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A High Affinity Glutamate/Aspartate Transport System in Pancreatic Islets of Langerhans Modulates Glucose-stimulated Insulin Secretion

1998; Elsevier BV; Volume: 273; Issue: 3 Linguagem: Inglês

10.1074/jbc.273.3.1647

1083-351X

C. David Weaver, Vidar Gundersen, Todd A. Verdoorn,

Genetics and Neurodevelopmental Disorders

To examine the role of glutamatergic signaling in the function of pancreatic islets, we have characterized a high affinity glutamate/aspartate uptake system in this tissue. The islet [3H]glutamate uptake activity was Na+-dependent, and it was blocked byl-trans-pyrrolidine-2,4-dicarboxylic acid, a blocker of neuronal and glial glutamate transporters. Islet glutamate transport activity exhibited a V max of 8.48 ± 1.47 fmol/min/islet (n = 4), which corresponds to 102.2 ± 17.7 pmol/min/mg islet protein. The apparent K m of islet glutamate transport activity depended on the glucose concentration used in the assay. In the presence of glucose concentrations that do not stimulate insulin secretion (2.8 mm), the apparent K m was 34.7 ± 7.8 μm (n = 3). However, in high glucose (16.7 mm) the apparent K m increased to 112.7 ± 16.5 μm (n = 3) with little or no change in V max. Like most known plasma membrane glutamate transporters, islet glutamate transporters also transported d-aspartate. Anti-d-aspartate immunoreactivity showed that the islet glutamate/aspartate transport activity was localized to the non-β cell islet mantle. In perifusion experiments with isolated islets in the absence of exogenous amino acids,l-trans-pyrrolidine-2,4-dicarboxylic acid in the presence of 8.3 mm glucose potentiated insulin secretion 23.3 ± 2.3% (n = 3) compared with 8.3 mm glucose alone. This effect was abolished in the presence of the α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione. Furthermore, 6-cyano-7-nitroquinoxaline-2,3-dione alone inhibited glucose-stimulated insulin secretion in isolated islets by 15.9 ± 5.9% (n = 3). Taken together these data suggest that a high affinity glutamate transport system exists in pancreatic islets and that this system contributes to a glutamatergic signaling pathway that can modulate glucose-inducible insulin secretion. To examine the role of glutamatergic signaling in the function of pancreatic islets, we have characterized a high affinity glutamate/aspartate uptake system in this tissue. The islet [3H]glutamate uptake activity was Na+-dependent, and it was blocked byl-trans-pyrrolidine-2,4-dicarboxylic acid, a blocker of neuronal and glial glutamate transporters. Islet glutamate transport activity exhibited a V max of 8.48 ± 1.47 fmol/min/islet (n = 4), which corresponds to 102.2 ± 17.7 pmol/min/mg islet protein. The apparent K m of islet glutamate transport activity depended on the glucose concentration used in the assay. In the presence of glucose concentrations that do not stimulate insulin secretion (2.8 mm), the apparent K m was 34.7 ± 7.8 μm (n = 3). However, in high glucose (16.7 mm) the apparent K m increased to 112.7 ± 16.5 μm (n = 3) with little or no change in V max. Like most known plasma membrane glutamate transporters, islet glutamate transporters also transported d-aspartate. Anti-d-aspartate immunoreactivity showed that the islet glutamate/aspartate transport activity was localized to the non-β cell islet mantle. In perifusion experiments with isolated islets in the absence of exogenous amino acids,l-trans-pyrrolidine-2,4-dicarboxylic acid in the presence of 8.3 mm glucose potentiated insulin secretion 23.3 ± 2.3% (n = 3) compared with 8.3 mm glucose alone. This effect was abolished in the presence of the α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione. Furthermore, 6-cyano-7-nitroquinoxaline-2,3-dione alone inhibited glucose-stimulated insulin secretion in isolated islets by 15.9 ± 5.9% (n = 3). Taken together these data suggest that a high affinity glutamate transport system exists in pancreatic islets and that this system contributes to a glutamatergic signaling pathway that can modulate glucose-inducible insulin secretion. Although the role of glutamate as a signaling molecule is well established in the central nervous system, a similar role in the periphery has only recently been suggested. We (1Weaver C.D. Yao T.L. Powers A.C. Verdoorn T.A. J. Biol. Chem. 1996; 271: 12977-12984Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar) and others (2Inagaki N. Kuromi H. Gonoi T. Okamoto Y. Ishida H. Seino Y. Kaneko T. Iwanaga T. Seino S. FASEB J. 1995; 9: 686-691Crossref PubMed Scopus (177) Google Scholar) have detected functional glutamate receptors in the pancreatic islets of Langerhans. These miniature organs, found dispersed throughout the exocrine pancreas, are composed of four major cell types as follows: the insulin-secreting β cell, the glucagon-secreting α cell, the pancreatic polypeptide-secreting PP cell, and the somatostatin-secreting δ cell. The electrically excitable β cells are stimulated to secrete insulin in response to changes in serum glucose concentrations. Secretion of insulin, and the three other major peptide hormones found in islets, is also believed to be affected by other metabolic and neuronal signals (reviewed in Refs. 3Boyd A.E. J. Cell. Biochem. 1992; 48: 234-241Crossref Scopus (54) Google Scholar and 4Ashcroft F.M. Proks P. Smith P.A. Ämmälä C. Bokvist K. Rorsman P. J. Cell. Biochem. 1994; 55: 54-65Crossref PubMed Scopus (244) Google Scholar). Bertrand et al. (5Bertrand G. Gross R. Puech R. Loubatières-Mariani M.M. Bockaert J. Br. J. Pharmacol. 1992; 106: 354-359Crossref PubMed Scopus (97) Google Scholar, 6Bertrand G. Gross R. Puech R. Loubatières-Mariani M.-M. Bockaert J. Eur. J. Pharmacol. 1993; 237: 45-50Crossref PubMed Scopus (86) Google Scholar) have shown that α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) 1The abbreviations used are: AMPA, α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione;l-trans-PDC,l-trans-pyrrolidine-2,4-dicarboxylic acid; PBS, phosphate-buffered saline; TBS, Tris-buffered saline. receptor agonists can potentiate both insulin and glucagon secretion from a perfused pancreas preparation and that oral or intravenous glutamate can increase insulin secretion and glucose tolerance in vivo (7Bertrand G. Puech R. Loubatieres-Mariani M.M. Bockaert J. Am. J. Physiol. 1995; 269: E551-E556PubMed Google Scholar). We have localized AMPA-type glutamate receptors to β, α, and PP cells and kainate receptors in α cells using immunohistochemistry and electrophysiology (1Weaver C.D. Yao T.L. Powers A.C. Verdoorn T.A. J. Biol. Chem. 1996; 271: 12977-12984Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). To elucidate the role of glutamatergic signaling in islet physiology, we examined islets for the presence of a high affinity uptake system similar to those described in the central nervous system. Glutamate transporters in the central nervous system allow glutamate to act as a specific signaling molecule despite its relatively high concentration in the cerebrospinal fluid because they reduce the concentration of glutamate in the vicinity of receptors. Earlier studies have suggested that islets do not possess a high capacity for glutamate uptake and utilization; however, these studies were focused on the possible role of glutamate as a carbon source or as a fuel secretagogue (8Sehlin J. Hormones. 1972; 3: 156-166Crossref PubMed Scopus (13) Google Scholar). The millimolar concentrations of glutamate used in these studies did not adequately address the possibility of a high affinity glutamate uptake system in islets, the presence of which might serve to support a role for glutamate receptors in islet signal transduction. By using a [3H]glutamate uptake assay, we detected glutamate transport activity in isolated rat pancreatic islets. The uptake observed in islets had properties similar to those of central nervous system transporters. It had high affinity for glutamate, was Na+-dependent, and was blocked byl-trans-pyrrolidine-2,4-dicarboxylic acid (l-trans-PDC), a compound that blocks neuronal and glial transporters. Furthermore, the apparent K m of islet glutamate uptake was markedly increased by increasing glucose concentrations. Visualization of d-aspartate uptake with anti-d-aspartate antibodies showed that the transporter activity was located in the α cell-rich islet mantle. The blockade of glutamate transport in isolated islets byl-trans-PDC potentiated glucose-induced insulin secretion, whereas blockade of AMPA receptors with 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) resulted in inhibition of insulin secretion. These observations indicate that glutamate transporters are important components of a glutamatergic signaling system within the pancreatic islets of Langerhans. Percoll was purchased from Pharmacia Biotech Inc. Culture dishes were purchased from Corning. Ham's F-12 tissue culture media, fetal bovine serum, horse serum, penicillin, and streptomycin were purchased from Life Technologies, Inc.l-[3,4-3H]Glutamate andd-[2,3-3H]aspartate were purchased from NEN Life Science Products. l-trans-PDC, bicuculline methiodide, and CNQX were purchased from Research Biochemicals.l-Cystine was purchased from Sigma. Alkaline phosphatase-conjugated goat anti-rabbit antibody was purchased from DAKO. Alkaline phosphatase-conjugated donkey anti-guinea pig antibody was purchased from Jackson ImmunoResearch. Triton X-100 Surfact-Amps was purchased form Pierce. Electron microscopy grade glutaraldehyde, paraformaldehyde, and tissue freezing medium were purchased from Electron Microscopy Sciences. Poly(A)qua/Mount was purchased from Polysciences. All other chemicals were of reagent grade or higher. Pregnant Sprague-Dawley rats were obtained from Harlan. Islets were isolated and cultured by a modification of the method described (9Hegre O.D. Marshall S. Schulte B.A. Hickey G.E. Williams F. Sorenson R.L. Serie J.R. In Vitro. 1983; 19: 611-620Crossref PubMed Scopus (44) Google Scholar). Pancreata were harvested from 5 to 10, 4- or 5-day-old neonatal rat pups and placed in 4 ml of Ham's F-12 medium containing 5% (v/v) fetal bovine serum, 100 IU/ml penicillin, and 100 μg/ml streptomycin (culture medium). The pancreata were diced with fine iris scissors until the pieces were approximately 0.5 mm3. During the process of dicing, the pieces of pancreas were washed 5 times with fresh culture medium. Diced pancreas tissue was placed in 100-mm diameter polystyrene culture dishes at a density of approximately three diced pancreata per dish in 6 ml of culture medium. The pieces were incubated overnight in a 5% CO2 incubator at 37 °C. The following day, the medium was removed from the pancreas explants and replaced with fresh culture medium. The explants were then incubated for 3 days in the 5% CO2 incubator after which time the medium was changed again. Two days following the last medium change the islet explants were dislodged from the bottom of the polystyrene dishes with gentle trituration and transferred to a 50-ml polypropylene conical tube. The explants were pelleted by centrifugation at 2000 rpm for 2 min in a Beckman TJ-6 centrifuge. The explant pellet was then resuspended in 5 ml of fresh culture medium. The resuspended islet pellet was layered onto a Percoll gradient prepared as described (10Brunstedt J. Diabetes Metab. Rev. 1980; 6: 87-89Google Scholar) with the exception that the Percoll solutions were made up in culture medium. The gradients were centrifuged at 5000 rpm for 15 min in a Beckman TJ-6 centrifuge. The islets were harvested from the 1.060/1.082 g/ml Percoll interface, and the remaining acinar tissue was found at the medium 1.060 g/ml Percoll interface. Five ml of culture medium was added to the harvested islets in a 15-ml conical tube, and the sample was mixed by inversion. The islets were pelleted by centrifugation at 500 ×g for 3 min in a clinical centrifuge and washed with an additional 10 ml of culture medium. The islets were pelleted as above and resuspended in culture medium. The islets were plated at a density of approximately 1000–2000 islets per 100-mm diameter polystyrene culture dish in 6 ml of culture medium per dish and subsequently cultured in a 5% CO2 incubator overnight at 37 °C. The following day the islets were harvested from the culture dish using a 5-ml polystyrene pipette leaving behind the remainder of the fibroblasts adhering to the culture dish. The islets were pelleted as described above and resuspended in 6 ml of culture medium where 25% (v/v) horse serum was used in place of 5% (v/v) fetal bovine serum. The islets were incubated overnight in a 5% CO2 incubator and used for l-glutamate/d-aspartate uptake or insulin release assays the next day. Islets isolated as described above were hand counted using a Gilson P-10 micropipetor and an Olympus CK2 inverted microscope. Thirty to fifty islets were transferred to 1.5-ml polypropylene microcentrifuge tubes containing 100 μl of a buffer consisting of (in mm) 140 NaCl, 4.7 KCl, 1.2 KH2PO4, 1.2 MgSO4, 2.2 CaCl2, 10 HEPES, pH 7.3 (uptake buffer), containing 11.2 mm glucose. In some studies choline was used in place of Na+ and gluconate was used in place of Cl− in the uptake buffer. After harvest 1 ml of uptake buffer was added to the tubes containing the islets. The islets were pelleted in a swinging bucket microcentrifuge at 500 × g for 15 s. The buffer was removed by careful aspiration, and the islets were resuspended in 150 μl of uptake buffer containing varying glucose concentrations and bicuculline, as appropriate, at either 37 or 0 °C. The islets were then incubated for 5 min at either 37 °C or on ice. Following the preincubation, 150 μl of uptake buffer was added containing the appropriate glucose concentration, bicuculline (where appropriate), and twice the desired final concentration ofl-[3H]glutamate,d-[3H]aspartate, l-cystine, andl-trans-PDC, as appropriate. Samples were incubated for 5–9 min at either 37 °C or on ice. The uptake assay was stopped by the addition of 1 ml of ice-cold uptake buffer. The islets were pelleted by centrifugation as described above. The supernatant solution was aspirated, and the islets were resuspended in 1 ml of ice-cold uptake buffer. This wash procedure was repeated a total of three times. After the final wash, the islets were lysed in 100 μl of 0.5 m NaOH for 5 min with occasional vortexing. The samples were centrifuged as above and then transferred to a vial containing 3 ml of aqueous scintillation mixture, mixed by inversion, and counted using a Packard model 1600 TR liquid scintillation counter. Values obtained from the scintillation counter were converted into uptake activity units using l-[3H]glutamate or d-[3H]aspartate standards. For calculation of K m and V max, values obtained at 0 °C were treated as a blank and subtracted from the values obtained at 37 °C. Uptake data were analyzed using Igor (Wavemetrics), Excel (Microsoft), and Instat (GraphPad Software). Statistical comparisons were paired two-tailed t tests.l-trans-PDC inhibition curves were fit, andl-trans-PDC IC50 was calculated using the logistic equation. Figures were constructed using Igor. Two to five hundred islets were placed in a 50-μl perifusion chamber. The islets were washed for 30 min at a flow rate of 100 μl/min with oxygenated uptake buffer containing 2.8 mm glucose at 37 °C. Following the wash the islets were perifused for 15 min at the same flow rate and temperature with uptake buffer containing 2.8 mm glucose (control) or uptake buffer containing 2.8 mm glucose and 100 μmd-aspartate. The islets were washed for 15 min at 200 μl/min with ice-cold uptake buffer. The islets were then fixed at room temperature in the chamber by perifusion with uptake buffer containing 2.5% (v/v) glutaraldehyde and 1% (v/v) paraformaldehyde at 200 μl/min for 5 min. The perifusion was halted, and the islets were incubated for 1 h in the fixative at room temperature. At the end of the incubation the chambers were disassembled, and the frits with fixed islets attached were removed. The islet-bearing frits were transferred to 1.5-ml centrifuge tubes containing uptake buffer with 0.25% (v/v) glutaraldehyde and 0.1% (v/v) paraformaldehyde and incubated overnight at 4 °C. The frits were then washed 3 times for 20 min in phosphate-buffered saline (PBS) composed of (in mm) 137 NaCl, 2.7 KCl, 10.1 Na2HPO4, 1.5 KH2PO4, and 2 times for 30 min in PBS containing 30% (w/v) sucrose. The sucrose equilibrated islet-containing frits were placed face down in a mold containing tissue freezing medium (OCT compound) and frozen on dry ice. Five-μm sections were cut and thaw-mounted onto charge slides. The sections were allowed to dry for 10 min and were rehydrated in PBS. Sections were permeabilized for 10 min in PBS containing 0.2% (v/v) Triton X-100 and blocked for 1 h in 5% (v/v) normal goat serum (for the anti-d-aspartate antibody) or 5% (v/v) normal donkey serum (for the anti-glucagon antibody) in PBS. The blocked sections were incubated overnight at 4 °C with a 1:250 dilution of anti-glucagon antibody or a 1:1000 dilution of anti-d-aspartate antibody. The anti-d-aspartate antibody was raised as described (11Storm-Mathisen J. Leknes A.K. Bore A. Vaaland J.L. Edminson P. Haug F.M.S. Ottersen O.P. Nature. 1983; 301: 517-520Crossref PubMed Scopus (775) Google Scholar) and thoroughly tested as described (12Gundersen V. Danbolt N.C. Ottersen P.O. Storm-Mathisen J. Neuroscience. 1993; 57: 97-111Crossref PubMed Scopus (128) Google Scholar). The antibodies were diluted in PBS plus 1% (v/v) normal goat serum (for the anti-d-aspartate antibody) or 1% (v/v) normal donkey serum (for the anti-glucagon antibody) and 0.1% (v/v) Triton X-100. Following incubation the sections were washed 1 time for 10 min in PBS plus 0.1% (v/v) Triton X-100 and 2 times for 10 min/wash in Tris-buffered saline (TBS) composed of (in mm) 25 Tris, pH 8.0, 137 NaCl, 2.7 KCl plus 0.1% (v/v) Triton X-100. For detection of tissue-bound antibody, washed sections were incubated for 1–3 h at room temperature with a 1:100 dilution of alkaline phosphatase-conjugated goat anti-rabbit antibody (for the anti-d-aspartate antibody) or a 1:500 dilution of alkaline phosphatase-conjugated donkey anti-guinea pig antibody (for the anti-glucagon antibody) diluted in TBS plus 1% (v/v) normal goat serum or 1% (v/v) normal donkey serum, respectively, and 0.1% (v/v) Triton X-100. Following incubation slides were washed 3 times for 10 min/wash in TBS plus 0.1% Triton X-100 and 1 time for 5 min with a buffer containing (in mm) 100 Tris, pH 9.5, 100 NaCl, 5 MgCl2 (alkaline phosphatase buffer) plus 0.1 mmlevamisole. Immunoreactivity was visualized with nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate in alkaline phosphatase buffer containing 0.1 mm levamisole. Slides were mounted with Poly(A)qua/Mount. Photomicrographs were taken using a Leica Laborlux S microscope equipped with a Wild MPS52 camera attachment, a Wild MPS46 photoautomat, and Kodak T max 100 film. Prints were produced on Ilford Multigrade IV RC Deluxe paper. 100 islets per experimental condition were placed in a 50-μl perifusion chamber. The islets were perifused for 30 min at a flow rate of 100 μl/min at 37 °C with a buffer containing (in mm) 128 NaCl, 1.19 MgSO4, 18 NaHCO3, 2.54 CaCl2, 1.19 KH2PO4, 4.74 KCl, and 0.1% (w/v) bovine serum albumin (perifusion buffer) plus 2.8 mm glucose (bubbled for 1 h prior to perifusion with 95% O2/5% CO2). Following this wash, the perifusion was stopped for 5 min. At the end of the 5 min, a fraction was collected by perifusing the islets at 1 ml/min for 30 s. Perifusion was allowed to continue at 100 μl/min for 2.5 min after which the flow was stopped for an additional 5 min. Another fraction was collected as above. The islets were then perifused for 2.5 min at 100 μl/min with perifusion buffer containing 2.8 mm glucose or perifusion buffer containing 2.8 mm glucose plus either 500 μml-trans-PDC, 10 μm CNQX, or bothl-trans-PDC, and CNQX. Flow was stopped and fractions were collected as above. Following this treatment, the islets were perifused for 2.5 min at 100 μl/min with perifusion buffer containing 8.3 mm glucose or perifusion buffer containing 8.3 mm glucose plus eitherl-trans-PDC, CNQX, or bothl-trans-PDC and CNQX. Flow was stopped and fractions were collected as above. Insulin concentrations in the perifusate were determined by a core facility using the Linco insulin radioimmunoassay with appropriate standards. Data were analyzed using Instat and Excel. Statistical comparisons are from paired two-tailedt tests. We have identified a high affinity glutamate uptake system in isolated, intact pancreatic islets using a [3H]glutamate uptake assay. Islet glutamate uptake exhibited saturable kinetics (Fig.1) and an affinity for [3H]glutamate that was nearly identical to glutamate transporter activities observed in the central nervous system. Lineweaver-Burk replots of glutamate uptake activity revealed an apparent K m of 34.7 ± 7.8 μm(n = 3) in the presence of 2.8 mm glucose. This [3H]glutamate uptake activity displayed aV max of 8.48 ± 1.47 fmol/min/islet (corresponding to 102.2 ± 17.7 pmol/min/mg islet protein) (n = 4). Islet glutamate uptake activity showed other properties similar to central nervous system glutamate transport activities. In particular, islet [3H]glutamate uptake activity was dependent on temperature. Uptake at 0 °C averaged only 13.1 ± 4.1% (n = 3) of that measured at 37 °C. Islet [3H]glutamate uptake was also strongly dependent on the presence of Na+ in the uptake buffer. In experiments where Na+ was replaced with choline, the glutamate uptake activity was 28.7 ± 3.0% (n = 3) that measured in the presence of normal external Na+. A Na+-independent, Cl−-dependent glutamate transport activity has been reported in the periphery (13Bannai S. Kitamura E. J. Biol. Chem. 1980; 255: 2372-2376Abstract Full Text PDF PubMed Google Scholar,14Bannai S. J. Biol. Chem. 1986; 261: 2256-2263Abstract Full Text PDF PubMed Google Scholar) and in neurons (15Murphy T.H. Miyamoto M. Sastre A. Schnaar R.L. Coyle J.T. Neuron. 1989; 2: 1547-1558Abstract Full Text PDF PubMed Scopus (852) Google Scholar). This activity, designated system Xc−, is inhibited by excess cystine. Islet glutamate uptake decreased only modestly (6.1%,n = 2) when Cl− was replaced by gluconate in the uptake assay buffer. However, the addition of 500 μm cystine in Na+-free conditions further decreased [3H]glutamate activity to 11.3% of control. Since system Xc− is believed to be primarily involved in the uptake of cystine, we focused our study on the more prominent Na+-dependent [3H]glutamate uptake activity in islets. To determine which islet cell types were responsible for the observed glutamate uptake activity, we examined transport ofd-aspartate. As in the case of most other known plasma membrane glutamate transporters, isletd-[3H]aspartate transport was quite similar to l-[3H]glutamate transport for bothK m and V max (Fig.2).d-[3H]Aspartate uptake was also sensitive to temperature and to the removal of external Na+ (Fig. 2,inset). The similarities betweenl-[3H]glutamate andd-[3H]aspartate uptake suggested that both were transported by the same transporter proteins and therebyd-aspartate uptake offered a way to determine which islet cells harbored glutamate transporters. In these experiments islets were incubated in the presence and absence of 100 μmd-aspartate and fixed with glutaraldehyde and paraformaldehyde to covalently bind amino acids in a form accessible for immunostaining. This technique was first demonstrated as indicated in Ref. 11Storm-Mathisen J. Leknes A.K. Bore A. Vaaland J.L. Edminson P. Haug F.M.S. Ottersen O.P. Nature. 1983; 301: 517-520Crossref PubMed Scopus (775) Google Scholar, and the degree of retention of labeledl-glutamate and d-aspartate by different fixatives was determined (16Storm-Mathisen J. Ottersen O.P. J. Histochem. Cytochem. 1990; 38: 1733-1743Crossref PubMed Scopus (102) Google Scholar). Samples were stained with the selectived-aspartate antibodies (12Gundersen V. Danbolt N.C. Ottersen P.O. Storm-Mathisen J. Neuroscience. 1993; 57: 97-111Crossref PubMed Scopus (128) Google Scholar, 17Gundersen V. Shupliakov O. Brodin L. Ottersen O.P. StormMathisen J. J. Neurosci. 1995; 15: 4417-4428Crossref PubMed Google Scholar) and with anti-peptide hormone antibodies for the identification of the cell types that concentrated d-aspartate. As shown in Fig.3 A, anti-d-aspartate immunoreactivity was restricted to the cells of the islet mantle which, in our preparation, are primarily glucagon-containing α cells (Fig. 3 B). The islet mantle may also contain a small percentage of δ and PP cells (data not shown), and thus our data cannot exclude the presence ofd-aspartate uptake activity in these cell types. Anti-d-aspartate staining was not observed in islets that had not been incubated in the presence of d-aspartate (Fig.3 C).Figure 3Islet d-aspartate (l-glutamate) uptake is localized to the cells of the islet mantle. Shown are light micrographs of 2.5% (v/v) glutaraldehyde, 1% (v/v) paraformaldehyde-fixed, isolated islet sections.A, islet incubated with 100 μmd-aspartate prior to fixation and probed with anti-d-aspartate antibody. B, islet probed with anti-glucagon antibody. Note the similar staining pattern of the islet mantle cells with both the anti-d-aspartate and anti-glucagon antibodies. C, islet incubated in the absence of d-aspartate prior to fixation, probed with anti-d-aspartate antibody. Scale bar, 25 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Because islets are glucose-sensitive organs, we have also investigated the effect of d-glucose on [3H]glutamate uptake in islets. We found that islet glutamate uptake activity was significantly inhibited (32.0 ± 3.8%, n = 5,p = 0.0013) in the presence of a glucose concentration that stimulates insulin secretion (16.7 mmd-glucose) compared with uptake in lowd-glucose (2.8 mm) (Fig.4 A). The inhibitory effect of glucose on glutamate transport was not due to an increase in the osmolarity of the uptake buffer since non-metabolizablel-glucose (16.7 mm) failed to result in an inhibition of glutamate uptake (102.06 ± 4.2% of control) (Fig.4 A). Replots of the uptake data revealed that inhibition byd-glucose was the result of a change in the apparentK m for glutamate and not a change in theV max (Fig. 4 B). At low glucose, islet glutamate uptake had an apparent K m of 34.7 ± 7.8 μm (n = 3). However, in the presence of 16.7 mmd-glucose (Fig. 4 B) the apparent K m for glutamate shifted to 112.7 ± 16.5 μm (n = 3). These data are consistent with a competitive mode of inhibition byd-glucose at a concentration that stimulates insulin secretion. It has been suggested that down-regulation of glucagon secretion in α cells exposed to high glucose may be mediated by GABAAreceptors expressed in these cells (18Rorsman P. Berggren P.-O. Bokvist K. Ericson H. Möhler H. Östenson C.-G. Smith P.A. Nature. 1989; 341: 233-236Crossref PubMed Scopus (376) Google Scholar, 19Rorsman P. Ashcroft F.M. Berggren P.-O. Biochem. Pharmacol. 1991; 41: 1783-1790Crossref PubMed Scopus (38) Google Scholar). The proposed source of GABA is β cells, which may coordinately release it with insulin (20Gerber J.C. Hare T.A. Brain Res. Bull. 1980; 5: 341-346Crossref Scopus (36) Google Scholar). Since glutamate/aspartate uptake activity was predominantly in α cells, we tested whether glucose inhibition of glutamate transport might be mediated through this mechanism. To do this we repeated the uptake experiments with 2.8 and 16.7 mmd-glucose in the presence of the GABA receptor antagonist, bicuculline. However, the degree of inhibition (31.7 ± 1.7%,n = 3) was the same as that measured in the absence of bicuculline (Fig. 4 A). The addition of 50 μmbicuculline alone had no effect on [3H]glutamate uptake (102.7 ± 11.2% of uptake in the absence of bicuculline,n = 3). Previously, Bertrand et al. (5Bertrand G. Gross R. Puech R. Loubatières-Mariani M.M. Bockaert J. Br. J. Pharmacol. 1992; 106: 354-359Crossref PubMed Scopus (97) Google Scholar, 7Bertrand G. Puech R. Loubatieres-Mariani M.M. Bockaert J. Am. J. Physiol. 1995; 269: E551-E556PubMed Google Scholar) have demonstrated that AMPA receptor agonists potentiate glucose-induced insulin secretion in a perfused pancreas preparation (5Bertrand G. Gross R. Puech R. Loubatières-Mariani M.M. Bockaert J. Br. J. Pharmacol. 1992; 106: 354-359Crossref PubMed Scopus (97) Google Scholar) and in vivo (7Bertrand G. Puech R. Loubatieres-Mariani M.M. Bockaert J. Am. J. Physiol. 1995; 269: E551-E556PubMed Google Scholar). To identify potential roles of glutamate transporters in islets, we examined the effects of glutamate transport inhibition on glucose-stimulated insulin secretion.l-trans-PDC is an inhibitor of glutamate transport in the central nervous system (21Price C.J. Raymond L.A. Mol. Pharmacol. 1996; 50: 1665-1671PubMed Google Scholar) and showed an IC50 of 129.2 μm against islet [3H]glutamate transport. In the presence of 35 μm glutamate the maximum amount of inhibition we observed in islets was 66.0% with 2 mml-trans-PDC. The average inhibition of glutamate transport (35 μm) was 55.0 ± 3.9% (n = 4) in the presence of 500 μml-trans-PDC (Fig.5 A). This concentration ofl-trans-PDC has been shown to potentiate glutamate receptor activity in hippocampal CA1 neurons (22Isaacson J.S. Nicoll R.A. J. Neurophysiol. 1993; 70: 2187-2191Crossref PubMed Scopus (131) Google Scholar). Fig. 5 B shows the results from a representative experiment where isolated islets, in the absence of exogenous amino acids, were exposed to 2.8 and 8.3 mmd-glucose in the absence and presence of either 500 μml-trans-PDC, the AMPA receptor antagonist CNQX (10 μm), or both l-trans-PDC and CNQX. We have previously demonstrated that 10 μm CNQX is sufficient