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INTRODUCTION |
Thirty years has elapsed since the initial demonstration that the
imidazoline compound phentolamine stimulated glucose-induced insulin
release in humans (1-3). Good evidence exists that the insulinotropic
effects of imidazoline compounds do not result from antagonism of
2-adrenergic receptors but rather from inhibition of
ATP-sensitive K+
(KATP)1 channels
in the
-cell plasma membrane (6-9), resulting in membrane depolarization, stimulation of Ca2+ influx, and exocytosis.
In addition, imidazoline compounds also stimulate insulin release by a
direct interaction with the exocytotic machinery (10).
Recent evidence suggests that imidazoline compounds stimulate not only
insulin release but also somatostatin release while suppressing
glucagon secretion (11). The mechanism underlying the inhibitory action
of imidazoline compounds on glucagon release is not clear but may
involve either a direct or a paracrine effect on the
-cells
(11-13). Here we have combined the patch clamp technique with
capacitance measurements of exocytosis to explore the effects of
different imidazoline compounds on exocytosis in single rat pancreatic
-cells. We thereby provide the first direct evidence that
imidazoline compounds inhibit Ca2+-dependent
exocytosis of glucagon via Gi2-dependent
activation of the serine/threonine protein phosphatase calcineurin.
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EXPERIMENTAL PROCEDURES |
Preparation of Islets and
-Cells--
Male Lewis rats
(250-300 g; Møllegaard, Lille Skensved, Denmark) were anesthetized by
pentobarbital (100 mg/kg intraperitoneally), and the pancreas was
removed. The experimental procedures were approved by the local ethical
committee. Islets were isolated by collagenase digestion and dispersed
into single cells using dispase. Pancreatic
-cells were separated by
fluorescence-activated cell sorting as described elsewhere (14). Based
on the hormone contents and their glucose sensitivity, we estimate that
the preparations contain >80%
-cells and <3%
-cells (14, 15).
The cell suspension was plated on 35-mm diameter Petri dishes and
incubated in a humidified atmosphere for up to 3 days in RPMI 1640 tissue culture medium (Life Technologies Ltd., Paisley, United Kingdom)
supplemented with 10% (v/v) heat-inactivated fetal calf serum, 100 IU/ml penicillin, and 100 µg/ml streptomycin.
Electrophysiology--
Pipettes were pulled from borosilicate
glass, coated with Sylgard near their tips, and fire-polished. When
filled with pipette solutions, the electrodes had a resistance of 3-4
megaohms. The whole-cell KATP conductance was estimated by
applying 10-mV hyper- and depolarizing voltage pulses (duration, 200 ms; pulse interval, 2 s) from a holding potential of
70 mV using
either the perforated patch or standard whole-cell configuration. The
currents were recorded using an Axopatch 200B patch clamp amplifier,
digitized, and stored in a computer using the Digidata AD converter and
the software pClamp (Axon Instruments, Foster City, CA). The
-cell membrane potential was recorded using the perforated patch whole-cell configuration. Exocytosis was measured as increases in cell capacitance using, except for Fig. 3, an EPC-9 patch clamp amplifier and the Pulse
software (version 8.30; HEKA Elektronik, Lamprecht/Pfalz, Germany). The
interval between two successive points was 0.2 s. All measurements
of cell capacitance, except those in Fig. 3 in which the perforated
patch whole-cell configuration was used, have been performed using the
standard whole-cell recording mode. In Fig. 3, changes in cell
capacitance were elicited by 500-ms voltage clamp depolarizations to 0 mV from a holding potential of
70 mV using an EPC-7 patch clamp
amplifier (List Elektronik, Darmstadt, Germany) and in-house software
written in AxoBasic (Axon Instruments, Foster City, CA) as detailed
elsewhere (16). The volume of the recording chamber was 0.4 ml, and the
solution entering the bath (1.5-2 ml/min) was maintained at
33 °C.
Solutions--
The extracellular medium consisted of 138 mM NaCl, 5.6 mM KCl, 2.6 mM
CaCl2, 1.2 mM MgCl2, 5 mM HEPES (pH 7.4 with NaOH), and 0 or 5 mM
D-glucose. The extracellular solution used for measurements of cell capacitance evoked by voltage clamp depolarizations contained 118 mM NaCl, 20 mM tetraethylammonium
chloride, 5.6 mM KCl, 2.6 mM
CaCl2, 1.2 mM MgCl2, 5 mM HEPES (pH 7.40 with NaOH), and 5 mM glucose.
Tetraethylammonium chloride was included in the medium to block
the outward delayed rectifying K+ current, which otherwise
obscures the smaller Ca2+ current (17). The pipette
solution used for the infusion experiments consisted of 125 mM potassium glutamate, 10 mM KCl, 10 mM NaCl, 1 mM MgCl2, 8 mM CaCl2, 3 mM Mg-ATP, 10 mM EGTA, and 5 mM HEPES (pH 7.15 with KOH). The
free Ca2+ concentration of the resulting buffer was 0.87 µM using the binding constants of Martell and Smith (18).
The pipette solution used for measurements of membrane potential and
KATP channel activity, using the perforated patch
configuration, was composed of 76 mM K2SO4, 10 mM KCl, 10 mM
NaCl, 1 mM MgCl2, 5 mM HEPES (pH
7.35 with KOH). For measurements of exocytosis using voltage clamp depolarizations, K2SO4 was replaced with
Cs2SO4 in the pipette solution. Electrical
contact was established by adding 0.24 mg/ml amphotericin B to the
pipette solution (16). Perforation required a few minutes, and the
voltage clamp was considered satisfactory when the
Gseries was stable and >35-40 nS. The pipette
solution used for recording of KATP channel activity using
the standard whole-cell configuration contained 125 mM KCl,
30 mM KOH, 10 mM EGTA, 1 mM
MgCl2, 5 mM HEPES, 0.3 mM Mg-ATP,
and 0.3 mM K-ADP (pH 7.15). Pertussis toxin was obtained
from RBI (Natick, MA). Deltamethrin and its inactive analog permethrin
were from Alomone Labs (Jerusalem, Israel). All other chemicals were
purchased from Sigma.
Antisense and Sense Oligonucleotides--
Single
-cells were
incubated for 24 h with a 20 µM concentration of the
following antisense and sense oligonucleotides, obtained from TAG
Copenhagen (Copenhagen, Denmark): antisense-G
i1,
5'-CATGGTGGCCGACGTCGCCCGCCCTCGGCGCCGGGGCCG-3' (this sequence is
based on the 5'-noncoding sequence upstream of the initiation codon of
the rat G
i1 cDNA (19));
antisense-G
i2, 5'-CATCCTGCCGTCCGCCGGCCCGGCCTGGCCCCCACCACG-3';
sense-G
i2, 5'-CGTGGTGGGGGCCAGGCCGGGCCGGCGGACGGCAGGATG-3' (from the leader sequence just before the initiation codon, based on the rat G
i2 cDNA sequence (19, 20));
antisense-G
i3,
5'-CATGACGGCGGCCGGAGAGGGGACCGGGCCCTGGCTCCAC-3' (from the leader
sequence just before the initiation codon, based on the rat
G
i3 cDNA sequence (19, 20)); and
antisense-G
o, 5'-CATGGTGGCCCCTTCCCTGCCACAGCCCGCACGACTCGG-3' (from the leader sequence just before the initiation codon, based on the rat
G
o cDNA sequence; this sequence is common for
G
o1 and G
o2 (19, 21)).
Glucagon Release--
Glucagon release was measured at 37 °C
in static incubation. Groups of 10 size-matched rat islets were
preincubated for 30 min in 200 µl of extracellular solution
consisting of 138 NaCl, 5.6 mM KCl, 2.6 mM
CaCl2, 1.2 mM MgCl2, 5 mM HEPES (pH 7.4 with NaOH) and 0-20 mM
D-glucose in 96-well Durapore membrane plates (Millipore,
Molsheim, France). The medium was aspirated using a vacuum control pump
(Millipore) and discarded. The islets were resuspended in 200 µl of
extracellular solution in the absence and presence of test compounds
and the indicated glucose concentration. At the end of the test
incubation (1 h), the medium was aspirated and assayed immediately for
glucagon using a glucagon radioimmune assay kit (GL-32K; Linco
Research, St. Charles, MO).
Data Analysis--
In the infusion experiments, the exocytotic
rate is presented as the increase in cell capacitance occurring during
the first 60 s following establishment of the whole-cell
configuration, excluding any rapid changes occurring during the initial
~10 s required for equilibration of the pipette solution with
cytosol. Results are presented as mean values ± S.E. for the
indicated number of experiments. Statistical significance was evaluated using Student's t test for paired or unpaired observations
or Dunnett's test for multiple comparisons with a single control.
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RESULTS |
Effects of Phentolamine on Electrical Activity and KATP
Channels in Rat
-Cells--
Fig. 1
illustrates electrical activity recorded from a single rat
-cell
using the perforated patch whole-cell configuration in the absence of
glucose. Spontaneous electrical activity was observed in >80% of the
tested cells (n > 80 cells), as expected for an
-cell-rich preparation. The application of phentolamine (0.1 mM) did not affect the ability of the
-cells to fire
action potentials (Fig. 1A), whereas the subsequent addition
of diazoxide (0.1 mM), which activates KATP
channels in rat
-cells, was associated with a reversible inhibition
of electrical activity (Fig. 1B).

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Fig. 1.
Effects of phentolamine and diazoxide on
electrical activity. Spontaneous electrical activity recorded from
an individual rat pancreatic -cell in the absence of glucose.
Phentolamine and diazoxide (both 0.1 mM) were added to the
bath solution during the periods indicated by the bars. The
asteriks in B indicate a period of 1 min. The recording is
continuous and representative of five cells.
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Fig. 2A shows measurements of
the whole-cell KATP current from an intact
-cell using
the perforated patch whole-cell configuration. In the absence of
glucose, the 10-mV voltage steps applied from a holding potential of
70 mV elicited currents with amplitudes of 2 pA, corresponding to an
input conductance of 0.2 nS. In a series of five experiments, the input
conductance averaged 0.3 ± 0.1 nS. The application of
phentolamine or the sulfonylurea tolbutamide (both 0.1 mM)
did not affect the current amplitude (phentolamine: 0.4 ± 0.1 nS,
n = 5; tolbutamide: 0.4 ± 0.2 nS, n = 5), whereas the KATP channel opener
diazoxide (0.1 mM) produced a 500% increase in the
membrane current, and a specific conductance of 1.8 ± 1.1 nS
(n = 5) was observed. The lack of effect of tolbutamide suggests that the KATP channels are already closed in the
absence of glucose in the bathing solution. The imidazoline compound
RX871024 (0.1 mM) likewise failed to affect electrical
activity and KATP channel activity in intact rat
-cells
(data not shown).

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Fig. 2.
Effects of phentolamine on whole-cell
KATP channel activity in rat pancreatic
-cells. Whole-cell KATP currents
were measured in response to 10-mV de- and repolarizing voltage pulses
from a holding potential of 70 mV using the perforated patch
configuration (A) or the standard whole-cell configuration
(B) and a pipette solution with 0.3 mM
ATP and 0.3 mM ADP to activate the channels. Phentolamine,
tolbutamide, and diazoxide (all 0.1 mM) were applied for 2 min. The traces are representative of four (standard whole-cell) and
five cells (perforated patch configuration).
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We used the standard whole-cell patch configuration and intracellular
dialysis with 0.3 mM ATP and 0.3 mM ADP to
activate the KATP channels to evaluate whether the
KATP channels expressed in rat
-cells are sensitive to
phentolamine. Fig. 2B shows that phentolamine (0.1 mM) reduced the whole-cell KATP current by
60%. The inhibitory effect of phentolamine on the whole-cell
KATP current was prompt and amounted on average to 55 ± 5% (p < 0.001; n = 5). The
subsequent addition of 0.1 mM tolbutamide caused a complete but reversible block of the whole-cell K+ conductance
(97 ± 1% inhibition; p < 0.001;
n = 5).
Effects of Phentolamine on Depolarization-evoked
Exocytosis--
Fig. 3A
illustrates whole-cell Ca2+ currents and the associated
changes in cell capacitance elicited by 500-ms depolarizations from
70 mV to 0 mV in an intact rat
-cell using the perforated patch
configuration. In the presence of forskolin, which elevates cytoplasmic
cAMP levels, the integrated Ca2+ current amounted to 4.8 picocoulombs, and a capacitance increase of 87 fF was evoked. Two
minutes after inclusion of 0.1 mM phentolamine in the
bathing solution, the same membrane depolarization produced an
integrated Ca2+ current of 4.7 picocoulombs and a
capacitance increase of 16 fF (82% inhibition). The depolarizations
and increases in cell capacitance were not associated with any changes
in cell conductance, and the capacitance measurements are accordingly
likely to report exocytosis. On average (Fig. 3B),
phentolamine produced 89 ± 16% (p < 0.01;
n = 5) reversible inhibition of exocytosis, which
was not associated with a change of the integrated Ca2+
current (Fig. 3C).

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Fig. 3.
Phentolamine inhibition of
depolarization-induced exocytosis in rat
-cells. A, Ca2+
currents (top), capacitance increases (middle),
and membrane conductances (bottom) evoked by 500-ms
depolarizations from 70 to 0 mV before and 2 min after the addition
of 0.1 mM phentolamine and 4 min after removal of the
imidazoline compound from the bath solution. Histograms
summarize the average effects of phentolamine on increases in cell
capacitance ( Cm; B) and the integrated
Ca2+ current (QCa; C).
The data are mean ± S.E. of five cells. *, p < 0.01.
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Phentolamine Inhibits Exocytosis Evoked by Intracellular Infusion
of Ca2+--
The effects of phentolamine on exocytosis
were further investigated in standard whole-cell experiments in which
secretion was evoked by intracellular dialysis with a
Ca2+-EGTA buffer with a free Ca2+ concentration
of 0.87 µM. Following establishment of the whole-cell configuration, exocytosis was observed as a gradual capacitance increase (Fig. 4A,
control). In general, cell capacitance reached a new
steady-state level within 3-5 min. It is clear that inclusion of 0.1 mM phentolamine in the pipette solution exerted a strong inhibition of the increase in cell capacitance (Fig. 4A,
phentolamine). On average, phentolamine evoked a 83%
inhibition of the rate of capacitance increase measured over the first
60 s (excluding the first ~10 s) after the establishment of the
whole-cell configuration (p < 0.01; n = 10 (control) and n = 4 (phentolamine)).

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Fig. 4.
Intracellular application of phentolamine
inhibits exocytosis in rat -cells.
A, increases in cell capacitance elicited by intracellular
infusion with a Ca2+-EGTA buffer with a free
Ca2+ concentration of 0.87 µM in the absence
(control) or presence of 0.1 mM phentolamine in the pipette
solution observed during the first 2 min after establishment of the
standard whole-cell configuration. Throughout the recording, the cell
was clamped at 70 mV in order to avoid activation of the
voltage-dependent Ca2+ channels that would
otherwise interfere with the measurement. B, dose response
of phentolamine-induced inhibition of exocytosis. The rates of
capacitance increase ( Cm/ t) were
measured over the first 60 s after establishment of the whole-cell
configuration. The line is the best fit of the average data
to the Hill equation. Data are mean values ± S.E. of 3-10
different cells.
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The effect of phentolamine on exocytosis was dependent on dose (Fig.
4B). No inhibition of exocytosis was observed at
3
µM. At higher concentrations, phentolamine decreased the
rate of capacitance increase by 42-78%. Approximating the average
data points of the inhibitory effect of phentolamine on exocytosis to
the Hill equation yielded values of the half-maximal inhibitory
concentration (IC50) and cooperativity factor of 9 µM and 3, respectively. The maximal effects were seen at
phentolamine concentrations of
100 µM (Fig. 4B).
Table I shows that the inhibitory action
of phentolamine on exocytosis was mimicked by RX871024 and efaroxan.
When applied at a concentration of 0.1 mM, exocytosis was
decreased by >70% for both compounds (p < 0.01;
n = 5). The inhibitory effect of imidazoline compounds
on exocytosis does not result from
2-adrenergic antagonistic activity or binding to I1 or I2
receptors, since clonidine (
2-adrenergic agonist), AGN
192403 (I1 ligand), or BU-224 (I2 ligand)
failed to affect exocytosis (Table I). Furthermore, an irreversible
blockade of either
2-adrenergic receptors with benextramine or I2 receptors with clorgyline did not affect
the ability of phentolamine to inhibit exocytosis (Table I). These data
suggest that the inhibitory action of phentolamine on exocytosis does
not involve
2-adrenergic, I1, or
I2 receptors.
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Table I
Imidazoline compounds inhibit exocytosis independently of
2-adrenergic, I1 and I2 receptors
Increases in cell capacitance were elicited by intracellular infusion
of a pipette solution with a free Ca2+ concentration of 0.87 µM. Throughout the recording, the cell was clamped at
70 mV in order to avoid activation of the
voltage-dependent Ca2+ channels that would
otherwise interfere with the measurement. The rates of capacitance
increase ( Cm/ t) were measured over
the first 60 s after establishment of the whole-cell configuration
(excluding the initial ~10 s). The following concentrations were
used: phentolamine, RX871024, efaroxan, BU-224, and clonidine, 100 µM; clorgyline, 50 µM; benextramine, 10 µM; AGN 192403, 1 µM.
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Table II shows that the inhibitory action
of phentolamine on exocytosis was associated with suppression of
glucagon release from batches of 10 size-matched rat islets.
Phentolamine reduced glucagon release independently of the ambient
glucose concentration, with the most pronounced effect at 2.5 mM glucose (54% inhibition). At this glucose
concentration, phentolamine reduced glucagon release dose-dependently (Table III)
with an IC50 at 1.2 µM, which is in fair
agreement with that observed for inhibition of exocytosis.
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Table II
Phentolamine produces glucose-independent inhibition of glucagon
release from rat islets
Glucagon release was measured from freshly isolated batches of 10 size-matched islets exposed for 1 h to the indicated glucose (G)
concentrations in the absence or presence of 0.1 mM
phentolamine.
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Table III
Phentolamine produces dose-dependent inhibition of glucagon
release from rat islets
Glucagon release was measured from freshly isolated batches of 10 size-matched islets exposed to the indicated phentolamine concentration
for 1 h in an extracellular medium with 2.5 mM
glucose.
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Phentolamine Evokes Gi2 Protein-dependent
Inhibition of Exocytosis--
We explored whether the ability of
phentolamine to inhibit exocytosis involved activation of GTP binding
proteins. Fig. 5A shows that
inclusion of a 1 mM concentration of the stable GDP analog
GDP
S in the pipette solution abolished the inhibitory effect of
phentolamine (0.1 mM) on exocytosis, and the
exocytotic response amounted to 90% (n = 5) of the
control level. The effect of phentolamine on exocytosis was probably
mediated by activation of inhibitory G proteins of the
Gi/Go type, since pretreatment of the
-cells
with pertussis toxin (100 ng/ml for >20 h) abolished the inhibitory
action of phentolamine (Fig. 5B).

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Fig. 5.
Phentolamine produces Gi2
protein-dependent inhibition of exocytosis in rat
-cells. Changes in cell capacitance were
elicited by intracellular dialysis of single cells with 0.87 µM free Ca2+ as described in the legend to
Fig. 4. A (left), increases in cell capacitance
in the absence and presence of 0.1 mM phentolamine using a
pipette solution supplemented with 1 mM GDP S.
B (left), effects of phentolamine in cells
pretreated with pertussis toxin (PTX; 100 ng/ml for >20 h).
Histograms (right) show average rates of increase
in cell capacitance ( Cm/ t) measured
over the first 60 s after establishment of the whole-cell
configuration ± S.E. of five or six different experiments.
C, histogram depicting average rates of increase
in cell capacitance ( Cm/ t) measured
over the first 60 s after establishment of the whole-cell
configuration under control conditions and in the presence of 0.1 mM phentolamine in untreated cells and in cells pretreated
with 20 µM of the indicated antisense or sense
oligonucleotides for 24 h. Data are mean values ± S.E. of
five different cells. *, p < 0.05; **,
p < 0.001.
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To determine the type of pertussis toxin-sensitive G protein, we used
antisense oligonucleotides against G
i1-3 and G
o. Fig. 5C shows that
-cells pretreated
for 24 h with antisense oligonucleotides against
G
i1, G
i3, or G
o did not
affect the ability of phentolamine to inhibit exocytosis. In contrast,
phentolamine did not suppress exocytosis in cells pretreated with
antisense oligonucleotides against G
i2. In cells treated
with sense oligonucleotides against G
i2, phentolamine
decreased the rate of capacitance comparable with that observed in
control cells (Fig. 5C). This suggests that Gi2
proteins mediate the inhibitory action of phentolamine on
-cell exocytosis.
Inhibitory Effect of Phentolamine on Exocytosis Involves Activation
of Calcineurin--
Dephosphorylation catalyzed by the
serine/threonine protein phosphatase calcineurin (PP2B) underlies
inhibition of exocytosis produced by adrenaline, somatostatin, and ATP
in pancreatic
-cells (22, 23). As illustrated in Fig.
6A, this may also apply with regard to phentolamine, since the immunosuppressant cyclosporin A, an
inhibitor of calcineurin, abolished the inhibitory action of this
imidazoline compound on exocytosis. A similar abolition of
phentolamine-evoked inhibition of exocytosis was observed with the
calcineurin inhibitor deltamethrin (Fig. 6B) but not in the presence of its inactive analogue permethrin (Fig. 6C). On
the contrary, okadaic acid (an inhibitor of type 1, 2A, and 3 serine/threonine protein phosphatases) failed to counteract the
inhibitory action of phentolamine (Fig. 6D). On average,
phentolamine reduced the exocytotic response by 81% (p < 0.05; n = 5), similar to that observed in the
absence of okadaic acid.

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Fig. 6.
Phentolamine-induced inhibition of exocytosis
involves activation of the protein phosphatase calcineurin.
Increases in cell capacitance were elicited in single rat pancreatic
-cells by intracellular dialysis with a pipette solution with free
Ca2+ concentration of 0.87 µM as described in
the legend to Fig. 4. Effects of phentolamine (0.1 mM) in
cells pretreated with cyclosporin A (1 µM for >20 min)
(A), deltamethrin (20 nM for >1h)
(B), its inactive analog permethrin (20 nM for
>1 h) (C), or okadaic acid (100 nM for >10
min) (D) are shown. The histograms to the
right show average rates of increase in cell capacitance
( Cm/ t) measured over the first
60 s after establishment of the whole-cell configuration under
control conditions ( ) or in the presence of phentolamine (+). Data
are given as the mean values ± S.E. of 3-7 cells. *,
p < 0.01.
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To ascertain that the decrease in exocytosis evoked by phentolamine
infusion indeed reflects activation of calcineurin, we measured
glucagon release from islets pretreated with inhibitors of this protein
phosphatase. Table IV clearly
demonstrates that deltamethrin and cyclosporin A prevented the
inhibitory action of phentolamine on glucagon release. Under these
conditions, glucagon release in the presence of phentolamine amounted
to 95% (deltamethrin) and 93% (cyclosporin A) of the control level.
On the contrary, phentolamine reduced glucagon release by >50%
(p < 0.01; n = 5) in islets pretreated
with either permethrin or okadaic acid (Table IV). Finally, no
inhibition of glucagon release was observed in islets pretreated
overnight with pertussis toxin.
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Table IV
Calcineurin mediates the inhibitory effect of phentolamine on glucagon
release from rat islets
Glucagon release was measured from batches of 10 size-matched islets in
the absence or presence of 0.1 mM phentolamine for 1 h
in an extracellular medium with 2.5 mM glucose. Islets were
pretreated with deltamethrin and permethrin (20 nM for
1 h), cyclosporin A (1 µM for 1 h), okadaic
acid (100 nM for 30 min), and pertussis toxin (100 ng/ml
for 20 h).
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DISCUSSION |
Imidazoline compounds have been shown not only to stimulate
insulin release but also to improve insulin sensitivity (10, 24), which
constitutes two main defects underlying glucose intolerance in type 2 diabetic patients. Since patients with type 2 diabetes also exhibit
exaggerated glucagon secretion, our present finding that phentolamine
inhibits exocytosis of glucagon may constitute the basis for an
additional target for the antidiabetogenic action of this class of
compounds. The inhibitory action of phentolamine on exocytosis was not
associated with a change in the activity of plasma membrane
KATP channels, the activity of voltage-gated Ca2+ channels, or changes in cytoplasmic free
Ca2+ levels (data not shown) but results from a direct
interference with the exocytotic machinery, an effect mediated by the
protein phosphatase calcineurin.
In this study, we extend previous observations in
-cells (6, 7, 25)
by showing that phentolamine blocked KATP channel activity
in standard whole-cell patch clamp experiments. This is consistent with
the observation that rat
-cells are equipped with KATP
channels identical to those expressed in
-cells (26, 27). The
-
and
-cell KATP channel is a complex of two proteins: a
pore-forming subunit, Kir6.2, and the sulfonylurea receptor, SUR1 (26,
28, 29). It has recently been demonstrated that phentolamine block of
KATP channels is mediated by Kir6.2 and results from a
voltage-independent reduction in channel activity (9). Kir6.2 is also
expressed in the heart, which may explain why native cardiac and
-cell KATP channels share a similar sensitivity to
phentolamine (6, 30).
In keeping with previous observations (15, 26), we found rat
-cells
to be spontaneously active in the absence of glucose. Exposure of the
-cells to phentolamine in the absence of glucose was not associated
with increased electrical activity. The failure of phentolamine to
affect electrical activity is consistent with the inability of the
imidazoline compound to reduce KATP channel activity in
metabolically intact cells. This suggests that the KATP
channels are already maximally inhibited in the absence of glucose and
is consistent with the observation that tolbutamide failed to reduce
channel activity under these conditions. Little information is
available on how glucose inhibits glucagon secretion in rat
-cells,
except that inhibition is mediated by glucose metabolism (31).
Our data suggest that dephosphorylation of components regulating
exocytosis underlies the inhibitory action of phentolamine on glucagon
secretion from intact islets and the perfused rat pancreas (11). This
is likely to be mediated by activation of the protein phosphatase
calcineurin, since the action of phentolamine was abolished by
maneuvers that suppressed the activity of the phosphatase. Calcineurin
has been identified in rat pancreatic
-cells (32). Our data
demonstrate that the ability of phentolamine to inhibit
Ca2+-dependent exocytosis is rapid and readily
reversible, suggesting that the magnitude of the secretory response
depends principally upon phosphorylation of as yet unidentified
exocytotic proteins. Since activation of protein kinase A leads to
enhancement of Ca2+-dependent exocytosis in rat
-cells (15), it could be argued that suppression of exocytosis by
phentolamine is the result of reduced cAMP levels and inhibition of
protein kinase A-mediated exocytosis. However, this possibility seems
unlikely, since the ability of phentolamine to suppress exocytosis
remained observable in experiments where the cytoplasmic cAMP
concentration was elevated using forskolin or by inclusion of the
cyclic nucleotide in the pipette solution dialyzing the cells (data not shown).
Measurements of adenine nucleotide content in purified rat
-cells
have revealed that they have a high ATP/ADP ratio already at 1 mM glucose and that it does not change significantly during glucose stimulation (33). This contrasts with the situation in the
-cells, where the ATP/ADP ratio increases severalfold following an
elevation in the glucose concentration. The constant high ATP/ADP ratio
in rat
-cells is likely to provide the energy to maintain the cells
in a phosphorylated state and consequently to enable phentolamine to
inhibit exocytosis in a glucose-independent manner.
Our results show that Gi2 proteins mediate the inhibition
of exocytosis by phentolamine in rat
-cells. This is indeed
consistent with the observation that Gi2 proteins have been
identified in rat pancreatic islets (34). However, it remains to be
established whether calcineurin activity is controlled by direct
interaction of the Gi2 protein or whether intermediate
proteins are responsible for signal transduction. Recent studies have
revealed that Gi and Go proteins are involved
in the regulation of intracellular transport processes, adding novel
targets to the list of effectors for these versatile molecular
switches. Heterotrimeric Gi and Go proteins
have been found on chromaffin granules and small vesicles from rodent
and bovine brain (35, 36). Interestingly, these heterotrimeric
G-proteins differ in their composition of
-subunits. G
o1, G
o2, G
i1, and
G
i2 were detected on small synaptic vesicles, whereas
chromaffin granules only contain G
o2 (36-38). These G proteins are in an ideal position for controlling transport processes across the granular membrane and the priming and fusion steps regulating exocytosis. Indeed, Go proteins are involved in
the exocytotic priming step in chromaffin cells (38), whereas
Gi3 proteins regulate swelling of zymogen granules, a
potentially important prerequisite for granule fusion (39). These
considerations raise the interesting possibility that phentolamine
inhibits glucagon exocytosis by interfering with granular associated
Gi2 proteins.