Physiology BIOPAC complete lab solutions
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Physiology 22: 113-121, 2007; doi:10.1152/physiol.00047.2006
1548-9213/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Web of Science (1)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by MacDonald, P. E.
Right arrow Articles by Rorsman, P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by MacDonald, P. E.
Right arrow Articles by Rorsman, P.
Physiology, Vol. 22, No. 2, 113-121, April 2007
© 2007 Int. Union Physiol. Sci./Am. Physiol. Soc.

REVIEW

The Ins and Outs of Secretion from Pancreatic ß-Cells: Control of Single-Vesicle Exo- and Endocytosis

Patrick E. MacDonald1 and Patrik Rorsman2

1 Department of Pharmacology, University of Alberta, Edmonton, Alberta, Canada; and
2 Oxford Centre for Diabetes, Endocrinology and Metabolism, Churchill Hospital, University of Oxford, Oxford, United Kingdom pmacdonald{at}bcell.org


   Abstract
 
Exocytosis of insulin-containing secretory vesicles in pancreatic ß-cells is crucial to maintenance of plasma glucose levels. They fuse with the plasma membrane in a regulated manner to release their contents and are subsequently recaptured either intact or through conventional clathrin-mediated endocytosis. Here, we discuss these mechanisms in ß-cells at the single-vesicle level.


   Introduction
Top
 Introduction
Insulin Release by Exocytosis
 Membrane Recapture by...
 Monitoring Single Vesicles in...
 The Regulated Exocytosis and...
 Recapture of Membranes by...
 Kiss-and-Run Exocytosis of...
 Conclusion and Future...
 References
 
Insulin regulates whole body energy balance. Its secretion from the endocrine pancreatic islet ß-cells is stimulated by increases in blood glucose and modulated by neuronal and hormonal inputs (2, 24, 50). Insulin acts primarily at liver, muscle, and fat to promote energy storage. The major effects of insulin include the upregulation of glucose uptake and storage and the downregulation of glycogen breakdown. Along with insulin, ß-cells secrete a host of peptides and small molecules of largely unknown physiological relevance (34, 87). Among these, the small transmitter molecules ATP and {gamma}-aminobutyric acid (GABA) may mediate important auto- and paracrine communication within the islets of Langerhans (14, 77, 84, 101). Secretion of these various peptides and small molecules occurs via the regulated fusion of vesicles with the plasma membrane, a process termed exocytosis.

In diabetes, the insulin-producing ß-cells are either destroyed (Type 1) or demonstrate a reduced functional capacity (Type 2). In either case, this leads to a relative reduction in circulating insulin and increased blood glucose levels. The pathophysiology of reduced insulin secretion in diabetes is not well understood, but some evidence implicates defects in the distal mechanisms of insulin release (56, 67, 98, 100). Thus an understanding of how individual vesicles fuse with the plasma membrane, release their content, and are ultimately recaptured may be of importance in determining causes and treatment of diabetes. Although several excellent recent articles have reviewed ß-cell exocytosis at the single-cell level (12, 78, 79), in the present review, we will focus on exocytosis and vesicle recapture in the pancreatic ß-cells at the single-vesicle level.


   Insulin Release by Exocytosis
Top
 Introduction
 Insulin Release by Exocytosis
Membrane Recapture by...
 Monitoring Single Vesicles in...
 The Regulated Exocytosis and...
 Recapture of Membranes by...
 Kiss-and-Run Exocytosis of...
 Conclusion and Future...
 References
 
In pancreatic islet ß-cells, insulin is stored in a crystalline (30), Zn2+-complexed (10, 23) form within dense-core vesicles (DCVs), named so due to their dense appearance in electron micrographs (FIGURE 1AGo). When blood glucose is elevated, insulin-containing DCVs are stimulated to fuse with the plasma membrane and release their contents into circulation. The mechanism coupling the glucose stimulus to DCV exocytosis (summarized in FIGURE 1BGo) has been extensively reviewed (50, 76) and will not be considered here. In order for DCVs to undergo exocytosis, they must be in close proximity to the plasma membrane. Indeed, a subset of insulin-containing DCVs within the ß-cell appears physically "docked" with the plasma membrane, in close association with the voltage-dependent Ca2+ channels (VDCCs) that mediate Ca2+ entry (8, 9, 52). This results from a physical interaction of the channels with exocytotic SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) proteins (102). These well conserved and essential components of exocytosis are expressed in neuronal and endocrine cells, including ß-cells, and have recently been reviewed in Physiology (80). This pool of vesicles, accounting for ≤1% of all ß-cell DCVs, likely underlies the rapid first phase of insulin secretion (79). It is worth noting that in human Type 2 diabetes the first phase of insulin release is reduced (28), often before overt disease onset. Furthermore, the expression of several exocytotic proteins is downregulated in cultured islets exposed to hyperglycemia (1), animal models of diabetes (56, 68), and islets from humans with Type 2 diabetes (67), supporting a role for reduced exocytosis in diabetes.


Figure 1
View larger version (44K):
[in this window]
[in a new window]

  		FIGURE 1. Pancreatic ß-cells release insulin and other molecules via the regulated exocytosis of secretory vesicles

A: two parallel pathways for regulated exocytosis in ß-cells are represented by the presence of large dense-core vesicles (DCV) and small synaptic-like vesicles (SLV) in rat ß-cells. Right: a mouse ß-cell DCV is seen to undergo exocytosis. B: the mechanism for glucose-stimulated insulin secretion. The metabolism of glucose leads to closure of ATP-sensitive K+ (KATP) channels. This depolarizes the membrane, leading to opening of voltage-dependent Ca2+ channels (VDCCs) and influx of Ca2+. The influx of Ca2+ is the main trigger for the exocytosis of DCVs in the ß-cell. The regulation of SLV exocytosis is less well understood but may involve a similar mechanism.

 

   Membrane Recapture by Endocytosis
Top
 Introduction
 Insulin Release by Exocytosis
 Membrane Recapture by...
Monitoring Single Vesicles in...
 The Regulated Exocytosis and...
 Recapture of Membranes by...
 Kiss-and-Run Exocytosis of...
 Conclusion and Future...
 References
 
By definition, exocytosis results in addition of vesicular membrane to the plasma membrane. To maintain membrane homeostasis, the added membrane must eventually be recaptured. Indeed, stimulated insulin secretion is coupled to the reuptake of vesicles (65). Whether this occurs rapidly at the site of exocytosis (by kiss-and-run; see below) or through the upregulation of conventional endocytosis is a matter of debate. The so-called "conventional" endocytosis (reviewed in Refs. 17, 83, 91) involves recruitment of clathrin, which, along with a host of adaptor proteins, initiates an inward curvature of the plasma membrane. The GTPase dynamin is then thought to form a ring structure that constricts to mediate the ultimate scission of the endocytotic vesicle from the plasma membrane. Although the exact mechanism by which dynamin catalyzes membrane fission has yet to be elucidated (91), the role for this enzyme in endocytosis is undisputed.

This conventional endocytotic pathway can be coupled to stimulated exocytosis through its upregulation by Ca2+ (17, 18, 86). Calcium acts through the Ca2+-dependent phosphatase calcineurin, which dephosphorylates a number of endocytotic proteins collectively termed the dephosphins (17). Dephosphrylation of several of these, including dynamin and the clathrin adaptor protein AP180, appears crucial to the upregulation of endocytosis (18). Recent work suggests that many of the dephosphins are subsequently rephosphorylated by Cdk5 (92, 96). Although little is known about the upregulation of conventional endocytosis in ß-cells, several endocytotic proteins, including the dephosphins, are expressed in insulinoma cells and pancreatic islets (32, 39, 66).


   Monitoring Single Vesicles in the ß-Cell
Top
 Introduction
 Insulin Release by Exocytosis
 Membrane Recapture by...
 Monitoring Single Vesicles in...
The Regulated Exocytosis and...
 Recapture of Membranes by...
 Kiss-and-Run Exocytosis of...
 Conclusion and Future...
 References
 
An elegant way of monitoring single insulin release events is to develop electrodes that sense insulin. Such measurements have been performed (33, 38) and allow the millisecond resolution of individual secretory events (FIGURE 2AGo; reviewed in Ref. 55). This work importantly demonstrated that frequency changes in single-vesicle exocytosis correspond well with known insulin secretory responses (33). Notably, transient insulin secretion in response to depolarization with high extracellular K+ evoked a brief burst of exocytotic activity, whereas the prolonged secretory response to glucose resulted from a sustained period of stimulated DCV exocytosis. Ultrastructural analysis has revealed that stimulation with high K+ alone leads to the depletion of the pool of docked granules, an effect that is prevented in the presence of glucose (64). Clearly, glucose has the capacity to maintain the size of the docked vesicle pool by recruitment of new granules (63). The amperometric detection of insulin itself has not been widely adopted, however, largely due to the difficulty in producing insulin-oxidizing electrodes. Improved methods for producing these will undoubtedly prove useful (69, 74, 85, 107).


Figure 2
View larger version (41K):
[in this window]
[in a new window]

  		FIGURE 2. Methods for the examination of single-vesicle exocytosis in endocrine cells

Amperometry (A1) allows the detection of the quantal release of readily oxidized vesicle contents. Pancreatic ß-cells are generally preloaded with serotonin (5-HT), the release of which from a single vesicle is detected as an amperometric spike (A2). Fluorescence imaging (B1) using either confocal microscopy or total internal reflection fluorescence (TIRF) microscopy allows single tagged vesicles to be tracked as they undergo exocytosis and ultimately release their fluorescent cargo (seen as the disappearance of punctate fluorescence as highlighted by the circle in frames ~1.5 s apart; B2). The overexpression of ionotropic receptors (C1) can be used to create an "auto-synapse" that detects the quantal release of ligand. In this example, the P2X2 receptor has been expressed in a ß-cell to detect quantal ATP release as current (It,ATP) spike (C2). Note that, in both A2 and C2, the large current spike (reflecting full fusion of the granules with the plasma membrane) is preceded by a pedestal to a lower current level (arrows) that reflects exit of the serotonin and ATP via the fusion pore. Cell-attached capacitance measurements (D1) monitor the surface area of small membrane patches on the cell. Exocytosis of single vesicles can be observed as step-wise increases in capacitance (D2).

 
The majority of amperometric studies of single-vesicle secretion from ß-cells do not measure quantal insulin release but instead rely on preloading of cells with serotonin (5-HT) (108), which is much more easily oxidized than insulin. Although preloading with 5-HT may alter ß-cell secretory activity (105), this methodology is thought to faithfully report the exocytosis of single DCVs (5, 6). This work has demonstrated the direct coupling between membrane depolarization (108), bursts of ß-cell action potentials (11), and DCV exocytosis; an important role for ATP and cAMP in increasing the number of rapidly releasable vesicles (89); and compound or multi-vesicle exocytosis (11). The occurrence of the latter is supported by fluorescence imaging studies (42), although more recent work in intact islets suggests that this is a rare occurrence in ß-cells (88). Comparison of unitary 5-HT and insulin release from ß-cells has also revealed important aspects of membrane fusion kinetics. However, for obvious reasons, amperometic measurements of 5-HT release do not provide any information about what happens to vesicles after fusion with the plasma membrane.

Advances in fluorescence imaging approaches (FIGURE 2BGo) have made it possible to track a single DCV within the ß-cell before, during, and in some cases after exocytosis. These methods include the use of lipophilic dyes (42), DCV-targeted fluorescent proteins (61, 70, 71, 99), fluorescent extracellular probes (47, 88, 90), and fluorescent Zn2+ indicators (27, 72, 73). Combination of these markers with confocal microscopy, two-photon imaging (37), or total internal reflection fluorescence microscopy (TIRF-M) (60) has resulted in a number of seminal observations (80). These include evidence supporting the direct and intact recapture of insulin-containing DCVs following exocytosis (59, 70, 82, 97, 99); the compound or sequential exocytosis of DCVs (40, 42, 88); and a role for DCV recruitment to the membrane as an underlying mechanism for sustained exocytosis (57, 63). Perhaps more importantly, this recent work has raised numerous questions, and indeed the above conclusions are not universally accepted. Two major areas of contention remain to be resolved in our view: 1) what is the contribution and physiological role of the direct and intact recapture of vesicles, via a kiss-and-run mechanism, to the recycling of ß-cell DCVs (47, 88, 90); and 2) does DCV exocytosis occur at physically stable "hot spots," perhaps defined by SNARE protein clusters (62)? An elegant three-dimensional imaging approach has recently provided evidence against the latter (81).

More recently, we have employed two patch-clamp-based methodologies to approach the issue of single vesicle exo- and endocytosis. The first employs the overexpression of ionotropic P2X2 purinoreceptors or GABAA receptors to detect the unitary release of ATP and GABA, respectively (13, 59) (FIGURE 2CGo). Thus ATP or GABA released from a single vesicle in the ß-cell under study is detected as a current spike resulting from the autocrine activation of the ionotropic receptors. This technique is somewhat comparable to amperometry in that the quantal release of transmitter is detected but provides the advantage of measuring all events over the surface of the cell. Since ATP is stored in ß-cell DCVs and co-secreted with insulin (41), ATP spikes in these cells are good indicators of DCV exocytosis. GABA is stored within ß-cell SLVs (75, 93), and thus the detection of GABA spikes in ß-cells expressing GABAA receptors may reflect single SLV exocytotic events (13), but it is important to mention that some ß-cell DCVs also contain GABA (26) and that they are thus likely to contribute to the unitary GABA release events observed with this technique.

In a second approach, we utilized a variation of the capacitance technique (reviewed in Ref. 36) that monitors the capacitance (i.e., membrane surface area) of small cell-attached membrane patches rather than the entire cell (46) (FIGURE 2DGo). This method increases the resolution of capacitance measurements by >100-fold, allowing single exo- and endocytotic vesicles to be observed as discrete capacitance steps. The diameter of single vesicles undergoing exo- or endocytosis can be calculated from the magnitude of the capacitance step (46). Using this method, we were able to monitor the exocytosis of vesicles as small as 50 nm in diameter in INS-1 and rat ß-cells and thus could distinguish between the exocytosis of single SLVs and LDCVs based on their respective sizes (48, 51). Furthermore, the ability to record cell-attached patch capacitance in electrically and metabolically intact cells makes it possible to investigate the response of single vesicles to physiological stimuli such as glucose. Finally, this technique allows the simultaneous measurement of changes in membrane patch electrical conductance, and therefore the analysis of single small fusion pore kinetics in a manner similar to that of a single ion channel (19).


   The Regulated Exocytosis and Rapid Recapture of ß-Cell SLVs
Top
 Introduction
 Insulin Release by Exocytosis
 Membrane Recapture by...
 Monitoring Single Vesicles in...
 The Regulated Exocytosis and...
Recapture of Membranes by...
 Kiss-and-Run Exocytosis of...
 Conclusion and Future...
 References
 
In addition to DCVs, pancreatic ß-cells contain small (~70 nm in diameter), clear SLVs that actively accumulate GABA (75, 93) and share many similarities with neuronal synaptic vesicles (94) (FIGURE 1Go). Until recently, it was unclear whether endocrine SLVs were even capable of undergoing regulated exocytosis, and most evidence in support of this idea was indirect (44). We sought to address this issue through the overexpression of GABAA-receptors in ß-cells and thus observed Ca2+-regulated exocytosis of GABA-containing vesicles (13). Further work demonstrated that the release of GABA via this pathway acts to inhibit glucagon secretion from neighboring {alpha}-cells (101) and to inhibit insulin secretion via an autocrine pathway (14). However, the recent ultra-structural finding (26) that some DCVs also contain GABA complicates the interpretation of the data.

Using single-vesicle on-cell capacitance measurements, we have obtained direct evidence for exocytosis of small SLVs. Like ß-cell DCVs, the exocytosis of SLVs can be stimulated by glucose through a pathway that is dependent on L-type VDCCs (51). We further demonstrated glucose-stimulated SLV exocytosis at a rate slightly lower than that of DCV exocytosis (40 vs. 60 vesicles · cell–1 · min–1). Data from insulinoma cells and rat ß-cells showed that the membrane docked pool of SLVs is larger than that for DCVs (100 vs. 67 vesicles/cell) (51) and that a greater fraction of SLVs (1.2% min–1) than DCVs (0.6% min–1) undergo exocytosis on stimulation.

The relatively smaller reserve pool of ß-cell SLVs may be offset by the mechanism of SLV recycling. The so called "conventional" view of vesicle recycling is described above and shown in FIGURE 3AGo. More recently, synaptic vesicle recycling by a "kiss-and-run" mechanism has been intensively investigated. In this mechanism (FIGURE 3BGo), the vesicle fuses with the plasma membrane only transiently and is reinternalized intact. The vesicle can then be refilled directly with neurotransmitter, ready for another round of exocytosis. This requires the presence of transporters for these transmitters in the ß-cell secretory vesicle membrane and such have indeed been documented in ultrastructural studies. However, whether ß-cell DCVs can be refilled following partial release has not been studied. In chromaffin granules, ATP-dependent uptake of ATP has been documented and requires a positive intra-granular membrane potential set up by a bafilomycin-sensitive proton pump and appears to involve DIDS-sensitive Cl– channels (7). The relative contribution of the kiss-and-run and conventional recycling mechanisms is still a matter of debate (29, 31). However we recently demonstrated kiss-and-run as a mechanism for ß-cell SLV recycling (48, 51). This occurred via the transient opening of small fusion pores averaging 0.8 nm in diameter and sufficient to allow the release of most of the GABA content (48). Furthermore, although kiss-and-run accounted for approximately 25% of the exocytotic events in the intact cell, this value approached 70% when we examined membrane-docked SLVs exclusively [using excised inside-out patches (51)], suggesting that predocked vesicles may preferentially undergo kiss-and-run recycling. It seems possible that these events are followed by the refilling of ß-cell SLVs, since these can actively accumulate GABA in an ATP-dependent manner (93).


Figure 3
View larger version (42K):
[in this window]
[in a new window]

  		FIGURE 3. Electrophysiological capacitance and conductance measurements correspond to several modes of secretory vesicle exocytosis

A: the so called "conventional" exocytosis proceeds via the initial opening of a small fusion pore, which then expands to incorporate the vesicle membrane into the plasma membrane. This can be observed as a stepwise increase in cell-attached patch capacitance when the vesicle first fuses with the plasma membrane. The early fusion pore can occasionally be detected as an increase in conductance, but usually rapidly expands beyond the limit of detection. This may be followed by conventional endocytosis, where clathrin and its adaptor proteins generate an inward curvature of the plasma membrane that is "pinched off" by the GTPase dynamin. This is observable as the reverse process in the capacitance measurements where a stepwise decrease in capacitance reflects endocytosis and the formation, constriction, and eventual closure of the fission pore can be seen as a reduction in pore conductance. B: kiss-and-run exocytosis may be mediated by the opening and closure of a very small fusion pore. In the cell-attached capacitance measurements, this is observed as an upward followed by a downward capacitance step of similar magnitude. Coincident with this, rather than expanding, it closes (seen as a decrease in pore conductance). C: occasionally, we observe stable fusion pores that neither expand fully nor reclose. This was observed almost exclusively in the absence of raised cAMP, perhaps suggesting a role for this in regulating the fusion pore.

 

   Recapture of Membranes by Conventional Endocytosis
Top
 Introduction
 Insulin Release by Exocytosis
 Membrane Recapture by...
 Monitoring Single Vesicles in...
 The Regulated Exocytosis and...
 Recapture of Membranes by...
Kiss-and-Run Exocytosis of...
 Conclusion and Future...
 References
 
Although kiss-and-run appears to account for the reinternalization of a relatively large fraction of exocytotic SLVs, particularly membrane docked SLVs, it does not account for the recycling of all SLVs. A bigger issue is the recapture of DCV membranes, which account for as much as 99% of the cell surface area increase during stimulated exocytosis (13). Although some DCVs may be recaptured through a kiss-and-run mechanism (discussed below), this is responsible at the very most for 50–60% of vesicles. Therefore, a mechanism must exist to retrieve exocytosed membranes following stimulation to maintain membrane homeostasis and cell size.

The stimulated upregulation of compensatory endocytosis is well documented and described above. Not surprisingly then, in ß-cells, endocytosis is triggered in a stimulation- and Ca2+-dependent manner (25), involves a calcineurin-dependent step and the formation of phosphatidylinositol 4,5-bisphosphate (32), which may play an important role in the recruitment of endocytotic proteins and stimulated endocytosis (22). Endocytosis itself has received little attention at the single vesicle level in ß-cells, in part due to the lack of high-resolution techniques to monitor it. We have found (49) that the vast majority (98%) of endocytotic events in INS-1 insulinoma cells are not the result of direct retrieval of exocytotic vesicles, and furthermore these were comparatively small (70-nm diameter). Although whole-cell membrane retrieval was indeed upregulated in a glucose- and Ca2+-dependent manner, the frequency of endocytotic events did not change significantly. The increase in membrane retrieval was due solely to a 60% increase in endocytotic vesicle size concomitant with an increased rate of fission pore constriction.

Few studies have addressed the regulation of endocytotic vesicle size. Certain manipulations, such as disruption of the actin cytoskeleton (16) and excision of membrane patches (20), are known to alter endocytotic vesicle size. More interesting perhaps, mutation of the AP180 homolog in C. elegans and Drosophila increases synaptic vesicle size as a result of altered vesicle biogenesis at the point of endocytosis (58, 106). AP180 is also known to regulate the size of clathrin cages in vitro (104) and thus represents an interesting potential target for the Ca2+-dependent regulation of endocytotic vesicle size, particularly since this clathrin adaptor protein is dephosphorylated in a Ca2+-dependent manner by calcineurin. The same is true for dynamin, which may contribute to the Ca2+-dependent increase in fission pore constriction observed. The specific mechanism mediating upregulation of endocytosis in ß-cells remains largely unclear. Although we provide some evidence for a stimulated increase in endocytotic vesicle size, further work is needed to clarify the role and mechanism for the upregulation of conventional endocytosis in these cells.


   Kiss-and-Run Exocytosis of ß-Cell DCVs
Top
 Introduction
 Insulin Release by Exocytosis
 Membrane Recapture by...
 Monitoring Single Vesicles in...
 The Regulated Exocytosis and...
 Recapture of Membranes by...
 Kiss-and-Run Exocytosis of...
Conclusion and Future...
 References
 
Kiss-and-run as a mechanism for DCV exocytosis and recycling is controversial. Strong evidence suggests that chromaffin cell DCVs undergo kiss-and-run (20, 45) and, furthermore, that the transient opening of the fusion pore during these events allows catecholamine release (3, 21). The vesicles are presumably refilled directly with catecholamine following these events (95). The physiological rationale for kiss-and-run of peptide-containing DCVs, such as in ß-cells, is less clear since these must return to an endosomal compartment to be refilled with peptide cargo. Thus it seems unlikely that the kiss-and-run of ß-cell DCVs would serve the purpose of vesicle recycling at the membrane following insulin release. Other possible roles for the kiss-and-run of ß-cell DCVs remain, including the maintenance of vesicular membrane protein and lipid composition, vesicle priming via alterations in luminal pH, and the selective release of small transmitter molecules through the fusion pore. It is the latter possibility that we have become the most interested in.

In ß-cells, the frequency of kiss-and-run, its apparent mechanism, and indeed its definition depend on the method by which it is studied. The amperometric method has provided valuable information regarding formation and expansion of the fusion pore before full vesicle fusion (33, 108) and can report kiss-and-run exocytosis as the gradual release of transmitter through a restrictive fusion pore without a rapid full-release spike (4). However, this method has not been used to report the frequency of ß-cell DCV kiss-and-run due to the difficulty in discriminating between kiss-and-run events and full fusion events occurring far from the carbon fiber electrode. Examination of cytosolic marker exclusion (47, 88, 90) generally reports a low frequency (>7%) of DCV kiss-and-run, whereas studies utilizing fluorescent DCV cargo or membrane markers have produced results in striking contrast (as much as 60%) (59, 82, 97, 99). Our own electrophysiological measurements present intermediate results, suggesting that DCV kiss-and-run occurs 20–30% of the time in response to glucose stimulation (48, 51). Furthermore, direct imaging of insulin crystal dissolution demonstrated that approximately 33% of insulin release events occurred via a slow release form (54), which may correlate to release by a kiss-and-run mechanism.

These approaches represent fundamentally differing measurements, however, making direct comparisons difficult. Studies examining tagged DCV membrane proteins provide the clearest evidence that the ß-cell DCVs frequently remain intact following exocytosis and are often associated with the retention of peptide and closure of the fusion pore (59, 97). Monitoring the release of different sized cargoes, it was inferred that multiple forms of kiss-and-run exist (99), which may be related to different functional states and different stable diameters of the fusion pore (FIGURE 3, B AND CGo). Indeed, kiss-and-run fusion pores of ß-cell DCVs have been reported as either small (1.4 nm or less) (48, 90), of intermediate diameter (97), or so large as to be practically nonexistent (70, 82, 97, 99). These measurements are not without their drawbacks, however, and expression of bulky granule-targeted probes may alter the biophysical properties of the exocytotic release event (53).

If the relative occurrence of transient fusion of DCVs in the ß-cell is not well established, the mechanistic basis for this is even less so. Recent work demonstrated the recruitment of dynamin to putative DCV kiss-and-run sites in insulin-secreting cells (97), suggesting a mechanism whereby dynamin restricts expansion of the fusion pore before the vesicle can fully fuse with the plasma membrane. Other work suggests a role for the Ca2+-sensing exocytotic protein synaptotagmin 1, since genetic knockdown of this resulted in a reduction in fast endocytosis often taken to represent kiss-and-run (103). Our own work suggests that a stepwise opening and closing of a fusion pore, not much larger than an ion channel (1.4-nm diameter), mediates transient DCV fusion and acts to slow the release of ATP and likely to restrict or even prevent the release of insulin (48). This may explain the different results obtained by imaging of extracellular markers vs. granule-targeted probes. The former rely on extracellular markers ≥1.4 nm in diameter, whereas the latter often depend on transient changes in vesicle pH. It seems clear that measurements relying on the flux of the smaller marker (that is H+: diameter ~0.1 nm) would detect a proportionally greater number of fusions mediated by small pores. Our own measurements of the early fusion pore demonstrate that permeation of the pore increases with decreasing transmitter size (14a). The sequence GABA > 5-HT > ATP echoes the size of these molecules, suggesting that their flux may be determined by the dimension of the pore.

Larger fusion pores have been associated with long-lasting "kiss-and-stay," "kiss-and-glide," or "cavicapture" events responsible for the release of peptides of 5- to 10-nm diameter (97). We ourselves have detected DCV fusion pores on this scale that failed to close over the course of an experiment (48). These became apparent at the expense of the smaller, transient fusion pores in the absence of forskolin, suggesting a role for cAMP as a regulator of the kiss-and-run fusion pore. The nature of the fusion pore, whether it is proteinaceous or lipidic, remains unclear (for a review of these, see Refs. 15, 35, 43). The answer may be "both"; i.e., that the protein pore accounts for the initial small (ion channel-like pore), whereas the cavicapture-type pore is predominantly lined by lipids.


   Conclusion and Future Perspective
Top
 Introduction
 Insulin Release by Exocytosis
 Membrane Recapture by...
 Monitoring Single Vesicles in...
 The Regulated Exocytosis and...
 Recapture of Membranes by...
 Kiss-and-Run Exocytosis of...
 Conclusion and Future...
References
 
A consensus has yet to be reached in regard to specific details of the exocytotic and endocytotic process in ß-cells of the pancreas. Several interesting lines of research have been generated by the study of single vesicle exocytosis through a variety of approaches. Many discrepancies remain to be investigated and may only be resolved through the combination of elecrophysiological and imaging approaches. Recent imaging experiments have revealed that the insulin secretion defect that develops following long-term exposure of ß-cells to high glucose results from insufficient expansion of the fusion pore rather than a reduction of exocytosis per se (98). It is an exciting possibility that similar defects may contribute to the pathophysiology of Type 2 diabetes in humans.


   Acknowledgments
 
We thank Dr. M. Braun and Anne Clark for providing some of the original data presented in the Figures.

This review was supported by grants from the Alberta Heritage Foundation for Medical Research and Canadian Institutes of Health Research to P. E. MacDonald (MOP 81350) and the Wellcome Trust to P. Rorsman. P. E. MacDonald is a Canadian Diabetes Association Scholar, Alberta Heritage Foundation for Medical Research Scholar, and the Canada Research Chair in Islet Biology. P. Rorsman is a Wolfson-Royal Society Merit Award Research Fellow.


   References
Top
 Introduction
 Insulin Release by Exocytosis
 Membrane Recapture by...
 Monitoring Single Vesicles in...
 The Regulated Exocytosis and...
 Recapture of Membranes by...
 Kiss-and-Run Exocytosis of...
 Conclusion and Future...
 References
 

  1. AbderrahmaniA, Cheviet S, Ferdaoussi M, Coppola T, Waeber G, Regazzi R. ICER induced by hyperglycemia represses the expression of genes essential for insulin exocytosis. EMBO J 25: 977–986, 2006.[CrossRef][Web of Science][Medline]
  2. AhrenB. Autonomic regulation of islet hormone secretion: implications for health and disease. Diabetologia 43: 393–410, 2000.[CrossRef][Web of Science][Medline]
  3. AlbillosA, Dernick G, Horstmann H, Almers W, Alvarez de Toledo G, Lindau M. The exocytotic event in chromaffin cells revealed by patch amperometry. Nature 389: 509–512, 1997.[CrossRef][Medline]
  4. Alvarez de ToledoG, Fernandez-Chacon R, Fernandez JM. Release of secretory products during transient vesicle fusion. Nature 363: 554–558, 1993.[CrossRef][Medline]
  5. AspinwallCA, Brooks SA, Kennedy RT, Lakey JR. Effects of intravesicular H+ and extracellular H+ and Zn2+ on insulin secretion in pancreatic beta cells. J Biol Chem 272: 31308–31314, 1997.[Abstract/Free Full Text]
  6. AspinwallCA, Huang L, Lakey JR, Kennedy RT. Comparison of amperometric methods for detection of exocytosis from single pancreatic beta-cells of different species. Anal Chem 71: 5551–5556, 1999.[Medline]
  7. BankstonLA, Guidotti G. Characterization of ATP transport into chromaffin granule ghosts. Synergy of ATP and serotonin accumulation in chromaffin granule ghosts. J Biol Chem 271: 17132–17138, 1996.[Abstract/Free Full Text]
  8. BargS, Eliasson L, Renstrom E, Rorsman P. A subset of 50 secretory granules in close contact with L-type Ca2+ channels accounts for first-phase insulin secretion in mouse ß-cells. Diabetes 51, Suppl 1: S74–S82, 2002.[Abstract/Free Full Text]
  9. BargS, Ma X, Eliasson L, Galvanovskis J, Gopel SO, Obermuller S, Platzer J, Renstrom E, Trus M, Atlas D, Striessnig J, Rorsman P. Fast exocytosis with few Ca2+ channels in insulin-secreting mouse pancreatic B cells. Biophys J 81: 3308–3323, 2001.[Web of Science][Medline]
  10. BlundellTL, Cutfield JF, Dodson EJ, Dodson GG, Hodgkin DC, Mercola DA. The crystal structure of rhombohedral 2 zinc insulin. Cold Spring Harb Symp Quant Biol 36: 233–241, 1972.[Abstract/Free Full Text]
  11. BokvistK, Holmqvist M, Gromada J, Rorsman P. Compound exocytosis in voltage-clamped mouse pancreatic beta-cells revealed by carbon fibre amperometry. Pflügers Arch 439: 634–645, 2000.[CrossRef][Web of Science][Medline]
  12. Bratanova-TochkovaTK, Cheng H, Daniel S, Gunawardana S, Liu YJ, Mulvaney-Musa J, Schermerhorn T, Straub SG, Yajima H, Sharp GW. Triggering and augmentation mechanisms, granule pools, and biphasic insulin secretion. Diabetes 51, Suppl 1: S83–S90, 2002.[Abstract/Free Full Text]
  13. BraunM, Wendt A, Birnir B, Broman J, Eliasson L, Galvanovskis J, Gromada J, Mulder H, Rorsman P. Regulated exocytosis of GABA-containing synaptic-like microvesicles in pancreatic ß-cells. J Gen Physiol 123: 191–204, 2004.[Abstract/Free Full Text]
  14. BraunM, Wendt A, Buschard K, Salehi A, Sewing S, Gromada J, Rorsman P. GABAB-receptor activation inhibits exocytosis in rat pancreatic ß-cells by G-protein-dependent activation of calcineurin. J Physiol 559: 397–409, 2004. 14a. Braun M, Wendt A, Karanauskaite J, Galvanovskis J, Clark A, Macdonald PE, Rorsman P. Corelease and differential exit via the fusion pore of GABA, serotonin, and ATP from LDCV in rat pancreatic ß-cells. J Gen Physiol 129: 221–231, 2007.[Abstract/Free Full Text]
  15. ChernomordikLV, Kozlov MM. Membrane hemifusion: crossing a chasm in two leaps. Cell 123: 375–382, 2005.[CrossRef][Web of Science][Medline]
  16. ChowdhuryHH, Kreft M, Zorec R. Distinct effect of actin cytoskeleton disassembly on exo- and endocytic events in a membrane patch of rat melanotrophs. J Physiol 545: 879–886, 2002.[Abstract/Free Full Text]
  17. CousinMA. Synaptic vesicle endocytosis: calcium works overtime in the nerve terminal. Mol Neurobiol 22: 115–128, 2000.[CrossRef][Web of Science][Medline]
  18. CousinMA, Robinson PJ. The dephosphins: dephosphorylation by calcineurin triggers synaptic vesicle endocytosis. Trends Neurosci 24: 659–665, 2001.[CrossRef][Web of Science][Medline]
  19. DebusK, Lindau M. Resolution of patch capacitance recordings and of fusion pore conductances in small vesicles. Biophys J 78: 2983–2997, 2000.[Web of Science][Medline]
  20. DernickG, Alvarez dT, Lindau M. Exocytosis of single chromaffin granules in cell-free inside-out membrane patches. Nat Cell Biol 5: 358–362, 2003.[CrossRef][Web of Science][Medline]
  21. DernickG, Gong LW, Tabares L, de Toledo GA, Lindau M. Patch amperometry: high-resolution measurements of single-vesicle fusion and release. Nat Methods 2: 699–708, 2005.[CrossRef][Web of Science][Medline]
  22. Di PaoloG, Moskowitz HS, Gipson K, Wenk MR, Voronov S, Obayashi M, Flavell R, Fitzsimonds RM, Ryan TA, De Camilli P. Impaired PtdIns(4,5)P2 synthesis in nerve terminals produces defects in synaptic vesicle trafficking. Nature 431: 415–422, 2004.[CrossRef][Medline]
  23. DodsonEJ, Dodson GG, Hodgkin DC, Reynolds CD. Structural relationships in the two-zinc insulin hexamer. Can J Biochem 57: 469–479, 1979.[Web of Science][Medline]
  24. Drucker DJ. Biological actions and therapeutic potential of the glucagon-like peptides. Gastroenterology 122: 531–544, 2002.[CrossRef][Web of Science][Medline]
  25. EliassonL, Proks P, Ammala C, Ashcroft FM, Bokvist K, Renstrom E, Rorsman P, Smith PA. Endocytosis of secretory granules in mouse pancreatic beta-cells evoked by transient elevation of cytosolic calcium. J Physiol 493: 755–767, 1996.[Abstract/Free Full Text]
  26. GammelsaeterR, Froyland M, Aragon C, Danbolt NC, Fortin D, Storm-Mathisen J, Davanger S, Gundersen V. Glycine, GABA and their transporters in pancreatic islets of Langerhans: evidence for a paracrine transmitter interplay. J Cell Sci 117: 3749–3758, 2004.[Abstract/Free Full Text]
  27. GeeKR, Zhou ZL, Qian WJ, Kennedy R. Detection and imaging of zinc secretion from pancreatic beta-cells using a new fluorescent zinc indicator. J Am Chem Soc 124: 776–778, 2002.[CrossRef][Web of Science][Medline]
  28. GerichJE. Is reduced first-phase insulin release the earliest detectable abnormality in individuals destined to develop type 2 diabetes? Diabetes 51, Suppl 1: S117–S121, 2002.[Abstract/Free Full Text]
  29. GransethB, Odermatt B, Royle SJ, Lagnado L. Clathrin-mediated endocytosis is the dominant mechanism of vesicle retrieval at hippocampal synapses. Neuron 51: 773–786, 2006.[CrossRef][Web of Science][Medline]
  30. GreiderMH, Howell SL, Lacy PE. Isolation and properties of secretory granules from rat islets of Langerhans. II. Ultrastructure of the beta granule. J Cell Biol 41: 162–166, 1969.[Abstract/Free Full Text]
  31. HarataNC, Aravanis AM, Tsien RW. Kiss-and-run and full-collapse fusion as modes of exo-endocytosis in neurosecretion. J Neurochem 97: 1546–1570, 2006.[CrossRef][Web of Science][Medline]
  32. HoyM, Efanov AM, Bertorello AM, Zaitsev SV, Olsen HL, Bokvist K, Leibiger B, Leibiger IB, Zwiller J, Berggren PO, Gromada J. Inositol hexa-kisphosphate promotes dynamin I-mediated endocytosis. Proc Natl Acad Sci USA 99: 6773–6777, 2002.[Abstract/Free Full Text]
  33. HuangL, Shen H, Atkinson MA, Kennedy RT. Detection of exocytosis at individual pancreatic beta cells by amperometry at a chemically modified microelectrode. Proc Natl Acad Sci USA 92: 9608–9612, 1995.[Abstract/Free Full Text]
  34. HuttonJC, Penn EJ, Peshavaria M. Low-molecular-weight constituents of isolated insulin-secretory granules. Bivalent cations, adenine nucleotides and inorganic phosphate. Biochem J 210: 297–305, 1983.[Web of Science][Medline]
  35. JacksonMB, Chapman ER. Fusion pores and fusion machines in Ca2+-triggered exocytosis. Annu Rev Biophys Biomol Struct 35: 135–160, 2006.[CrossRef][Web of Science][Medline]
  36. KannoT, Ma X, Barg S, Eliasson L, Galvanovskis J, Gopel S, Larsson M, Renstrom E, Rorsman P. Large dense-core vesicle exocytosis in pancreatic beta-cells monitored by capacitance measurements. Methods 33: 302–311, 2004.[CrossRef][Web of Science][Medline]
  37. KasaiH, Kishimoto T, Nemoto T, Hatakeyama H, Liu TT, Takahashi N. Two-photon excitation imaging of exocytosis and endocytosis and determination of their spatial organization. Adv Drug Delivery Res 58: 850–877, 2006.[CrossRef][Web of Science][Medline]
  38. KennedyRT, Huang L, Atkinson MA, Dush P. Amperometric monitoring of chemical secretions from individual pancreatic beta-cells. Anal Chem 65: 1882–1887, 1993.[Medline]
  39. KlumpermanJ, Kuliawat R, Griffith JM, Geuze HJ, Arvan P. Mannose 6-phosphate receptors are sorted from immature secretory granules via adaptor protein AP-1, clathrin, and syntaxin 6-positive vesicles. J Cell Biol 141: 359–371, 1998.[Abstract/Free Full Text]
  40. KwanEP, Gaisano HY. Glucagon-like peptide 1 regulates sequential and compound exocytosis in pancreatic islet ß-cells. Diabetes 54: 2734–2743, 2005.[Abstract/Free Full Text]
  41. LeitnerJW, Sussman KE, Vatter AE, Schneider FH. Adenine nucleotides in the secretory granule fraction of rat islets. Endocrinology 96: 662–677, 1975.[Abstract/Free Full Text]
  42. LeungYM, Sheu L, Kwan E, Wang G, Tsushima R, Gaisano H. Visualization of sequential exocytosis in rat pancreatic islet beta cells. Biochem Biophys Res Commun 292: 980–986, 2002.[CrossRef][Web of Science][Medline]
  43. LindauM, Alvarez de Toledo G. The fusion pore. Biochim Biophys Acta 1641: 167–173, 2003.[Medline]
  44. LlonaI. Synaptic like microvesicles: do they participate in regulated exocytosis? Neurochem Int 27: 219–226, 1995.[CrossRef][Web of Science][Medline]
  45. LollikeK, Borregaard N, Lindau M. Capacitance flickers and pseudoflickers of small granules, measured in the cell-attached configuration. Biophys J 75: 53–59, 1998.[Web of Science][Medline]
  46. LollikeK, Lindau M. Membrane capacitance techniques to monitor granule exocytosis in neutrophils. J Immunol Methods 232: 111–120, 1999.[CrossRef][Web of Science][Medline]
  47. MaL, Bindokas VP, Kuznetsov A, Rhodes C, Hays L, Edwardson JM, Ueda K, Steiner DF, Philipson LH. Direct imaging shows that insulin granule exocytosis occurs by complete vesicle fusion. Proc Natl Acad Sci USA 101: 9266–9271, 2004.[Abstract/Free Full Text]
  48. MacDonaldPE, Braun M, Galvanovskis J, Rorsman P. Release of small transmitters through kiss-and-run fusion pores in rat pancreatic beta cells. Cell Metab 4: 283–290, 2006.[CrossRef][Web of Science][Medline]
  49. MacDonaldPE, Eliasson L, Rorsman P. Calcium increases endocytotic vesicle size and accelerates membrane fission in insulin secreting INS-1 cells. J Cell Sci 118: 5911–5920, 2005.[Abstract/Free Full Text]
  50. MacDonaldPE, Joseph JW, Rorsman P. Glucose-sensing mechanisms in pancreatic ß-cells. Phil Trans Royal Soc B Biol Sci 360: 2211–2225, 2005.[CrossRef]
  51. MacDonaldPE, Obermuller S, Vikman J, Galvanovskis J, Rorsman P, Eliasson L. Regulated exocytosis and kiss-and-run of synaptic-like microvesicles in INS-1 and primary rat ß-cells. Diabetes 54: 736–743, 2005.[Abstract/Free Full Text]
  52. MearsD. Regulation of insulin secretion in islets of Langerhans by Ca2+ channels. J Membr Biol 200: 57–66, 2004.[CrossRef][Web of Science][Medline]
  53. MichaelDJ, Geng X, Cawley NX, Loh YP, Rhodes CJ, Drain P, Chow RH. Fluorescent cargo proteins in pancreatic beta-cells: design determines secretion kinetics at exocytosis. Biophys J 87: L03–L05, 2004.[CrossRef][Medline]
  54. MichaelDJ, Ritzel RA, Haataja L, Chow RH. Pancreatic ß-cells secrete insulin in fast- and slow-release forms. Diabetes 55: 600–607, 2006.[Abstract/Free Full Text]
  55. MosharovEV, Sulzer D. Analysis of exocytotic events recorded by amperometry. Nat Methods 2: 651–658, 2005.[CrossRef][Web of Science][Medline]
  56. NagamatsuS, Nakamichi Y, Yamamura C, Matsushima S, Watanabe T, Ozawa S, Furukawa H, Ishida H. Decreased expression of t-SNARE, syntaxin 1, and SNAP-25 in pancreatic ß-cells is involved in impaired insulin secretion from diabetic GK rat islets: restoration of decreased t-SNARE proteins improves impaired insulin secretion. Diabetes 48: 2367–2373, 1999.[Abstract]
  57. NagamatsuS, Ohara-Imaizumi M, Nakamichi Y, Kikuta T, Nishiwaki C. Imaging docking and fusion of insulin granules induced by antidiabetes agents: sulfonylurea and glinide drugs preferentially mediate the fusion of newcomer, but not previously docked, insulin granules. Diabetes 55: 2819–2825, 2006.[Abstract/Free Full Text]
  58. NonetML, Holgado AM, Brewer F, Serpe CJ, Norbeck BA, Holleran J, Wei L, Hartwieg E, Jorgensen EM, Alfonso A. UNC-11, a Caenorhabditis elegans AP180 homologue, regulates the size and protein composition of synaptic vesicles. Mol Biol Cell 10: 2343–2360, 1999.[Abstract/Free Full Text]
  59. ObermullerS, Lindqvist A, Karanauskaite J, Galvanovskis J, Rorsman P, Barg S. Selective nucleotide-release from dense-core granules in insulin-secreting cells. J Cell Sci 118: 4271–4282, 2005.[Abstract/Free Full Text]
  60. Ohara-ImaizumiM, Nagamatsu S. Insulin exocytotic mechanism by imaging technique. J Biochem (Tokyo) 140: 1–5, 2006.[Abstract/Free Full Text]
  61. Ohara-ImaizumiM, Nakamichi Y, Tanaka T, Katsuta H, Ishida H, Nagamatsu S. Monitoring of exocytosis and endocytosis of insulin secretory granules in the pancreatic ß-cell line MIN6 using pH-sensitive green fluorescent protein (pHluorin) and confocal laser microscopy. Biochem J 363: 73–80, 2002.[CrossRef][Web of Science][Medline]
  62. Ohara-ImaizumiM, Nishiwaki C, Kikuta T, Kumakura K, Nakamichi Y, Nagamatsu S. Site of docking and fusion of insulin secretory granules in live MIN6 beta cells analyzed by TAT-conjugated anti-syntaxin 1 antibody and total internal reflection fluorescence microscopy. J Biol Chem 279: 8403–8408, 2004.[Abstract/Free Full Text]
  63. Ohara-ImaizumiM, Nishiwaki C, Kikuta T, Nagai S, Nakamichi Y, Nagamatsu S. TIRF imaging of docking and fusion of single insulin granule motion in primary rat pancreatic ß-cells: different behaviour of granule motion between normal and Goto-Kakizaki diabetic rat beta-cells. Biochem J 381: 13–18, 2004.[CrossRef][Web of Science][Medline]
  64. OlofssonCS, Gopel SO, Barg S, Galvanovskis J, Ma X, Salehi A, Rorsman P, Eliasson L. Fast insulin secretion reflects exocytosis of docked granules in mouse pancreatic ß-cells. Pflügers Arch 444: 43–51, 2002.[CrossRef][Web of Science][Medline]
  65. OrciL, Malaisse-Lagae F, Ravazzola M, Amherdt M, Renold AE. Exocytosis-endocytosis coupling in the pancreatic beta cell. Science 181: 561–562, 1973.[Abstract/Free Full Text]
  66. OrciL, Ravazzola M, Amherdt M, Brown D, Perrelet A. Transport of horseradish peroxidase from the cell surface to the Golgi in insulin-secreting cells: preferential labelling of cisternae located in an intermediate position in the stack. EMBO J 5: 2097–2101, 1986.[Web of Science][Medline]
  67. OstensonCG, Gaisano H, Sheu L, Tibell A, Bartfai T. Impaired gene and protein expression of exocytotic soluble N-ethylmaleimide attachment protein receptor complex proteins in pancreatic islets of type 2 diabetic patients. Diabetes 55: 435–440, 2006.[Abstract/Free Full Text]
  68. PartonLE, McMillen PJ, Shen Y, Docherty E, Sharpe E, Diraison F, Briscoe CP, Rutter GA. Limited role for SREBP-1c in defective glucose-induced insulin secretion from Zucker diabetic fatty rat islets: a functional and gene profiling analysis. Am J Physiol Endocrinol Metab 291: E982–E994, 2006.[Abstract/Free Full Text]
  69. PikulskiM, Gorski W. Iridium-based electrocatalytic systems for the determination of insulin. Anal Chem 72: 2696–2702, 2000.[Medline]
  70. PouliAE, Emmanouilidou E, Zhao C, Wasmeier C, Hutton JC, Rutter GA. Secretory-granule dynamics visualized in vivo with a phogrin-green fluorescent protein chimaera. Biochem J 333: 193–199, 1998.[Web of Science][Medline]
  71. PouliAE, Kennedy HJ, Schofield JG, Rutter GA. Insulin targeting to the regulated secretory pathway after fusion with green fluorescent protein and firefly luciferase. Biochem J 331: 669–675, 1998.[Web of Science][Medline]
  72. QianWJ, Aspinwall CA, Battiste MA, Kennedy RT. Detection of secretion from single pancreatic beta-cells using extracellular fluorogenic reactions and confocal fluorescence microscopy. Anal Chem 72: 711–717, 2000.[Medline]
  73. QianWJ, Gee KR, Kennedy RT. Imaging of Zn2+ release from pancreatic beta-cells at the level of single exocytotic events. Anal Chem 75: 3468–3475, 2003.[Medline]
  74. QuF, Yang M, Lu Y, Shen G, Yu R. Amperometric determination of bovine insulin based on synergic action of carbon nanotubes and cobalt hexa-cyanoferrate nanoparticles stabilized by EDTA. Anal Bioanal Chem 386: 228–234, 2006.[CrossRef][Web of Science][Medline]
  75. ReetzA, Solimena M, Matteoli M, Folli F, Takei K, De Camilli P. GABA and pancreatic ß-cells: colocalization of glutamic acid decarboxylase (GAD) and GABA with synaptic-like microvesicles suggests their role in GABA storage and secretion. EMBO J 10: 1275–1284, 1991.[Web of Science][Medline]
  76. RorsmanP. The pancreatic beta-cell as a fuel sensor: an electrophysiologist’s viewpoint. Diabetologia 40: 487–495, 1997.[CrossRef][Web of Science][Medline]
  77. RorsmanP, Berggren PO, Bokvist K, Ericson H, Mohler H, Ostenson CG, Smith PA. Glucose-inhibition of glucagon secretion involves activation of GABAA-receptor chloride channels. Nature 341: 233–236, 1989.[CrossRef][Medline]
  78. RorsmanP, Eliasson L, Renstrom E, Gromada J, Barg S, Gopel S. The cell physiology of biphasic insulin secretion. News Physiol Sci 15: 72–77, 2000.[Abstract/Free Full Text]
  79. RorsmanP, Renstrom E. Insulin granule dynamics in pancreatic beta cells. Diabetologia 46: 1029–1045, 2003.[CrossRef][Web of Science][Medline]
  80. RutterGA, Hill EV. Insulin vesicle release: walk, kiss, pause . . . then run. Physiology Bethesda 21: 189–196, 2006.[CrossRef][Medline]
  81. RutterGA, Loder MK, Ravier MA. Rapid three-dimensional imaging of individual insulin release events by Nipkow disc confocal microscopy. Biochem Soc Trans 34: 675–678, 2006.[CrossRef][Web of Science][Medline]
  82. RutterGA, Tsuboi T. Kiss and run exocytosis of dense core secretory vesicles. Neuroreport 15: 79–81, 2004.[CrossRef][Web of Science][Medline]
  83. RyanTA. A pre-synaptic to-do list for coupling exocytosis to endocytosis. Curr Opin Cell Biol 18: 416–421, 2006.[CrossRef][Web of Science][Medline]
  84. SalehiA, Quader SS, Grapengiesser E, Hellman B. Inhibition of purinoceptors amplifies glucose-stimulated insulin release with removal of its pulsatility. Diabetes 54: 2126–2131, 2005.[Abstract/Free Full Text]
  85. SalimiA, Roushani M, Haghighi B, Soltanian S. Amperometric detection of insulin at renewable sol-gel derived carbon ceramic electrode modified with nickel powder and potassium octa-cyanomolybdate(IV). Biosens Bioelectron 22: 220–226, 2006.[CrossRef][Web of Science][Medline]
  86. SmillieKJ, Cousin MA. Dynamin I phosphorylation and the control of synaptic vesicle endocytosis. Biochem Soc Symp 72: 87–97, 2005.[Web of Science][Medline]
  87. SopwithAM, Hales CN, Hutton JC. Pancreatic B-cells secrete a range of novel peptides besides insulin. Biochim Biophys Acta 803: 342–345, 1984.[Medline]
  88. TakahashiN, Hatakeyama H, Okado H, Miwa A, Kishimoto T, Kojima T, Abe T, Kasai H. Sequential exocytosis of insulin granules is associated with redistribution of SNAP25. J Cell Biol 165: 255–262, 2004.[Abstract/Free Full Text]
  89. TakahashiN, Kadowaki T, Yazaki Y, Ellis-Davies GC, Miyashita Y, Kasai H. Post-priming actions of ATP on Ca2+-dependent exocytosis in pancreatic beta cells. Proc Natl Acad Sci USA 96: 760–765, 1999.[Abstract/Free Full Text]
  90. TakahashiN, Kishimoto T, Nemoto T, Kadowaki T, Kasai H. Fusion pore dynamics and insulin granule exocytosis in the pancreatic islet. Science 297: 1349–1352, 2002.[Abstract/Free Full Text]
  91. TakeiK, Yoshida Y, Yamada H. Regulatory mechanisms of dynamin-dependent endocytosis. J Biochem (Tokyo) 137: 243–247, 2005.[Abstract/Free Full Text]
  92. TanTC, Valova VA, Malladi CS, Graham ME, Berven LA, Jupp OJ, Hansra G, McClure SJ, Sarcevic B, Boadle RA, Larsen MR, Cousin MA, Robinson PJ. Cdk5 is essential for synaptic vesicle endocytosis. Nat Cell Biol 5: 701–710, 2003.[CrossRef][Web of Science][Medline]
  93. Thomas-ReetzA, Hell JW, During MJ, Walch-Solimena C, Jahn R, De Camilli P. A {gamma}-aminobutyric acid transporter driven by a proton pump is present in synaptic-like microvesicles of pancreatic ß cells. Proc Natl Acad Sci USA 90: 5317–5321, 1993.[Abstract/Free Full Text]
  94. Thomas-ReetzAC, De Camilli P. A role for synaptic vesicles in non-neuronal cells: clues from pancreatic ß-cells and from chromaffin cells. FASEB J 8: 209–216, 1994.[Abstract]
  95. TollL, Gundersen CB Jr, Howard BD. Energy utilization in the uptake of catecholamines by synaptic vesicles and adrenal chromaffin granules. Brain Res 136: 59–66, 1977.[CrossRef][Web of Science][Medline]
  96. TomizawaK, Sunada S, Lu YF, Oda Y, Kinuta M, Ohshima T, Saito T, Wei FY, Matsushita M, Li ST, Tsutsui K, Hisanaga S, Mikoshiba K, Takei K, Matsui H. Cophosphorylation of amphiphysin I and dynamin I by Cdk5 regulates clathrin-mediated endocytosis of synaptic vesicles. J Cell Biol 163: 813–824, 2003.[Abstract/Free Full Text]
  97. TsuboiT, McMahon HT, Rutter GA. Mechanisms of dense core vesicle recapture following "kiss and run" ("cavicapture") exocytosis in insulin-secreting cells. J Biol Chem 279: 47115–47124, 2004.[Abstract/Free Full Text]
  98. TsuboiT, Ravier MA, Parton LE, Rutter GA. Sustained expolsure to high glucose concentrations modifies glucose signaling and the mechanics of secretory vesicle fusion in primary rat pancreatic ß-cells. Diabetes 55: 1057–1065, 2006.[Abstract/Free Full Text]
  99. TsuboiT, Rutter GA. Multiple forms of "kiss-and-run" exocytosis revealed by evanescent wave microscopy. Curr Biol 13: 563–567, 2003.[CrossRef][Web of Science][Medline]
  100. TsunodaK, Sanke T, Nakagawa T, Furuta H, Nanjo K. Single nucleotide polymorphism (D68D, T to C) in the syntaxin 1A gene correlates to age at onset and insulin requirement in Type II diabetic patients. Diabetologia 44: 2092–2097, 2001.[CrossRef][Web of Science][Medline]
  101. WendtA, Birnir B, Buschard K, Gromada J, Salehi A, Sewing S, Rorsman P, Braun M. Glucose inhibition of glucagon secretion from rat {alpha}-cells is mediated by GABA released from neighboring ß-cells. Diabetes 53: 1038–1045, 2004.[Abstract/Free Full Text]
  102. WiserO, Trus M, Hernandez A, Renstrom E, Barg S, Rorsman P, Atlas D. The voltage sensitive Lc-type Ca2+ channel is functionally coupled to the exocytotic machinery. Proc Natl Acad Sci USA 96: 248–253, 1999.[Abstract/Free Full Text]
  103. XiongX, Zhou KM, Wu ZX, Xu T. Silence of synaptotagmin I in INS-1 cells inhibits fast exocytosis and fast endocytosis. Biochem Biophys Res Commun 347: 76–82, 2006.[CrossRef][Web of Science][Medline]
  104. YeW, Lafer EM. Bacterially expressed F1-20/AP-3 assembles clathrin into cages with a narrow size distribution: implications for the regulation of quantal size during neurotransmission. J Neurosci Res 41: 15–26, 1995.[CrossRef][Web of Science][Medline]
  105. ZawalichWS, Tesz GJ, Zawalich KC. Are 5-hydroxytryptamine-preloaded beta-cells an appropriate physiologic model system for establishing that insulin stimulates insulin secretion? J Biol Chem 276: 37120–37123, 2001.[Abstract/Free Full Text]
  106. ZhangB, Koh YH, Beckstead RB, Budnik V, Ganetzky B, Bellen HJ. Synaptic vesicle size and number are regulated by a clathrin adaptor protein required for endocytosis. Neuron 21: 1465–1475, 1998.[CrossRef][Web of Science][Medline]
  107. ZhangM, Mullens C, Gorski W. Insulin oxidation and determination at carbon electrodes. Anal Chem 77: 6396–6401, 2005.[Medline]
  108. ZhouZ, Misler S. Amperometric detection of quantal secretion from patch-clamped rat pancreatic beta-cells. J Biol Chem 271: 270–277, 1996.[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Web of Science (1)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by MacDonald, P. E.
Right arrow Articles by Rorsman, P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by MacDonald, P. E.
Right arrow Articles by Rorsman, P.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2007 by the Int. Union Physiol. Sci./Am. Physiol. Soc.