Characterization of vesicular glutamate transporter in pancreatic alpha - and beta -cells and its regulation by glucose

Liqun Bai1,2, Xiaohong Zhang2, and Fayez K. Ghishan1,2

Departments of 1 Pediatrics and 2 Physiology, Steele Memorial Children's Research Center, University of Arizona Health Sciences Center, Tucson, Arizona 85724


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha - and beta -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 alpha - and beta -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 alpha - and beta -cells, whereas VGLUT1 is only expressed in beta -cells. High (12.8 mM) and low (2.8 mM) concentrations of glucose increased vesicular glutamate transport in beta - and alpha -cells, respectively. VGLUT2 mRNA was significantly increased in beta - and alpha -cells by high and low glucose concentration, respectively. This increase in VGLUT2 mRNA was suppressed by actinomycin D. We conclude that both alpha - and beta -cells possess functional vesicular glutamate transporters regulated by alteration in glucose concentration, partly via the transcriptional mechanism.

diabetes; insulin; glucagon; transcriptional regulation


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta - and alpha -cells, respectively. In the model of glucose-induced insulin secretion, increased cytosolic Ca2+ concentration by the opening of voltage-sensitive Ca2+ channels, constitutes the main trigger of insulin exocytosis. However, the Ca2+ 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 Ca2+-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 Ca2+ channels, raising intracellular Ca2+ concentration and triggering insulin exocytosis. Glutamate is packaged in vesicles with insulin by the pancreatic cells, and the glutamate sensitizes the release of insulin.

Neuronal cells utilize L-glutamate as an intracellular signaling molecule via glutamatergic systems comprising the storage of glutamate in synaptic vesicles and its exocytosis, glutamate receptor, and glutamate reuptake mechanism. It has been suggested that Langerhans islets have their own glutamatergic system as do neurons. Multiple glutamate receptors have been found in the pancreas (14, 15, 18, 33) and glutamate has been found to stimulate insulin and glucagon release via alpha -amino-3-hydroxy-5-methylisoxazole-4-propionic acid subtypes of the glutamate receptor (5, 6). A sodium-dependent glutamate transporter has been identified as the glutamate reuptake system on the plasma membrane of the pancreas (34). However, vesicular glutamate transporter, the protein responsible for the accumulation of glutamate from cytoplasm into the vesicles, has not been characterized in the pancreas.

Glutamate uptake into the secretory granules by vesicular glutamate transporter is a rate-limiting step for glutamate release. 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- concentration, and on temperature (13, 21-23). Vesicular glutamate transport processes depend on the proton electrochemical gradient (Delta µH+) generated by the Mg2+-activated vacuolar H+-ATPase (V-ATPase) on the vesicular membrane (12). When protons are pumped into the vesicular lumen, a proton gradient (Delta pH) and a membrane potential (Delta phi) occur across the membrane to form Delta µH+, which favors the exchange of luminal protons for the cytoplasmic transmitter (8, 21, 23). Two vesicular glutamate transporters, VGLUT1 and -2, with 75% homology, have been recently identified from synaptic vesicles of neurons (3, 4, 30). Both transporters have functional characteristics of the synaptic vesicle glutamate transporter, including ATP dependence, chloride stimulation, substrate specificity, and substrate affinity. These molecular and functional advances led us to functionally characterize and identify vesicular glutamate transporter(s) from the pancreatic alpha - and beta -cells and to study the regulation of these transporters in response to the changes of glucose concentration.

This is the first study to functionally characterize the vesicular glutamate transporter in the pancreatic cells. We found that both alpha - and beta -cells contained functional vesicular glutamate transporters similar to the cloned VGLUT1 and -2 regarding ATP dependence, substrate specificity, kinetics, and chloride dependence. VGLUT2 is expressed in both alpha - and beta -cells and is regulated by alteration of extracellular glucose concentration.


    MATERIAL AND METHODS
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ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. N,N'-dicyclohexylcarbodiimide (DCCD) was purchased from Fisher Scientific (Pittsburgh, PA). All other chemicals were obtained from Sigma (St. Louis, MO).

Cell culture, preparation of vesicular membranes, and glutamate uptake. Two alpha -cell lines (mouse alpha -TC-1-9 and human HPAC), two beta -cell lines (mouse beta -TC-6 and rat RIN-m), and a rat pheochromocytoma cell line (PC-12) were purchased from American Type Culture Collection (Manassas, VA). Cells were maintained in the manufacturer's suggested culture medium (Ham's F-12K medium for PC-12 cells, a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium for HPAC cells, Dulbecco's modified Eagle's medium for alpha -TC-1-9 and beta -TC-6 cells, and RPMI 1640 medium for RIN-m cells). Various glucose concentrations will be indicated in the figure legends for the individual experiment. For experiments dealing with glucose regulation, alpha -TC-1-9 and beta -TC-6 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 7.5 mM glucose, 10% fetal bovine serum, and 1% penicillin and streptomycin. At 70% confluence, the cells were incubated in Dulbecco's modified Eagle's medium containing either 12.8 (beta -TC-6 cells) or 2.8 mM (alpha -TC-1-9 cells) glucose for 12 h before they were harvested. For transcriptional assays, the cells were pretreated with actinomycin D (5 µg/ml) (Calbiochem-Novabiochem, San Diego, CA) for 2 h and then were treated with 12.8 or 2.8 mM glucose medium in the presence of actinomycin D before they were harvested.

Vesicular membranes were prepared as described previously (3). Briefly, the cells were washed twice with ice-cold PBS and were collected in 0.32 M sucrose and 10 mM HEPES-KOH (pH 7.4) buffer containing protease inhibitors (2 µg/ml leupeptin, 1 µg/ml pepstatin, 0.2 mM diisopropylfluorphosphate, 2 µg/ml aprotinnin, and 1.25 mM Mg EDTA). The cells were then homogenized by using a ball bearing homogenizer (10 µM clearance) for 15 passes. The resulting homogenate was pelleted at 1,000 g for 5 min to remove the nuclei and at 27,000 g for 35 min to remove the heavier membranes. The supernatant containing lighter membranes, mainly vesicular membrane, was sedimented at 210,000 g for 1 h, and the pellet was resuspended in 0.32 M sucrose, 4 mM MgSO4, and 10 mM HEPES-KOH, pH 7.4 (uptake buffer) at a final concentration of 10 mg protein/ml.

To start the transport assay, 100 µg of vesicular membrane in uptake buffer were mixed with 4 mM ATP, 4 mM KCl, and 50 µM L-[3H]glutamate (potassium salt, 0.4 Ci/mmol; Pharmacia) at room temperature for varying times with other additions when indicated. Kinetic experiments were performed with different glutamate concentrations as indicated in the Fig. 2 legend. The uptake was stopped by rapid filtration, followed by four immediate washes with 2 ml of ice-cold 0.15 M KCl. Radioactivity was determined by using a liquid scintillation spectrophotometer.

mRNA isolation and semiquantitative RT-PCR. mRNAs were isolated from four types of cells incubated at different glucose concentrations by using the Fast-Track mRNA purification kit (Invitrogen, Carlsbad, CA). First-strand cDNAs were synthesized by using oligo(dT) primer. Subsaturated levels of cDNA templates that were needed to produce a dose-dependent amount of PCR products were defined in the initial experiments by testing a range of template concentrations. Subsequent PCR was carried out with subsaturated level of RT reaction with specific primers for VGLUT1 and -2 of mouse, human, and rat species and beta -actin in separate reactions for 35 cycles (94°C for 1 min, 55°C for 1 min, and 72°C for 1 min). The primers were designed to amplify a 453-bp region corresponding to amino acids 368-519 for human VGLUT1 (GenBank accession no. AB032436) and an 870-bp region corresponding to amino acids 290-580 for VGLUT2 (GenBank accession no. AF 324864). The sequences of primers are mouse VGLUT1: sense strand 5'-CACATAATGTCCACTACCAA-3', antisense strand 5'-CACTGCCAGCCAGCTGGTCG-3'; human VGLUT1: sense strand 5'-CGCATCATGTCCACCACCAA-3', antisense strand 5'-CACTGCCAGCCAGCTGGTCA-3'; mouse VGLUT2: sense strand 5'-ATCTGCTAGGTGCAATGGAA-3', antisense strand 5'-AATCATCTCGGTCCTTATAG-3'; and human VGLUT2: sense strand 5'-ATCTTTTAGGTGCAATGGAA-3', antisense strand 5'-AATCAACTCGGTCCTTATAG-3'. We used mouse primers to amplify rat VGLUT1 and -2 message, because the sequences of the primers are identical between these two spices. PCR products from the same cell line were loaded on 1% agarose gels and visualized with ethidium bromide. PCR products were sequenced at the University of Arizona sequencing facility to ensure the correct sequences.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

Functional characterization of vesicular glutamate transport in cultured pancreatic alpha - and beta -cell lines. Vesicular glutamate transport has been extensively characterized in the neuronal synaptic vesicles. The neuronal vesicular ATP-dependent glutamate transporter is specific for glutamate, is stimulated by millimolar concentrations of chloride, and has a low affinity for uptake (Km, ~1-2 mM) (16, 24). We first tested two alpha -cell lines (mouse alpha -TC-1-9 and human HPAC) and two beta -cell lines (mouse beta -TC-6 and rat RIN-m) for vesicular glutamate uptake. Figure 1A shows that vesicle membranes from these four cell lines exhibited four- to fivefold increased uptake of [3H]glutamate than that of PC-12 cell, a vesicular glutamate transporter null cell (3). We then further characterized vesicular glutamate transport in alpha -TC-1-9 and beta -TC-6 cells.


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Fig. 1.   Functional characteristics of vesicular glutamate transport in cultured pancreatic alpha - and beta -cell lines. Glutamate uptake was performed in uptake buffer containing (in mM) 4 ATP, 4 KCl, and 4 MgSO4 and 50 µM L-[3H]glutamate (pH 7.4), unless specifically indicated below. All experiments were performed in triplicate with different membrane preparations. Error bars indicate SD A: ATP-dependent vesicular glutamate uptake from 4 types of pancreatic and PC-12 cells was performed for 5 min. *P < 0.001. B: glutamate uptake was determined in the presence or absence of 4 mM ATP or ATPase inhibitors for 5 min. Inhibitors for different types of ATPases including bafalomycin A1 (Baf A1, 100 nM), N,N'-dicyclohexylcarbodiimide (DCCD; 50 µM), oligomycin B (5 µM), and ouabain (2 mM) were used in transport assays. *P < 0.01. C: substrate specificity of VGLUT2 was determined by competition of [3H]glutamate uptake with 10 mM unlabeled substrate for 5 min. D: glutamate uptake was measured at various concentrations of chloride for 5 min.

Vesicular glutamate transport in alpha -TC-1-9 and beta -TC-6 cells is ATP-dependent, because removal of ATP abolished the uptake (Fig. 1B). To specify the types of ATPase involved in glutamate uptake by these cells, we tested inhibitors for different types of ATPases in transport assays. DCCD and bafalomycin A1, inhibitors for vacuolar Mg2+-ATPase dramatically inhibited glutamate uptake. The plasma membrane and mitochondrial ATPase inhibitors oligomycin B and ouabain had no effect on glutamate uptake in both cells (Fig. 1B), which is in agreement with previous works (3, 4, 30).

Substrate specificity was also determined in alpha -TC-1-9 and beta -TC-6 cells. Vesicular transporter recognized only glutamate, but not aspartate. Uptake of glutamate by both cells was partially inhibited by D-glutamate, but not by a number of other compounds including aspartate and GABA (Fig. 1C), which was consistent with known characteristics of glutamate transport by the vesicular membranes. Evans blue, a potent inhibitor of vesicular glutamate transport, significantly blocked the uptake mediated by alpha -TC-1-9 and beta -TC-6 cells.

Another feature of the vesicular glutamate transporter that segregates it from other neurotransmitter transporters is the marked stimulation seen in the presence of low concentrations of chloride (3, 10, 11, 24, 29). At low concentrations (1-5 mM), chloride stimulates transport, whereas at higher concentrations (above 10 mM), chloride inhibits transport. Figure 1D showed that low concentrations of chloride (1-4 mM) stimulated glutamate uptake by both alpha -TC-1-9 and beta -TC-6 cells, whereas concentrations higher than 10 mM attenuated the stimulatory effect. This is consistent with the characteristics of VGLUT1 and -2. The above result suggested that the transport characteristics of the two types of cells were very similar and resembled that of the neuronal vesicular glutamate transporter and the cloned VGLUT1 and -2 (3, 4).

Kinetic experiments indicated that vesicular glutamate transport in both cells was dose-dependent and saturable (Fig. 2). In three separate batches of membranes, the Vmax was 5,124 ± 411 pmol · min-1 · mg protein for alpha -TC-1-9 cells and 3,683 ± 241 pmol · min-1 · mg protein for beta -TC-6 cells. Km for glutamate was 1.44 ± 0.36 mM for alpha -TC-1-9 cells and 1.63 ± 0.32 mM for beta -TC-6 cells. The Km was similar to that of glutamate transport by neuronal synaptic vesicles (~1-2 mM) (3, 4, 16, 30).


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Fig. 2.   Kinetics of vesicular glutamate transport from alpha -TC-1-9 and beta -TC-6 cells. The initial uptake rate by vesicular membranes of alpha -TC-1-9 cells (A) and beta -TC-6 cells (B) was measured at 1.5 min in the presence of various concentrations of glutamate (20 µM to 10 mM). One representative of 3 experiments from each cell is shown.

mRNA expression of VGLUT1 and -2 in cultured pancreatic alpha - and beta -cells. VGLUT1 and -2 were originally cloned from the brain and VGLUT2 protein was shown to be expressed in the pancreatic islets (14). However, the mRNA expression of these two transporters in individual cells of islets has not been determined. Because functional vesicular glutamate transport was detected in the two alpha -cell lines (mouse alpha -TC-1-9 and human HPAC) and two beta -cell lines (mouse beta -TC-6 and rat RIN-m) (Fig. 1A), we measured endogenous mRNA expression of VGLUT1 and -2 in these cells by RT-PCR by using human-, mouse-, or rat-specific primers corresponding to different cell types. RT-PCR was performed with a subsaturated dose of mRNA and PCR cycle, as determined by measuring beta -actin mRNA (data not shown) (2). VGLUT2 was expressed in all types of tested cells, although relatively less expression was observed in the two beta -cell lines (Fig. 3, lanes 1-4). In contrast, VGLUT1 was only expressed in the two beta -cell lines (Fig. 3, lanes 3 and 4). All PCR products were confirmed by automatic sequencing. Neither VGLUT1 nor -2 was detected in RT reaction-free negative control (data not shown). These results suggested that VGLUT2 could play a functional role in the regulation of vesicular glutamate uptake in both alpha - and beta -cells, whereas VGLUT1 might play a role in a beta -cells but not in an alpha -cells.


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Fig. 3.   mRNA expression of VGLUT1 and -2 in cultured pancreatic alpha - and beta -cells. PCR fragments of VGLUT1 and -2 were indicated (left). Lanes 1-5 indicate mouse alpha -TC-1-9 cells, human HPAC cells, mouse beta -TC-6 cells, rat RIN-m cells, and 1-kb DNA ladder (GIBCO).

Regulation of vesicular glutamate transport by changing of extracellular glucose concentration. Under normal physiological conditions, changes of serum glucose concentration, i.e., hyperglycemia or hypoglycemia, result in secretion of insulin or glucogan from the pancreas, respectively. Vesicular glutamate transporter in pancreatic islet cells controls accumulation and storage of glutamate in secretory vesicles. Therefore, we studied the regulation of pancreatic vesicular glutamate transporter by different glucose concentrations. Time courses of vesicular glutamate transport in alpha -TC-1-9 and beta -TC-6 cells in response to high (12.8 mM) and low (2.8 mM) glucose concentrations were shown in Fig. 4. Cells were maintained at physiological glucose concentration (7.5 mM) before changing glucose concentration. Under low glucose concentration (Fig. 4A), vesicular glutamate uptake by alpha -TC-1-9 cells was significantly increased after 12 h exposure and remained steady until 48 h. However, vesicular glutamate uptake by beta -TC-6 cells was not changed by the same exposure. Interestingly, under high glucose concentration (Fig. 4B), vesicular glutamate uptake by beta -TC-6 cells was significantly increased after 12 h exposure, whereas vesicular glutamate uptake by alpha -TC-1-9 cells was not changed by the same exposure.


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Fig. 4.   Time course of vesicular glutamate uptake in alpha -TC-1-9 and beta -TC-6 cells in response to different glucose concentrations. Cells were cultured in Dulbecco's modified Eagle's medium supplemented with physiological glucose level (7.5 mM), 10% fetal bovine serum, 1% penicillin, and streptomycin. The medium glucose level was then changed to 2.8 or 12.8 mM, and cells were incubated until the indicated time course. Vesicular membrane was prepared from these cells for [3H]glutamate uptake. A: cells were incubated at a low glucose level (2.8 mM). B: cells were incubated at a high glucose level (12.8 mM).

Potential mechanism of glucose-mediated regulation of vesicular glutamate transport in cultured pancreatic alpha - and beta -cells. Because increased vesicular glutamate transport was observed after 12 h exposure to low or high glucose concentrations, we tested the potential mechanism for this regulation. As we have shown in Fig. 3, alpha -TC-1-9 cells expressed VGLUT2 but not VGLUT1. We first determined VGLUT2 mRNA change in alpha -TC-1-9 cells in response to low glucose level by the semiquantitative RT-PCR technique (Fig. 5). The expression of VGLUT2 mRNA was significantly increased in alpha -TC-1-9 cells by low glucose (2.8 mM) stimulation, and the increased expression was suppressed by pretreatment of actinomycin D. 


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Fig. 5.   Detection of VGLUT2 mRNA levels in alpha -TC-1-9 cells by semiquantitative RT-PCR. A: mRNA prepared from alpha -TC-1-9 cells grown in 7.5 mM glucose medium (lane 1), 2.8 mM medium (lane 2) and 2.8 mM medium with pretreatment of actinomycin D (5 µg/ml) (lane 3) for 12 h was used for 1st-strand cDNA synthesis. Subsequent PCR amplification was performed with VGLUT2 or beta -actin-specific oligonucleotide primers in separate reactions. PCR products for VGLUT2 and beta -actin for each sample were loaded on the same gel lane and visualized with ethidium bromide. B: summary of densitometry data from semiquantitative RT-PCR. *P < 0.05 with the other two groups.

A similar approach was used to determine the mechanism of increased vesicular glutamate transport in beta -TC-6 cells in response to chronic high glucose treatment. As shown in Fig. 6, expression of VGLUT2 mRNA was significantly increased in beta -TC-6 cells by high glucose, whereas VGLUT1 mRNA was not changed by the same treatment. The increase of VGLUT2 mRNA was also suppressed by actinomycin D. Taken together, the increased VGLUT2 expression in both alpha - and beta -cells in response to changes of glucose concentration suggested that transcriptional regulation accounted, at least partially, for the increased transport function.


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Fig. 6.   Expression of VGLUT1 and -2 mRNA in beta -TC-6 cells in response to high glucose. A: mRNA prepared from beta -TC-6 cells grown in 7.5 mM glucose medium (lanes 1 and 4), 12.8 mM medium (lanes 2 and 5), and 12.8 mM medium with pretreatment of actinomycin D (5 µg/ml) (lanes 3 and 6) for 12 h was used for 1st-strand cDNA synthesis. Semiquantitative RT-PCR was performed with VGLUT2 (lanes 1 to 3) and VGLUT1 (lanes 4 to 6) or beta -actin-specific oligonucleotide primers in separate reactions. PCR products for each sample were loaded on the same gel lane and visualized with ethidium bromide. B: summary of densitometry data from semiquantitative RT-PCR. *P < 0.05 to lanes 1 and 3.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

Glutamate plays an important role in glucose-induced insulin secretion in pancreatic beta -cells and glucagon secretion in pancreatic alpha -cells by sensitizing Ca2+-induced hormone exocytosis. Packaging and storage of glutamate into specialized secretory vesicles in neurons and endocrine cells ensure its regulated release. A unique group of proteins found on secretory vesicles is the transporter needed for the accumulation of glutamate from cytoplasm into these vesicles. Although Langerhans islets were believed to equip their own glutamatergic systems, the functional characteristics of vesicular glutamate transport in the pancreas has not been characterized. In this report, we functionally characterized vesicular glutamate transporters in pancreatic alpha - and beta -cells, which appear to have the same major characteristics as their neuronal counterpart. VGLUT1 is expressed in beta -cells, whereas VGLUT2 is expressed in both alpha - and beta -cells. VGLUT2 is upregulated by changes of glucose concentration in both cells. We conclude that both alpha - and beta -cells contained functional vesicular glutamate transporter(s), which can be regulated by alteration of glucose concentration.

Vesicular glutamate transport has been extensively characterized in neuronal synaptic vesicles. This vesicular transport processes depend on the Delta µH+ generated by a Mg2+-activated V-ATPase on the vesicular membrane (12). The vesicular ATP-dependent glutamate transporter is specific for glutamate, is stimulated by millimolar concentrations of chloride, and has a low affinity for uptake (Kmapprox 1-2 mM) (16, 24). Endocrine cells, such as pancreatic alpha - and beta -cells, contain synaptophysin-containing vesicles in which classical neurotransmitters are accumulated (31). These vesicles contain transporters for specific neurotransmitters and the transport relies on the presence of ATP. Consistent with this, vesicular glutamate transport in cultured pancreatic cells is specific for glutamate and is ATP dependent. Bafalomycin A1 and DCCD, inhibitors for vacuolar Mg2+-ATPase, dramatically inhibited glutamate uptake. The plasma membrane and mitochondrial ATPase inhibitors, oligomycin and ouabain, had no effect on glutamate uptake. Furthermore, pancreatic vesicular glutamate transport is saturable with a Km of 1.4 and 1.6 mM for alpha -TC-1-9 cells and beta -TC-6 cells, respectively.

The requirement of low chloride concentration is a significant property of vesicular glutamate transport. Presence of chloride at low concentrations (1-5 mM) is essential for the uptake of glutamate, with substantially lower transporter activity observed at higher and lower levels (10, 21, 29). This biphasic effect of chloride has been observed in glutamate uptake from both alpha -TC-1-9 and beta -TC-6 cells. The intracellular chloride concentration is within 2-15 mM under normal physiological conditions (1, 27). Thus the low extravesicular concentration of chloride favoring vesicular glutamate uptake is considered physiologically relevant in the cells. We did not test the concentration of chloride in the pancreatic cells. However, the chloride concentration in these cells is likely comparable to that of neurons, because these endocrine cells have the major characteristics of neurons, such as neurotransmitter systems. These results suggested that the pancreatic vesicular glutamate transporter possessed similar characteristics to their neuronal counterpart including ATP dependence, transport kinetics, substrate specificity, and chloride dependence.

Because transport characteristics of the pancreatic vesicular glutamate transporter are also similar to the two recently cloned neuronal vesicular glutamate transporters, VGLUT1 and -2, expression of VGLUT1 and -2 was determined in the cultured pancreatic cells. VGLUT1 was expressed only in beta -cells, whereas VGLUT2 was expressed in both alpha - and beta -cells, although a lesser level expression was found in beta -cells. Similar to our study, another group (14) using double immunostaining with antibodies against VGLUT2 and glucagon found that VGLUT2 protein is expressed in the pancreatic alpha -cell. However, they failed to detect VGLUT2 protein in beta -cells by using double staining with antibody against VGLUT2 and insulin. This difference is probably due to lesser sensitivity of immunohistochemistry for detection of a protein with lower expression in the cells. The differential expression of VGLUT1 and -2 implies that VGLUT2 may be the vesicular glutamate transporter involved in regulation of glucagon secretion from alpha -cells, because VGLUT1 is not present in alpha -cells.

Considerable evidence indicates that the biosynthesis and transport of neurotransmitters undergo regulation by physiological or pathophysiological factors. For example, vesicular monoamine transporter isoform 2 (VMAT2) is an important vesicular transporter for histamine. Recent work has demonstrated that VMAT2 gene expression may be regulated by gastrin (32) and ovarian hormones (26). Intracellular calcium increases vesicular monoamine transporter through a transcriptional activation mechanism in chromaffin cells (9). We have shown that VGLUT2 mRNA is upregulated by high concentration of glucose in beta -cells and by low concentration of glucose in alpha -cells. In contrast, expression of VGLUT1 was not changed by high glucose, although its expression was more predominant than VGLUT2 in beta -cells. These findings suggest that chronic exposure to low glucose concentration stimulates glutamate uptake into secretory vesicles in alpha -cells, which favors glutamate-evoked glucagon release, whereas chronic exposure to high glucose concentration stimulates glutamate uptake into secretory vesicles in beta -cells, which favors glutamate-evoked insulin release. However, the exact mechanisms of this regulatory processes are not clear. The suppression of glucose-induced upregulation of VGLUT2 by actinomycin D suggested that transcriptional mechanism was at least partially involved. Further studies of regulation of VGLUT2 promoter by glucose will help to elucidate the mechanism.


    ACKNOWLEDGEMENTS

This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant 2R01-R37-DK-33209.


    FOOTNOTES

Address for reprint requests and other correspondence: F. K. Ghishan, Professor and Head, Dept. of Pediatrics, Director, Steele Memorial Children's Research Center, 1501 N. Campbell Ave., Tucson, AZ 85724 (E-mail: fghishan{at}peds.arizona.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published November 20, 2002;10.1152/ajpgi.00333.2002

Received 8 August 2002; accepted in final form 18 November 2002.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
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