Electrophysiological effects of GABA on cat pancreatic
neurons
L.
Sha,
S. M.
Miller, and
J. H.
Szurszewski
Department of Physiology and Biophysics, Mayo Clinic and Mayo
Foundation, Rochester, Minnesota 55905
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ABSTRACT |
In mammalian peripheral sympathetic ganglia GABA acts
presynaptically to facilitate cholinergic transmission and
postsynaptically to depolarize membrane potential. The GABA effect on
parasympathetic pancreatic ganglia is unknown. We aimed to determine
the effect of locally applied GABA on cat pancreatic ganglion neurons.
Ganglia with attached nerve trunks were isolated from cat pancreata.
Conventional intracellular recording techniques were used to record
electrical responses from ganglion neurons. GABA pressure microejection
depolarized membrane potential with an amplitude of 17.4 ± 0.7 mV. Electrically evoked fast excitatory postsynaptic potentials were
significantly inhibited (5.4 ± 0.3 to 2.9 ± 0.2 mV) after
GABA application. GABA-evoked depolarizations were mimicked by the
GABAA receptor agonist muscimol and abolished by the
GABAA receptor antagonist bicuculline and the
Cl
channel blocker picrotoxin. GABA was taken up and
stored in ganglia during preincubation with 1 mM GABA;
-aminobutyric
acid application after GABA loading significantly (P < 0.05) increased depolarizing response to GABA (15.6 ± 1.0 vs.
7.8 ± 0.8 mV without GABA preincubation). Immunolabeling with
antibodies to GABA, glial cell fibrillary acidic protein, protein gene
product 9.5, and glutamic acid decarboxylase (GAD) immunoreactivity
showed that GABA was present in glial cells, but not in neurons, and
that glial cells did not contain GAD, whereas islet cells did. The data
suggest that endogenous GABA released from ganglionic glial cells acts
on pancreatic ganglion neurons through GABAA receptors.
-Aminobutyric acid A receptors; glial cells;
-aminobutyric
acid release; electrophysiology; immunohistochemistry
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INTRODUCTION |
GABA WAS DISCOVERED
OVER 40 years ago as a key inhibitory neurotransmitter in the
brain (5, 25). Since then, evidence has accumulated that
this amino acid may function as a neurotransmitter not only in the
central nervous system but also in the peripheral nervous system,
including the myenteric plexus (3, 17, 42), major pelvic
ganglia (2), and sympathetic ganglia, encompassing the rat
superior cervical ganglion (7, 20, 45) and abdominal prevertebral ganglia (18, 33, 41).
The mammalian pancreas, like the gut wall, has an intrinsic nervous
system consisting of ganglia, interconnecting intrinsic nerve fibers,
and extrinsic parasympathetic and sympathetic nerves (6, 22, 23,
27-30, 38). Histological studies suggest that pancreatic
ganglia resemble enteric ganglia anatomically and are considered to be
a subclass of parasympathetic ganglia (40). Pancreatic
ganglion neurons receive input from cholinergic (4), adrenergic (26), serotonergic (23),
peptidergic (12, 26), and nitrergic nerves (24,
37).
GABA is present in pancreatic islets in concentrations comparable to
that found in the brain (10, 19, 31, 32), and GABA is
cosecreted with insulin from
-cells (11, 34). Although there is as yet no evidence for the existence of GABA-containing nerves
in the pancreas (15, 43), there is nevertheless evidence that GABA and GABAA receptors modulate endocrine function
(14, 21, 35, 43). There is no information on whether GABA
receptors are present on pancreatic ganglion neurons or on the effect
of exogenous GABA on pancreatic ganglion neuron excitability. Thus the
present study was designed to determine in vitro the effect of GABA and
GABA receptor-modulating drugs locally applied to pancreatic ganglion neurons.
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MATERIALS AND METHODS |
Adult cats of either sex were anesthetized with an intramuscular
injection of ketamine hydrochloride (100 mg; Bristol Laboratories) and
then killed by an intraperitoneal injection of pentobarbital sodium (325 mg; Fort Dodge Laboratories). The use of cats and the
method of euthanasia used in these studies were approved by the Mayo
Institutional Animal Care and Use Committee. The cats used in this
study also provided tissues for experiments conducted by other
investigators. Through a midline abdominal incision, the pancreas was
rapidly removed and a section of pancreas (1.5 × 1.8 cm) from the
head or body region was removed and pinned to the Sylgard-coated
floor of a recording chamber. The chamber was superfused with normal
Krebs solution equilibrated with 97% O2-3%
CO2 at 34-36°C. The composition of the solution was
(in mM) 137.4 Na+, 5.9 K+, 2.5 Ca2+, 1.2 Mg2+, 124 Cl
, 15.5 HCO
, 1.2 H2PO
, and 11.5 glucose.
Pancreatic ganglia and their attached nerve trunks were identified in
the interlobular connective tissue using a microscope (×6 to ×30
magnification) and then removed from the surrounding pancreatic
parenchyma. Nerve trunks were designated as being either central or
peripheral in origin, as described previously (22).
Glass microelectrodes filled with 3 M KCl or 3 M KAc (resistance:
50-80 M
) were used to record intracellularly from ganglion neurons. The membrane potentials and intracellular current injections were displayed on an oscilloscope (Tektronix 513), and permanent records were made on a chart recorder (Gould Brush 220) and an FM tape
recorder (Hewlett Packard 3968A). Satisfactory impalements resulted in
a stable rest membrane potential equal to or more negative than
40 mV.
Nerve trunks were stimulated using bipolar platinum-wire electrodes
(0.5 mm between the 2 poles) connected to a stimulator (Grass S88) and
stimulus isolation unit (Grass SIU5). Stimuli of 5-40 V with pulse
durations of 0.5 ms were used to evoke fast excitatory synaptic
potentials. Stimuli of 100 V with pulse duration of 0.5 ms at 20 Hz and
train duration of 3 s were used to evoke slow excitatory synaptic potentials.
The following drugs dissolved in Krebs solution were applied by
pressure microejection (2 × 103 Torr, 5-20 ms)
from a glass micropipette placed close to the ganglia: GABA (5 mM), ACh
(20 mM), muscimol (10 mM), baclofen (10 mM), and
-aminobutyric acid
(BABA; 30 mM). Glass micropipettes filled with drug solution were
brought as close as possible to the site of the recording
microelectrode. Bicuculline (10 µM), picrotoxin (20 µM), TTX (10 µM), hexamethonium bromide (10 µM), atropine sulfate (5 µM), and
nipecotic acid (0.5 mM) were dissolved in Krebs solution and applied by
bath perfusion. In some experiments, GABA in concentrations of 0.1, 0.5, or 1.0 mM was applied by bath perfusion. All drugs were obtained
from Sigma Chemical. A low-Ca2+,
high-Mg2+solution was made by decreasing the
Ca2+ concentration to 1 mM and increasing the
Mg2+ concentration to 15 mM.
Immunohistochemical staining for GABA was performed on ten ganglia from
two pancreata. The ganglia were fixed overnight at 4°C in 4%
paraformaldehyde. The tissues were then rinsed in 0.1 M PBS (pH 7.4),
incubated overnight in PBS containing 30% sucrose, and frozen in
isopentane at
40°C to
50°C. Cryostat sections (12 µm thick)
were cut, mounted on glass slides, and blocked for 60 min in PBS
containing 10% normal donkey serum (NDS) and 0.3% Triton X-100.
Tissue sections were incubated overnight in GABA antiserum (rabbit
polyclonal; Sigma Chemical) diluted 1:200 in PBS containing 5% NDS and
0.3% Triton X-100 at room temperature. After being rinsed for 15 min
in PBS, sections were incubated in fluorescein- or Cy3-conjugated
donkey anti-rabbit secondary antiserum (Chemicon) diluted 1:100 in PBS
containing 2.5% NDS and 0.3% Triton X-100 for 90 min at room
temperature. Some sections were double immunostained for GABA and
protein gene product 9.5 (PGP 9.5; mouse monoclonal; Biogenesis) to
label neurons or glial fibrillary acidic protein (GFAP; mouse
monoclonal; Boehringer Mannheim) to label glial cells. The secondary
antibodies for double labeling were mixtures of donkey anti-rabbit
fluorescein and donkey anti-mouse Cy3 or donkey anti-rabbit Cy3 and
donkey anti-mouse fluorescein. The presence of glutamic acid
decarboxylase (GAD), the biosynthetic enzyme for GABA, in glial cells
and islets was examined using a rabbit polyclonal GAD antibody
(Chemicon) at 1:500-1:1,000 dilution. Islets were identified using
a mouse monoclonal synaptophysin antibody (16) at 1:20
dilution (Dako, Carpinteria, CA). Tissues were examined by
epifluorescence and by laser scanning confocal microscopy. Control
tissues, omitting primary antibodies or using mismatched secondary
antibodies, showed no immunoreactivity for GABA, PGP 9.5, GFAP, or GAD.
All data are expressed as means ± SE and compared using
Student's t-test. P < 0.05 was considered
significant, and n represents the number of neurons tested.
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RESULTS |
Effects of exogenous GABA.
Intracellular recordings were made from 84 neurons in pancreatic
ganglia of 51 cats. These neurons had a mean resting membrane potential
of
49.1 ± 0.7 mV and a mean input resistance of 61.2 ± 3.4 M
. All 84 neurons responded with a depolarization of the membrane potential on microejection (5 mM at 2 × 10
3 Torr for 5-20 ms) of GABA. The GABA-evoked
response consisted of a fast rising depolarization followed by a slow
repolarization. During the depolarizing response, action potentials
were evoked in eight (~10%) neurons. In 61 neurons, the recording
microelectrodes were filled with 3 M KCl. The mean amplitude of the
GABA-evoked depolarization was 17.4 ± 0.7 mV, and the duration
was 47.3 ± 4.0 s. The membrane input resistance during the
GABA-evoked response (n = 25) significantly
(P < 0.05) decreased to 36.8 ± 3.8 M
compared with 63.2 ± 4.8 M
observed in the same neurons before GABA
application. In 23 other neurons, when the recording microelectrode was
filled with 3 M KAc, the GABA-evoked depolarization was 7.7 ± 0.5 mV in amplitude and 35.7 ± 4.3 s in duration. These values
are significantly (P < 0.05) less compared with the
corresponding values obtained when KCl-filled microelectrodes were
used. During application of GABA, the membrane input resistance
significantly decreased to 43.7 ± 4.1 from 59.5 ± 4.8 M
observed in the same neurons before applying GABA.
The amplitude of GABA-evoked depolarizations was dependent on the
membrane potential. The more positive the membrane potential, the
smaller the amplitude of the GABA-evoked depolarization. The depolarization to GABA was abolished when the membrane potential was
30.2 ± 0.3 mV (n = 6), and a hyperpolarization
was evoked by GABA when the membrane potential was held at
15.7 ± 0.2 mV (n = 6; Fig.
1).

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Fig. 1.
Effect of conditioning depolarization and hyperpolarization on
GABA-evoked depolarization in a pancreatic neuron. The resting membrane
potential (RMP) was 42 mV. Note that the reversal potential for the
GABA-evoked depolarization was 16 mV.
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The concentration dependence of GABA-evoked depolarizations was tested
in four experiments. Bath superfusion with 0.1 mM GABA depolarized the
membrane potential by 1.7 ± 0.2 mV. The depolarization persisted
for the duration of bath superfusion (15-25 min). An additional
depolarizing response was evoked when GABA was applied by pressure
ejection (5 mM for 20 ms). When 0.5 mM GABA was superfused, the
membrane potential depolarized by 13.2 ± 0.6 mV in the first minute. During continuous superfusion with 0.5 mM GABA, the membrane potential repolarized. After 15 to 20 min, it remained depolarized by
3.1 ± 0.3 mV above the control resting membrane potential. During
this steady-state depolarization, pressure ejection of GABA (5 mM for
20 ms) could not evoke further depolarization. When the bath was
superfused with 1.0 mM GABA, the membrane potential depolarized by
21.1 ± 1.3 mV in the first minute. During continuous superfusion,
the membrane potential partially repolarized but remained depolarized
by 7.7 ± 0.5 mV above the control resting membrane potential for
the duration of GABA application. Further depolarization could not be
evoked by pressure ejection of GABA until GABA was washed out.
To eliminate the possible involvement of cholinergic nicotinic and
muscarinic receptors in the GABA response, the nicotinic receptor
antagonist hexamethonium and the muscarinic receptor antagonist
atropine were used to block cholinergic transmission. Superfusing
ganglia with hexamethonium (10 µM; n = 8 neurons) for
30 min inhibited ACh-evoked fast nicotinic responses but did not affect
GABA-evoked depolarizations (Fig.
2B). Superfusing ganglia with
atropine (5 µM; n = 6 neurons) for 30 min abolished ACh-evoked slow muscarinic depolarization but did not affect
GABA-evoked depolarizations (Fig. 2D). These results
indicate that neither cholinergic nicotinic receptors nor cholinergic
muscarinic receptors were involved in GABA-evoked depolarizations.

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Fig. 2.
Lack of effect of cholinergic receptor antagonists on
GABA-evoked depolarizations. A-D:
upper traces show the response to pressure application to
ACh; lower traces show the response to pressure application
of GABA. Note that in normal Krebs solution, the response to ACh
consisted of a fast depolarization followed by a lower amplitude slow
depolarization (A and C). Hexamethonium blocked
the fast depolarization to ACh (B) but had no effect on the
GABA response. Atropine blocked the slow depolarization, but had no
affect on the depolarizing response to GABA. Recordings in A
and B were from the same neuron; recordings in C
and D were from another neuron. Downward deflections in
traces in A and B are membrane potential changes
in response to intracellular injection of hyperpolarizing current
(i).
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To determine whether an increase in membrane permeability to sodium was
involved in the GABA-evoked depolarization, TTX, a sodium channel
blocker, was used. Superfusing ganglia with TTX (5 µM) for 30 min did
not affect GABA-evoked depolarizations (Fig. 3). The mean amplitude of GABA-evoked
depolarizations was 19.6 ± 0.8 mV in normal Krebs solution
(n = 2) and 20.1 ± 0.7 mV when TTX was present.
These values were not significantly different (P > 0.05). These data suggest that GABA-evoked depolarizations in
pancreatic neurons did not involve an increase in membrane permeability
to sodium.

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Fig. 3.
Response in the same pancreatic neuron to application of
GABA in normal Krebs solution (A) and in the presence of TTX
(B). TTX (1 µM) had no effect on the GABA-evoked
depolarization.
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To determine whether GABA acted postsynaptically to evoke
depolarization, low-Ca2+, high-Mg2+ solution
was used to block synaptic transmission. The mean amplitude of
GABA-evoked depolarizations in normal Krebs solution and in the
low-Ca2+, high-Mg2+ solution was similar
(18.1 ± 0.9 vs. 17.8 ± 1.0 mV; P > 0.05; n = 6 neurons). An example of the GABA-evoked
depolarization under the two test conditions is shown in Fig.
4.

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Fig. 4.
Response to GABA in normal Krebs solution (A) and in a
low-Ca2+ (1 mM), high-Mg2+ (15 mM) solution
(B) used to block synaptic transmission. Note that the
low-Ca2+, high-Mg2+ solution had no effect on
the GABA-evoked depolarization. Downward deflections are membrane
potential responses to intracellular injection of hyperpolarizing
current. Recordings were from the same neuron.
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Twenty-two neurons were tested to determine whether either fast
nicotinic or slow synaptic transmission could be affected by
application of GABA. In 15 neurons tested, the amplitude of the fast
excitatory postsynaptic potential (fEPSP) in normal Krebs solution was
5.4 ± 0.3 mV; the duration was 26.8 ± 1.8 ms. After pressure ejection of GABA, the amplitude of fEPSP decreased
significantly to 2.9 ± 0.2 mV (P < 0.01). This
inhibitory effect of GABA on the nicotinic fEPSP lasted from 1 to 3 min
in the different experiments. In seven other experiments, ganglia were
preincubated with 1 mM GABA for 1 h and the slow excitatory
postsynaptic potentials (sEPSP) were evoked by repetitive nerve
stimulation (20 Hz for 3 s). Under these conditions, the amplitude
of the sEPSP was 5.5 ± 0.4 mV. In the same experiments,
bicuculline was added to determine whether endogenously released GABA
acting through GABAA receptors affected the amplitude of
the sEPSP. After superfusion of bicuculline (10 µM) for 15 min, the
amplitude of the sEPSP was reduced to 4.8 ± 0.4 mV, but this was
not statistically significant (P > 0.05) compared with
control measurements.
Effect of GABA receptor agonist and antagonist.
Pressure microejection (5-20 ms) of baclofen (10 mM), a
GABAB receptor agonist, had no significant effect on
resting membrane potential (62.5 ± 4.0 vs. 63.8 ± 4.5 mV;
P > 0.05; n = 6). In contrast,
pressure microejection (5-30 ms) of muscimol (10 mM), a
GABAA receptor agonist, evoked a membrane depolarization in 15 of 15 neurons tested (Fig. 5).
Muscimol-evoked depolarizations had a mean amplitude of 17.1 ± 1.9 mV and a mean duration of 44.3 ± 5.7 s, similar to
GABA-evoked depolarizations. Muscimol also significantly decreased
membrane input resistance (66.3 ± 4.9 m
before application vs.
37.0 ± 5.4 m
in presence of muscimol; P < 0.05; n = 5).

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Fig. 5.
Effect of muscimol, a GABAA receptor agonist,
on the membrane potential of a pancreatic neuron. Muscimol evoked a
depolarization (upper) that was similar in amplitude and
duration to GABA-evoked depolarization recorded from the same neuron
(lower). Downward deflections in both traces are membrane
potential changes in response to intracellular injection of
hyperpolarizing current.
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Bicuculline was used to determine whether GABAA receptors
mediated GABA-evoked depolarizations. After recordings were made in
normal Krebs solution, the ganglia were superfused with Krebs solution
containing bicuculline (10 µM) for 10-15 min, followed by
washout of bicuculline with normal Krebs solution. During superfusion of bicuculline and washing out, GABA was applied by pressure
microejection every 4-5 min. In 10 of 10 neurons tested, after
10-15 min of superfusion with bicuculline, GABA-evoked
depolarizations were completely blocked. GABA-evoked
depolarizations partially recovered 2 min after washout of bicuculline
and completely recovered after 10 min (Fig.
6).

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Fig. 6.
Effect of bicuculline, a GABAA receptor
antagonist, on GABA-evoked depolarizations. The antagonist blocked
GABA-evoked depolarizations (B). The effect was reversible
on washout of the antagonist (C and D). Downward
deflections in A-D are membrane potential
changes in response to intracellular injection of hyperpolarizing
current. Recordings were made from the same neuron.
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To determine whether GABA-evoked depolarizations were dependent on
Cl
, the effect of picrotoxin, a Cl
channel
blocker, was tested. In 8 of 8 neurons studied, superfusion of
picrotoxin (20 µM) almost completely inhibited the depolarizing response to GABA (Fig. 7).

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Fig. 7.
Effect of picrotoxin, a Cl channel blocker,
on GABA evoked-depolarizations. Picrotoxin blocked GABA-evoked
depolarizations (B); picrotoxin had no effect on the resting
membrane potential. Downward deflections in A and
B are membrane potential responses to intracellular
injection of hyperpolarizing current. The resting membrane potential of
52 mV applies to both A and B.
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GABA uptake and release.
To determine whether GABA was taken up by and released from pancreatic
ganglion glial cells, the effect of BABA was tested before and after
prolonged superfusion with a GABA-containing Krebs solution. BABA, a
weak GABA receptor agonist, is a substrate for the GABA carrier in
glial cells; BABA stimulates efflux of GABA from glial cells
(9). In 12 neurons tested, microejection of BABA (30 mM
for 5-6 ms) evoked a depolarization that had an amplitude of
7.8 ± 0.8 mV and a duration of 16.7 ± 1.2 s. The membrane input resistance decreased from 60.4 ± 4.7 to 45.2 ± 4.1 M
during the BABA-evoked depolarization, a reduction similar to that recorded during GABA-evoked depolarizations. In 11 other neurons, the ganglia (n = 9) were pretreated by
superfusing Krebs solution containing 1 mM GABA for 1 h, as
required to potentiate the action of BABA (9). After 30 min washout of the GABA-containing solution, neurons were impaled and
tested for their response to BABA. In these neurons, BABA (30 mM for
5-6 ms) evoked a depolarization that was 15.6 ± 1.0 mV in
amplitude and 35.5 ± 2.3 s in duration. These values are
significantly (P < 0.01) greater compared with values
recorded without GABA preincubation. An example of the response to BABA
with and without GABA preincubation is shown in Fig.
8.

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Fig. 8.
The effect of preincubation with GABA on -aminobutyric
acid (BABA)-evoked depolarizations. BABA evoked a lower amplitude
depolarization compared with the GABA-evoked depolarization in the same
neuron (A). In a ganglion pretreated with 1 mM GABA, the
BABA-evoked depolarization was of higher amplitude and similar in
amplitude to the GABA-evoked depolarization (B). Downward
deflections in A and B are membrane potential
responses to intracellular injection of hyperpolarizing current. All
recordings were made from the same neuron.
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In another series of experiments (n = 9), the GABA
uptake inhibitor nipecotic acid (0.5 mM) was added to Krebs solution
containing 1 mM GABA. The ganglia were superfused for 1 h with the
solution containing nipecotic acid and GABA. After a 30-min washout,
neurons were impaled and tested for their response to BABA. The
amplitude of the BABA-evoked depolarization was 4.7 ± 0.6 mV
(n = 9), a value significantly lower (P < 0.05) compared with the amplitude of the BABA-evoked depolarization
seen in the neurons pretreated with only GABA.
Immunohistochemistry.
GABA immunoreactivity was present in pancreatic ganglia (Fig.
9). Double immunolabeling for GABA and
the glial cell marker GFAP and double labeling for GABA and the nerve
cell marker PGP 9.5 showed that GABA immunoreactivity was colocalized
with GFAP but not with PGP 9.5. An example of colocalization of
GABA and GFAP is shown in Fig. 9. These results indicate that
GABA was present in glial cells but absent from ganglion neurons.
However, immunoreactivity for GAD, the biosynthetic enzyme for GABA,
was absent in pancreatic ganglia (Fig.
10). Pancreatic islets, identified with
synaptophysin antibody (44), contained
immunoreactivity for GAD (Fig. 10), suggesting that islets but
not glial cells synthesize GABA in cat pancreas.

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Fig. 9.
Micrograph, obtained using a laser scanning confocal microscope, of
a section of cat pancreatic ganglion that was double immunostained for
GABA and glial fibrillary acidic protein (GFAP). GABA immunoreactivity
was colocalized with GFAP immunoreactive glial cells (arrowheads).
Principal ganglion neurons (*) were not immunoreactive for GABA. Scale
bar = 100 µm.
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Fig. 10.
Glutamic acid decarboxylase (GAD)-like immunoreactivity in cat
pancreas. Tissue was double-immunolabeled with antibodies to GAD and
synaptophysin. A and B: pancreatic islet.
GAD-like immunoreactivity was present in synaptophysin-positive cells
at the periphery of the islet. C and D:
pancreatic ganglion. Synaptophysin immunoreactive varicosities but no
GAD immunoreactivity was detected (*). Micrograph was obtained using a
laser scanning confocal microscope. Scale bars: A and
B = 100 µm; C and D = 50 µm.
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DISCUSSION |
The two key observations made in the present study were
that 1) GABA was found by immunohistochemistry in glial
cells in pancreatic ganglia and 2) GABAA
receptors were located on cell bodies of pancreatic ganglion neurons.
The presence of GABA in glial cells and the absence of GABA
immunoreactivity in ganglion neurons and nerve fibers and endings
suggest that GABA in pancreatic ganglia functions as a paracrine
messenger molecule rather than as a neurotransmitter substance. The
absence of GABA in intrapancreatic nerve structures is in agreement
with previous studies (15, 43). It has been shown that
GABA is present in pancreatic islets (10, 19, 31, 32) and
that GABA is cosecreted with insulin (11, 34). Our study
also shows that GAD immunoreactivity was not present in glial cells but was found in pancreatic islets. Based on these observations and the ones made in the present study, it can be suggested that release of GABA from islet cells could act directly on
ganglion nerve cell bodies or be stored for later release from glial cells.
It is known that GABA is transported in both directions across the
glial cell membrane (9) and that glial cells in superior cervical ganglia take up GABA at concentrations as low as 1 µM (8, 46). Normally, the outward transport rate of GABA from glial cells is submaximal. However, when BABA is present on the outside
surface of the cell membrane, the inward transport of BABA increases
the turnover rate of the carrier (8) especially when glial
cells are loaded with GABA by preincubation with exogenous GABA. The
present study showed that BABA, a substrate with high affinity for the
GABA carrier located in glial cells (9), evoked a membrane
depolarization that was significantly potentiated when ganglia were
preincubated with GABA. These data suggest that the increased
turnover rate of the carrier by BABA accelerated the efflux of
GABA, thereby evoking a larger amplitude GABA-evoked depolarization. If
BABA acted via release of GABA through a transport process in glial
cells, then pretreatment with nipecotic acid should block the effect of
BABA. The results obtained show that nipecotic acid significantly
reduced the effect of BABA. The remaining BABA-evoked depolarization
most likely was due to BABA acting on GABAA receptors
because BABA is a weak GABAA receptor agonist (9).
The GABA-evoked depolarization observed in pancreatic ganglia in the
present study was mediated by postsynaptic GABA receptors because the
GABA response was well maintained in the presence of a
low-Ca2+, high-Mg2+ solution to block synaptic
vesicle release. Similarly, in the major pelvic ganglion, there is no
evidence for presynaptically located GABA receptors (2).
The absence of any presynaptic effect of GABA in pancreatic ganglia and
in the pelvic ganglia (a mixed sympathetic, parasympathetic ganglia) is
in sharp contrast to prevertebral sympathetic ganglia in which GABA
acts on presynaptic GABAA receptors (33, 41)
as well as on postsynaptic GABAA receptors. There was no
evidence for the existence of GABAB receptors on pancreatic
ganglion neurons, because the GABAB receptor agonist baclofen was without effect.
The present results suggest that GABAA receptors mediated
the GABA depolarizing response, because muscimol, a GABAA
receptor agonist, mimicked the GABA response and bicuculline, a
GABAA receptor antagonist, blocked it. Recently
(2), it was shown in the major pelvic ganglion that GABA
can evoke an early and late depolarizing response mediated through
GABAA and GABAC receptors, respectively, and
that the latter response is bicuculline insensitive. In the present
study, the GABA-evoked depolarization was completely blocked by
bicuculline. These data indicate that GABAC receptors are
not present in pancreatic ganglia.
Although GABA and nicotine are similar in structure (36),
GABA did not interact with nicotinic receptors because the response to
exogenously applied GABA was not affected by nicotinic blockade with
hexamethonium. Furthermore, the GABA response was also not affected by
muscarinic receptor blockade with atropine, ruling out the possibility
that GABA may have released ACh, which in turn acted postsynaptically
on muscarinic receptors.
Previous studies (1, 13, 18, 41) on sympathetic ganglion
neurons show that exogenously applied GABA acts on GABAA receptors to increase Cl
conductance, thereby evoking
membrane depolarization. The present results suggest that GABA
increases Cl
conductance also in pancreatic ganglion
neurons because the Cl
blocker picrotoxin inhibited the
GABA depolarizing response. This conclusion is supported by the
observation in the present study that the reversal potential for the
GABA-evoked depolarization was approximately
30 mV, a value
close to the Cl
equilibrium potential in autonomic
ganglion neurons (18).
Although we observed a statistically significant inhibitory effect of
GABA on the fEPSP, it was not so clear whether the depolarizing response to GABA also inhibited the sEPSP.
In conclusion, the present study shows the following: pancreatic
ganglion neurons contain GABAA receptors; exogenously added GABA acts through GABAA receptors to cause depolarization,
inhibiting fEPSPs; and ganglionic glial cells store and can release
endogenous GABA under the experimental conditions used in this study.
It remains to be determined whether in vivo GABA originating from glial
cells or islet cells alters synaptic transmission in pancreatic ganglia.
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ACKNOWLEDGEMENTS |
Some of the results of this study were published previously in
abstract form (39).
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FOOTNOTES |
Address for reprint requests and other correspondence: J. H. Szurszewski, Dept. of Physiology and Biophysics, Mayo Clinic and
Mayo Foundation, 200 First St. SW, Rochester, MN 55905 (E-mail: gijoe{at}mayo.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 11 January 2000; accepted in final form 25 September 2000.
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