Phentolamine Inhibits Exocytosis of Glucagon by Gi2 Protein-dependent Activation of Calcineurin in Rat Pancreatic alpha -Cells*

Marianne Høy, Krister Bokvist, Weng Xiao-Gang, John Hansen, Kirstine Juhl, Per-Olof BerggrenDagger , Karsten Buschard§, and Jesper Gromada

From the Laboratory of Islet Cell Physiology, Novo Nordisk A/S, Novo Alle, DK-2880 Bagsvaerd, § Bartholin Instituttet, Kommunehospitalet, Øster Farimagsgade 5, DK-1353 Copenhagen, Denmark and Dagger  The Rolf Luft Center for Diabetes Research, Department of Molecular Medicine, Karolinska Institutet, Karolinske Hospital L1:02, S-171 76 Stockholm, Sweden

Received for publication, August 18, 2000



    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Capacitance measurements were used to investigate the molecular mechanisms by which imidazoline compounds inhibit glucagon release in rat pancreatic alpha -cells. The imidazoline compound phentolamine reversibly decreased depolarization-evoked exocytosis >80% without affecting the whole-cell Ca2+ current. During intracellular application through the recording pipette, phentolamine produced a concentration-dependent decrease in the rate of exocytosis (IC50 = 9.7 µM). Another imidazoline compound, RX871024, exhibited similar effects on exocytosis (IC50 = 13 µM). These actions were dependent on activation of pertussis toxin-sensitive Gi2 proteins but were not associated with stimulation of ATP-sensitive K+ channels or adenylate cyclase activity. The inhibitory effect of phentolamine on exocytosis resulted from activation of the protein phosphatase calcineurin and was abolished by cyclosporin A and deltamethrin. Exocytosis was not affected by intracellular application of specific alpha 2, I1, and I2 ligands. Phentolamine reduced glucagon release (IC50 = 1.2 µM) from intact islets by 40%, an effect abolished by pertussis toxin, cyclosporin A, and deltamethrin. These data suggest that imidazoline compounds inhibit glucagon secretion via Gi2-dependent activation of calcineurin in the pancreatic alpha -cell. The imidazoline binding site is likely to be localized intracellularly and probably closely associated with the secretory granules.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha 2-adrenergic receptors but rather from inhibition of ATP-sensitive K+ (KATP)1 channels in the beta -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 alpha -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 alpha -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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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Preparation of Islets and alpha -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 alpha -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% alpha -cells and <3% beta -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 alpha -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 alpha -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-Galpha i1, 5'-CATGGTGGCCGACGTCGCCCGCCCTCGGCGCCGGGGCCG-3' (this sequence is based on the 5'-noncoding sequence upstream of the initiation codon of the rat Galpha i1 cDNA (19)); antisense-Galpha i2, 5'-CATCCTGCCGTCCGCCGGCCCGGCCTGGCCCCCACCACG-3'; sense-Galpha i2, 5'-CGTGGTGGGGGCCAGGCCGGGCCGGCGGACGGCAGGATG-3' (from the leader sequence just before the initiation codon, based on the rat Galpha i2 cDNA sequence (19, 20)); antisense-Galpha i3, 5'-CATGACGGCGGCCGGAGAGGGGACCGGGCCCTGGCTCCAC-3' (from the leader sequence just before the initiation codon, based on the rat Galpha i3 cDNA sequence (19, 20)); and antisense-Galpha o, 5'-CATGGTGGCCCCTTCCCTGCCACAGCCCGCACGACTCGG-3' (from the leader sequence just before the initiation codon, based on the rat Galpha o cDNA sequence; this sequence is common for Galpha o1 and Galpha 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.


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Effects of Phentolamine on Electrical Activity and KATP Channels in Rat alpha -Cells-- Fig. 1 illustrates electrical activity recorded from a single rat alpha -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 alpha -cell-rich preparation. The application of phentolamine (0.1 mM) did not affect the ability of the alpha -cells to fire action potentials (Fig. 1A), whereas the subsequent addition of diazoxide (0.1 mM), which activates KATP channels in rat alpha -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 alpha -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.

Fig. 2A shows measurements of the whole-cell KATP current from an intact alpha -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 alpha -cells (data not shown).



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Fig. 2.   Effects of phentolamine on whole-cell KATP channel activity in rat pancreatic alpha -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).

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 alpha -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 alpha -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 alpha -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 (Delta Cm; B) and the integrated Ca2+ current (QCa; C). The data are mean ± S.E. of five cells. *, p < 0.01.

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 alpha -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 (Delta Cm/Delta 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.

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 alpha 2-adrenergic antagonistic activity or binding to I1 or I2 receptors, since clonidine (alpha 2-adrenergic agonist), AGN 192403 (I1 ligand), or BU-224 (I2 ligand) failed to affect exocytosis (Table I). Furthermore, an irreversible blockade of either alpha 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 alpha 2-adrenergic, I1, or I2 receptors.


                              
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Table I
Imidazoline compounds inhibit exocytosis independently of alpha 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 (Delta Cm/Delta 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.

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.

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 GDPbeta 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 alpha -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 alpha -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 GDPbeta 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 (Delta Cm/Delta 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 (Delta Cm/Delta 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.

To determine the type of pertussis toxin-sensitive G protein, we used antisense oligonucleotides against Galpha i1-3 and Galpha o. Fig. 5C shows that alpha -cells pretreated for 24 h with antisense oligonucleotides against Galpha i1, Galpha i3, or Galpha 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 Galpha i2. In cells treated with sense oligonucleotides against Galpha 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 alpha -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 beta -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 alpha -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 (Delta Cm/Delta 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.

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).



    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 beta -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 alpha -cells are equipped with KATP channels identical to those expressed in beta -cells (26, 27). The alpha - and beta -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 beta -cell KATP channels share a similar sensitivity to phentolamine (6, 30).

In keeping with previous observations (15, 26), we found rat alpha -cells to be spontaneously active in the absence of glucose. Exposure of the alpha -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 alpha -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 alpha -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 alpha -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 alpha -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 beta -cells, where the ATP/ADP ratio increases severalfold following an elevation in the glucose concentration. The constant high ATP/ADP ratio in rat alpha -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 alpha -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 alpha -subunits. Galpha o1, Galpha o2, Galpha i1, and Galpha i2 were detected on small synaptic vesicles, whereas chromaffin granules only contain Galpha 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.


    FOOTNOTES

* This study was supported by grants from the Swedish Diabetes Association, the Novo Nordisk Foundation, the Nordic Insulin Foundation committee, and the Swedish Medical Research Council Grants 72X-00034, 72XS-12708, and 72X-09890 (to P.-O. B.).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.

To whom correspondence and reprint requests should be addressed: Lilly Research Laboratories, Lilly Forschung GmbH, Essener Strasse 93, D-22419 Hamburg, Germany. Tel.: 49-40-5-27-24-323; Fax: 49-40-5-27-24-615; E-mail: Gromada_Jesper@lilly.com.

Published, JBC Papers in Press, September 19, 2000, DOI 10.1074/jbc.M007562200


    ABBREVIATIONS

The abbreviations used are: KATP, ATP-sensitive K+ channels; nS, nanosiemens; fF, femtofarads; RRP, readily releasable pool; TEA, tetraethylammonium; GDPbeta S, guanosine 5'-O-2thiodiphosphate.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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