons, pinealocytes may fire and terminate glutamate signals simultaneously.…”

Section: Immunological Localization Of the Glt-1-type Transportermentioning

confidence: 99%

Functional Expression of a GLT‐1 Type Na⁺‐Dependent Glutamate Transporter in Rat Pinealocytes

Yamada¹,

Yatsushiro²,

Yamamoto

et al. 1997

Journal of Neurochemistry

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Pinealocytes, the neuroendocrine cells that produce melatonin, accumulate glutamate in microvesides through a specific vesicular transporter energetically coupled with vacuolar-type proton ATPase. The glutamate is secreted into the extracellular space through microvesicle-mediated exocytosis and then stimulates neighboring pinealocytes, resulting in inhibition of norepinephrine-dependent melatonin synthesis. In this study, we identified and characterized the plasma membranetype glutamate transporter in rat pinealocytes. The [ 3H]-glutamate uptake by cultured pinealocytes was driven by extracellular Nat, saturated with the [3H]glutamate concentration used, and significantly inhibited by L-glutamate, L-aspartate, ß-threo-hydroxyaspartate, pyrrolidine dicarboxylate, and L-cysteine sulfinate, substrates or inhibitors of the plasma membrane glutamate transporter. Consistently, the clearance of extracellular glutamate, as measured by HPLC, was also dependent on Na~and inhibited by /9-threo-hydroxyaspartate and L-cysteine sulfinate. Immunological studies with site-specific antibodies against three isoforms of the Nat-dependent glutamate transporter (GLT-1, GLAST, and EAAC1) revealed the expression of only the GLT-1 type transporter in pineal glands. Expression of the GLT-1 type transporter in pineal glands was further demonstrated by means of reverse transcription-polymerase chain reaction with specific DNA probes. Immunohistochemical analysis indicated that the immunological counterpart(s) of the GLT-1 is localized in pinealocytes. These results suggested that the GLT-1 -type Nat-dependent transporter is expressed and functions as a reuptake system for glutamate in rat pinealocytes. The physiological role of the transporter in the termination of the glutamate signal in the pineal gland is discussed. Key Words: Na-dependent glutamate transporter-Pinealocyte-Pineal gland -L-Glutamate-GLT-1 -Reuptake-Microvesicle-Endocrine cell.

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“…Vesicular glutamate transporter has been characterized in neurons and pinealocytes, which possess an active glutamate-specific transporter dependent on ATP-generated electrochemical proton gradient across the vesicle membrane, on extravesicular Cl Ϫ concentration, and on temperature (13,(21)(22)(23).…”

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Characterization of vesicular glutamate transporter in pancreatic α- and β-cells and its regulation by glucose

Bai

Zhang

2003

American Journal of Physiology-Gastrointestinal and Liver Physi

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.-Glutamate has been suggested to play an important role in the release of insulin and glucagon from pancreatic cells via exocytosis. Vesicular glutamate transporter is a rate-limiting step for glutamate release and is involved in the glutamate-evoked exocytosis. Two vesicular glutamate transporters (VGLUT1 and -2) have recently been cloned from the brain. In this report, we first functionally characterized vesicular glutamate transporter in cultured pancreatic ␣-and ␤-cells, and then detected mRNA expression of VGLUT1 and -2 in these cells. We also investigated the effect of high or low level of glucose on vesicular glutamate transport in cultured pancreas cells. Our results suggest that both ␣-and ␤-cells contain functional vesicular glutamate transporter. The transport characteristics are similar to the cloned neuronal VGLUT1 and -2 in regard to ATP dependence, substrate specificity, kinetics, and chloride dependence. VGLUT2 mRNA is expressed in both ␣-and ␤-cells, whereas VGLUT1 is only expressed in ␤-cells. High (12.8 mM) and low (2.8 mM) concentrations of glucose increased vesicular glutamate transport in ␤-and ␣-cells, respectively. VGLUT2 mRNA was significantly increased in ␤-and ␣-cells by high and low glucose concentration, respectively. This increase in VGLUT2 mRNA was suppressed by actinomycin D. We conclude that both ␣-and ␤-cells possess functional vesicular glutamate transporters regulated by alteration in glucose concentration, partly via the transcriptional mechanism. diabetes; insulin; glucagon; transcriptional regulation L-GLUTAMATE IS THE MAJOR EXCITATORY neurotransmitter in the mammalian central nervous system and plays important roles in many neuronal processes, such as fast synaptic transmission and neuronal plasticity. More recently, it has been suggested that glutamate is a functional molecule in nonneuronal tissues including pancreas, bone, stomach, intestine, liver, lung, kidney, and skin (28). Glutamate has been found to stimulate insulin (5, 7) and glucagon (6, 17) secretion in the pancreatic ␤-and ␣-cells, respectively. In the model of glucose-induced insulin secretion, increased cytosolic Ca 2ϩ concentration by the opening of voltage-sensitive Ca 2ϩ channels, constitutes the main trigger of insulin exocytosis. However, the Ca 2ϩ signal alone is not sufficient for the full development of biphasic insulin secretion (see Ref. 20 for review). Maechler and Wollheim (19) recently provided evidence that glutamate acts downstream of the mitochondria by sensitizing the Ca 2ϩ -mediated exocytotic process. In that model, a rise in extracellular glucose causes elevation of intracellular glucose followed by a subsequent increase in glycolysis and tricarboxylic acid activity. These metabolic changes lead to an increase in cellular ATP that closes ATP-sensitive potassium channels causing depolarization of the plasma membrane potential. Subsequently, depolarization opens voltage-sensitive Ca 2ϩ channels, raising intracellular Ca 2ϩ concentration and triggering insulin exocytosis. Glutamate is pack...

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Microvesicles isolated from bovine pineal gland specifically accumulate l‐glutamate

Cited by 55 publications

References 26 publications

Vesicular L-Glutamate Transporter in Microvesicles from Bovine Pineal Glands

Vesicular L-Glutamate Transporter in Microvesicles from Bovine Pineal Glands

Functional Expression of a GLT‐1 Type Na⁺‐Dependent Glutamate Transporter in Rat Pinealocytes

Characterization of vesicular glutamate transporter in pancreatic α- and β-cells and its regulation by glucose

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Microvesicles isolated from bovine pineal gland specifically accumulate l‐glutamate

Cited by 55 publications

References 26 publications

Vesicular L-Glutamate Transporter in Microvesicles from Bovine Pineal Glands

Vesicular L-Glutamate Transporter in Microvesicles from Bovine Pineal Glands

Functional Expression of a GLT‐1 Type Na+‐Dependent Glutamate Transporter in Rat Pinealocytes

Characterization of vesicular glutamate transporter in pancreatic α- and β-cells and its regulation by glucose

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Functional Expression of a GLT‐1 Type Na⁺‐Dependent Glutamate Transporter in Rat Pinealocytes