Noradrenaline inhibits exocytosis via the G protein βγ subunit and refilling of the readily releasable granule pool via the α_i1/2 subunit

Abstract

The molecular mechanisms responsible for the ‘distal’ effect by which noradrenaline (NA) blocks exocytosis in the β-cell were examined by whole-cell and cell-attached patch clamp capacitance measurements in INS 832/13 β-cells. NA inhibited Ca²⁺-evoked exocytosis by reducing the number of exocytotic events, without modifying vesicle size. Fusion pore properties also were unaffected. NA-induced inhibition of exocytosis was abolished by a high level of Ca²⁺ influx, by intracellular application of antibodies against the G protein subunit Gβ and was mimicked by the myristoylated βγ-binding/activating peptide mSIRK. NA-induced inhibition was also abolished by treatment with BoNT/A, which cleaves the C-terminal nine amino acids of SNAP-25, and also by a SNAP-25 C-terminal-blocking peptide containing the BoNT/A cleavage site. These data indicate that inhibition of exocytosis by NA is downstream of increased [Ca²⁺]_i and is mediated by an interaction between Gβγ and the C-terminus of SNAP-25, as is the case for inhibition of neurotransmitter release. Remarkably, in the course of this work, a novel effect of NA was discovered. NA induced a marked retardation of the rate of refilling of the readily releasable pool (RRP) of secretory granules. This retardation was specifically abolished by a Gα_i1/2 blocking peptide demonstrating that the effect is mediated via activation of Gα_i1 and/or Gα_i2.

Introduction

In pancreatic β-cells, a variety of inhibitory agonists, such as NA, somatostatin and PGE₁ reduce insulin secretion via activation of G protein-coupled receptors (Sharp, 1996). This inhibition of insulin release is sensitive to pertussis toxin (PTX), indicating the involvement of heterotrimeric G_i and/or G_o proteins (Katada & Ui, 1979; Komatsu et al. 1995a). The mechanisms of G_i/G_o-mediated inhibition include the activation of K_ATP channels and inhibition of adenylyl cyclase, and a so-called ‘distal action’ at a point late in stimulus–secretion coupling (Sharp, 1996; Lang, 1999). The latter, which occurs downstream of increased [Ca²⁺]_i and blocks exocytosis per se, is the most powerful of the individual inhibitory mechanisms (Sharp et al. 1989; Drews et al. 1994; Komatsu et al. 1995b). Studies on the mechanism of serotonin-mediated inhibition of the presynaptic synapse in the lamprey (Blackmer et al. 2001; Gerachshenko et al. 2005) and on permeabilized PC12 cells (Blackmer et al. 2005) have shown that Gβγ subunits bind to synaptosomal-associated protein 25 (SNAP-25) and impair vesicle fusion mediated by the SNARE (‘SNAP (soluble NSF attachment protein) receptors’) complex. However, in insulin-secreting cells it has been proposed that the protein phosphatase calcineurin mediates the distal inhibition (Renstrom et al. 1996). In view of the evidence that the inhibition of neurotransmitter release downstream of increased [Ca²⁺]_i is due to the interaction of Gβγ with the SNARE complex and blockade of synaptotagmin binding, we investigated the possibility that this mechanism was also operative in the β-cell. In doing so, we found that Gβγ is indeed the mediator of the distal effect and that inhibition is due to a reduction in the number of exocytotic events without any change in vesicle size. Moreover, we also uncovered a novel effect of noradrenaline, namely to slow the refilling of the RRP.

Methods

Cell culture

INS 832/13 beta-cells (a kind gift of Dr C. B. Newgard) were cultured in complete RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 μg ml⁻¹ streptomycin, and 100 U ml⁻¹ penicillin at 37°C in a 95% air–5% CO₂ atmosphere. The cells (passage numbers 60–66) were divided once a week by treatment with trypsin and the medium was changed twice between divisions. The measurements were performed 1–2 days after cell plating.

Whole-cell patch-clamp and capacitance measurements

Data were acquired using a PULSE 8.75-controlled EPC-10 amplifier (HEKA electronics). Exocytosis was elicited by voltage-clamp depolarizations (from −70 mV to +10 mV, the pulse duration and pulse frequencies varied according to the different stimulation protocols) and detected as changes in cell capacitance estimated by the Lindau–Neher technique as implemented by the ‘Sine+DC’ feature of the lock-in software module (sine wave stimulus: 500 Hz, 40 mV peak-to-peak amplitude, DC-holding potential −70 mV). The membrane capacitance (C_m) was analysed with the customized IgorPro routines (WaveMetrics). The standard extracellular solution contained (in mm): 120 NaCl, 20 TEA-Cl, 2.6 CaCl₂, 5.6 KCl, 1.2 MgCl₂, 10 glucose and 10 Hepes-NaOH (pH 7.4). The pipette solution contained (in mm): 145 caesium glutamate, 8 NaCl, 0.18 CaCl₂, 0.28 BAPTA, 1 MgCl₂, 2 ATP-Mg, 0.5 GTP-Na₂, 0.3 cAMP, 10 Hepes-CsOH (pH 7.3), and 300 nm calculated free [Ca²⁺]_i. Na⁺ currents were blocked by 500 nm TTX. NA was freshly prepared before experiments and added into the extracellular solution at a final concentration of 5 μm. mSIRK (Calbiochem Corporation) was dissolved in DMSO and stored at −20°C as stock. The antibodies of Gβ and Gα_i,o,t,z,gust used in the studies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). The peptides containing the same sequences as the last 13 amino acids of the C-termini of Gα_i1/2, Gα_i3, Gα_o1, Gα_o2 and scrambled peptide (as control) were purchased from GenScript Corporation and had greater than 90% purity. They were dissolved in ddH₂O and stored at −20°C as stocks and added to the pipette solutions at a concentration of 60 μm on the day of the experiments. Botulinum toxin A (BoNT/A) light chain was purchased from List Biological Laboratories, Inc. It was dissolved in intracellular solution at a final concentration of 1 μm. Before stimulation, ∼5 min was allowed for the antibodies, peptides or BoNT/A light chain to reach the equilibrium between the pipette solution and cytosol. The recordings were performed at temperatures between 32 and 35°C.

Cell-attached capacitance measurements

After wax-coating and fire-polishing, the pipette tips had resistances of >1 MΩ when filled with pipette solution containing (in mm): 50 NaCl, 100 TEA-Cl, 5 KCl, 1 MgCl₂, 5 CaCl₂, 10 Hepes-NaOH (pH 7.3). The cells were plated on an 8 mm coverslip and continuously bathed in a ∼70 μl solution containing (in mm): 130 NaCl, 5 KCl, 1 MgCl₂, 5 CaCl₂, 10 glucose, and 10 Hepes-NaOH (pH 7.3). The holding potential 0 mV was added to the sine wave (50 mV r.m.s., 20 kHz) from the lock-in amplifier and fed into the stimulus input of the patch-clamp amplifier. The C-slow compensation of the EPC-7 (HEKA electronics) was set to 0.2 pF, the G-series compensation to 0.2 pS. The pipette current was filtered by the built-in 10 kHz filter of the patch-clamp amplifier and scaled down by a factor of 10 before input to the lock-in amplifier. The correct phase for the lock-in amplifier was found by utilizing a capacitance dither switch on the patch-clamp amplifier. At the correct phase, the lock-in amplifier computed the real (Re) and the imaginary part (Im) of the pipette current, which was recorded by the 16-bit ADC as Y1 and Y2, respectively. The output filter of the lock-in amplifier was set to 1 ms time constant, 24 dB octave⁻¹. After the recording, the calibration pulses were used to convert units of the Y1 and Y2 traces from raw counts into pS and fF, respectively. Individual vesicle capacitance step size values were converted to vesicle diameters assuming spherical geometry and a specific vesicle capacitance of 9 fF μm⁻².

Data analysis

To minimize the variations due to different cell sizes, all the ΔC_m values were normalized by the cell sizes (fF pF⁻¹). Data are presented as means ±s.e.m. Significance was determined either by Student's t test or by ANOVA followed by Fisher's least significant difference test as appropriate.

Results

Increased Ca²⁺ entry abolishes the inhibitory effects of NA on insulin exocytosis

In the whole-cell patch-clamp configuration (Lindau & Neher, 1988), exocytosis was stimulated by depolarizing pulses (from −70 mV to +10 mV) in INS 832/13 cells and was identified as the capacitance increase of cell membrane (ΔC_m). The NA-mediated inhibition of insulin secretion was evaluated by monitoring ΔC_m in response to varying pulse durations at a constant extracellular Ca²⁺ concentration ([Ca²⁺]_o) of 2.6 mm. The test cells were incubated in extracellular solution containing NA for ∼2–5 min before the recordings. The averaged traces of ΔC_m and I_Ca, evoked by depolarizing pulses with different durations (100, 300 and 500 ms) in control and NA-treated cells, are presented in Fig. 1A, B and C. Ca²⁺ currents were recorded while the capacitance measurements were interrupted and Ca²⁺ entry was quantified by the Ca²⁺ current integral. The absolute values of ΔC_m (fF) and of charge integrals (pQ) were normalized by cell size (pF) and defined as fF pF⁻¹ and pQ pF⁻¹, respectively. When pulses of increasing duration were used to stimulate the cells, such that Ca²⁺ entry was progressively increased, a parallel increase in exocytosis was observed. As shown in the statistical analysis (Fig. 1D, upper panel), with a pulse duration of 100 ms (Fig. 1A), NA exhibited strong inhibition of exocytosis (control, 8.5 ± 0.6 fF pF⁻¹, n = 41 cells; NA, 2.8 ± 0.3 fF pF⁻¹, n = 30 cells, P < 0.01, ∼67% inhibition). With a longer pulse of 300 ms (Fig. 1B) a larger increase in exocytosis occurred but the inhibitory effect of NA was significantly reduced (control, 25.9 ± 2.9 fF pF⁻¹, n = 15 cells; NA, 18.7 ± 1.9 fF pF⁻¹, n = 14 cells, P < 0.05, ∼28% inhibition). When the pulse duration was prolonged up to 500 ms, such that Ca²⁺ entry was further increased, the ΔC_m in control cells was 31.6 ± 2.2 fF pF⁻¹ (n = 34 cells), 3.7-fold greater than that seen with the 100 ms pulse (P < 0.01) and significantly greater than that of the 300 ms pulse (P < 0.05) (Fig. 1D). However, with the 500 ms pulse, NA-induced inhibition of exocytosis was abolished (NA, 27.4 ± 2.0 fF pF⁻¹, n = 28 cells, n.s.). Ca²⁺ influx was strongly increased by the pulses of longer duration but was not affected by NA (Fig. 1D, lower panel) (control/NA, 100 ms: 5.5 ± 0.3/5.2 ± 0.4 pQ pF⁻¹, n.s.; 300 ms: 12.5 ± 2.5/12.4 ± 2.3 pQ pF⁻¹, n.s.; 500 ms: 17.8 ± 2.7/16.6 ± 2.4 pQ pF⁻¹, n.s.). These results indicate that the inhibitory effect of NA on insulin secretion can be counteracted by a high level of Ca²⁺ entry. The effect of NA on exocytosis was mediated by activation of G_i/G_o proteins, since pretreatment of the cells with PTX (150 ng ml⁻¹, >24 h) abolished the inhibitory effects (PTX, 9.1 ± 1.1 fF pF⁻¹, n = 12 cells; PTX+NA, 8.8 ± 0.9 fF pF⁻¹, n = 11 cells, n.s.) (Fig. 1E).

Figure 1. Increasing the Ca²⁺ influx by the prolongation of the depolarizing pulse can alleviate and/or abolish the inhibitory effects of NA on exocytosis.

Using standard whole-cell capacitance measurements, the exocytosis elicited by depolarizing pulses (from −70 mV to +10 mV) with various pulse durations was monitored in control and hormone-treated cells. During the depolarizing pulses, the Ca²⁺ currents were recorded simultaneously. A, B and C, the recording traces of capacitance and I_Ca in control and NA-treated cells stimulated with 100, 300 and 500 ms pulses, respectively. D, the summaries of capacitance changes (upper) and calcium influxes (lower) in A, B and C, control (filled columns) and NA (open columns). E, the summaries of the capacitance changes on PTX-treated cells. In A, B and C, the arrows indicate I_Ca, C_m and the different experimental conditions. The traces shown in the figure were averaged from 14–41 cells. In D and E the values of ΔC_m and charge integrals were normalized by cell size (fF pF⁻¹ and pQ pF⁻¹). *P < 0.05, **P < 0.01, #n.s.

In similar experiments designed to increase Ca²⁺ influx by increasing [Ca²⁺]_o at a fixed pulse duration of 100 ms, NA-induced inhibition of exocytosis was apparent at 1.1 mm (control, 4.7 ± 0.8 fF pF⁻¹, n = 11 cells; NA, 1.6 ± 0.3 fF pF⁻¹, n = 14 cells, P < 0.05) and 2.6 mm[Ca²⁺]_o (control, 7.0 ± 0.6 fF pF⁻¹, n = 27 cells; NA, 2.7 ± 0.4 fF pF⁻¹, n = 32 cells, P < 0.01) (Fig. 2A, B and D), but not at 10 mm[Ca²⁺]_o (control, 10.2 ± 1.5 fF pF⁻¹, n = 18 cells; NA, 9.0 ± 1.0 fF pF⁻¹, n = 19 cells, n.s.) and there was no effect of NA on Ca²⁺ influx (Fig. 2C and D) demonstrating again that a high Ca²⁺ influx blocks the inhibitory effect of NA.

Figure 2. The inhibition of depolarization-evoked exocytosis by NA can also be abolished by increasing [Ca²⁺]_o.

Under the standard whole-cell configuration, the exocytosis elicited by depolarizing pulses (from −70 to +10 mV) was monitored in control and NA-treated cells. A, B and C, control and NA-treated cells were stimulated with 100 ms pulses at [Ca²⁺]_o of 1.1, 2.6 and 10 mm, respectively. Significant inhibition of exocytosis by NA was observed when [Ca²⁺]_o was 1.1 or 2.6 mm. The inhibition was abolished when [Ca²⁺]_o was increased to 10 mm. The data in A, B and C are summarized in D, control (filled columns) and NA (open columns). The traces shown in this figure were averaged from 11–32 cells, and the arrows indicate the different experimental conditions. The values of ΔC_m and charge integrals were normalized by cell size. *P < 0.05, **P < 0.01, #n.s.

NA inhibits insulin secretion by reducing the number of exocytotic events, without changing the size of exocytotic vesicles

To determine whether the change of capacitance was due to a decrease in exocytotic vesicle size or due to a reduction in the number of exocytotic events, cell-attached capacitance measurements were performed (Debus & Lindau, 2000). The cells were incubated in the extracellular solution containing 5 μm NA or without NA for ∼5 min before the pipette was sealed onto a membrane patch, and the capacitance was recorded for ∼10 min. Upward capacitance steps indicating individual exocytotic events were detected (see example in Fig. 3A, Im). Narrow, low conductance fusion pores lead to transient increases in the real part of the patch admittance (Fig. 3A, Re), which are evident for the larger events (e.g. Fig. 3A, left trace) but were usually undetectable for small events (e.g. Fig. 3A, right), presumably due to the low signal-to-noise ratio of these events, or due to a very rapid (<4.5 ms) increase of fusion pore conductance beyond our detection limit (Debus & Lindau, 2000). The mean step size and derived mean diameter of vesicles with a detectable change in the Re trace was ∼0.8 fF (control, 0.81 ± 0.08 fF/172.2 ± 8.3 nm, 21.7% of the total events; NA, 0.82 ± 0.08 fF/174.1 ± 9.3 nm, 22.0%). The capacitance steps lacking a detectable transient in the Re trace had a mean size of ∼0.5 fF (control, 0.52 ± 0.08 fF/137.6 ± 8.5 nm, 78.3%; NA, 0.53 ± 0.07 fF/138.9 ± 7.6 nm, 78.0%). NA reduced the number of exocytotic events per cell by ∼65% (Fig. 3B), consistent with the ∼70% inhibition observed in whole-cell recordings (Fig. 1A). The frequency distributions of exocytotic capacitance step size (Fig. 3C), as well as the mean values of the vesicle size (Fig. 3D) were similar in control and NA-treated conditions (control, 0.58 ± 0.10 fF/145.2 ± 11.2 nm, n = 318 events; NA, 0.59 ± 0.09 fF/146.7 ± 10.5 nm, n = 227 events, n.s.). Thus, inhibition of exocytosis by NA is due to a decrease in the number of events without a significant change in vesicle size and affects exocytotic vesicles of different sizes similarly. The diameter of most large dense core vesicles is >60 nm, and the corresponding capacitance step sizes are >0.1 fF (MacDonald et al. 2005), which is threefold larger than the peak-to-peak noise of our cell-attached capacitance measurements. The mean vesicle size of the control cells was slightly larger than that reported by MacDonald et al. The difference is probably due to the different lock-in amplifier filter applied. The lock-in amplifier setting used here (1 ms) allows a higher time resolution at the expense of a twofold higher recording noise. Therefore, the synaptic-like microvesicles, which produce small (0.01–0.1 fF) capacitance steps were not reliably detected due to the capacitance noise. Consequently, they were not included in the analysis here. However, the fast filter allowed us to detect the kinetics of the fusion pore. In cell-attached capacitance measurement, the fusion pore conductance and fusion pore lifetime provide information on fusion pore properties (Fang et al. 2008). As summarized from large events (Fig. 3E and F), control and NA-treated cells show similar mean values of fusion pore duration (control, 36.5 ± 7.8 ms, n = 69 events; NA, 40.1 ± 8.2 ms, n = 50 events, n.s.) and of the initial fusion pore conductance (control, 0.28 ± 0.06 nS, n = 69 events; NA, 0.25 ± 0.08 nS, n = 50 events, n.s.). These data indicate that the fusion pore properties are not modulated by NA.

Figure 3. NA reduces the number of exocytotic events in INS-832/13 cells, without affecting vesicle size or fusion pore properties.

A, two types of individual exocytotic events were detected using cell-attached capacitance measurements, the small event without detectable fusion pore conductance change (right panel), and the large event with detectable fusion pore conductance change (G_p; left panel). C_v, vesicle capacitance. B, summary of the number of exocytotic events occurring in control and NA-treated cells. C and D, the size distributions of exocytotic vesicles calibrated to the vesicle diameters, and the mean values of vesicle size, respectively, in control and NA-treated cells. E and F, summaries of the fusion pore duration and initial fusion pore conductance, respectively. *P < 0.05, **P < 0.01, #n.s.

Inhibition of exocytosis by NA is mediated by Gβγ subunits

To test the possibility that in the β-cell the distal inhibition of exocytosis is mediated by Gβγ subunits, we used the cell-permeable myristoylated βγ-binding peptide mSIRK that promotes G protein subunit dissociation to release free βγ subunits without activating the α subunits in intact cells (Goubaeva et al. 2003). In the whole-cell configuration, cells pretreated with mSIRK (30 μm, ∼30 min) exhibited ∼65% reduction of exocytosis (Fig. 4Aa and b), similar to that produced by NA (Fig. 1A) under the same stimulation protocol with 100 ms pulses (Fig. 4Ab, control, 10.7 ± 0.9 fF pF⁻¹, n = 23 cells; mSIRK, 3.9 ± 0.3 fF pF⁻¹, n = 25 cells, P < 0.01). Moreover, the inhibition induced by NA or mSIRK alone was not further enhanced by the combination of the two (Fig. 4Ab, NA+mSIRK, 4.0 ± 0.4 fF pF⁻¹, n = 25 cells, n.s.). To test the hypothesis that NA and mSIRK share the same signalling pathway in the regulation of exocytosis, polyclonal antibodies raised against Gβ subunits (anti-Gβ) were added to the pipette solution and dialysed into the cells. Consistent with the hypothesis, the inhibition of exocytosis in NA-treated cells was almost abolished in the presence of anti-Gβ (Fig. 4Ba). In contrast, polyclonal antibodies raised against Gα subunits of heterotrimeric G_i,o,t,z,gust proteins (anti-Gα_common) had no effect on the NA-induced inhibition (Fig. 4Ba). As summarized in Fig. 4Bb, the inhibition induced by NA was abolished by anti-Gβ, but neither by anti-Gα_common nor by other non-specific antibodies, indicating that the inhibition of exocytosis by NA is mediated by Gβγ subunits (control, 8.5 ± 0.6 fF pF⁻¹, n = 41 cells; NA+anti-Gβ, 7.5 ± 0.5 fF pF⁻¹, n = 38 cells, n.s.; NA+anti-Gα_common, 2.8 ± 0.2 fF pF⁻¹, n = 26 cells, P < 0.01; NA+anti-goat serum, 3.0 ± 0.3 fF pF⁻¹, n = 22 cells, P < 0.05; NA+anti-rabbit serum, 2.8 ± 0.3, n = 20 cells, P < 0.01). Importantly, the inhibition induced by NA and mSIRK could both be overcome, when the Ca²⁺ influx was increased by prolongation of the stimulating pulses to 500 ms (Fig. 4Ca and b control, 31.6 ± 2.2 fF pF⁻¹, n = 34 cells; NA, 27.4 ± 2.0 fF pF⁻¹, n = 28 cells, n.s.; mSIRK, 32.3 ± 2.4 fF pF⁻¹, n = 25 cells, n.s.) further supporting the conclusion that NA and mSIRK act by similar mechanisms.

Figure 4. Gβγ subunits are involved in the inhibitory effect of NA on exocytosis.

The stimulating protocols applied in A and B are the same as that performed in Fig. 1A. A, averaged traces of ΔC_m (a) recorded under the conditions indicated, and the summaries (b) normalized by cell size. B, averaged traces of ΔC_m (a) recorded under the conditions indicated. In the presence of NA, either anti-Gβ or anti-Gα_common was added to the pipette solutions. To exclude non-specific effects, anti-Gβ and anti-Gα_common were replaced by IgG (not shown). All the analyses are summarized in Bb (normalized by cell size). C, averaged traces of ΔC_m (Ca) evoked by a single 500 ms depolarizing pulse (from −70 mV to +10 mV) under the conditions indicated, and the summaries (Cb) normalized by cell size. *P < 0.05, **P < 0.01, #n.s. To exclude possible steric effects of IgG and non-specific effects of anti-Gα_common/anti-Gβ, the studies were controlled by testing the effects of anti-goat IgG, anti-rabbit IgG, anti-Gα_common and anti-Gβ alone, in the absence of NA. No effects were observed (data not shown).

NA inhibits Ca²⁺-evoked exocytosis via an interaction between Gβγ subunits and the C-terminus of SNAP-25

The Ca²⁺-dependent binding of synaptotagmin to the C-terminal domain of SNAP-25 is essential for Ca²⁺-triggered exocytosis of dense core vesicles (Zhang et al. 2002). This binding is competitively blocked by the binding of Gβγ subunits to the SNARE complex (Blackmer et al. 2005; Gerachshenko et al. 2005). BoNT/A cleaves off nine amino acids at the C-terminus of SNAP-25 (Binz et al. 1994) rendering it incapable of interacting with synaptotagmin. When BoNT/A light chain (1 μm) was included in the pipette solution and dialysed into the cells, exocytosis in response to a 100 ms stimulation pulse was reduced by 40% (control, 10.7 ± 0.9 fF pF⁻¹, n = 23 cells; BoNT/A, 6.5 ± 0.4 fF pF⁻¹, n = 37 cells, P < 0.05) but not completely abolished, consistent with other reports (Xu et al. 1998; Blackmer et al. 2005). However, when SNAP-25 was cleaved by BoNT/A, NA-induced inhibition was abolished (Fig. 5Aa and b; NA, 2.8 ± 0.2 fF pF⁻¹, n = 33 cells, P < 0.01; BoNT/A+NA, 6.8 ± 0.7 fF pF⁻¹, n = 25 cells, P < 0.05). In the presence of BoNT/A, there was no difference in exocytosis between NA-treated and -untreated cells, while BoNT/A reduced exocytosis compared to control cells. Exocytosis in BoNT/A+NA-treated cells was still higher than in cells treated with NA alone. These results indicate that Gβγ interacts with the C-terminus of SNAP-25 to mediate the NA-induced inhibition of exocytosis. If the C-terminus of SNAP-25 represents the target for Gβγ, then peptides mimicking this binding region should block NA-mediated inhibition. To test this prediction, a 14 amino acid peptide containing the BoNT/A cleavage site in the C-terminus of SNAP-25 (SNAP-25^193−206, 60 μm) was added to the intracellular solution and dialysed into the cells (Fig. 5B). In the presence of SNAP-25^193−206, the amplitude of evoked ΔC_m was 9.2 ± 0.9 fF pF⁻¹ (n = 17 cells), similar to that in the presence of a scrambled control peptide with the amino acids 193–206 in random order (10.2 ± 1.0 fF pF⁻¹, n = 19 cells) and to that of control cells (Fig. 5Ab). However, in the presence of SNAP-25^193−206, NA-induced inhibition was reduced to ∼16% (SNAP-25^193−206+NA, 7.7 ± 0.7 fF pF⁻¹, n = 26 cells, P < 0.05), but was unchanged from its normal value when the scrambled peptide was used (Fig. 5Bb, NA+scrambled peptide, 3.3 ± 0.3 fF pF⁻¹, n = 24 cells, P < 0.01).

Figure 5. The inhibition of exocytosis by NA can be attenuated by BoNT/A and by a synthesized peptide mimicking the C-terminus of SNAP-25.

A and B, the traces of ΔC_m (Aa, Ba) were measured under the conditions indicated, and the corresponding analyses (Ab, Bb) normalized by cell size. *P < 0.05, **P < 0.01, #n.s.

NA retards the refilling of the RRP via Gα_i1/2 subunits

As shown in Figs 1A and 5A, exocytosis stimulated by a single depolarizing pulse of 100 ms was inhibited by NA and the inhibition was largely abolished by anti-Gβ (Fig. 4B). However, when exocytosis was triggered by a pulse train, the control and NA+anti-Gβ-treated cells did still exhibit an obvious difference in ΔC_m (Fig. 6A), indicating that under these conditions antibodies against Gβ did not fully abolish the NA-induced inhibition of exocytosis. As shown, the amplitudes of ΔC_m elicited by the 1st pulse in control and NA+anti-Gβ-treated cells were very similar as before. However, a strong reduction of exocytosis was observed in NA+anti-Gβ-treated cells in response to the subsequent stimuli (Fig. 6A). Only when the NA-treated cells were stimulated in the presence of both anti-Gβ and anti-Gα_common the capacitance trace responding to the whole pulse train closely resembled the trace in control cells. The cumulative ΔC_m, evoked by the 15 individual pulses, were added up (ΔC_{m total}) and quantitatively analysed in Fig. 6B. The average ΔC_{m total} in control cells was 29.1 ± 1.6 fF pF⁻¹ (n = 41 cells) and was significantly reduced by NA in the presence of NA+anti-Gβ (17.4 ± 0.9 fF pF⁻¹, n = 38 cells, P < 0.01), and NA+anti-goat and NA+anti-rabbit groups (10.2 ± 1.0 fF pF⁻¹ and 11.3 ± 1.4 fF pF⁻¹, n = 22 and 18 cells, respectively, P < 0.01 for both). In contrast, NA did not significantly inhibit cumulative exocytosis stimulated by the pulse train when both antibodies were present NA+anti-Gβ+anti-Gα_common (27.4 ± 2.5 fF pF⁻¹, n = 38 cells, n.s.). Exocytosis in response to the later pulses depends not only on stimulation and release from the RRP but also on the rate at which the RRP is refilled. To estimate the size of the RRP, we applied the dual stimulation protocol (Gillis et al. 1996), consisting of two 250 ms pulses separated by a 100 ms interpulse interval (Fig. 7A). The short interpulse interval is required to minimize refilling of the pool between the first and second stimulations. In this protocol, pool depletion due to the first stimulation (ΔC_m1) is manifest when the response to the second pulse (ΔC_m2) is small, as is the case in Fig. 7A. In 23 cells, the mean normalized RRP size was 34.1 ± 3.2 fF pF⁻¹. As the average exocytosis stimulated by a single 500 ms pulse was 31.6 ± 2.2 fF pF⁻¹ (Fig. 1C), this single 500 ms depolarizing pulse is sufficient to essentially deplete the RRP in INS 832/13 cells. The fact that in NA-treated cells the ΔC_m stimulated by a 500 ms pulse was unchanged compared to controls (Fig. 4C) means that the size of the RRP in NA-treated cells at the start of the recordings was unchanged by the treatment.

Figure 6. Effects of anti-Gβ and anti-Gα_common antibodies on NA-induced inhibition of exocytosis due to a depolarizing pulse chain.

A, the cells in different experimental groups responded differentially to the depolarizing pulse train (100 ms pulse duration from −70 mV to +10 mV, 300 ms pulse interval). B, analysis of ΔC_m responses in A to the 1st and total pulses, respectively. To exclude possible steric effects of IgG and non-specific effects of anti-Gα_common/anti-Gβ, the studies were controlled by testing the effects of anti-goat IgG, anti-rabbit IgG, anti-Gα_common and anti-Gβ alone, in the absence of NA. No effects were observed (data not shown). Data similar to these for control and NA-treated cells were obtained when the experiments were repeated under the perforated patch configuration.

Figure 7. Measurement of the size of the RRP and its refilling rates in the presence of blocking peptides specific for the Gα_i and Gα_o proteins.

A, the averaged capacitance traces obtained in control cells by using a dual-pulse protocol (two 250 ms pulses with an interval of 100 ms). The RRP size was estimated according to the equation, . B, in different cell groups, the refilling rates of the RRP were estimated by using a dual-pulse protocol with various intervals from 1 to 40 s. The stimulatory pulses from −70 mV to +10 mV were 500 ms in duration in order to block the inhibitory effect of NA on exocytosis and to release all the granules present in the RRP. The ratios of 2nd ΔC_m/1st ΔC_m were plotted versus the pulse intervals, as shown. Studies were performed under control and NA-treated conditions, and under NA-treated conditions with the pipette solutions containing different blocking peptides as indicated. The scrambled peptide (Gir) was applied as a control. *P < 0.05, **P < 0.01, #n.s.

To measure the rates of pool refilling directly, a double pulse protocol with different interpulse intervals was applied. Because a single 500 ms pulse (from −70 mV to +10 mV) is capable of depleting the RRP and abolishes the direct inhibitory effect of NA on exocytosis, the duration of the stimulation in the dual-pulse protocol was set to 500 ms. The 2nd pulse was applied after different intervals from 1 to 40 s. The ΔC_m resulting from the 1st (ΔC_m1) and the 2nd (ΔC_m2) depolarizing pulses were determined. In Fig. 7B the ratio of ΔC_m2/ΔC_m1 is plotted versus the corresponding interpulse intervals and expressed as a percentage of the initial pool size. The refilling of the RRP in control cells increased from less than 20% after 1 s to ∼90% after 40 s. In the presence of NA the refilling rate was markedly reduced such that the pool refilled only to ∼5% after 1 s and ∼50% after 40 s. To determine if NA treatment impairs the refilling of the RRP via Gα_i/o, synthetic peptides containing the last 11 amino acids of the α-subunits of G_i/o proteins were tested. These peptides are known to block the interaction between G proteins and their activated receptors (Gilchrist et al. 2002) and we have used them successfully previously (Zhao et al. 2008). When the blocking peptide for Gα_i1/2 was included in the pipette solution the rate of recovery for NA-treated cells was similar to that of the control cells. In contrast, in the presence of the blocking peptides for Gα_o1, Gα_o2, Gα_i3 or the scrambled peptide G_ir, the recovery at each time point was similarly inhibited by NA treatment as in the absence of any peptides (Fig. 7B). These results show that refilling of the RRP was retarded by NA via activation of Gα_i1/2.

Discussion

Physiological inhibitors of insulin secretion such as NA, acting on the α₂-adrenergic receptor, have multiple effects on their target cells. These effects, exerted on ion channels, enzymes and exocytosis, were widely thought to be mediated by the PTX-sensitive heterotrimeric G_i/G_o proteins (Komatsu et al. 1995a; Sharp, 1996) until the recent demonstration of PGE₁-induced inhibition of adenylyl cyclase via the PTX-insensitive G_Z (Kimple et al. 2008). In the endocrine pancreatic β-cell, the overall effect of NA is to inhibit stimulated insulin secretion and its potentiation by agents such as GLP-1 that act by raising cyclic AMP levels. This is achieved by activation of K⁺ channels including the important K_ATP channel (Zhao et al. 2008), inhibition of adenylyl cyclase (Wiedenkeller & Sharp, 1983) and inhibition of exocytosis per se (Sharp, 1996). The latter is usually referred to as the distal inhibitory effect. Additionally, there are reports that in some clonal cell lines the inhibitors can directly reduce the activity of the L-type Ca²⁺ channels (Homaidan et al. 1991; Hsu et al. 1991). This has not been observed in primary β-cells, nor is it seen in the INS 832/13 cell line that we have used here. We show now that NA has two distinct distal inhibitory effects, a direct inhibition of exocytosis from the RRP and a slowing of the refilling of the RRP.

NA inhibition of exocytosis is mediated by Gβγ/SNAP-25 interaction

The direct effect of NA to inhibit the release of insulin is due to a decreased number of exocytotic events without a change in vesicle size or fusion pore properties. This indicates that the inhibitory mechanism is exerted at a site common to the mechanisms of exocytosis of the granules following a stimulatory increase in [Ca²⁺]_i. In previous studies on the distal inhibitory effect it was suggested that the inhibition was mediated by activation of the protein phosphatase calcineurin. The basis for this was that deltamethrin, a well-known inhibitor of calcineurin, blocked the inhibitory effect of somatostatin and the α₂-adrenergic agonist clonidine (Renstrom et al. 1996). Similarly, in the same study, a calcineurin auto-inhibitory peptide corresponding to amino acids 457–482 (the calmodulin-binding domain) blocked the inhibitory effect of somatostatin. These results suggested that the mechanism of distal inhibition was due to calcineurin-mediated dephosphorylation of a protein critical for exocytosis. However, studies on a similar distal mechanism, inhibition of neurotransmitter release downstream of elevated [Ca²⁺]_i demonstrated that Gβγ is involved and provided a convincing mechanism of action, i.e. Gβγ binding to the C-terminus of SNAP-25 and competitive blockade of the interaction between the calcium sensor protein synaptotagmin and SNAP-25 on the SNARE complex, thus inhibiting exocytosis (Blackmer et al. 2001, 2005; Gerachshenko et al. 2005). Therefore, we looked into the possibility that the same mechanism was present in the β-cell. We found that anti-Gβ antibodies blocked NA inhibition of exocytosis and also that the βγ-activating peptide mSIRK inhibited exocytosis to the same extent as NA. Furthermore, the combination of NA and mSIRK caused no additional inhibition relative to either of them alone and the inhibitory effects of both were overcome by increased Ca²⁺ influx. These results provide strong evidence that both NA and mSIRK act by releasing Gβγ. Further, BoNT/A, which cleaves off the C-terminus of SNAP-25 prevented NA from inhibiting exocytosis, and a C-terminal SNAP-25 peptide, that contains the BoNT/A cleavage site also blocked the inhibitory effect of NA. These results strongly suggest that NA inhibits exocytosis by activating Gβγ and binding of the released Gβγ to the C-terminus of SNAP-25. This would be similar to the inhibition of neurotransmitter release and is not in accord with the idea that calcineurin is involved in the distal inhibition of exocytosis.

Stimulation of exocytosis in insulin-secreting cells involves not only the SNARE proteins but also synaptotagmin (Gauthier & Wollheim, 2008), complexin (Abderrahmani et al. 2004), and other proteins. It has recently been proposed that complexin interacts with the C-terminal part of the SNARE domains, thereby activating and also clamping the SNARE complex such that synaptotagmin can reverse the clamping function upon Ca²⁺ binding (Maximov et al. 2009). Release of the clamping function is followed by zippering of the SNARE domains and a force transfer to the membranes that lead to fusion. The Gβγ activated by NA inhibits exocytosis via its interaction with the SNAP-25 C-terminus. Gβγ may thus prevent complexin from activating the SNARE complex, inhibit the synaptotagmin/SNAP-25 interaction that releases the clamping function, interferes with the force transfer to the membranes, the C-terminal zippering and fusion pore formation. It seems likely that Gβγ could interfere with all these steps due to its binding to SNAP-25.

The inhibition of exocytosis by NA in response to a 100 ms depolarizing pulse is substantial but not complete. When exocytosis is stimulated by a 500 ms pulse, generating larger Ca²⁺ influx and extending the duration of the Ca²⁺ rise, NA-induced inhibition is abolished, as is inhibition by release of Gβγ by mSIRK. Thus, all the granules in the RRP can undergo exocytosis even in the presence of the released Gβγ, if the Ca²⁺ stimulus is sufficiently high. As reported, in the presence of a very high [Ca²⁺]_i, synaptotagmin is able to compete successfully with Gβγ for SNAP-25 (Blackmer et al. 2005; Gerachshenko et al. 2005). Fusion of a secretory vesicle involves activation of multiple SNARE complexes although the number of SNARE complexes required for a single fusion event is not known. It thus seems likely that some fraction of the available SNARE complexes is rendered inactive by Gβγ binding but that in response to a sufficiently large Ca²⁺ stimulus a sufficient number of SNARE complexes can still be activated by synaptotagmin such that fusion of the vesicle is fully enabled.

NA inhibits refilling of the RRP via Gα_i1/2

In addition to the direct inhibition of exocytosis via Gβγ, NA retarded the rate of refilling of the RRP. This provides a possible explanation for a previous report that high Ca²⁺ influx (using 500 ms depolarizing pulses from −70 mV to 0 mV) did not block the effects of inhibitors (Renstrom et al. 1996) as we have observed here with a slightly larger depolarization. As their method was essentially a dual-pulse protocol (depolarization under control conditions followed by treatment with somatostatin and a second depolarization), the reduced response to somatostatin seen and interpreted as inhibition of exocytosis may reflect retardation of refilling of the granule pool after its depletion by the first pulse. The recruitment and priming of secretory granules from reserve pools into RRPs is needed to enable exocytosis to occur following discharge of the RRP (Bittner & Holz, 1992; Neher & Zucker, 1993; Straub & Sharp, 2004). The NA-induced retardation of RRP refilling is inhibited by a Gα_i1/2 blocking peptide but not by blocking peptides for Gα_o1, Gα_o2, Gα_i3 or the scrambled G_ir, indicating that the retardation of RRP refilling is mediated by Gα_i1, Gα_i2, or both. Regulation of the RRP size is complex. Several signalling pathways can influence the RRP, apparently by altering the rate of recruitment of vesicles. Activation of protein kinases such as cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) modulate Ca²⁺-triggered exocytosis from both primary and clonal β-cells and thus provide mechanisms for hormonal regulation of insulin release through second messengers (Ammala et al. 1994; Jones & Persaud, 1998; Rosengren et al. 2002). PKA activation potentiates insulin secretion by increasing the total number of vesicles that are available for release (Renstrom et al. 1997; Rorsman et al. 2000). PKC activation has also been linked to priming of Ca²⁺-mediated insulin secretion (Eliasson et al. 1996; Efanov et al. 1997). PKA and PKC are also found to increase the size of a highly Ca²⁺-sensitive vesicle pool in INS-1 cells (Yang & Gillis, 2004). The direct interactions of PKA and PKC with the secretory machinery have also been suggested in other cell types, such as chromaffin cells and hippocampal neurons, where the size of RRP and its rate of replenishment is increased (Smith et al. 1998; Stevens & Sullivan, 1998). As one of the mechanisms underlying NA-mediated inhibition of insulin secretion is a decrease in cAMP levels and consequent reduction of PKA activity (Sharp, 1996), it is conceivable that this could account for the retarded refilling rate. However, in our experiments, the [cAMP]_i was buffered by the presence of cAMP in the intracellular (pipette) solution. Therefore, it is unlikely that Gα_i1/2 modified the kinetics of RRP refilling via inhibition of adenylyl cyclase (McDermott & Sharp, 1995) and interference with the cAMP–PKA signalling pathway (Kwan et al. 2007). While inhibition of PKC activity could be involved, there is no evidence in the literature for such an effect of NA. Similarly, while members of the Ras superfamily of GTPases also play a role in regulating the RRP (Ngsee et al. 1991; Bielinski et al. 1993; Mark et al. 1996; Geppert & Sudhof, 1998; Lonart & Sudhof, 2000; Polzin et al. 2002), there is no obvious connection to NA or Gα_i1/2. Thus the mechanism by which Gα_i1/2 impairs the refilling of the RRP remains to be elucidated.

Physiologically, it is of interest that NA inhibits both of the major pathways by which glucose induces biphasic insulin secretion. The first phase of glucose-stimulated release is due to the K_ATP channel-dependent or ‘triggering’ pathway that involves closure of K_ATP channels, depolarization of the β-cell, increased Ca²⁺ influx and increased [Ca²⁺]_i. This is blocked by the effect of NA to activate the K_ATP channel and thereby re-polarize the cell (Sharp, 1996). The second phase of release is due to the K_ATP channel-independent or ‘amplifying’ pathway and is caused by an increased rate of refilling of the RRP (Straub & Sharp, 2002, 2004). We have shown here for the first time that NA retards the refilling of this pool and that this retardation is mediated by activation of Gα_i1/2.

In summary, NA has two distal effects that result in the inhibition of insulin secretion. One is to inhibit exocytosis by βγ blockade of the binding of synaptotagmin to SNAP-25 in the SNARE complex. The other is to retard the refilling of the RRP.

Glossary

Abbreviations

BoNT/A

botulinum toxin A

Im

imaginary part of the patch admittance

mSIRK

myristoylated βγ-binding/activating peptide

NA

noradrenaline

PTX

pertussis toxin

Re

real part of the patch admittance

RRP

readily releasable pool

SNAP-25

synaptosomal-associated protein 25

SNARE

SNAP (soluble NSF attachment protein) receptors

Author contributions

Y.Z. and Q.F. contributed equally to this work. All authors contributed to the conception and design of the experiments and to the writing of the manuscript. They have all approved the final version for publication. The work was performed in the Department of Molecular Medicine and the School of Applied and Engineering Physics, Cornell University, Ithaca, NY 14853, USA.