DIABETES-INSULIN-GLUCAGON-GASTROINTESTINAL
Characteristics and Functions of ␣-Amino-3-Hydroxy5-Methyl-4-Isoxazolepropionate Receptors Expressed
in Mouse Pancreatic ␣-Cells
Jung-Hwa Cho, Liangyi Chen, Mean-Hwan Kim, Robert H. Chow, Bertil Hille,
and Duk-Su Koh
Departments of Life Sciences (J.-H.C.) and Physics (M.-H.K., D.-S.K.), Pohang University of Science and
Technology, Pohang 790-784, Republic of Korea; Institute of Biophysics (L.C.), Academy of Science,
Beijing 100864, China; Department of Physiology and Biophysics (J.-H.C., R.H.C.), Keck School of
Medicine, University of Southern California, Los Angeles, California 90089; and Department of
Physiology and Biophysics (B.H., D.-S.K.), University of Washington, Seattle, Washington 98195-7290
Pancreatic islet cells use neurotransmitters such as L-glutamate to regulate hormone secretion. We
determined which cell types in mouse pancreatic islets express ionotropic glutamate receptor
channels (iGluRs) and describe the detailed biophysical properties and physiological roles of these
receptors. Currents through iGluRs and the resulting membrane depolarization were measured
with patch-clamp methods. Ca2⫹ influx through voltage-gated Ca2⫹ channels and Ca2⫹-evoked
exocytosis were detected by Ca2⫹ imaging and carbon-fiber microamperometry. Whereas iGluR2
glutamate receptor immunoreactivity was detected using specific antibodies in immunocytochemically identified mouse ␣- and ␤-cells, functional iGluRs were detected only in the ␣-cells. Fast
application of L-glutamate to cells elicited rapidly activating and desensitizing inward currents at
⫺60 mV. By functional criteria, the currents were identified as ␣-amino-3-hydroxy-5-methyl-4isoxazolepropionate (AMPA) receptors. They were activated and desensitized by AMPA, and were
activated only weakly by kainate. The desensitization by AMPA was inhibited by cyclothiazide, and
the currents were blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Islet iGluRs showed
nonselective cation permeability with a low Ca2⫹ permeability (PCa/PNa ⫽ 0.16). Activation of the
AMPA receptors induced a sequence of cellular actions in ␣-cells: 1) depolarization of the membrane by 27 ⫾ 3 mV, 2) rise in intracellular Ca2⫹ mainly mediated by voltage-gated Ca2⫹ channels
activated during the membrane depolarization, and 3) increase of exocytosis by the Ca2⫹ rise. In
conclusion, iGluRs expressed in mouse ␣-cells resemble the low Ca2⫹-permeable AMPA receptor in
brain and can stimulate exocytosis. (Endocrinology 151: 1541–1550, 2010)
lutamate, the predominant excitatory neurotransmitter in the mammalian central nervous system (CNS),
mediates fast synaptic transmission by acting on ionotropic
glutamate receptors (iGluRs) (1, 2). The endocrine pancreas
is one of the very few places outside the CNS where glutamate-mediated signaling is implicated (3–5). In the pancreatic islet of Langerhans, L-glutamate together with inhibitory
␥-aminobutyric acid (GABA) are proposed as intercellular
paracrine signals that regulate the hormone secretion involved in glucose homeostasis (6).
G
Pancreatic islets have all components required for glutamatergic transmission: glutamate sources, receptors,
and clearance mechanisms. The glucagon-secreting ␣-cells
express vesicular glutamate transporter subtypes 1 and 2,
accumulate L-glutamate into large dense-core granules
containing glucagon, and secrete both of them in parallel
in low-glucose conditions (7). The secreted extracellular
L-glutamate is sequestered by high-affinity glutamate/aspartate transporters in non-␤-cells (8, 9). Nevertheless, the
expression of iGluRs in islets seems complicated and even
ISSN Print 0013-7227 ISSN Online 1945-7170
Printed in U.S.A.
Copyright © 2010 by The Endocrine Society
doi: 10.1210/en.2009-0362 Received March 24, 2009. Accepted January 13, 2010.
First Published Online February 26, 2010
Abbreviations: AMPA, ␣-Amino-3-hydroxy-5-methyl-4-isoxazolepropionate; Cm, membrane capacitance; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; CNS, central nervous
system; iGluR, ionotropic glutamate receptor; I-V, current-voltage; NMDG, N-methyl-Dglucamine; VMAT, vesicular monoamine transporter.
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controversial (4). ␣-Amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-type iGluRs have been reported
both in dissociated glucagon-secreting ␣-cells and in insulin-secreting ␤-cells (10 –14), and their stimulation enhanced the secretion of glucagon and insulin in intact rat
islets and perfused pancreas (12, 13, 15–17). Somatostatin-secreting ␦-cells of rat expressed a newly identified
splice variant of AMPA receptors that promotes somatostatin release (18). Expression of kainate-type iGluRs
was reported in dissociated rat ␣-cells, ␦- cells, and islets
(10, 12, 13), but their functional roles have not been examined. N-methyl-D-aspartic acid (NMDA)-type iGluR
immunoreactivity was also detected in ␤-cells and rat islets
(13, 14), and NMDA receptor activation elicited insulin
secretion (12). In contrast, L-glutamate inhibited glucagon
secretion from rat islets via metabotropic GluR subtypes
such as metabotropic GluR2, 4, and 8 that couple to Gi/Go
G proteins (19, 20).
In sum, it has been reported that several subtypes of
iGluRs exist in pancreatic islet cells and that their activation promotes secretion of islet hormones. However,
many of these studies were carried out in unidentified and
mixed islet cells, intact islets, or whole pancreas, making
mechanistic interpretations difficult. Does L-glutamate act
directly on the cell type of interest or indirectly via paracrine signaling from other cells? Therefore, it is necessary
to test for functional iGluRs using single isolated cells.
Here, we identified cell types based on size, hormone-specific antibodies, single-cell RT-PCR, and investigated
iGluR expression. In addition, we investigated the electrophysiological and pharmacological properties and the
effects of iGluR activation on intracellular free Ca2⫹ concentration ([Ca2⫹]i) and on exocytosis of secretory vesicles
using single-cell techniques.
Materials and Methods
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7.3 with CsOH). For current-clamp experiments, K⫹-rich internal solution contained: 140 KCl, 4 NaCl, 1 CaCl2, 1 MgCl2, 5
EGTA, 2 MgATP, and 5 HEPES (pH 7.3 with KOH). For [Ca2⫹]i
measurements and amperometric experiments, the external solution contained: 140 NaCl, 5.6 KCl, 2.5 CaCl2, 1 MgCl2, 3.3
glucose, and 10 HEPES (pH 7.3 with NaOH). In Na⫹-free solutions, NaCl was replaced by equimolar N-methyl-D-glucamine (NMDG). Fura 2-AM (Molecular Probes, Eugene, OR),
R,S-AMPA, kainate, 6-cyano-7-nitroquinoxaline-2,3-dione
(CNQX), and cyclothiazide (Tocris, Ellisville, MO) were freshly
dissolved before experiments. Culture medium and serum were
from Invitrogen (Carlsbad, CA) and all other chemicals from
Sigma (St. Louis, MO).
Immunocytochemistry
Islet cells were fixed with 2% paraformaldehyde in a Na⫹rich solution, permeabilized with 0.2% Triton X-100, and
treated with a blocking solution (Pierce, Rockford, IL) to reduce
nonspecific binding of antibodies (Fig. 1). Cells were incubated
with primary antibodies at 4 C overnight. After washing, cells
were incubated with secondary antibodies at 22 C for 1 h. Antihormone antibodies used were as follows: antiinsulin (1:75
dilution; catalog no. 250-2788, Ventana, Tucson, AZ), antiglucagon (1:75 dilution; catalog no. A0565, DakoCytomation,
Carpinteria, CA), and antisomatostatin (1:100 dilution; catalog
no. sc-13099, Santa Cruz Biotechnology, Santa Cruz, CA); secondary antibodies for antiinsulin antibody [fluorescein isothiocyanate (FITC)-conjugated goat antimouse, 1:100 dilution; catalog no. 115-095-003, Jackson ImmunoResearch, West Grove,
PA; or Alexa 488, 1:1000 dilution; catalog no. A11008, Molecular Probes] and for antiglucagon and antisomatostatin antibody [tetramethylrhodamine B isothiocyanate (TRITC)-conjugated goat antirabbit, 1:100 dilution; catalog no. 111-025003, Jackson ImmunoResearch]. The coverslips were mounted
on slides using Gel/Mount (Biomeda, Foster city, CA). To determine which cell types express iGluR, islet cells were first tested
for kainate-induced [Ca2⫹]i response and then stained with hormone-specific antibodies as in Fig. 2. A coverslip with etched
alphanumeric coordinates (Bellco, Vineland, NJ) was used to
match the cells in the two experiments. Images were collected
using a Deltavision SA3.1 deconvolution microscope (Applied
Precision, Issaquah, WA) or a Leica SP1/MP confocal microscope (Leica Microsystems, Inc., Bannockburn, IL).
Cell preparation
The preparation of islets and single cells was previously reported (21). Animal care followed the University of Washington
Animal Medicine guidelines. Islets of Langerhans were isolated
from 5- to 10-wk-old male BALB/c mice euthanized by CO2.
Isolated cells plated on coverslips coated with poly-L-ornithine
were cultured in RPMI 1640 culture medium for 1–2 d before
use.
Solutions and chemicals
For whole-cell voltage-clamp experiments, Na⫹-rich external
solution contained (in mmol/liter): 135 NaCl, 2.5 KCl, 2.5
CaCl2, 1 MgCl2, 3.3 glucose, 10 TEA, and 10 HEPES (pH 7.3
with NaOH). Ca2⫹-rich external solution contained: 100 CaCl2,
1 MgCl2, 3.3 glucose, 10 TEA, and 5 HEPES [pH 7.3 with
Ca(OH)2]. Patch pipettes were filled with: 125 CsCl, 4 NaCl, 1
CaCl2, 1 MgCl2, 5 EGTA, 2 MgATP, 15 TEA, and 5 HEPES (pH
Single-cell RT-PCR
After membrane capacitance (Cm) measurement, each cell’s
cytoplasm was sucked into the recording patch pipette and
stored in RT-PCR reaction buffer included in a one-step RT-PCR
kit (QIAGEN, Valencia, CA) (Fig. 3). Intron-spanning specific
mouse glucagon primer pairs were as described by Vignali et al.
(forward 5⬘-GACTTCCCAGAAGAAGTCGCCAT-3⬘; reverse_
5⬘-CTACGGTTACCAGGTGGTCATGT-3⬘) (22). Two samples served as negative controls: 1) a minus-RT control where an
aliquot of without reverse transcriptase was used for RT reaction, and 2) a minus-template PCR, containing all of the reaction
components but a single cell. For the amplification of insulin
messengers, intron-spanning specific mouse primer pairs were
used (forward primer_5⬘-CAGCAAGCAGGTCATTGTTT-3⬘;
reverse_5⬘-CAGTAGTTCTCCAGCTGGTAGA-3⬘) (22).
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Ca2ⴙ measurement and amperometry
Cytoplasmic free Ca2⫹ concentration ([Ca2⫹]i) was monitored with Fura 2 Ca2⫹-sensitive dye. Cells were loaded with 2
␮mol/liter Fura 2-AM in a Na⫹-rich solution containing 3 or 5
mmol/liter glucose for 30 min at 37 C (25). The dye was excited
alternately at 340 and 380 nm, and the fluorescence signals were
recorded every 1 sec at 510 nm using a charge-coupled device
camera (Roper, Tucson, AZ). Most Ca2⫹ measurements were
done at approximately 22 C (with the exception of those in Supplemental Fig. 7). For measuring exocytosis, cells were preincubated in culture medium that was supplemented with 2 mmol/
liter serotonin for 4 –16 h (26). The carbon-fiber amperometric
electrode (27) was connected to an EPC-9 patch clamp amplifier
and held at 600 mV. Quantal serotonin release from individual
granules was detected as single spikes of oxidation current by the
electrode. All amperometric recordings (Fig. 6) were performed
at 33–36 C.
FIG. 1. Preferential expression of iGluRs in small islet cells. A, Isolated
single islet cells were immunostained with antiinsulin antibody. A low
magnification image is present to illustrate the contrast between an
insulin-negative (left) and an insulin-positive cell (right) in the same
visual field. Shown as an inverted grayscale image (fluorescence is
dark). The cell boundary of the insulin-negative cell is shown with
broken line. Scale bar, 5 ␮m. Diameter of cells was measured after
digital zooming. B, Histogram of cell diameters fitted by two Gaussian
distributions (smooth lines) (n ⫽ 202). C, Time course of ionic current
in two cells with different whole-cell capacitance in response to rapid
application of 1 mmol/liter L-glutamate at a holding potential of ⫺60
mV. D, Peak current amplitude activated by 1 mmol/liter L-glutamate
plotted against whole-cell capacitance (n ⫽ 76).
Electrophysiology
Gigaseal clamp measurements were performed in the wholecell configuration (23). After formation of the whole-cell configuration, negative pressure (⬃⫺200 mbar) was applied to the
pipette for approximately 2 min to attract the cell nucleus to the
glass tip. The access resistance increased but by less than 25 M⍀.
With the support of the nucleus, single islet cells could be detached from the glass bottom to permit faster application of
agonists. The optimal pipette size for lifting cells was 3–5 M⍀
when filled with the pipette solution. For single-channel recordings, excised outside-out patches were pulled out after the formation of the whole-cell configuration. For fast agonist application experiments, a double-barreled perfusion pipette was
fabricated from ␪-glass tubing and attached to a piezo-electric element (ceramic multilayer bender 5 from Noliac, Kvistgaard, Denmark). The solution exchange time (10 –90%) measured electrically with an open patch-pipette tip during a switch from
100% Na⫹ to 10% Na⫹ solution was about 0.5–1 msec. For
the experiments of Figs. 2, 4C, 5, and 6 and Supplemental Fig.
7 (see Supplemental Figs. 1–7 published on The Endocrine
Society’s Journals Online web site at http://endo.endojournals.org), solutions were applied through a multibarreled perfusion system that had a solution exchange time less than 0.5
sec (24). Membrane currents were recorded with an EPC-9
amplifier (HEKA Elektronik, Lambrecht/Pfalz, Germany), filtered at 1 kHz with an 8-pole, low-pass Bessel filter, and sampled at 5
kHz, unless noted. Cm before and after glutamate stimulation was
measured with the Lindau-Neher technique implemented as the “sine
⫹ dc” mode of the software lock-in extension of Pulse (HEKA Elektronik), in which a 1-kHz, 25-mV sinusoidal voltage stimulus (50 mV,
peak to peak) is superimposed onto a holding potential of ⫺80 mV.
Data analysis
Membrane current and voltage traces shown in the figures
are averages of two to five single traces. Desensitization time
constants of the AMPAR-mediated component were determined by fitting the decay phases with a single-exponential
function. Concentration-response curves were fitted with a
Hill function:
F(c) ⫽ 1/[1 ⫹ (EC50 /c)n]
(1)
where c, EC50, and n denote concentration of agonists, halfmaximal effective concentration, and Hill coefficient, respectively. Data points of current-voltage (I-V) relations were fitted
with a second-order polynomial, from which the reversal potentials were determined by interpolation. Rectification index was
calculated by dividing the conductance at ⫹40 mV by that at
⫺40 mV. Ca2⫹ permeability ratio was calculated from the modified Goldman-Hodgkin-Katz voltage equation (28):
PCa/PNa ⫽ 0.25aNa/aCa关exp兵共2VrevCa ⫺ VrevNa兲F/(RT)}
⫹ exp{(VrevCa ⫺ VrevNa兲F/(RT)}] (2)
where aNa and aCa represent the activities of Na⫹ and Ca2⫹
in the extracellular solutions, respectively. R, T, and F have their
conventional thermodynamic meaning. Reversal potentials measured under high Na⫹ or Ca2⫹ external solutions (VrevNa and
VrevCa) were corrected for liquid junction potentials of 4.5 and
10.3 mV, respectively, in Supplemental Fig. 5D. Data were analyzed in Igor Pro (WaveMetrics, Lake Oswego, OR). All averaged values were given as mean ⫾ SEM. Student’s t test was used
for statistical tests, and P values less than 0.05 were considered
statistically significant.
Results
␣-Cells respond to glutamate
Using dissociated islet cells, we determined the cell-size
dependence of insulin expression and the cell-size dependence of functional responses to iGluR agonists. Insulinsecreting ␤-cells tend to be larger than glucagon-secreting
␣-cells or somatostatin-secreting ␦-cells in purified rat islet
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FIG. 2. Expression of iGluRs in immunocytochemically identified
cells. A, An image of a single cell immunostained with antiinsulin
antibody (scale bar, 5 ␮m) and the time course of Ca2⫹ response to
0.5 mmol/liter kainate (KA) for 20 sec. The cell was kainate
unresponsive, relatively small, and insulin-positive. Insulin granules
are labeled as punctate structures outside the nucleus. B, An image
of another cell immunostained with antiglucagon antibody and its
Ca2⫹ response to kainate. The cell was kainate responsive, small,
and glucagon-positive. C, Summary bar graph showing the number
of cells responding to kainate stimulation (KA⫹, black) or not
responding (KA⫺, white). All the cells were sorted by their
immunocytochemical identification as positive (⫹) or negative (⫺)
for the indicated peptides. The numbers in parentheses indicated
the total number of tested single cells.
preparations (29). We measured the size distribution of
mouse insulin-positive and insulin-negative cells using antiinsulin antibody (Fig. 1A). These two categories of cells
fell into diameter classes that could be approximated by
two Gaussian distributions (Fig. 1B). Insulin-positive cells
had larger average diameters (10.7 ⫾ 0.2 ␮m, n ⫽ 134
cells) than insulin-negative cells (8.3 ⫾ 0.2 ␮m, n ⫽ 68).
The two distributions overlapped in the 7- to 10-␮m
range. Electrical responses of iGluRs were assessed by applying 1 mmol/liter L-glutamate rapidly to single patchclamped cells lifted from the glass bottom (Fig. 1C; also,
see Materials and Methods), and cell size was estimated as
membrane electrical capacitance, Cm. L-glutamate consistently evoked transient inward currents in small cells (53
tested cells with Cm ⱕ 4.0) but not in large cells (23 tested
cells with Cm ⱖ 5.5). Inward currents are shown as downward deflections by convention. For rough comparison
Endocrinology, April 2010, 151(4):1541–1550
FIG. 3. Expression of iGluRs in cells identified with single-cell RT-PCR.
A and B, Representative Cm traces in response to 100 ␮M cyclothiazide
(CTZ) and 1 mM L-glutamate from a single identified ␣ (A) and ␤ (B)
cell. After the Cm measurement, the cell type was identified using
single-cell RT-PCR with either glucagon (Gluca⫹)- or insulin (Ins⫹)specific primers (lane 6 in panel D and lane 2 in panel E, respectively).
C, Percent increase of Cm from ␣ (n ⫽ 4) and ␤-cells (n ⫽ 6). Data are
shown as mean ⫾ SEM. **, P ⬍ 0.01. D, Single-cell RT-PCR for
glucagon expression. Each cell’s cytoplasm was harvested after
capacitance measurement using glucagon primer pairs. For negative
controls, reverse transcriptase (⫺RT) or cell content (⫺Cell) was omitted.
Glucagon PCR products were found in only four cells (1, 3, 6, 7). A second
image of lane 3 is shown at the end with enhanced contrast. Lane 8 was
a cell whose cytoplasm was collected for single-cell RT-PCR without Cm
measurement. E, Single-cell RT-PCR for insulin expression. Six cells
(lane 1– 6) showed the insulin PCR products after Cm measurement.
Lanes 7 and 8 are from two single cells whose cytoplasm was
collected for single-cell RT-PCR without Cm measurement.
with the diameter measurements, a cell with 4 pF of Cm
would have a plasma membrane surface area of 400 ␮m2,
and if this were a smooth perfect sphere, the diameter
would be 11.3 ␮m, but a real, somewhat convoluted cell
would be smaller. Figure 1D plots the peak current amplitudes evoked by L-glutamate in different cells showing
peak currents ranging from approximately 10 pA to approximately 150 pA. Thus, the glutamate-responsive cells
are small, whereas the immunocytochemistry in separate
experiments shows that insulin-containing islet cells are
large.
Because of some overlap of size distributions, it seemed
worthwhile to test for iGluR function and hormone content in the same individual cells. In this case, function was
tested using calcium imaging in a field of islet cells. The
cells were challenged with 0.5 mM kainate to measure
[Ca2⫹]i responses, and then stained with an antihormone
antibody (Fig. 2A, see also Fig. 4C for [Ca2⫹]i rise mediated by kainate-activated iGluR). No insulin-positive
␤-cells (n ⫽ 0/18) responded to kainate (Fig. 2C). As expected, some cells with diameters of 7–10 ␮m were identified as ␤-cells (Fig. 1B), and they were not responsive to
kainate (Fig. 2A). Thus, in this middle diameter range, the
identification of cell types solely based on cell size would
not be reliable. Most of the insulin-negative, non-␤-cells
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FIG. 4. Pharmacological properties of AMPA receptors. A, Time course
of currents evoked by 1 mmol/liter L-glutamate in the absence or
presence of 100 ␮mol/liter cyclothiazide (CTZ) or 10 ␮mol/liter CNQX
in the same cell. Membrane potential was held at ⫺60 mV. B, Change
of membrane potential induced by a 200-msec pulse of 1 mmol/liter
L-glutamate using current-clamp recording mode. The peak
depolarization was 42 mV in this experiment (upper trace). Current
response was measured in the voltage-clamp recording mode at ⫺60
mV from the same cell (lower trace). C, The [Ca2⫹]i response evoked
by 0.5 mmol/liter L-glutamate was potentiated by inhibition of AMPAR
desensitization using 100 ␮mol/liter cyclothiazide (n ⫽ 9). Block of
AMPAR by 10 ␮mol/liter CNQX abolished [Ca2⫹]i response to 0.5
mmol/liter kainate (n ⫽ 3).
responded to kainate (26/30 cells). Among the islet cells
that stained with antiglucagon antibody after the [Ca2⫹]i
measurement, most showed a positive response to kainate
(35/39 glucagon-positive cells). The exact identity of two
glucagon-negative cells out of 13 cells is not known. They
could represent a small percentage of non-␣-cells expressing iGluRs or ␣-cells that stained weakly with the antiglucagon antibody. Finally, islet cells were stained with
antisomatostatin antibody. When [Ca2⫹]i measurements
and immunocytochemistry were done in 87 cells in parallel, only four cells were positive to antisomatostatin antibody. None of these four cells responded to kainate. Of
the 83 somatostatin-negative cells, 29 were responsive to
kainate. We note that the proportions of each cell type in
dissociated single-cell cultures was quite variable between
batches. We suggest that this is an artifact of detachment
of some larger ␤-cells from the glass chips during the transfers from the culture dish to the measuring chamber and
during processing for immunohistochemistry.
In addition to size distribution and immunocytochemistry, we also identified hormone expression of single cells
by single-cell RT-PCR. We paired this assay with tests for
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FIG. 5. Effect of extracellular Na⫹ removal and of Ca2⫹ channel
blockers on kainate-induced [Ca2⫹]i response. A, [Ca2⫹]i responses to
0.5 mmol/liter kainate (KA) for 10 sec in the presence and absence of
Na⫹ (n ⫽ 5). The 135 mM extracellular Na⫹ was replaced by equimolar
NMDG⫹ to prevent depolarization by Na⫹ influx through iGluRs. The
[Ca2⫹]i trace in this figure is the average for all measured cells, and has
error bars. B–E, [Ca2⫹]i with several Ca2⫹ channel blockers. Kainateevoked [Ca2⫹]i increase was measured in the presence of 200 ␮M
CdCl2 as a nonselective blocker of Ca2⫹ channels (n ⫽ 9), 10 ␮M
nimodipine (“Nimo”) for blocking L-type Ca2⫹ channels (n ⫽ 12), 2
␮mol/liter ␻-conotoxin GIVA (“␻-CTX”) for N-type Ca2⫹ channels
(n ⫽ 18), and 100 nmol/liter SNX482 for R-type Ca2⫹ channels (n ⫽ 11).
F, Percent inhibition of kainate-induced [Ca2⫹]i peaks by each condition.
Data are shown as mean ⫾ SEM.
functional iGluRs in the same individual cell. In this experiment, functional activity was gauged by measurements of Cm changes. The reasoning was as follows. Islet
iGluRs have a small but measurable Ca2⫹ permeability
(see below), and activated receptors would then raise Ca2⫹
and induce exocytosis. If we observed an increase of Cm
after agonist addition that would indicate a plasma membrane surface area increase from addition of secretory
vesicles (exocytosis) in response to Ca2⫹ influx through
activated receptors. For this test, we maximized this Ca2⫹
influx by eliminating iGluR desensitization with cyclothiazide and by holding the membrane at a very negative potential ⫺80 mV. Indeed, there was an increase of Cm
in some cells (Fig. 3A), mainly small ones, and not in other
cells (Fig. 3B). Next, the cytoplasm of each cell was harvested into the patch pipette for hormone identification by
single-cell RT-PCR with either glucagon- or insulin-specific primers. Glucagon PCR products were detected in
four tested cells (lanes 1, 3, 6, and 7 in Fig. 3D), and insulin
PCR products were detected in six other tested cells (lanes
1– 6 in Fig. 3E). The capacitance change of the four glucagon-positive cells was significantly larger than that of
six insulin-positive cells (Fig. 3C).
We also looked for iGluR2 immunoreactivity in immunohistochemically identified mouse islet cells (Supplemen-
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FIG. 6. Increase of exocytosis by AMPA-receptor activation in single
islet cells. A, Raw time course of amperometric current obtained in
kainate (1 mmol/liter, 1 min) or glucose (16.7 mmol/liter, 5 min). Inset,
One exocytotic event, marked with an asterisk, is displayed on an
expanded time scale to show typical quantal exocytotic release of
oxidizable molecules. B and C, Averaged rate of exocytosis for small
islet cells (B, n ⫽ 17) and large islet cells (C, n ⫽ 7). Data are expressed
as mean ⫾ SEM. For the transient kainate response, significance levels
are indicated as *, P ⬍ 0.01 and **, P ⬍ 0.0005. These experiments
were performed at 35 C, because exocytosis from ␤-cells is reduced at
lower temperatures (50).
tal Fig. 1). Unexpectedly, but similar to a previous study
(10) in rat islets, both the mouse ␣- and ␤-cells, but not the
␦-cells, showed immunoreactivity for iGluR2. Thus,
whereas the results in Figs. 1 and 2 reveal functional
iGluRs only in ␣-cells, immunostaining suggests that there
is nonfunctional iGluR2 protein in ␤-cells as well.
␣-Cells express AMPA receptors
We analyzed the basic functional characteristics of islet
iGluRs and found that they corresponded to the AMPA
subtype as shown in the supplemental figures. For these,
studies L-glutamate, AMPA, and kainate were applied rap-
Endocrinology, April 2010, 151(4):1541–1550
idly to islet cells (Supplemental Fig. 2A). Inward currents
activated by 100-msec pulses of 1 and 10 mmol/liter Lglutamate or AMPA rose to a peak within 2 msec and
desensitized within 10 msec. The average exponential time
constant for desensitization was 5.1 ⫾ 0.1 msec (10 mmol/
liter L-glutamate; n ⫽ 10). To test whether the agonist
application might be retarded by the whole cell in the front
of a patch pipette, we also measured kinetics using outsideout patches (Supplemental Fig. 3). The desensitization
time constant obtained from ensemble averages of these
single-channel recordings was 6.1 ⫾ 0.3 msec (n ⫽ 6). The
excised-patch experiments indicated a single-channel current of 0.68 ⫾ 0.03 pA (n ⫽ 12 from three different
patches) at ⫺60 mV and a chord conductance of 11.3 pS.
As is characteristic of AMPA receptors, the currents
with kainate were smaller and showed primarily a nondesensitizing component. The concentration dependence
of the evoked currents was estimated for each agonist
(Supplemental Fig. 2, B–D). The half-maximal activating
concentrations (EC50) for activation of peak current were
similar: 720 ␮mol/liter for AMPA, 841 ␮mol/liter for Lglutamate, and 882 ␮mol/liter for kainate. Steady-state
current was evoked with an EC50 value of 530 ␮mol/liter
kainate.
The I-V relation for L-glutamate-activated peak currents in Na⫹-rich external solution showed a reversal potential at 1.2 ⫾ 0.7 mV (n ⫽ 5), indicating an approximately equal permeability for the extracellular Na⫹ and
intracellular Cs⫹ ions (Supplemental Fig. 4). Similarly, the
reversal potentials measured with AMPA and kainate
were 0.1 ⫾ 2.1 mV (n ⫽ 7) and ⫺1.9 ⫾ 1.0 mV (n ⫽ 9),
respectively. The I-V relation was linear when iGluRs
were activated with glutamate or AMPA but strongly outwardly rectifying when activated by kainate. When external monovalent cations were entirely replaced by Ca2⫹,
the reversal potential for glutamate-activated peak current
shifted to very negative values, ⫺60.1 ⫾ 1.1 mV (n ⫽ 10,
Supplemental Fig. 5C) (corrected for liquid junction potentials). From Eq. 2, the relative Ca2⫹ permeability of
iGluRs was therefore low (PCa/PNa ⫽ 0.16 ⫾ 0.01; Supplemental Fig. 4D, n ⫽ 10) compared with highly Ca2⫹
permeable iGluRs (30).
To address again whether AMPA- or kainate-type
iGluRs contribute to the current activated by L-glutamate,
we investigated the effect of cyclothiazide, a blocker of
desensitization with high specificity for AMPA-type over
kainate-type iGluRs (30, 31). Cyclothiazide reversibly
blocked the desensitization of iGluR currents in pancreatic
islet cells (Fig. 4A) and doubled the peak current (186 ⫾
10% compared with control, n ⫽ 7). Cyclothiazide also
potentiated the increase of intracellular free Ca2⫹ concentration ([Ca2⫹]i) evoked by 1 mmol/liter L-glutamate (Fig.
Endocrinology, April 2010, 151(4):1541–1550
4C). The [Ca2⫹]i increase evoked by 1 mmol/liter L-glutamate together with 100 ␮M cyclothiazide was 720 ⫾ 186
nmol/liter (n ⫽ 9), whereas the change with L-glutamate
alone was only 94 ⫾ 18 nmol/liter. Currents evoked by 1
mmol/liter L-glutamate were completely and reversibly
blocked by 10 ␮M CNQX, an antagonist of AMPA and
kainate-type iGluRs (n ⫽ 3, Fig. 4A). Similarly, CNQX
also reduced [Ca2⫹]i responses evoked by 0.5 mmol/liter
kainate by up to 96% (n ⫽ 16, Fig. 4C). We conclude that
the kainate responses we see in islet cells are due to its
well-known nondesensitizing activation of AMPA receptors rather than to activation of true kainate receptors.
Activation of AMPAR depolarizes the membrane
and activates voltage-gated Ca2ⴙ channels
In neurons, iGluR-mediated inward currents depolarize the cell membrane to produce excitatory postsynaptic
potentials. The same occurred in islet cells. Recording in
current-clamp mode (Fig. 4B), islet cells had a resting
membrane potential of ⫺67 ⫾ 5 mV (n ⫽ 8) and depolarized transiently by 27 ⫾ 3 mV (n ⫽ 8) upon application
of 1 mmol/liter L-glutamate. The membrane potential decayed to the resting level with a time constant of 68 ⫾ 7
msec (n ⫽ 8, much longer than the decay time constant of
iGluR currents (5.7 ⫾ 0.1 msec, n ⫽ 9).
Such membrane depolarizations should also activate voltage-gated Ca2⫹ channels as in neurons. Indeed, both kainate
and glutamate increased [Ca2⫹]i levels (Fig. 4C). As expected, the response to the nondesensitizing agonist kainate
was larger than that with desensitizing L-glutamate and
AMPA. Kainate triggered detectable [Ca2⫹]i rises in 84%
(366 of 435 cells), whereas L-glutamate and AMPA triggered
[Ca2⫹]i rises in 34% (71 of 209 cells) and 56% (126 of 226
cells) of cells, respectively. The experiments presented in Fig.
5 further show that these Ca2⫹ elevations are primarily due
to opening of voltage-gated Ca2⫹ channels by the prolonged
depolarization. When the depolarization was prevented by
replacing extracellular Na⫹ with NMDG (Fig. 5A), which is
not measurably permeant in AMPA receptors (32, 33), no
[Ca2⫹]i increases were evoked by kainate (0.5 mmol/liter,
n ⫽ 5). When all subtypes of voltage-gated Ca2⫹ channels
were blocked with nonselective CdCl2, the [Ca2⫹]i rises were
reduced by 84% (n ⫽ 9, Fig. 5B). The residual small Ca2⫹
signal appears to be mediated by Ca2⫹ influx through the
iGluRs themselves. The [Ca2⫹]i rise was partially reduced by
more selective blockers of L-, N-, and R-subtype Ca2⫹ channels, suggesting involvement of several voltage-activated
Ca2⫹ channel types (Fig. 5, C–F).
Activation of AMPA receptors triggers exocytosis
in ␣-cells
Pancreatic islet cells secrete endocrine hormones and
neurotransmitters when [Ca2⫹]i increases (5, 6). There-
endo.endojournals.org
1547
fore, we tested whether the [Ca2⫹]i rises evoked by AMPA
receptor activation would be sufficient to trigger exocytosis. As noted in Fig. 1, the size distributions of ␤- and
non-␤-cells overlap, especially in the diameter range between 7 and 10 ␮m (Fig. 1). Therefore, we used cells
smaller than 7 ␮m for non-␤-cells and cells larger than 10
␮m for ␤-cells. Based on these criteria, the probability of
correct cell identification was more than 90% from the
size distribution shown in Fig. 1B. To measure exocytosis
with carbon-fiber amperometry, we loaded islet cells
with exogenous oxidizable serotonin (see Ref. 26 and
references therein). To confirm the loading of serotonin
into islet cells, the cytoplasmic accumulation was assessed indirectly using its fluorescent analog 5,7-dihydroxytryptamine (Supplemental Fig. 6A). All islet cells
including cells smaller than 7 ␮m gradually became fluorescent. In addition, immunostaining with vesicular
monoamine transporter (VMAT) antibodies revealed that
both ␣- and ␤-cells expressed VMAT2 (Supplemental Fig.
6B) but not VMAT1 (data not shown). After the serotonin
loading, fusion of individual vesicles was detected as
spikes of oxidation current (Fig. 6A) (see Ref. 26). Upon
application of kainate, the average rate of exocytosis, defined as the number of spikes per 30-sec time-bin, transiently increased within 30 sec in small islet cells (diameter,
⬍7 ␮m, n ⫽ 17; Fig. 6B). These small cells did not respond
to 16.7 mmol/liter glucose. As expected, a group of large
cells (⬎11 ␮m), which should lack functional iGluRs (Fig.
1), secreted in response to 16.7 mmol/liter glucose but not
to kainate (n ⫽ 7, Fig. 6C). Thus, we conclude that in
␣-cells, the activation of AMPA receptors can elicit
exocytosis.
Discussion
Cell types expressing AMPAR in mouse islets
It is now well accepted that some pancreatic islet cells
express functional glutamate receptors and that their activation modulates hormone secretion (4). Many studies
have investigated the function of iGluRs by measuring
secretion of islet hormones in intact islets or in perfused
pancreas (6, 15, 16). Such data should be interpreted carefully, however, due to possible paracrine or autocrine interactions between the cells. For example, insulin secretion
might be stimulated by glucagon (34) released by the activation of iGluRs in ␣-cells even if ␤-cells themselves
lacked functional iGluRs. Therefore, Gromada et al. (5)
suggested that tests of glutamate actions might have to be
done in isolated single cells to avoid potentially confounding paracrine interactions. Some groups have reported Lglutamate-induced currents or membrane depolarization
in single ␤-cells. The cells were identified either by [Ca2⫹]i
1548
Cho et al.
AMPA Receptors in Pancreatic ␣-Cells
rises in high glucose or by their relatively large size and the
predominance of ␤-cells in islet-cell preparations. However, even though islet cells fall into two size classes, the
intermediate range of cell sizes, where ␤- and non-␤-cells
overlap, may have obscured cell identification (Fig. 1B).
Our initial single-cell screens based on cell size suggested that functional iGluRs are found only in non-␤-cells
(Fig. 1). A similar conclusion was drawn in experiments in
which we distinguished cell types based on intrinsic Ca2⫹
responses to glucose (Supplemental Fig. 7). Other laboratories have reported with cultured ␤-cells that [Ca2⫹]i oscillates in high glucose but not in low glucose (e.g. a cell
shown in Supplemental Fig. 7A) (35). None of such cells
responded to kainate (23 cells out of 79 tested cells).
␣-Cells are known to be unresponsive to high concentrations of glucose (e.g. a cell shown in Supplemental Fig. 7B)
(36, 37). However, we observed that some of the dispersed
single ␣-cells, under culture conditions, strangely responded to high glucose level as well (e.g. a cell shown in
Supplemental Fig. 7C) (38, 39). Those presumed ␣-cells
showed Ca2⫹ increases upon kainate application (36 cells
for the cell groups as Supplemental Fig. 7B and 20 cells for
the cell groups as Supplemental Fig. 7C). In spite of some
uncertainty, the identification of islet cell types using the
glucose response supports our hypothesis that non-␤-cells
but not ␤-cells express iGluRs. More definitive conclusions for the cell type expressing iGluRs came from our
parallel [Ca2⫹]i measurement and hormone immunocytochemistry in single cells, indicating that ␣-cells, but not
␤- or ␦-cells, express functional iGluRs in mouse islets (Fig.
2), and from measurement of the change of Cm in islet cells
identified using single-cell RT-PCR (Fig. 3). This conclusion is in line with a recent study that was published during
the preparation of this paper (16).
Our study and previous ones suggest that AMPA receptor immunoreactivity is present in ␤ cells as well (10).
We do not fully understand why iGluRs in ␤-cells show no
function. Possibly iGluRs recycle between the pools in the
plasma membrane and cytoplasm as at neuronal synapses
(40), and most iGluRs of ␤-cells are in the cytoplasmic
pool. This assertion is supported by the presence of cytoplasmic iGluR2 in our immunostaining (Supplemental
Fig. 1). It would be interesting to address whether iGluRs
in ␤-cells acquire functionality in certain circumstances
such as elevated glucose concentration.
The subtype of iGluRs expressed in ␣-cells
We found that the iGluRs of pancreatic islets are AMPA
receptors rather than kainate receptors on the basis of
three criteria: their preferential activation and desensitization by AMPA, weak- and nondesensitizing activation
by kainate, and the block of desensitization by cyclothia-
Endocrinology, April 2010, 151(4):1541–1550
zide. The mRNA transcript for iGluR2 subunits in adult
brains is frequently edited so that a glutamine codon initially present in the RNA is altered to an arginine codon
(41). In neurons, channels with higher iGluR2 content in
the edited form are characterized by a linear I-V relation
and a low Ca2⫹ permeability, whereas channels lacking
iGluR2 subunits show a doubly rectifying I-V relation and
a high Ca2⫹ permeability (30, 42). Therefore, islet AMPA
receptors probably include edited iGluR2 subunits. Previous
immunohistochemical studies concluded that AMPA receptors are composed mainly of iGluR2/3 in ␣- and ␤-cells but
not ␦-cells of rat intact islets (10). Our data using mouse
islets are in line with those findings (Supplemental Fig. 1).
The AMPA receptors expressed in islet cells desensitize
rapidly and completely with L-glutamate. Such 5-msec desensitization is a functional signature of AMPA receptors
in GABAergic interneurons (43, 44) and distinct from the
receptors on neocortical glutamatergic neurons, which
have slower kinetics (␶ ⫽ 10 –16 msec) (33). In addition to
the subunit composition, flip-flop splice variants are another determinant of receptor desensitization (45, 46). Of
them, the “flop” variants are relatively faster in desensitization (␶ ⫽ 5– 6 msec) with less than 1% nondesensitizing current, similar to what we saw for islet AMPA receptors (45).
Functions of AMPA receptors expressed in mouse
islets
In CNS neurons, excitatory neurotransmitters are released from presynaptic terminals, diffuse 10 –20 nm
across the synaptic cleft, and activate postsynaptic receptors to evoke excitatory postsynaptic potentials (47). The
time course of L-glutamate concentration in the synaptic
cleft is determined by receptor binding and by clearance by
glutamate transporters. In pancreatic islets, the release of
L-glutamate and localization of iGluRs would be less well
organized than in the synapse, so activation of the receptors might be less effective. Nevertheless, morphological
studies reveal tight junctions and gap junctions between
islet cells with narrow intercellular spaces of only 15–20
nm (48). Future studies on L-glutamate release and diffusion in the extracellular space will be critical to determine
how rapidly desensitizing iGluRs elicit cellular reactions
as we demonstrated in this study. In our mouse islet cells,
the ultrafast application of L-glutamate rapidly depolarized the membrane of small cells by about 30 mV. This
membrane depolarization lasted 12 times longer than the
decay time constant of iGluR-mediated current measured
under voltage-clamp mode and lasted much longer than
the expected passive decay of small electrical signals for
such cells. For example, our responsive cells had a Cm of
3– 4 pF and an input resistance (Rm) of 1 G⍀ at ⫺70 mV,
Endocrinology, April 2010, 151(4):1541–1550
giving an expected passive electrical decay time constant
(RmCm) of only 3– 4 msec. Therefore, the depolarization
must be secondarily prolonged by the induced activation of other depolarizing channels such as voltagegated Ca2⫹ channels (Fig. 5). The final event of iGluR
activation in ␣-cells is glucagon secretion as determined
by our single cell methods and by glucagon released
from islets (16, 17).
The ␣-cells are regarded as the site of L-glutamate production in the pancreas because glutaminase activity is
confined to the mantle of the islets (49). Thus glucagon
secretion would be potentiated by a glutamatergic autocrine positive feedback after low glucose stimulation in
␣-cells. Because glucagon is known to activate ␤-cells via
glucagon receptors (34), the glutamatergic signaling can
further regulate insulin secretion in a limited time window.
Therefore, our results define the iGluRs that fine-tune
blood glucose levels and expand our understanding of Lglutamate as an intercellular signal in peripheral systems.
endo.endojournals.org
6.
7.
8.
9.
10.
11.
12.
Acknowledgments
We thank Joseph G. Duman and Lindsey Burnett for helpful
advice about immunocytochemistry and Arnold Sipos for confocal microscopy.
Address all correspondence and requests for reprints to:
Duk-Su Koh, Department of Physiology and Biophysics, University of Washington School of Medicine, G-424 Health Science
Building, Box 357290, Seattle, Washington 98195-7290. E-mail:
[email protected].
Present address for J.-H.C. and R.H.C.: Department of Physiology and Biophysics, Keck School of Medicine, Zilkha Neurogenetic Institute, University of Southern California, Los Angeles, California 90089.
This work was supported by the Korea Science and Engineering Foundation Grant R01-2002-000-00285-0 (to D.-S.K.), National Institutes of Health Grants GM83913 (to B.H.) and
DK60623 and GM85791 (to R.H.C.), and by the Diabetes Endocrinology Research Center of the University of Washington.
Disclosure Summary: The authors have nothing to disclose.
13.
14.
15.
16.
17.
18.
19.
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