Proc. Natl. Acad. Sci. USAVol. 90, pp. 5317-5321, June 1993Cell Biology

A y-aminobutyric acid transporter driven by a proton pump ispresent in synaptic-like microvesicles of pancreatic f8 cellsANNETTE THOMAS-REETZ*t, JOHANNES W. HELLO§, MATTHEW J. DURING1,CHRISTIANE WALCH-SOLIMENA*II, REINHARD JAHN*t II, AND PIETRO DE CAMILLI*t*Howard Hughes Medical Institute and Departments of tCell Biology, lMedicine, and I1Pharmacology, Yale University School of Medicine, New Haven, CT06510; and tMax Planck Institute for Psychiatry, Martinsried, Munich, Germany

Communicated by Erminio Costa, February 25, 1993

ABSTRACT A variety of peptide-secreting endocrine cellscontain a population of reycling microvesicles that shareseveral major membrane polypeptides with neuronal synapticvesides (SVs). The function ofthese synaptic-like microvesicles(SLMVs) remains to be elucidated. It was previously suggestedthat SLMVs of pancreatic 13 cells may store and secreteraminobutyric acid (GABA). GABA, the major nonpeptideinhibitory neurotransmitter of the central nervous system, isstored in and secreted from SVs. GABA uptake into SVs ismediated by a transporter that is driven by a vacuolar protonATPase. GABA is also present at high concentration in theendocrine pancreas where it is selectively locWalied in insulin-secreting ,B cells, the core cells of pancreatic islets. GABA is notpresent in peripheral islet cells (mantle cells), representedprimarily by glucagon-secreting a cells. In this study, animmunoisolation procedure was used to puriy SLMVs fromcell lines derived from mouse ,B cells and a cells. SLMVsobtained from the 13-cell line, but not those obtained from thea-cell line, displayed a GABA-transport activity dependentupon a proton electrochemical gradient generated by a vacu-olar proton ATPase. These data support the hypotheses that (s)SLMVs have a secretory function similar to that of SVs and (ii)13cell SLMVs are involved in the secretion ofGABA, which inturn may have a paracrine function on mantle cells of the islet.

In the nerve terminal, nonpeptide neurotransmitters arestored in and secreted from highly specialized secretoryorganelles known as synaptic vesicles (SVs). Neurotransmit-ters are loaded into these vesicles by specific transportersthat are driven by an electrochemical gradient that is gener-ated by a proton vacuolar ATPase (V-ATPase) present in thevesicle membrane (1). Recent studies have shown that or-ganelles closely related to SVs and referred to as synaptic-like microvesicles (SLMVs) are present in a variety ofpeptide-secreting endocrine cells (2-4). Both SLMVs andSVs undergo cycles of exocytosis/endocytosis at the cellsurface (5-9). It is still unclear whether SLMVs, like SVs,store and secrete neurotransmitters or neurotransmitter-likemolecules. The pancreatic 8 cell is a particularly well-suitedmodel to address this question. 8 cells contain high concen-trations ofthe neurotransmitter -aminobutyric acid (GABA)and of the enzymes that catalyze the biosynthesis [glutamicacid decarboxylase (GAD)] and metabolism (GABA transam-inase) of GABA (10-14). Furthermore, a paracrine role ofGABA in the islets has been suggested (15-19). It has beendemonstrated that GAD, which in GABA-secreting neuronsis concentrated in close proximity to SVs, is also concen-trated in close proximity to SLMVs ofpancreatic (3cells (20).In this study, an immunoisolation procedure was used toobtain highly purified SLMVs from pancreatic (3- and a-celllines. SLMVs obtained from (3 cells, but not those obtained

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from a cells, contain a GABA transport activity driven by aV-ATPase that is similar to that of SVs.

MATERIALS AND METHODSAntibodies. The following antibodies were previously de-

scribed: affinity-purified polyclonal antibodies directedagainst p29 (21), monoclonal antibody (mAb) C.7.2 and poly-clonal antibodies directed against synaptophysin (2), polyclo-nal antibodies directed against synaptobrevin (22), humanserum containing autoantibodies directed against GAD (14),two antibodies directed against the P2p3 subunit oftheGABAAreceptor [mAb bd17 (23, 24) and mAb 62-3G1 (26, 27)], anantibody directed against ribophorin II (28), and an antibodydirected against SV2 (29). The following immunological re-agents were obtained from commercial sources: anti-insulin(chicken) and anti-chicken IgG (goat) sera (Sera Lab, West-bury, NY), anti-rabbit IgG (goat) and normal goat serum(Cappel Laboratories), and anti-mouse IgG (goat) (Sigma).

Pancreatic Cell Lines. ,BTC3 (30) and aTC9 cells (31) weregrown in Click's medium (Irvine Scientific, Santa Ana, CA)supplemented with 5% (vol/vol) fetal calf serum.

OrganeUle ImmunIsolation. Nonimmune bovine immuno-globulins (Sigma) or purified mAb C.7.2 directed againstsynaptophysin was conjugated to Eupergit CIZ methacrylatemicrobeads (Rohm Pharmaceuticals, Darmstadt, Germany) asdescribed (32, 33). (TC3 cells were grown to about 70%6confluency in either 100-mm tissue culture dishes (w5 x 106cells per dish) or 175-mm flasks (=15 x 106 cells per flask).Cells were detached from the dishes by a 10-min incubation inphosphate-buffered saline (PBS)/10 mM EDTA at 3TC, pel-leted by centrifugation, resuspended in 1 ml ofhomogenizationbuffer [250mM sucrose, 1 mM MgC2, 0.005%DNase (Sigma),4 mM Hepes at pH 7.4 containing the following proteaseinhibitors: 0.4 mM phenylmethylsulfonyl fluoride (BoehringerMannheim), 10 mM benzamidine (Sigma), and pepstatin A,leupeptin, antipain, and aprotinin (each at 4 pg/ml; Sigma)],and passed four to six times through a ball-bearing homoge-nizer (clearance of 0.0028 inch; Berni-Tech, Saratoga, CA).The homogenate was centrifuged (10,000 x g for 10 min at4°C), and the supernatant [low speed supernatant (LSS)] wasrecovered. EDTA was added to the LSS at a final concentra-tion of 1.5 mM. The LSS was incubated with immunobeads for60 min at 4°C with constant rotation. For electron microscopyand [3H]GABA uptake experiments, an excess ofLSS relativeto immunobeads was used. In this case, a LSS obtained from30 to 40 175-mm flasks was incubated with 200 id of animmunobead pellet. The beads were sedimented by centrifu-

Abbreviations: SV, synaptic vesicle; SLMV, synaptic-like microves-icle; V-ATPase, vacuolar proton pump ATPase; LSS, low speedsupernatant; GABA, yaminobutyric acid; FCCP, carbonylcyanidep-trifluoromethoxyphenylhydrazone; GAD, glutamic acid decarbox-ylase; AuH+, proton electrochemical gradient; mAb, monoclonalantibody.§Present address: Department of Pharmacology SJ30, University ofWashington School of Medicine, Seattle, WA 98195.

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gation at 5000 x g for 30-60 sec at 4°C, washed three times inhomogenization buffer, and further processed for gel electro-phoresis and Western blotting, electron microscopy, or ApHmeasurement and [3H]GABA uptake experiments as de-scribed below. At the end of each uptake experiment, therecovery of SLMVs on the beads was evaluated by gelelectrophoresis followed by immunoblotting for synapto-physin. This control was also used to confirm that comparablerecoveries of synaptophysin were obtained in different exper-iments and for both ,BTC3 and aTC9 cells. Typically, about40-60o of the synaptophysin in the LSS of both ,BTC3 andaTC9 cells was recovered on the immunobeads.Measurement ofApH. ApH was measured as described (33)

with minor modifications. Briefly, the washed immunobeadswere resuspended in 320 mM sucrose/10 mM Mops-KOH,pH 7.3, so that a 50-,ul bead suspension was equivalent to a1-,ul bead pellet. (This bead suspension was used for ApHmeasurements and [3H]GABA uptake experiments.) ApHwas measured using the pH-sensitive dye acridine orange.Changes in absorbance at 492 nm were measured with anAminco DW 2000 dual wavelength spectrophotometer, using530 nm as a reference wavelength. A 100-,lI bead suspensionwas added to 900 pul of standard assay buffer (160mM KCI/4mM MgCl2/10 mM Mops-KOH, pH 7.3/10 ,uM acridineorange) at 32°C. When H+-ATPase inhibitors were used (50,uM N-ethylmaleimide, 50 AM vanadate, and 5 ,M oligomy-cin B), they were added to standard assay buffer, and themixture was preincubated for 5 min at 32°C. After a stablebaseline was established, the reaction was initiated by theaddition of MgATP (2 mM final concentration). The acidifi-cation was reversed either by the addition of the protono-phore carbonylcyanide p-trifluoromethoxyphenylhydrazone(FCCP) (60 ,uM final concentration), which collapses theproton electrochemical gradient (AAH+), or by the additionof (NH4)2SO4 (10 mM final concentration), which eliminatesthe pH gradient but not the transmembrane potential.[3H]GABA Uptake. [3H]GABA uptake was measured as

described (34) with minor modifications. A 100-,l beadsuspension was preincubated for 3 min at 32°C. Then 50 Al of3 x uptake medium was added to the beads to obtain thefollowing final concentrations: 320 mM sucrose, 4 mMMgCl2, 4 mM KC1, 2 mM MgATP, 10 mM Mops-KOH (pH

7.3), 300 ,uM unlabeled GABA (present as a carrier mole-cule), 4 ,uCi (1 Ci = 37 GBq) of [3H]GABA and either (i) noadditions, (ii) 50mM unlabeled GABA, or (iii) 60 ,uM FCCP.After a 5-min incubation at 32°C, the beads were sedimentedby centrifugation for 30 sec at 5000 x g. The bead pellet wasimmediately resuspended in 500 ul of ice-cold 320 mMsucrose/10mM Mops-KOH, pH 7.3. The beads were washedrapidly three times at 0°C by resuspension, vortexing, andcentrifugation as described above. Scintillation fluid wasadded to the washed bead pellet, and 3H content was deter-mined by using a scintillation counter. The amount of[3H]GABA that was recovered on the immunobead fractionin the presence of ATP plus FCCP was taken as a baselinevalue because FCCP completely dissipates pL,H+, whichdrives GABA uptake into SVs. This baseline value representseither nonspecific binding to the immunoisolated vesicles orATP-independent transport of GABA.HPLC Determination ofGABA Content ofPTC3 and alC9

Cells. ,BTC3 cells or aTC9 cells were grown in 175-mm flasksfor 5 days until they were 70%o confluent. Cells were detachedfrom the flasks by incubation with PBS/10 mM EDTA,pelleted by centrifugation at 1000 x g for 10 min at roomtemperature, and resuspended in 150 mM NaCl/10 mM

a synaptocLSaNB beads

LSS NB B NB B

synaptophysin

p29

synaptobrevin

b LSS NB B LSS NB B

_ @* Z ~~~synaptophysin* * r

__q Wt ribophorin II

,STC3 aTC9

FIG. 1. Double-immunofluorescence micrographs showing colo-calization of the SV proteins synaptophysin (synapt.) and SV2 inTC3 (a and b) and aTC9 (c and d) cells. The two proteins have a

similar distribution in both cell lines. Immunoreactivity is scatteredthroughout the cells but is concentrated at a paranuclear area, whichcorresponds to the Golgi complex area. (Bar = 17 gm.)

FIG. 2. Specificity of the SLMV immunoisolation procedure. (a)Recovery of synaptophysin, synaptobrevin, and protein p29 in organ-elles bound by anti-synaptophysin immunobeads and control immu-nobeads incubated with a crude extract (LSS fraction) of f3TC3 cells.The starting material (LSS fraction), the resulting supernatant (NB,not bound material), and the immunoabsorbed material (B, boundmaterial) were analyzed by gel electrophoresis followed by immuno-blotting with antibodies directed against each of the three proteins.The immunoabsorbed material was resuspended to the original vol-ume of the LSS, and equal volumes of LSS, NB, and B were loadedonto the gel. Therefore, the intensity of the labeled bands closelyreflects the partition of the three proteins in the NB and B fractions.In this experiment an excess of immunobeads was used, and SLMVproteins were almost completely depleted from the starting LSS. Noneof SLMV proteins was recovered on control beads. (b) Western blotanalysis of immunoisolation experiments performed on LSS fractionsof a and 8 cells for the study ofGABA uptake. In these experimentsan amount of LSS was chosen to saturate the binding capacity of thebeads and therefore to maximize recovery of synaptophysin-positiveorganelles. Organelle binding was still highly specific as shown by thelack of any detectable immunoreactivity for the endoplasmic reticu-lum marker ribophorin II in the bead-bound fraction.

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Hepes, pH 7.3. A small aliquot was saved for proteindetermination. Perchloric acid was added to a final concen-tration of 1%, and the cells were solubilized by using a tipsonicator at 50% power for 3 min. Insoluble particles wereremoved by centrifugation, and the resulting supernatant wasadjusted to pH 8.0 with 1 M NaOH and stored at -70°C untilused for HPLC analysis. Determination of amino acid con-tent was accomplished by precolumn derivatization witho-phthalaldehyde-3-mercaptopropionic acid followed by re-verse-phase HPLC and fluorimetric detection (35).

Miscellaneous Procedures. Proteins were measured by theBCA assay (Pierce). SDS/PAGE was performed according toLaemmli (36) and Western blotting was according to Towbinet al. (37) using 1251-labeled protein A (Amersham). Doubleimmunofluorescence was performed as described (20). Elec-tron microscopy of the immunobead fraction was performedas described (8, 32).

RESULTSIn this study, cell lines that are derived from pancreatictumors of transgenic mice expressing the simian virus 40large tumor antigen under the control of either the insulin(3-cell lines) or glucagon (a-cell lines) promoter (30, 31, 39)have been used. The 3-cell line 83TC3 expresses GAD andcontains immunohistochemically detectable levels ofGABA.However, levels of GAD and GABA in these cells areheterogeneous, and the relative abundance of GAD- andGABA-expressing cells varied in culture from different pas-sages (20). The a-cell line aTC9 was derived from the aTC1cell line (31), which is devoid of GAD and GABA immuno-reactivity (20). The GABA content of a 13TC3 extract, asdetermined by HPLC, was 4-fold higher (1.24 nmol/mg ofprotein) than that of an aTC9 extract (0.28 nmol/mg ofprotein). Since less than 20% of the P3TC3 cells used for theseexperiments were positive for GAD and GABA by immuno-cytochemistry, the value obtained for 3TC3 cells is clearly anunderestimate of the value expected from an homogeneouspopulation of GAD-expressing .8TC3 cells. The amount ofGABA measured by HPLC in aTC9 cells probably reflects

0.10

0)

0cn.0Cl).0

0.00

Time, min

FIG. 4. ATP-dependent acidification monitored by acridine or-ange in SLMVs immunoisolated from f3TC3 cells. A 100-/4 beadsuspension was incubated at 32°C with 900 A of acidification buffercontaining the pH-sensitive dye acridine orange. The absorbancewas measured at 492 nm using 530 nm as a reference wavelength.Once a stable baseline had been established, MgATP was added (2mM final concentration). The acidification ofthe vesicle is registeredas a decrease in absorbance. Acidification could be reversed by theaddition of the proton ionophore FCCP at 60 ,uM.

nonvesicular metabolic levels of GABA since these cells donot have concentrated stores of GABA detectable by immu-nocytochemistry (20). The amount of GABA in isolatedmouse islets was reported to be 3 nmol/mg of protein (40).Both ,BTC3 and aTC9 cells were found to express mem-

brane proteins characteristic of SVs and SLMVs, includingsynaptophysin and SV2 (Fig. 1) and synaptobrevin andprotein p29 (data not shown). This agrees with previousobservations made in intact islets (2, 20-22, 29). Theseproteins had a similar subcellular distribution in both 13 cellsand a cells, as shown in Fig. 1 for SV2 and synaptophysin.

_ 150..r4-L-.

-

0-0 00,~0D -

16 0<a)0 oo.co

< 0 100 .CD

Ir EV-.*

0

FIG. 3. Electron micrograph showing the morphology of organ-elles immunoisolated by anti-synaptophysin immunobeads from acrude extract (LSS fraction) of (TC3 cells. Bound organelles arerepresented by small round or elongated vesicles (arrows). The coreof plastic beads is labeled by asterisks. Note the lack of secretorygranules on the beads. (Bar = 300 nm.)

IPTC3 aTC9

ATP ATP +unlabeledGABA

I I

ATP +FCCP

ATP ATP +FCCP

FIG. 5. [3H]GABA uptake into SLMVs immunoisolated from/3TC3 or aTC9 cells. Aliquots of immunobead fractions were incu-bated with [3H]GABA with the additions indicated in the figure.[3H]GABA uptake is expressed as percent increase ofthe radioactivityrecovered in the immunobead fraction at the end of the incubationover the radioactivity recovered when FCCP(which dissipates j,.&H+)was present in the uptake medium. A FCCP-sensitive [3H]GABAuptake, which could be blocked by competition with nonradioactiveGABA, was observed only in SLMVs from 13TC3 cells. Each incu-bation condition was performed in duplicate. Results represent thecombined data offour independent experiments performed with 3TC3cells and two independent experiments performed with aTC9 cells.Standard error bars are indicated in the figure.

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In the following experiments, methacrylate beads cova-lently conjugated to anti-synaptophysin mAbs were used toimmunoisolate synaptophysin-containing organelles from aLSS fraction of a 83TC3 homogenate. The enrichment ofsynaptophysin in the immunoabsorbed fraction paralleled theenrichment of p29 and synaptobrevin, which confirms thecolocalization of these proteins on the same organelles (Fig.2a). None of the proteins were recovered in control beadfractions prepared with nonimmune IgGs (Fig. 2a). The spec-ificity of the immunoisolation procedure was further sup-ported by the absence of the endoplasmic reticulum markerribophorin II in the immunoabsorbed fraction (Fig. 2b).An electron microscopic analysis of organelles immunoiso-

lated from (BTC3 cells is shown in Fig. 3. Organelles bound tothe beads were represented by small vesicles and tubules in thesame size range as SVs. They are similar to the synaptophysin-positive organelles of 3-cells previously identified by in situelectron microscopy immunocytochemistry (20).GABA uptake into SVs is dependent on a AkLH+ generated

by a V-ATPase (34). In each GABA-uptake experiment,aliquots of SLMVs, isolated from either 13TC3 or aTC9 cells,were first tested for the presence of an active proton pump.Acidification ofSLMVs was monitored photometrically usingthe pH-sensitive dye acridine orange as an indicator. In thepresence of MgATP, the SLMVs acidified, as indicated by adecrease in absorbance of the dye (Fig. 4). The acidificationwas reversed by the addition of the protonophore FCCP,which confirms a proton-driven process (Fig. 4). Furthermore,acidification was completely abolished if SLMVs were prein-cubated with N-ethylmaleimide (inhibitor of V-ATPases) (ref.41; data not shown), but not with either vanadate (inhibitor ofthe plasma membrane ATPase) (ref. 41; data not shown) oroligomycin B (inhibitor of the FiFo-ATPase) (ref. 41; data notshown), indicating that acidification ofSLMVs is catalyzed bya vacuolar V-ATPase as observed in SVs.For the measurement ofGABA uptake, aliquots ofSLMVs

were incubated with [3H]GABA in a buffer known to supportGABA transport into SVs. In SLMVs isolated from 13TC3cells, GABA uptake that was dependent on ATP and sensi-tive to the protonophore FCCP was observed (Fig. 5). Thisresult indicates that 1-cell SLMVs contain aGABA transportsystem, similar to that present in SVs, which is dependent onA,tfH+ generated by a V-ATPase. [3H]GABA uptake wasreduced by competition by 50 mM unlabeled GABA, whichdemonstrates that GABA uptake is mediated by a saturable

transporter. As observed for SVs, competition was incom-plete, indicating the presence of a transporter with a lowsubstrate affinity. In SLMVs isolated from aTC9 cells, noATP-dependent uptake ofGABA was detected (Fig. 5). SinceSLMVs were isolated with equal efficiency from both /3 anda cells (Fig. 2b), the absence of GABA transport activity inorganelles isolated from aTC9 cells is probably due to theabsence of a GABA transporter.A paracrine role of GABA in pancreatic islets was previ-

ously suggested by the detection of GABAA receptor-mediated responses and ofGABAA receptor (-subunit immu-noreactivity (mAb bdl7) in glucagon-secreting and somatosta-tin-secreting cells (15-19). We have further investigated thelocalization of GABAA receptor immunoreactivity in the en-docrine pancreas in situ by using two mAbs directed againstthe 13233 isoforms ofthis receptor [mAb bdl7 (23, 24) and mAb62-3G1 (26, 27)]. In immunofluorescence experiments on ratpancreatic sections, both antibodies produced an identicalstaining pattern. Immunoreactivity was localized in peripheralislet cells and in a few scattered cells within the islet core (Fig.6 b and d). Counterstaining of the same sections for eitherinsulin (Fig. 6a) or GAD (Fig. 6c) demonstrated that virtuallyall GABAA receptor positive cells were non-(3 cells, althougha few (3cells that were also positive for GABAA receptor wereoccasionally observed (Fig. 6 and data not shown).

DISCUSSIONSLMVs of endocrine cells are biochemically similar to SVsin their membrane protein composition. It was previouslyhypothesized that SLMVs store and secrete neurotransmitteror neurotransmitter-like molecules (4, 20). To our knowl-edge, the present study provides the first demonstration thatSLMVs are capable of transporting neurotransmitter mole-cules in a manner similar to SVs.The function ofSLMVs was investigated by using pancreatic

(3 cells as model endocrine cells because they are known tocontain high levels of the inhibitory neurotransmitter GABA(10-13) and, furthermore, a paracrine role for GABA in pan-creatic islets has been proposed (15-19). Moreover, it waspreviously demonstrated that GABA and GAD immunoreac-tivity closely colocalize with SLMVs, suggesting that SLMVsare the storage organelle for GABA in (3 cells. A direct mea-surement of GABA content in SLMVs was not feasible due tothe limited number ofSLMVs that could be purified even froma large-scale preparation of cultured (3 cells, as used in this

FIG. 6. Localization of the 18 subunitof the GABAA receptor in a pancreaticislet. Two frozen sections of rat pancreaswere stained with mAbs bdl7 (b) or 62-3G1 (d) directed against the P2(3 isoformsof the f3 subunit of the GABAA receptor(GABAAR) and counterstained with anti-sera directed against insulin (a) or GAD(c). GABAA receptor immunoreactivity ispresent mainly in the peripheral cells ofthe islet, which are negative for GAD andfor insulin. (Bar = 30 ,um.)

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study. However, a prerequisite for the storage ofGABA withinSLMVs is the presence of a GABA transporter in SLMVscapable of transporting GABA in a substrate-specific manner.To discern if SLMVs contain a GABA transporter, a rapid

immunoisolation procedure was used that yielded SLMVs withhigh purity and that was previously demonstrated to preserveSVs in a metabolically active form (32, 42). While SLMVs arepresent both in a and (8 cells of pancreatic islets, GABAtransport activity was present only in (-cell SLMVs, which isconsistent with the selective localization in (3 cells of GABA,GAD, and the GABA-metabolizing enzyme GABA transami-nase (10-14). It will be of interest to determine the content ofSLMVs of a cells and of other mantle cells of the islets.A study ofGABA secretion from P3TC3 and its regulation

was attempted by using HPLC to quantitate the amount ofGABA released into the medium in response to variousstimuli. An effiux ofGABA was observed under depolarizingconditions (50 mM K+). However, it was paralleled by theefflux of other amino acids, was Ca2+-independent, and wasprobably due to nonvesicular release mediated by reversibleplasmalemma amino acid transporters. No detectable peaksof GABA secretion were observed after treatments (highglucose, isobutylmethylxanthine and arginine, or phorbolester and carbachol) that led to a substantial increase ofinsulin secretion into the medium. However, the levels ofGABA in the medium were at the limit of detectability witha sensitive HPLC procedure, and no firm conclusions couldbe drawn from these studies (A.T.-R., M.J.D., and P.D.C.,unpublished observations). Due to the absence of a high-affinity plasma membrane GABA transporter in (3TC3 cells(A.T.-R. and P.D.C., unpublished observations), releasecould not be measured by preloading the cells with radioac-tive GABA, a procedure commonly used to study GABArelease from nerve endings (43). A further investigation ofGABA secretion and of its regulation in (3 cells and ,B-celllines remains an important priority for future studies. (3-celllines that homogeneously express GABAergic features in situwill be very useful for this study.Although a direct study of GABA secretion from (TC3

cells was not feasible, the presence of GABAA receptors inmantle cells [i.e., in the cells that are immediately adjacent to( cells and are downstream in the capillary blood flow (44)]strongly supports the hypothesis that GABA acts as a para-crine signal molecule in pancreatic islets. The presence ofthese receptors was previously suggested by experimentscarried out in vivo as well as on freshly isolated pancreaticcells (15-19). GABA was found to produce a hyperpolariza-tion of dissociated a cells and to inhibit glucagon secretion(18, 19). In addition, subpopulations of dissociated a and 8cells were immunoreactive with the mAb bd17 directedagainst the P2133 subunits of the GABAA receptor (18). Thesedata have been complemented by the present study, whichdemonstrates an identical staining of mantle cells in situ withtwo different antibodies that recognize the P2133 subunits ofthe GABAA receptor. It is of interest to note that Zn2+ isstored together with insulin in secretory granules (25). Zn2+is a modulator of GABAA receptor function in the centralnervous system (38), and perhaps it also plays a modulatoryrole on pancreatic GABAA receptors. In conclusion, thepresent data strongly support the hypothesis that (3-cellSLMVs are involved in GABA storage and secretion andmore generally that SLMVs of endocrine cells may havefunctional similarity with neuronal SVs.Note Added in Proof. After the submission of this manuscript forreview, regulated GABA secretion from the amphicrine pancreaticcell line AR42J was reported (45).

Solimena for insightful discussions and Dr. P. Maycox for criticallyreading the manuscript. This work was supported by the NationalInstitutes of Health, the Klingenstein Foundation and the McKnightEndowment for the Neurosciences (P.D.C.), the Deutsche For-schungsgemeinschaft (R.J.), and a National Institute of MentalHealth predoctoral fellowship (A.T.-R.). P.D.C. and R.J. were therecipients of a Max-Planck Research Prize.

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43.44.45.

We are greatly indebted to Drs. D. Hanahan and S. Efrat for thegift of cell lines and to Drs. F. Mohler, R. De Blas, G. Kreibich, andK. Buckley for generous gifts of antibodies. We thank Dr. M.

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