Prostaglandin E1 inhibits endocytosis in the β-cell endocytosis

in Journal of Endocrinology
Authors:
Ying Zhao Department of Molecular Medicine, Cornell University, Ithaca, New York, USA
School of Applied and Engineering Physics, Cornell University, Ithaca, New York, USA
Laboratory for Nanoscale Cell Biology, Max-Planck-Institute for Biophysical Chemistry, Goettingen, Germany

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Qinghua Fang School of Applied and Engineering Physics, Cornell University, Ithaca, New York, USA
Laboratory for Nanoscale Cell Biology, Max-Planck-Institute for Biophysical Chemistry, Goettingen, Germany

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Susanne G Straub Department of Molecular Medicine, Cornell University, Ithaca, New York, USA

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Manfred Lindau School of Applied and Engineering Physics, Cornell University, Ithaca, New York, USA
Laboratory for Nanoscale Cell Biology, Max-Planck-Institute for Biophysical Chemistry, Goettingen, Germany

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Geoffrey W G Sharp Department of Molecular Medicine, Cornell University, Ithaca, New York, USA

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Prostaglandins inhibit insulin secretion in a manner similar to that of norepinephrine (NE) and somatostatin. As NE inhibits endocytosis as well as exocytosis, we have now examined the modulation of endocytosis by prostaglandin E1 (PGE1). Endocytosis following exocytosis was recorded by whole-cell patch clamp capacitance measurements in INS-832/13 cells. Prolonged depolarizing pulses producing a high level of Ca2+ influx were used to stimulate maximal exocytosis and to deplete the readily releasable pool (RRP) of granules. This high Ca2+ influx eliminates the inhibitory effect of PGE1 on exocytosis and allows specific characterization of the inhibitory effect of PGE1 on the subsequent compensatory endocytosis. After stimulating exocytosis, endocytosis was apparent under control conditions but was inhibited by PGE1 in a Pertussis toxin-sensitive (PTX)-insensitive manner. Dialyzing a synthetic peptide mimicking the C-terminus of the α-subunit of the heterotrimeric G-protein Gz into the cells blocked the inhibition of endocytosis by PGE1, whereas a control-randomized peptide was without effect. These results demonstrate that PGE1 inhibits endocytosis and Gz mediates the inhibition.

Abstract

Prostaglandins inhibit insulin secretion in a manner similar to that of norepinephrine (NE) and somatostatin. As NE inhibits endocytosis as well as exocytosis, we have now examined the modulation of endocytosis by prostaglandin E1 (PGE1). Endocytosis following exocytosis was recorded by whole-cell patch clamp capacitance measurements in INS-832/13 cells. Prolonged depolarizing pulses producing a high level of Ca2+ influx were used to stimulate maximal exocytosis and to deplete the readily releasable pool (RRP) of granules. This high Ca2+ influx eliminates the inhibitory effect of PGE1 on exocytosis and allows specific characterization of the inhibitory effect of PGE1 on the subsequent compensatory endocytosis. After stimulating exocytosis, endocytosis was apparent under control conditions but was inhibited by PGE1 in a Pertussis toxin-sensitive (PTX)-insensitive manner. Dialyzing a synthetic peptide mimicking the C-terminus of the α-subunit of the heterotrimeric G-protein Gz into the cells blocked the inhibition of endocytosis by PGE1, whereas a control-randomized peptide was without effect. These results demonstrate that PGE1 inhibits endocytosis and Gz mediates the inhibition.

Introduction

Prostaglandins inhibit insulin secretion in clonal β-cells (Robertson et al. 1987, Seaquist et al. 1989), rat pancreatic islets (D’Ocon et al. 1988), and human islets (Straub et al. 1998). Along with norepinephrine (NE), somatostatin, and several other agonists, prostaglandins inhibit the secretion of insulin by several mechanisms. These include (i) activation of K+ channels to repolarize or hyperpolarize the cell (Rorsman et al. 1991, Sharp 1996, Schermerhorn & Sharp 2000, Sieg et al. 2004, Dezaki et al. 2007), thereby reversing or preventing the opening of voltage-dependent Ca2+ channels; (ii) inhibition of adenylyl cyclase to prevent the augmentation of stimulated insulin release via incretins such as glucagon-like peptide 1 (Komatsu et al. 1995, Kimple et al. 2008); and (iii) they inhibit secretion by a ‘distal’ effect that occurs downstream of elevated [Ca2+]i that blocks exocytosis per se (Sharp et al. 1989, Ullrich & Wollheim 1989, Tang et al. 1995). This distal inhibition of exocytosis in β-cells is similar to that in the central nervous system where serotonin inhibits neurotransmitter release downstream of increased [Ca2+]i (Blackmer et al. 2001) and in pituitary lactotrophs where endothelin blocks prolactin release; also downstream of elevated [Ca2+]i (Andric et al. 2005). The mechanism involves Gβγ blockade of syntaxin binding to SNAP25 (Blackmer et al. 2005, Gerachshenko et al. 2005, Photowala et al. 2006). Although it was once believed that all physiological inhibitory effects on the β-cells were mediated by the Pertussis toxin-sensitive (PTX)-sensitive heterotrimeric Gi and Go proteins (Komatsu et al. 1995), it has been reported that inhibition of adenylyl cyclase by PGE1 is mediated via Gz (Kimple et al. 2005, 2008).

Exocytosis has to be followed by endocytosis to recapture the granule membrane that has been added to the plasma membrane of the cell in order to maintain cellular homeostasis and integrity (Rizzoli & Jahn 2007). The classification of the endocytotic mechanisms includes conventional endocytosis involving recruitment of clathrin and a group of adaptor proteins; rapid endocytosis, for example, by ‘kiss and run’; bulk endocytosis (Ales et al. 1999, Jahn et al. 2003, Harata et al. 2006, Rizzoli & Jahn 2007, Cheung et al. 2010); and excess endocytosis (Eliasson et al. 1996, Smith & Neher 1997). Endocytosis is Ca2+-dependent as has been shown in synaptic systems and neuroendocrine cells (Eliasson et al. 1996, Photowala et al. 2006, Rizzoli & Jahn 2007, Cheung et al. 2010), including β-cells (Jarousse & Kelly 2001, Murthy & De Camilli 2003, MacDonald et al. 2005, He et al. 2008). Here we demonstrate that PGE1 inhibits endocytosis in insulin-secreting cells by activating Gz.

Materials and methods

Cell culture

INS-832/13 cells, a kind gift from Dr C B Newgard, were cultured in complete RPMI-1640 medium supplemented with 10% fetal bovine serum, 100µg/mL streptomycin, and 100U/mL penicillin at 37°C in a 95% air/5% CO2 atmosphere.

Electrophysiological techniques

Standard whole-cell patch clamp recording was applied to monitor capacitance changes and Ca2+ currents. Patch pipettes were fabricated from borosilicate glass capillaries and had resistances of 2–3M& when filled with the intracellular solutions. Data were acquired using a PULSE 8.75-controlled EPC-10 amplifier (HEKA, Holliston, MA, USA). The change in cell capacitance was estimated by the Lindau–Neher technique (Lindau & Neher 1988) as implemented by the ‘sine+DC’ feature of the lock-in software module (500 Hz, 40 mV peak-to-peak amplitude, −70mV DC-holding nominal potential without liquid junction potential correction, estimated to be ≃18 mV for the solutions used here) and as used previously in β-cells (Zhao et al. 2010a). The current signals were digitized at 10 kHz. The data were analyzed with customized IGOR Pro routines (WaveMetrics, Inc., Lake Oswego, OR, USA). 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 Cs-glutamate, 8 NaCl, 0.18 CaCl2, 0.28 BAPTA, 1 MgCl2, 2 ATP-Mg, 0.5 GTP-Na2, 0.3 cyclic AMP and 10 HEPES-CsOH (pH 7.3), with 300nM calculated free [Ca2+]i. Knowing that PGE1 inhibits adenylyl cyclase (Kimple et al. 2008), excess cyclic AMP was included in the pipette solution to buffer and prevent any changes in cyclic AMP concentration from influencing the results. Na+ currents were blocked by 500nM TTX. All the solutions were freshly prepared before the experiments. The final extracellular concentration of PGE1 was 10µM. Before stimulation, ~5min were allowed after establishing the whole-cell configuration for intracellularly applied chemicals and peptides to reach equilibrium between the pipette solution and the cytosol. The synthesized peptides and PTX were purchased from GenScript (Piscataway, NJ, USA) and Sigma, respectively. The whole-cell cell membrane capacitance (Cm) recordings were performed at 32–35°C.

Statistical analysis

All values are presented as mean±s.e.m. Significance was determined either by ANOVA followed by Fisher’s least significant difference test or by Student’s t-test as appropriate.

Results

High Ca2+ influx abolishes the inhibitory effects of PGE1 on exocytosis

Using the whole-cell patch clamp configuration, exocytosis was stimulated by subjecting INS-832/13 cells to depolarizing pulses from –70 to +10mV and was measured as the capacitance increase of the cell membrane (ΔCm). The PGE1-mediated inhibition of insulin secretion was evaluated by monitoring the ΔCm in response to two different pulse durations with an extracellular 2.6mM Ca2+ concentration ([Ca2+]o). Test cells were incubated in the presence of the inhibitor for ~2–5min before the recordings. The averaged traces of ICa and ΔCm evoked by depolarizing pulses of 100 and 500 ms in control and PGE1-treated cells are presented in Fig. 1A and B, respectively. Ca2+ currents were recorded although the capacitance measurements were interrupted, and Ca2+ entry was quantified as the Ca2+ current integral. The data were normalized by cell size (pF) so that the absolute values of ΔCm (fF) and Ca2+ charge integrals (pQ) are converted to fF/pF and pQ/pF, respectively. As shown in the statistical analysis (Fig. 1C, left panel), with a pulse duration of 100 ms (Fig. 1A), PGE1 signifi­cantly inhibited exocytosis (control, 9.0±0.5fF/pF, n=30; PGE1, 3.3±0.3fF/pF, n=23, P<0.01, ~60% inhibition) as expected. When the pulse duration was prolonged to 500ms (Fig. 1B), such that Ca2+ entry was strongly increased, exocytosis in control cells was 3.3-fold greater than that induced by the 100 ms pulse, (30.3±2.6fF/pF, n=22) P<0.01. However, with the 500-ms pulse, PGE1-induced inhibition of exocytosis was abolished (PGE, Cm=31.9±2.3fF/pF, n=18, not significant (NS)). The summed Ca2+ influx, quantified by integrating the Ca2+ current, was unchanged by PGE1 (Fig. 1C, right panel) (control/PGE1, 100ms: 5.1±0.4/5.0±0.8 pQ/pF, NS; 500ms: 17.2±3.1/17.0±2.7 pQ/pF, NS). Thus, the inhibition of exocytosis by PGE1 was completely abolished by a high level of Ca2+ entry. It should also be pointed out that this single 500-ms pulse depletes the RRP of approximately 95% of its granules (Zhao et al. 2010a).

Figure 1
Figure 1

A high level of Ca2+ influx achieved by prolonging the depolarizing pulse abolishes the inhibitory effect of PGE1 on exocytosis. Using standard whole-cell capacitance measurements, the exocytosis elicited by depolarizing pulses (from −70 to +10mV) with two different pulse durations was monitored in control and hormone-treated cells. During the depolarizing pulses, the Ca2+ currents were recorded simultaneously. (A and B) The recording traces of ICa and capacitance in control and PGE1-treated cells stimulated with 100- and 500-ms pulses, respectively. The arrows indicate the different experimental conditions. (C) The summaries of capacitance changes (left) and calcium influxes (right) in A and B, control (black), and PGE1 (white). The traces shown in the figure were averaged from 18 to 30 cells. The values of ΔCm and Ca2+ charge integrals were normalized by cell size (fF/pF and pQ/pF). **P<0.01, #NS.

Citation: Journal of Endocrinology 229, 3; 10.1530/JOE-15-0435

Inhibition of endocytosis by PGE1

The abolition of the inhibitory effect of PGE1 on exocytosis by the high Ca2+ influx and the depletion of the RRP by the 500-ms depolarizing pulse (Zhao et al. 2010b) permitted us to investigate the effect of PGE1 on the endocytosis that follows exocytosis. The results are expressed in absolute terms and in rates. Control and PGE1-treated cells were stimulated by a short train of 500-ms pulses (five pulses, from −70 to +10mV with 300-ms interpulse intervals). The capacitance increases due to exocytosis evoked by the first pulses were large in both groups of cells (Fig. 2A). However, the capacitance increases responding to the subsequent pulses had much smaller amplitude because the first pulse depleted most of the vesicles in the RRP and the interpulse interval of 300ms was insufficient to allow RRP refilling (Zhao et al. 2010b). As a consequence, the subsequent capacitance changes can be equated completely with endocytosis. Following the stimulation of exocytosis, endocytosis was evident as a decrease of cell membrane capacitance (negative ΔCm). The endocytosis observed during the four interpulse intervals and during the 300-ms interval following the last of the five pulses was termed early endocytosis (ΔCm/early–endo shown with high time resolution in Fig. 2A (right)) and was quantified as the sum of the endocytosis occurring during the 300-ms intervals following the five individual stimulation pulses. After the pulse train, the subsequent endocytosis was continuously monitored for ~75s, while the cell membrane potential was clamped at –70mV (Fig. 2A, left). This was termed late endocytosis (ΔCm/late–endo). The late endocytosis was determined as the maximal decrease of cell membrane capacitance within the 75s period starting 300ms after the last stimulus pulse. Quantitative analysis revealed significant inhibition of both phases of endocytosis by PGE1 (Fig. 2B, ΔCm/early–endo: control, −28.6±3.0fF/pF, n=20; PGE1, −10.8±2.0fF/pF, n=19, P<0.05; ΔCm/late–endo: control, −63.7±7.34fF/pF; PGE1, −44.6±4.8fF/pF, P<0.05). Although both kinetic phases were reduced in amplitude, PGE1 inhibition of the early phase was more pronounced (~62%) than inhibition of the late phase (~30%).

Figure 2
Figure 2

PGE1 inhibits endocytosis. (A) A 500-ms pulse train with depolarizations from −70 to +10mV (with 300-ms interpulse intervals) followed by ~75s of recording with the cell membrane clamped at −70mV was applied in control and PGE1-treated cells. The entire recordings are presented on the left hand side of the figure, and the pulse-stimulating parts of the traces (marked by dashed box) are shown on the expanded timescale on the right. The arrow on the left indicates the resting cell capacitances before the pulse train. Arrows indicate the experimental conditions (PGE1, black; control, gray). On the left, horizontal lines indicate the starting and minimum levels for determination of the extent of late endocytosis (vertical arrows). The extent of early endocytosis was defined as ΔCm / early-endo = i=1 5 Δ Cm i using ΔCmi values as indicated on the right. (B) A summary of the effect of PGE1 on endocytosis (∆Cm normalized as fF/pF), respectively. (C) A summary of the rates of endocytosis. The early and late phases of endocytosis are indicated as white and gray, respectively. The traces in the figure were averaged from 18 to 21 cells. *P<0.05, **P<0.01.

Citation: Journal of Endocrinology 229, 3; 10.1530/JOE-15-0435

It can be seen in Fig. 2A (left) that the amplitude of negative ΔCm due to endocytosis greatly exceeded the amplitude of positive ΔCm of the preceding exocytosis (see control trace in Fig. 2A). Thus, excess endocytosis in addition to compensatory endocytosis occurs under these conditions. Excess endocytosis was previously described in chromaffin cells and insulin-secreting cells following a high level of Ca2+ influx (Eliasson et al. 1996, Smith & Neher 1997, Zhao et al. 2010a). Due to the excess membrane retrieval, the averaged traces show that the plasma membrane capacitance decreased to a value below the pre-stimulus baseline and remained there for the duration of the recordings. In chromaffin cells, Ca2+ entry with an integrated Ca2+ current of ~70–90 pC was the threshold for the stimulation of excess endocytosis (Eliasson et al. 1996, Smith & Neher 1997). The calculated Ca2+ influx in our study of ~200 pC (based on an analysis of the data in Fig. 1C and the average cell membrane capacitance of ~8pF) is well above the threshold that causes excess endocytosis in chromaffin cells.

PGE1 reduces the rates of endocytosis

In control cells, the capacitance trace appearing after the first pulse showed a brief phase of slight exocytosis preceding a subsequent phase of endocytosis. After the second pulse, only the endocytosis phase appeared at the end of the pulse. Therefore, the rate of early endocytosis was determined as the slope of the capacitance traces for the 300-ms period following the second pulse and normalized by cell size (−22.1±2.5fF/pF/s, n=20). Fitted with a decaying single exponential, the late endocytosis trace proceeded with a time constant (τ) of 4.1±0.4s, and the initial rate for the late endocytosis was estimated as the amplitude of retrieved membrane (ΔCm/late–endo) divided byτas −15.5±1.8fF/pF/s. In the presence of PGE1, the early endocytosis was not apparent until after the second pulse and showed a slower rate of −7.3±1.1fF/pF/s (n=19, P<0.01). Although the late phase proceeded with a similar time constant (τ=5.7±0.6s, NS), the initial rate of the late endocytosis was also lower in the cells pretreated with PGE1(−7.8±0.8fF/pF/s, P<0.05) because of the smaller amplitude of ΔCm/late–endo.

PGE1 inhibition of endocytosis is insensitive to PTX

As hormonal inhibition of insulin secretion is mediated mostly by PTX-sensitive heterotrimeric Gi and/or Go proteins (Sharp 1996), we examined the PTX sensitivity of the inhibition of endocytosis by PGE1. Batches of cells were used as controls or treated with PTX (150ng/mL, >24h) and examined for the inhibition of endocytosis. PTX pretreatment of the cells failed to block the effect of PGE1 on early or late endocytosis (Fig. 3A). The data are as follows (Fig. 3B): ΔCm/early–endo: PTX, −29.5±3.7fF/pF, n=17 (not significantly different from the controls); PTX+PGE1, −13.7±1.7fF/pF, n=18, P<0.05. ΔCm/late–endo: PTX, −54.9±4.1 fF/pF (not significantly different from the controls); PTX+ PGE1, −36.4±3.9fF/pF, P<0.05. Thus, the PTX-sensitive Gi/Go proteins were not involved in PGE1 inhibition of endocytosis. Furthermore, with the effect of PGE1 to inhibit insulin release blocked by the high Ca2+ influx, there is no interference of the data on endocytosis from PGE1 effects on exocytosis.

Figure 3
Figure 3

PGE1 inhibits endocytosis in a PTX-insensitive manner. The signaling pathways in the hormonally mediated inhibition of endocytosis were investigated in control and PTX-pretreated cells. (A) Endocytosis recorded in control and PTX-pretreated cells in the absence and presence of PGE1. (B) Summary of the endocytotic actions studied under the various test conditions (as labeled), in the early endocytosis (left panel) and late endocytosis (right panel) phases. The early and late phases of endocytosis are indicated as white and gray, respectively. Data were analyzed from 17 to 25 cells for each experimental condition. The control data and the protocols are the same as those reported in Fig. 2.

*P < 0.05, **P < 0.01, #NS.

Citation: Journal of Endocrinology 229, 3; 10.1530/JOE-15-0435

PGE1 significantly slowed the rates of early and late endocytosis even in the presence of PTX. (ΔCm/early–endo: PTX, −23.4±2.1fF/pF/s; PTX+PGE1, −9.8±1.0fF/pF/s, P<0.05; ΔCm/late–endo: PTX, −12.2±0.9fF/pF/s; PTX+PGE1, −7.4±0.8fF/pF/s, P<0.05).

PGE1 inhibition of endocytosis is abolished by a Gαz peptide

As the PTX-insensitive Gz is responsible for the inhibitory effect of NE on endocytosis (Zhao et al. 2010a), we determined whether Gz also mediated the effect of PGE1 on endocytosis. For this, a synthetic peptide mimicking the C-terminal 11 amino acids of the Gαz subunit was dialyzed into the cell via the patch pipette at a final concentration of 75µM. This concentration was chosen on the basis of previous experiments comparing 25 and 75µM peptide concentrations and produced optimal suppression of NE inhibition of endocytosis (Zhao et al. 2010a).

To exclude nonspecific peptide effects, experiments were performed using a scrambled peptide with the same 11 amino acids in random order (Gz/scrambled, 75µM). The scrambled peptide alone had no effect on endocytosis (P=0.8). As shown in Fig. 4A, the Gz blocking peptide significantly reduced PGE1 inhibition of both early and late endocytosis (ΔCm/early–endo: PGE1+Gz/scrambled, −13.0±2.8 fF/pF, n=17; PGE1+Gz/blocking, −26.5±2.7fF/pF, n=19, P<0.05. ΔCm/late–endo: PGE1+Gz/scrambled, −34.1±4.8fF/pF; PGE1+Gz/blocking, −54.6±5.8fF/pF, P<0.05). These results indicate that Gz mediates the inhibition of endocytosis by PGE1.

Figure 4
Figure 4

The heterotrimeric G-protein Gz is involved in the inhibitory effects of PGE1 on endocytosis. (A) PGE1-induced inhibition of endocytosis was studied in the presence of the Gz blocking peptide (Gz/blocking) or a scrambled peptide (Gz/scrambled) as a control. A second control was endocytosis in the presence of the Gz scrambled peptide. (B) Summary of the endocytotic actions studied in the test and control conditions, as labeled, in the early (left panel) and late (right panel) phases. The early and late phases of endocytosis are indicated as white and gray, respectively. Data were analyzed from 17 to 23 cells for each experimental condition. Protocols were analogous to those used in the experiments of Fig. 2. *P<0.05, **P<0.01, #NS.

Citation: Journal of Endocrinology 229, 3; 10.1530/JOE-15-0435

The effects of PGE1 in the absence and presence of the Gz blocking peptide and the control scrambled peptide on the rates of endocytosis

The Gz blocking peptide significantly restored the rates of early and late endocytosis, which were reduced by PGE1 (ΔCm/early–endo: Gz/scrambled, −28.9±2.6fF/pF/s; Gz/scrambled+PGE1, −10.6±1.2fF/pF/s, P<0.05; Gz/blocking+PGE1, −28.0±2.7fF/pF/s, NS; ΔCm/late–endo: Gz/scrambled, −11.9±1.5fF/pF/s; Gz/scrambled+PGE1, 6.7±0.9fF/pF/s, P<0.05; Gz/blocking+PGE1, −14.4±1.5fF/pF/s, NS).

Discussion

Hormonal inhibition of exocytosis in the β-cell has long been well documented (Sharp 1996). In this work it has been shown that inhibition of exocytosis by PGE1 is abolished by a high level of Ca2+ influx in accord with previous reports that increased Ca2+ overcomes both serotonin-induced inhibition of neurotransmitter release (Blackmer et al. 2005, Gerachshenko et al. 2005, Photowala et al. 2006) and NE-induced inhibition of insulin release (Zhao et al. 2010b). The inhibition of release by serotonin and NE is mediated by heterotrimeric G-protein βγ subunits that block the binding of synaptotagmin to SNAP25 and thereby block SNARE protein function and exocytosis. Increased Ca2+ influx overcomes the inhibition of exocytosis by an enhancement of the Ca2+-dependent synaptotagmin binding to SNAP25, thus preventing βγ from binding and/or displacing it and allowing exocytosis to proceed (Blackmer et al. 2005, Gerachshenko et al. 2005, Photowala et al. 2006).

Inhibition of endocytosis by PGE1

This work shows that PGE1 inhibits endocytosis. Since endocytosis in the β-cell was first reported (Orci et al. 1973), it has been accepted that exocytosis–endocytosis equivalence was essential to maintain a constant area of cell surface and normal β-cell function. The mechanisms of endocytosis following exocytosis have, therefore, received considerable attention (Nagamatsu et al. 2001, Ohara-Imaizumi et al. 2002, Kuliawat et al. 2004, Ma et al. 2004, Tsuboi et al. 2004, MacDonald et al. 2005, He et al. 2008, Kimura et al. 2008, Zhao et al. 2010a). The extent to which full fusion and collapse of the granule membrane into the cell membrane, and ‘kiss and run’ occur are still not known with clarity (MacDonald & Rorsman 2007), but it seems likely that the frequency of each mode may vary depending upon the physiological and activation state of the cell. In the INS 832/13 cells reported here, endocytosis occurred in two kinetically distinct phases reminiscent of those previously reported for chromaffin cells (Smith & Neher 1997). PGE1 inhibited both the early and late phases of endocytosis, although inhibition of the early phase was more pronounced. In contrast, PGE1 reduced the amplitude and initial rate of late endocytosis in parallel with no change in the time constant of the late phase. By analogy with what is known about the inhibitory effect of NE on endocytosis, which acts via Gz at a late step in the fission process (Zhao et al. 2010a), it seems certain that PGE1 also acts at this late step. Whether Gz acts directly or indirectly on the late step is unknown and, at present, there are no known targets for heterotrimeric G proteins on endocytosis.

Inhibition of endocytosis by PGE1 has obvious physiological significance. When β-cell exocytosis is stimulated by the elevation of [Ca2+]i, the increased [Ca2+]i activates calcineurin so that endocytosis is also stimulated. As a result, the addition of granule membrane to the plasma membrane is balanced by its removal (compensatory endocytosis), and the cell volume remains constant. There are conditions, however, under which [Ca2+]i is elevated, yet insulin secretion can be inhibited, for example, an arginine-stimulated cell in which secretion is blocked by NE or PGE1. With increased [Ca2+]i and exocytosis inhibited, stimulation of endocytosis by the raised [Ca2+]i must also be inhibited to prevent a reduction in cell volume.

In summary, PGE1 inhibits endocytosis in the β-cell by an action that is most likely to be a late step in the fission process. This previously unknown action of PGE1 is mediated by activation of Gz. These results reveal that control of endocytosis by PGE1 is a novel mechanism in insulin-secreting cells, but one that is likely to occur in many other cell types also.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

This work did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector.

Acknowledgments

This work was supported by the National Institutes of Health (NIH) grants R01-DK54243 (to G W G S) and R01-NS38200 (to M L), and a Career Development Award from the Juvenile Diabetes Foundation International (to S G S).

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  • Kimple ME, Nixon AB, Kelly P, Bailey CL, Young KH, Fields TA & Casey PJ 2005 A role for Gz in pancreatic islet beta-cell biology. Journal of Biological Chemistry 280 3170831713. (doi:10.1074/jbc.M506700200)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Kimple ME, Joseph JW, Bailey CL, Fueger PT, Hendry IA, Newgard CB & Casey PJ 2008 Gαz negatively regulates insulin secretion and glucose clearance. Journal of Biological Chemistry 283 45604567. (doi:10.1074/jbc.M706481200)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Kimura T, Kaneko Y, Yamada S, Ishihara H, Senda T, Iwamatsu A & Niki I 2008 The GDP-dependent Rab27a effector coronin 3 controls endocytosis of secretory membrane in insulin-secreting cell lines. Journal of Cell Science 121 30923098. (doi:10.1242/jcs.030544)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Komatsu M, McDermott AM, Gillison SL & Sharp GWG 1995 Time course of action of pertussis toxin to block the inhibition of stimulated insulin release by norepinephrine. Endocrinology 136 18571863.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Kuliawat R, Kalinina E, Bock J, Fricker L, McGraw TE, Kim SR, Zhong J, Scheller R & Arvan P 2004 Syntaxin-6 SNARE involvement in secretory and endocytic pathways of cultured pancreatic β-cells. Molecular Biology of the Cell 15 16901701. (doi:10.1091/
mbc.E03-08-0554)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Lindau M & Neher E 1988 Patch-clamp techniques for time-resolved capacitance measurements in single cells. Pflügers Archiv 411 137146. (doi:10.1007/BF00582306)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Ma L, Bindokas VP, Kuznetsov A, Rhodes C, Hays L, Edwardson JM, Ueda K, Steiner DF & Philipson LH 2004 Direct imaging shows that insulin granule exocytosis occurs by complete vesicle fusion. PNAS 101 92669271. (doi:10.1073/pnas.0403201101)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • MacDonald PE & Rorsman P 2007 The ins and outs of secretion from pancreatic β-cells: control of single-vesicle exo- and endocytosis. Physiology 22 113121. (doi:10.1152/physiol.00047.2006)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • MacDonald PE, Eliasson L & Rorsman P 2005 Calcium increases endocytotic vesicle size and accelerates membrane fission in insulin-secreting INS-1 cells. Journal of Cell Science 118 59115920. (doi:10.1242/jcs.02685)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Murthy VN & De Camilli P 2003 Cell biology of the presynaptic terminal. Annual Review of Neuroscience 26 701728. (doi:10.1146/annurev.neuro.26.041002.131445)

  • Nagamatsu S, Nakamichi Y, Watanabe T, Matsushima S, Yamaguchi S, Ni J, Itagaki E & Ishida H 2001 Localization of cellubrevin-related peptide, endobrevin, in the early endosome in pancreatic β cells and its physiological function in exo-endocytosis of secretory granules. Journal of Cell Science 114 219227.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Ohara-Imaizumi M, Nakamichi Y, Tanaka T, Katsuta H, Ishida H & Nagamatsu S 2002 Monitoring of exocytosis and endocytosis of insulin secretory granules in the pancreatic beta-cell line MIN6 using pH-sensitive green fluorescent protein (pHluorin) and confocal laser microscopy. Biochemical Journal 363 7380. (doi:10.1042/bj3630073)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Orci L, Malaisse-Lagae F, Ravazzola M, Amherdt M & Renold AE 1973 Exocytosis-endocytosis coupling in the pancreatic β-cell. Science 181 561562. (doi:10.1126/science.181.4099.561)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Photowala H, Blackmer T, Schwartz E, Hamm HE & Alford S 2006 G protein βγ-subunits activated by serotonin mediate presynaptic inhibition by regulating vesicle fusion properties. PNAS 103 42814286. (doi:10.1073/pnas.0600509103)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Rizzoli SO & Jahn R 2007 Kiss-and-run, collapse and ‘readily retrievable’ vesicles. Traffic 8 11371144. (doi:10.1111/j.1600-0854.2007.00614.x)

  • Robertson RP, Tsai P, Little SA, Zhang HJ & Walseth TF 1987 Receptor-mediated adenylate cyclase-coupled mechanism for PGE2 inhibition of insulin secretion in HIT cells. Diabetes 36 10471053. (doi:10.2337/diab.36.9.1047)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Rorsman P, Bokvist K, Ammala C, Arkhammar P, Berggren PO, Larsson O & Wahlander K 1991 Activation by adrenaline of a low-conductance G protein-dependent K+ channel in mouse pancreatic β cells. Nature 349 7779. (doi:10.1038/349077a0)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Schermerhorn T & Sharp GWG 2000 Norepinephrine acts on the KATP channel and produces different effects on [Ca2+]i in oscillating and non-oscillating HIT-T15 cells. Cell Calcium 27 163173. (doi:10.1054/ceca.2000.0107)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Seaquist ER, Walseth TF, Nelson DM & Robertson RP 1989 Pertussis toxin-sensitive G protein mediation of PGE2 inhibition of cAMP metabolism and phasic glucose-induced insulin secretion in HIT cells. Diabetes 38 14391445. (doi:10.2337/diab.38.11.1439)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Sharp GWG 1996 Mechanisms of inhibition of insulin release. American Journal of Physiology 271 C1781C1799.

  • Sieg A, Su J, Munoz A, Buchenau M, Nakazaki M, Aguilar-Bryan L, Bryan J & Ullrich S 2004 Epinephrine-induced hyperpolarization of islet cells without KATP channels. American Journal of Physiology: Endocrinology and Metabolism 286 E463E471.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Smith C & Neher E 1997 Multiple forms of endocytosis in bovine adrenal chromaffin cells. Journal of Cell Biology 139 885894. (doi:10.1083/jcb.139.4.885)

  • Straub SG, James RF, Dunne MJ & Sharp GWG 1998 Glucose activates both KATP channel-dependent and KATP channel-independent signaling pathways in human islets. Diabetes 47 758763. (doi:10.2337/diabetes.47.5.758)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Tang SH, Yaney GC & Sharp GW 1995 Unusual carbachol responses in RINm5F cells: evidence for a “distal” site of action in stimulus-secretion coupling. Molecular Pharmacology 47 863870.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Tsuboi T, McMahon HT & Rutter GA 2004 Mechanisms of dense core vesicle recapture following ‘kiss and run’ (‘cavicapture’) exocytosis in insulin-secreting cells. Journal of Biological Chemistry 279 4711547124. (doi:10.1074/jbc.M408179200)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Ullrich S & Wollheim CB 1989 Galanin inhibits insulin secretion by direct interference with exocytosis. FEBS Letters 247 401404. (doi:10.1016/0014-5793(89)81379-1)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Zhao Y, Fang Q, Straub SG, Lindau M & Sharp GWG 2010 aHormonal inhibition of endocytosis: novel roles for noradrenaline and Gz. Journal of Physiology 588 34993509.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Zhao Y, Fang Q, Straub SG, Lindau M & Sharp GWG 2010 bNoradrenaline inhibits exocytosis via Gβγ and refilling of the readily releasable granule pool via Gα–i-1/2. Journal of Physiology 588 34853498.

    • PubMed
    • Search Google Scholar
    • Export Citation

 

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  • A high level of Ca2+ influx achieved by prolonging the depolarizing pulse abolishes the inhibitory effect of PGE1 on exocytosis. Using standard whole-cell capacitance measurements, the exocytosis elicited by depolarizing pulses (from −70 to +10mV) with two different pulse durations was monitored in control and hormone-treated cells. During the depolarizing pulses, the Ca2+ currents were recorded simultaneously. (A and B) The recording traces of ICa and capacitance in control and PGE1-treated cells stimulated with 100- and 500-ms pulses, respectively. The arrows indicate the different experimental conditions. (C) The summaries of capacitance changes (left) and calcium influxes (right) in A and B, control (black), and PGE1 (white). The traces shown in the figure were averaged from 18 to 30 cells. The values of ΔCm and Ca2+ charge integrals were normalized by cell size (fF/pF and pQ/pF). **P<0.01, #NS.

  • PGE1 inhibits endocytosis. (A) A 500-ms pulse train with depolarizations from −70 to +10mV (with 300-ms interpulse intervals) followed by ~75s of recording with the cell membrane clamped at −70mV was applied in control and PGE1-treated cells. The entire recordings are presented on the left hand side of the figure, and the pulse-stimulating parts of the traces (marked by dashed box) are shown on the expanded timescale on the right. The arrow on the left indicates the resting cell capacitances before the pulse train. Arrows indicate the experimental conditions (PGE1, black; control, gray). On the left, horizontal lines indicate the starting and minimum levels for determination of the extent of late endocytosis (vertical arrows). The extent of early endocytosis was defined as ΔCm / early-endo = i=1 5 Δ Cm i using ΔCmi values as indicated on the right. (B) A summary of the effect of PGE1 on endocytosis (∆Cm normalized as fF/pF), respectively. (C) A summary of the rates of endocytosis. The early and late phases of endocytosis are indicated as white and gray, respectively. The traces in the figure were averaged from 18 to 21 cells. *P<0.05, **P<0.01.

  • PGE1 inhibits endocytosis in a PTX-insensitive manner. The signaling pathways in the hormonally mediated inhibition of endocytosis were investigated in control and PTX-pretreated cells. (A) Endocytosis recorded in control and PTX-pretreated cells in the absence and presence of PGE1. (B) Summary of the endocytotic actions studied under the various test conditions (as labeled), in the early endocytosis (left panel) and late endocytosis (right panel) phases. The early and late phases of endocytosis are indicated as white and gray, respectively. Data were analyzed from 17 to 25 cells for each experimental condition. The control data and the protocols are the same as those reported in Fig. 2.

    *P < 0.05, **P < 0.01, #NS.

  • The heterotrimeric G-protein Gz is involved in the inhibitory effects of PGE1 on endocytosis. (A) PGE1-induced inhibition of endocytosis was studied in the presence of the Gz blocking peptide (Gz/blocking) or a scrambled peptide (Gz/scrambled) as a control. A second control was endocytosis in the presence of the Gz scrambled peptide. (B) Summary of the endocytotic actions studied in the test and control conditions, as labeled, in the early (left panel) and late (right panel) phases. The early and late phases of endocytosis are indicated as white and gray, respectively. Data were analyzed from 17 to 23 cells for each experimental condition. Protocols were analogous to those used in the experiments of Fig. 2. *P<0.05, **P<0.01, #NS.

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  • Andric SA, Zivadinovic D, Gonzalez-Iglesias AE, Lachowicz A, Tomic M & Stojilkovic SS 2005 Endothelin-induced, long lasting, and Ca2+ influx-independent blockade of intrinsic secretion in pituitary cells by Gz subunits. Journal of Biological Chemistry 280 2689626903.

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  • Blackmer T, Larsen EC, Takahashi M, Martin TF, Alford S & Hamm HE 2001 G protein βγ subunit-mediated presynaptic inhibition: regulation of exocytotic fusion downstream of Ca2+ entry. Science 292 293297. (doi:10.1126/science.1058803)

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  • Blackmer T, Larsen EC, Bartleson C, Kowalchyk JA, Yoon EJ, Preininger AM, Alford S, Hamm HE & Martin TF 2005 G protein βγ directly regulates SNARE protein fusion machinery for secretory granule exocytosis. Nature Neuroscience 8 421425.

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  • Cheung G, Jupp OJ & Cousin MA 2010 Activity-dependent bulk endocytosis and clathrin-dependent endocytosis replenish specific synaptic vesicle pools in central nerve terminals. Journal of Neuroscience 30 81518161. (doi:10.1523/JNEUROSCI.0293-10.2010)

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  • Dezaki K, Kakei M & Yada T 2007 Ghrelin uses Gαι2 and activates voltage-dependent K+ channels to attenuate glucose-induced Ca2+ signaling and insulin release in islet beta-cells: novel signal transduction of ghrelin. Diabetes 56 23192327.

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  • Eliasson L, Proks P, Ammala C, Ashcroft FM, Bokvist K, Renstrom E, Rorsman P & Smith PA 1996 Endocytosis of secretory granules in mouse pancreatic beta-cells evoked by transient elevation of cytosolic calcium. Journal of Physiology 493 755767. (doi:10.1113/jphysiol.1996.sp021420)

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  • Gerachshenko T, Blackmer T, Yoon EJ, Bartleson C, Hamm HE & Alford S 2005 Gβγ acts at the C terminus of SNAP-25 to mediate presynaptic inhibition. Nature Neuroscience 8 597605. (doi:10.1038/nn1439)

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  • Harata NC, Aravanis AM & Tsien RW 2006 Kiss-and-run and full-collapse fusion as modes of exo-endocytosis in neurosecretion. Journal of Neurochemistry 97 15461570. (doi:10.1111/j.1471-4159.2006.03987.x)

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  • He Z, Fan J, Kang L, Lu J, Xue Y, Xu P, Xu T & Chen L 2008 Ca2+ triggers a novel clathrin-independent but actin-dependent fast endocytosis in pancreatic beta cells. Traffic 9 910923. (doi:10.1111/j.1600-0854.2008.00730.x)

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  • Jahn R, Lang T & Sudhof TC 2003 Membrane fusion. Cell 112 519533. (doi:10.1016/S0092-8674(03)00112-0)

  • Jarousse N & Kelly RB 2001 Endocytotic mechanisms in synapses. Current Opinion in Cell Biology 13 461469. (doi:10.1016/S0955-0674(00)00237-4)

  • Kimple ME, Nixon AB, Kelly P, Bailey CL, Young KH, Fields TA & Casey PJ 2005 A role for Gz in pancreatic islet beta-cell biology. Journal of Biological Chemistry 280 3170831713. (doi:10.1074/jbc.M506700200)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Kimple ME, Joseph JW, Bailey CL, Fueger PT, Hendry IA, Newgard CB & Casey PJ 2008 Gαz negatively regulates insulin secretion and glucose clearance. Journal of Biological Chemistry 283 45604567. (doi:10.1074/jbc.M706481200)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Kimura T, Kaneko Y, Yamada S, Ishihara H, Senda T, Iwamatsu A & Niki I 2008 The GDP-dependent Rab27a effector coronin 3 controls endocytosis of secretory membrane in insulin-secreting cell lines. Journal of Cell Science 121 30923098. (doi:10.1242/jcs.030544)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Komatsu M, McDermott AM, Gillison SL & Sharp GWG 1995 Time course of action of pertussis toxin to block the inhibition of stimulated insulin release by norepinephrine. Endocrinology 136 18571863.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Kuliawat R, Kalinina E, Bock J, Fricker L, McGraw TE, Kim SR, Zhong J, Scheller R & Arvan P 2004 Syntaxin-6 SNARE involvement in secretory and endocytic pathways of cultured pancreatic β-cells. Molecular Biology of the Cell 15 16901701. (doi:10.1091/
mbc.E03-08-0554)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Lindau M & Neher E 1988 Patch-clamp techniques for time-resolved capacitance measurements in single cells. Pflügers Archiv 411 137146. (doi:10.1007/BF00582306)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Ma L, Bindokas VP, Kuznetsov A, Rhodes C, Hays L, Edwardson JM, Ueda K, Steiner DF & Philipson LH 2004 Direct imaging shows that insulin granule exocytosis occurs by complete vesicle fusion. PNAS 101 92669271. (doi:10.1073/pnas.0403201101)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • MacDonald PE & Rorsman P 2007 The ins and outs of secretion from pancreatic β-cells: control of single-vesicle exo- and endocytosis. Physiology 22 113121. (doi:10.1152/physiol.00047.2006)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • MacDonald PE, Eliasson L & Rorsman P 2005 Calcium increases endocytotic vesicle size and accelerates membrane fission in insulin-secreting INS-1 cells. Journal of Cell Science 118 59115920. (doi:10.1242/jcs.02685)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Murthy VN & De Camilli P 2003 Cell biology of the presynaptic terminal. Annual Review of Neuroscience 26 701728. (doi:10.1146/annurev.neuro.26.041002.131445)

  • Nagamatsu S, Nakamichi Y, Watanabe T, Matsushima S, Yamaguchi S, Ni J, Itagaki E & Ishida H 2001 Localization of cellubrevin-related peptide, endobrevin, in the early endosome in pancreatic β cells and its physiological function in exo-endocytosis of secretory granules. Journal of Cell Science 114 219227.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Ohara-Imaizumi M, Nakamichi Y, Tanaka T, Katsuta H, Ishida H & Nagamatsu S 2002 Monitoring of exocytosis and endocytosis of insulin secretory granules in the pancreatic beta-cell line MIN6 using pH-sensitive green fluorescent protein (pHluorin) and confocal laser microscopy. Biochemical Journal 363 7380. (doi:10.1042/bj3630073)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Orci L, Malaisse-Lagae F, Ravazzola M, Amherdt M & Renold AE 1973 Exocytosis-endocytosis coupling in the pancreatic β-cell. Science 181 561562. (doi:10.1126/science.181.4099.561)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Photowala H, Blackmer T, Schwartz E, Hamm HE & Alford S 2006 G protein βγ-subunits activated by serotonin mediate presynaptic inhibition by regulating vesicle fusion properties. PNAS 103 42814286. (doi:10.1073/pnas.0600509103)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Rizzoli SO & Jahn R 2007 Kiss-and-run, collapse and ‘readily retrievable’ vesicles. Traffic 8 11371144. (doi:10.1111/j.1600-0854.2007.00614.x)

  • Robertson RP, Tsai P, Little SA, Zhang HJ & Walseth TF 1987 Receptor-mediated adenylate cyclase-coupled mechanism for PGE2 inhibition of insulin secretion in HIT cells. Diabetes 36 10471053. (doi:10.2337/diab.36.9.1047)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Rorsman P, Bokvist K, Ammala C, Arkhammar P, Berggren PO, Larsson O & Wahlander K 1991 Activation by adrenaline of a low-conductance G protein-dependent K+ channel in mouse pancreatic β cells. Nature 349 7779. (doi:10.1038/349077a0)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Schermerhorn T & Sharp GWG 2000 Norepinephrine acts on the KATP channel and produces different effects on [Ca2+]i in oscillating and non-oscillating HIT-T15 cells. Cell Calcium 27 163173. (doi:10.1054/ceca.2000.0107)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Seaquist ER, Walseth TF, Nelson DM & Robertson RP 1989 Pertussis toxin-sensitive G protein mediation of PGE2 inhibition of cAMP metabolism and phasic glucose-induced insulin secretion in HIT cells. Diabetes 38 14391445. (doi:10.2337/diab.38.11.1439)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Sharp GWG 1996 Mechanisms of inhibition of insulin release. American Journal of Physiology 271 C1781C1799.

  • Sieg A, Su J, Munoz A, Buchenau M, Nakazaki M, Aguilar-Bryan L, Bryan J & Ullrich S 2004 Epinephrine-induced hyperpolarization of islet cells without KATP channels. American Journal of Physiology: Endocrinology and Metabolism 286 E463E471.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Smith C & Neher E 1997 Multiple forms of endocytosis in bovine adrenal chromaffin cells. Journal of Cell Biology 139 885894. (doi:10.1083/jcb.139.4.885)

  • Straub SG, James RF, Dunne MJ & Sharp GWG 1998 Glucose activates both KATP channel-dependent and KATP channel-independent signaling pathways in human islets. Diabetes 47 758763. (doi:10.2337/diabetes.47.5.758)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Tang SH, Yaney GC & Sharp GW 1995 Unusual carbachol responses in RINm5F cells: evidence for a “distal” site of action in stimulus-secretion coupling. Molecular Pharmacology 47 863870.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Tsuboi T, McMahon HT & Rutter GA 2004 Mechanisms of dense core vesicle recapture following ‘kiss and run’ (‘cavicapture’) exocytosis in insulin-secreting cells. Journal of Biological Chemistry 279 4711547124. (doi:10.1074/jbc.M408179200)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Ullrich S & Wollheim CB 1989 Galanin inhibits insulin secretion by direct interference with exocytosis. FEBS Letters 247 401404. (doi:10.1016/0014-5793(89)81379-1)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Zhao Y, Fang Q, Straub SG, Lindau M & Sharp GWG 2010 aHormonal inhibition of endocytosis: novel roles for noradrenaline and Gz. Journal of Physiology 588 34993509.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Zhao Y, Fang Q, Straub SG, Lindau M & Sharp GWG 2010 bNoradrenaline inhibits exocytosis via Gβγ and refilling of the readily releasable granule pool via Gα–i-1/2. Journal of Physiology 588 34853498.

    • PubMed
    • Search Google Scholar
    • Export Citation