Evidence that the ability to respond to a calcium stimulus in exocytosis is determined by the secretory granule membrane: Comparison of exocytosis of injected bovine chromaffin granule membranes and endogenous cortical granules inXenopus laevis oocytes

Summary 1. To understand better the mechanisms which govern the sensitivity of secretory vesicles to a calcium stimulus, we compared the abilities of injected chromaffin granule membranes and of endogenous cortical granules to undergo exocytosis inXenopus laevis oocytes and eggs in response to cytosolic Ca²⁺. Exocytosis of chromaffin granule membranes was detected by the appearance of dopamine--hydroxylase of the chromaffin granule membrane in the oocyte or egg plasma membrane. Cortical granule exocytosis was detected by release of cortical granule lectin, a soluble constituent of cortical granules, from individual cells.2. Injected chromaffin granule membranes undergo exocytosis equally well in frog oocytes and eggs in response to a rise in cytosolic Ca²⁺ induced by incubation with ionomycin.3. Elevated Ca²⁺ triggered cortical granule exocytosis in eggs but not in oocytes.4. Injected chromaffin granule membranes do not contribute factors to the oocyte that allow calcium-dependent exocytosis of the endogenous cortical granules.5. Protein kinase C activation by phorbol esters stimulates cortical granule exocytosis in bothXenopus laevis oocytes andX. laevis eggs (Bement, W. M., and Capco, D. G.,J. Cell Biol. 108, 885–892, 1989). Activation of protein kinase C by phorbol ester also stimulated chromaffin granule membrane exocytosis in oocytes, indicating that although cortical granules and chromaffin granule membranes differ in calcium responsiveness, PKC activation is an effective secretory stimulus for both.6. These results suggest that structural or biochemical characteristics of the chromaffin granule membrane result in its ability to respond to a Ca²⁺ stimulus. In the oocytes, cortical granule components necessary for Ca²⁺-dependent exocytosis may be missing, nonfunctional, or unable to couple to the Ca²⁺ stimulus and downstream events.     

Coupling of metabolic, second messenger pathways and insulin granule dynamics in pancreatic beta-cells: a computational analysis

Insulin secretory responses to nutrient stimuli and hormonal modulators in pancreatic beta-cells are controlled by a variety of secondary messengers. We have analyzed numerous mechanisms responsible for regulated exocytosis in these cells and present an integrated mathematical model of cytosolic Ca²⁺, cAMP and granule dynamics. The insulin-containing granules in the beta-cell were divided into four classes: a large “reserve” granule pool, a smaller pool of the morphologically docked granules that is chemically ‘primed’ for release or the “readily releasable pool”, and a pool of “restless newcomer granules” that undergoes preferential exocytosis. The model incorporates glucose and other aspects of metabolism, the cAMP amplifying pathway, insulin granule dynamics and the exocyst concept for granule binding. The values of most of the model parameters were inferred from available experimental data. The model can generate both the fast first phase and slow biphasic insulin secretion found experimentally in response to a step increase of membrane potential or of glucose. The numerical simulations have also reproduced a variety of experimental conditions, such as periodic stimulation by high K⁺ and the potentiation induced in islets by pre-incubation with cAMP pathway activators. The explicit incorporation of Ca²⁺ channels, Ca²⁺ and cAMP dynamics allows the model to be further connected to current models for calcium and metabolic dynamics and provides an interpretation of the roles of the triggering and amplifying pathways of glucose-stimulated insulin secretion. The model may be important in the identification of pharmacological targets for improving insulin secretion in type 2 diabetes.     

Role of secretory granules in inositol 1,4,5-trisphosphate-dependent Ca(2+) signaling: from phytoplankton to mammals

The majority of secretory cell calcium is stored in secretory granules that serve as the major IP₃-dependent intracellular Ca²⁺ store. Even in unicellular phytoplankton secretory granules are responsible for the IP₃-induced Ca²⁺ release that triggers exocytosis. The number of secretory granules in the cell is directly related not only to the magnitude of IP₃-induced Ca²⁺ release, which accounts for the majority of the IP₃-induced cytoplasmic Ca²⁺ release in neuroendocrine cells, but also to the IP₃ sensitivity of the cytoplasmic IP₃ receptor (IP₃R)/Ca²⁺ channels. Moreover, secretory granules contain the highest IP₃R concentrations and the largest amounts of IP₃Rs in any subcellular organelles in neuroendocrine cells. Secretory granules from phytoplankton to mammals contain large amounts of polyanionic molecules, chromogranins being the major molecules in mammals, in addition to acidic intragranular pH and high Ca²⁺ concentrations. The polyanionic molecules undergo pH- and Ca²⁺-dependent conformational changes that serve as a molecular basis for condensation-decondensation phase transitions of the intragranular matrix. Likewise, chromogranins undergo pH- and Ca²⁺-dependent conformational changes with increased exposure of the structure and increased interactions with Ca²⁺ and other granule components at acidic pH. The unique physico-chemical properties of polyanionic molecules appear to be at the center of biogenesis, and physiological functions of secretory granules in living organisms from primitive to advanced species.     

Adrenaline Stimulates Glucagon Secretion in Pancreatic A-Cells by Increasing the Ca2+ Current and the Number of Granules Close to the L-Type Ca2+ Channels

We have monitored electrical activity, voltage-gated Ca²⁺ currents, and exocytosis in single rat glucagon-secreting pancreatic A-cells. The A-cells were electrically excitable and generated spontaneous Na⁺- and Ca²⁺-dependent action potentials. Under basal conditions, exocytosis was tightly linked to Ca²⁺ influx through ω-conotoxin-GVIA–sensitive (N-type) Ca²⁺ channels. Stimulation of the A-cells with adrenaline (via β-adrenergic receptors) or forskolin produced a greater than fourfold PKA-dependent potentiation of depolarization-evoked exocytosis. This enhancement of exocytosis was due to a 50% enhancement of Ca²⁺ influx through L-type Ca²⁺ channels, an effect that accounted for <30% of the total stimulatory action. The remaining 70% of the stimulation was attributable to an acceleration of granule mobilization resulting in a fivefold increase in the number of readily releasable granules near the L-type Ca²⁺ channels.     

Ca-regulated secretory granule exocytosis in pancreatic and parotid acinar cells

Protein secretion from acinar cells of the pancreas and parotid glands is controlled by G-protein coupled receptor activation and generation of the cellular messengers Ca²⁺, diacylglycerol and cAMP. Secretory granule (SG) exocytosis shares some common characteristics with nerve, neuroendocrine and endocrine cells which are regulated mainly by elevated cell Ca²⁺. However, in addition to diverse signaling pathways, acinar cells have large ∼1 μm diameter SGs (∼30 fold larger diameter than synaptic vesicles), respond to stimulation at slower rates (seconds versus milliseconds), demonstrate significant constitutive secretion, and in isolated acini, undergo sequential compound SG–SG exocytosis at the apical membrane. Exocytosis proceeds as an initial rapid phase that peaks and declines over 3 min followed by a prolonged phase that decays to near basal levels over 20–30 min. Studies indicate the early phase is triggered by Ca²⁺ and involves the SG proteins VAMP2 (vesicle associated membrane protein2), Ca²⁺-sensing protein synatotagmin 1 (syt1) and the accessory protein complexin 2. The molecular details for regulation of VAMP8-mediated SG exocytosis and the prolonged phase of secretion are still emerging. Here we review the known regulatory molecules that impact the sequential exocytic process of SG tethering, docking, priming and fusion in acinar cells.     

Regulation of exocytosis by purinergic receptors in pancreatic duct epithelial cells

In epithelial cells, several intracellular signals regulate the secretion of large molecules such as mucin via exocytosis and the transport of ions through channels and transporters. Using carbon fiber amperometry, we previously reported that exocytosis of secretory granules in dog pancreatic duct epithelial cells (PDEC) can be stimulated by pharmacological activation of cAMP-dependent protein kinase (PKA) or protein kinase C (PKC), as well as by an increase of intracellular free Ca²⁺ concentration ([Ca²⁺]_i). In this study, we examined whether exocytosis in these cells is modulated by activation of endogenous P2Y receptors, which increase cAMP and [Ca²⁺]_i. Low concentrations of ATP (<10 µM) induced intracellular Ca²⁺ oscillation but no significant exocytosis. In contrast, 100 µM ATP induced a sustained [Ca²⁺]_i rise and increased the exocytosis rate sevenfold. The contribution of Ca²⁺ or cAMP pathways to exocytosis was tested by using the Ca²⁺ chelator BAPTA or the PKA inhibitors H-89 or Rp-8-bromoadenosine 3',5'-cyclic monophosphorothioate. Removal of [Ca²⁺]_i rise or inhibition of PKA each partially reduced exocytosis; when combined, they abolished exocytosis. In conclusion, ATP at concentrations >10 µM stimulates exocytosis from PDEC through both Ca²⁺ and cAMP pathways. secretion; amperometry; photometry; calcium, adenosine 3',5'-cyclic monophosphate     

Bovine chromaffin granule membranes undergo Ca(2+)-regulated exocytosis in frog oocytes

We have devised a new method that permits the investigation of exogenous secretory vesicle function using frog oocytes and bovine chromaffin granules, the secretory vesicles from adrenal chromaffin cells. Highly purified chromaffin granule membranes were injected into Xenopus laevis oocytes. Exocytosis was detected by the appearance of dopamine-beta-hydroxylase of the chromaffin granule membrane in the oocyte plasma membrane. The appearance of dopamine-beta-hydroxylase on the oocyte surface was strongly Ca(2+)-dependent and was stimulated by coinjection of the chromaffin granule membranes with InsP3 or Ca2+/EGTA buffer (18 microM free Ca2+) or by incubation of the injected oocytes in medium containing the Ca2+ ionophore ionomycin. Similar experiments were performed with a subcellular fraction from cultured chromaffin cells enriched with [3H]norepinephrine-containing chromaffin granules. Because the release of [3H]norepinephrine was strongly correlated with the appearance of dopamine-beta-hydroxylase on the oocyte surface, it is likely that intact chromaffin granules and chromaffin granule membranes undergo exocytosis in the oocyte. Thus, the secretory vesicle membrane without normal vesicle contents is competent to undergo the sequence of events leading to exocytosis. Furthermore, the interchangeability of mammalian and amphibian components suggests substantial biochemical conservation of the regulated exocytotic pathway during the evolutionary progression from amphibians to mammals.     

Intergranular bridges in the anterior pituitary cell and their possible involvement in Ca2+-induced granule-granule fusion

Quick-freeze deep-etch electron microscopy showed the presence of bridge-like structures between adjacent secretory granules in rat anterior pituitary secretory cells. These intergranular bridges were variable in length and thickness. The finest bridges were 7–8 nm in length, while the longest ones were as long as 80 nm. Annexin II, one of the Ca²⁺-dependent phospholipid-binding proteins, is known to interlink between two membranes and induce aggregation of liposomes and chromaffin granules under the presence of Ca²⁺. In anterior pituitary cells, annexin II was detected by immunoelectron microscopy at the contact sites of secretory granules with other granules. The anterior pituitary cells treated under the presence of extracellular Ca²⁺ with Clostridium perfringens enterotoxin which induces Ca²⁺ influx showed multigranular exocytosis, i.e., multiple fusions of secretory granules with each other and with the plasma membrane. The granule-granule fusion in progress could be captured by the quick-freeze deep-etch technique. The membranes of adjacent secretory granules were partially fused at their contact sites where intergranular strands were no longer seen, while there existed intergranular strands between unfused portions of the granule membranes. From these results, we consider that the intergranular bridges, some of which may be composed of annexin II, are involved in Ca²⁺-induced granule-granule fusion in anterior pituitary cells.     

Calcium Current Inactivation Rather than Pool Depletion Explains Reduced Exocytotic Rate with Prolonged Stimulation in Insulin-Secreting INS-1 832/13 Cells

Impairment in beta-cell exocytosis is associated with reduced insulin secretion and diabetes. Here we aimed to investigate the dynamics of Ca²⁺-dependent insulin exocytosis with respect to pool depletion and Ca²⁺-current inactivation. We studied exocytosis, measured as increase in membrane capacitance (ΔC_m), as a function of calcium entry (Q) in insulin secreting INS-1 832/13 cells using patch clamp and mixed-effects statistical analysis. The observed linear relationship between ΔC_m and Q suggests that Ca²⁺-channel inactivation rather than granule pool restrictions is responsible for the decline in exocytosis observed at longer depolarizations. INS-1 832/13 cells possess an immediately releasable pool (IRP) of ∼10 granules and most exocytosis of granules occurs from a large pool. The latter is attenuated by the calcium-buffer EGTA, while IRP is unaffected. These findings suggest that most insulin release occurs away from Ca²⁺-channels, and that pool depletion plays a minor role in the decline of exocytosis upon prolonged stimulation.     

Time-resolved release of calcium from an epithelial cell monolayer during mucin secretion

A significant amount of Ca²⁺ is contained in secretory mucin granules. Exchange of Ca²⁺ for monovalent cations drives the process of mucin decondensation and hydration after fusion of granules with the plasma membrane. Here we report direct observation of calcium secretion with a Ca²⁺ ion-selective electrode (ISE) in response to apical stimulation with ATP from HT29-Cl.16E cells, a subclone of the human colonic cancer cell line HT29. No increase in Ca²⁺ level was seen for the sister cell line Cl.19A, which lacks mucin granules, or for Cl.16E cells after inhibition of granule fusion with wortmannin. Further, the measured concentration was used to estimate the time-resolved rate of release of Ca²⁺ from the cell monolayer, by use of a deconvolution-based method developed previously (Nair and Gratzl in Anal Chem 77:2875–2881, 2005). The results argue that Ca²⁺ release by Cl.16E cells is associated specifically with mucin secretion, i.e., that the measured Ca²⁺ increase in the apical solution is derived from granules after fusion and mucin exocytosis. The Ca²⁺ ISE in conjunction with deconvolution provides a minimally disturbing method for assessment of Ca²⁺ secretion rates. The release rates provide estimates of exocytosis rates and, when combined with earlier capacitance measurements, estimates of post-stimulation endocytosis rates also.     

PtdIns(4,5)P2 is not required for secretory granule docking

Phosphoinositides (PtdIns) play important roles in exocytosis and are thought to regulate secretory granule docking by co‐clustering with the SNARE protein syntaxin to form a docking receptor in the plasma membrane. Here we tested this idea by high‐resolution total internal reflection imaging of EGFP‐labeled PtdIns markers or syntaxin‐1 at secretory granule release sites in live insulin‐secreting cells. In intact cells, PtdIns markers distributed evenly across the plasma membrane with no preference for granule docking sites. In contrast, syntaxin‐1 was found clustered in the plasma membrane, mostly beneath docked granules. We also observed rapid accumulation of syntaxin‐1 at sites where granules arrived to dock. Acute depletion of plasma membrane phosphatidylinositol (4,5) bisphosphate (PtdIns(4,5)P₂) by recruitment of a 5′‐phosphatase strongly inhibited Ca²⁺‐dependent exocytosis, but had no effect on docked granules or the distribution and clustering of syntaxin‐1. Cell permeabilization by α‐toxin or formaldehyde‐fixation caused PtdIns marker to slowly cluster, in part near docked granules. In summary, our data indicate that PtdIns(4,5)P₂ accelerates granule priming, but challenge a role of PtdIns in secretory granule docking or clustering of syntaxin‐1 at the release site.      

Cyclic AMP is sufficient for triggering the exocytic recruitment of aquaporin-2 in renal epithelial cells

The initial response of renal epithelial cells to the antidiuretic hormone arginine vasopressin (AVP) is an increase in cyclic AMP. By applying immunofluorescence, cell membrane capacitance and transepithelial water flux measurements we show that cAMP alone is sufficient to elicit the antidiuretic cellular response in primary cultured epithelial cells from renal inner medulla, namely the transport of aquaporin-2 (AQP2)-bearing vesicles to, and their subsequent fusion with, the plasma membrane (AQP2 shuttle). The AQP2 shuttle is evoked neither by AVP-independent Ca²⁺ increases nor by AVP-induced Ca²⁺ increases. However, clamping cytosolic Ca²⁺ concentrations below resting levels at 25 nM inhibited exocytosis. Exocytosis was confined to a slow monophasic response, and readily releasable vesicles were missing. Analysis of endocytic capacitance steps revealed that cAMP does not decelerate the retrieval of AQP2 from the plasma membrane. Our data suggest that cAMP initiates an early step, namely the transport of AQP2-bearing vesicles towards the plasma membrane, and do not support a regulatory function for Ca²⁺ in the AQP2 shuttle.     

Distribution of calmodulin and calmodulin-binding proteins in bovine pituitary: association of myosin light chain kinase with pituitary secretory granule membranes

Calcium is necessary for secretion of pituitary hormones. Many of the biological effects of Ca²⁺ are mediated by the Ca²⁺-binding protein calmodulin (CaM), which interacts specifically with proteins regulated by the Ca²⁺-CaM complex. One of these proteins is myosin light chain kinase (MLCK), a Ca²⁺-calmodulin dependent enzyme that phosphorylates the regulatory light chains of myosin, and has been implicated in motile processes in both muscle and non-muscle tissues. We determined the content and distribution of CaM and CaM-binding proteins in bovine pituitary homogenates, and subcellular fractions including secretory granules and secretory granule membranes. CaM measured by radioimmunoassay was found in each fraction; although approximately one-half was in the cytosolic fraction, CaM was also associated with the plasma membrane and secretory granule fractions. CaM-binding proteins were identified by an ²⁵¹-CaM gel overlay technique and quantitated by densitometric analysis of the autoradiograms. Pituitary homogenates contained nine major CaM-binding proteins of 146, 131, 90, 64, 58, 56, 52, 31 and 22 kilodaltons (kDa). Binding to all the bands was specific, Cat+-sensitive, and displaceable with excess unlabeled CaM. Severe heat treatment (100°C, 15 min), which results in a 75% reduction in phosphodiesterase activation by CaM, markedly decreased ²⁵¹I-CaM binding to all protein bands. Secretory granule membranes showed enhancement for CaM-binding proteins with molecular weights of 184, 146, 131, 90, and 52000. A specific, affinity purified antibody to chicken gizzard MLCK bound to the 146 kDa band in homogenates, centrifugal subcellular fractions, and secretory granule membranes. No such binding was associated with the granule contents. The enrichment of MLCK and other CaM-binding proteins in pituitary secretory granule membranes suggests a possible role for CaM and/or CaM-binding proteins in granule membrane function and possibly exocytosis.     

Spatiotemporal analysis of exocytosis in mouse parotid acinar cells

Exocrine cells of the digestive system are specialized to secrete protein and fluid in response to neuronal and/or hormonal input. Although morphologically similar, parotid and pancreatic acinar cells exhibit important functional divergence in Ca²⁺ signaling properties. To address whether there are fundamental differences in exocytotic release of digestive enzyme from exocrine cells of salivary gland versus pancreas, we applied electrophysiological and optical methods to investigate spatial and temporal characteristics of zymogen-containing secretory granule fusion at the single-acinar cell level by direct or agonist-induced Ca²⁺ and cAMP elevation. Temporally resolved membrane capacitance measurements revealed that two apparent phases of exocytosis were induced by Ca²⁺ elevation: a rapidly activated initial phase that could not be resolved as individual fusion events and a second phase that was activated after a delay, increased in a staircaselike fashion, was augmented by cAMP elevation, and likely reflected both sequential compound and multivesicular fusion of zymogen-containing granules. Optical measurements of exocytosis with time-differential imaging analysis revealed that zymogen granule fusion was induced after a minimum delay of 200 ms, occurred initially at apical and basolateral borders of acinar cells, and under strong stimulation proceeded from apical pole to deeper regions of the cell interior. Zymogen granule fusions appeared to coordinate subsequent fusions and produced persistent structures that generally lasted several minutes. In addition, parotid gland slices were used to assess secretory dynamics in a more physiological context. Parotid acinar cells were shown to exhibit both similar and divergent properties compared with the better-studied pancreatic acinar cell regarding spatial organization and kinetics of exocytotic fusion of zymogen granules. membrane capacitance; differential imaging; zymogen; gland slice; exocrine cells     

Control of Granule Mobility and Exocytosis by Ca2+-Dependent Formation of F-Actin in Pancreatic Duct Epithelial Cells

Elevation of intracellular Ca²⁺ concentration ([Ca²⁺]_i) triggers exocytosis of secretory granules in pancreatic duct epithelia. In this study, we find that the signal also controls granule movement. Motions of fluorescently labeled granules stopped abruptly after a [Ca²⁺]_i increase, kinetically coincident with formation of filamentous actin (F-actin) in the whole cytoplasm. At high resolution, the new F-actin meshwork was so dense that cellular structures of granule size appeared physically trapped in it. Depolymerization of F-actin with latrunculin B blocked both the F-actin formation and the arrest of granules. Interestingly, when monitored with total internal reflection fluorescence microscopy, the immobilized granules still moved slowly and concertedly toward the plasma membrane. This group translocation was abolished by blockers of myosin. Exocytosis measured by microamperometry suggested that formation of a dense F-actin meshwork inhibited exocytosis at small Ca²⁺ rises <1 μ m . Larger [Ca²⁺]_i rises increased exocytosis because of the co-ordinate translocation of granules and fusion to the membrane. We propose that the Ca²⁺-dependent freezing of granules filters out weak inputs but allows exocytosis under stronger inputs by controlling granule movements.     

ANIMAL/VEGETAL DIFFERENCES IN CORTICAL GRANULE EXOCYTOSIS DURING ACTIVATION OF THE FROG EGG*

One of the more striking morphological events during egg activation is exocytosis of the cortical granules. In the frog egg, the wave of cortical granule exocytosis takes about 100 sec to traverse the animal half, and travels slower in the vegetal half. We examined cortical granule exoctyosis during activation with respect to this animal/vegetal difference. In eggs which were acquiring the ability to be activated (recovering from CO₂-intoxication or undergoing meiotic maturation), animal half cortical granules became capable of responding to activating stimuli prior to vegetal half ones. Since Ca²⁺ is involved in exocytosis, we examined the effect of Ca²⁺ on cortical granule breakdown in vitro. There was no difference in sensitivity to Ca²⁺ of cortical granules from immature vs. mature eggs, but animal half cortical granules were more sensistive to Ca²⁺ than vegetal half ones. Finally, we found that prick-activation of eggs at the vegetal pole was frequently unsuccessful but would occur when external Ca²⁺ was raised. These experiments show that there are regional differences in the frog egg with respect to cortical granule responsiveness, and they suggest that the differences are due to Ca²⁺ sensitivity.     

Cooling Reduces cAMP-Stimulated Exocytosis and Adiponectin Secretion at a Ca2+-Dependent Step in 3T3-L1 Adipocytes

We investigated the effects of temperature on white adipocyte exocytosis (measured as increase in membrane capacitance) and short-term adiponectin secretion with the aim to elucidate mechanisms important in regulation of white adipocyte stimulus-secretion coupling. Exocytosis stimulated by cAMP (included in the pipette solution together with 3 mM ATP) in the absence of Ca²⁺ (10 mM intracellular EGTA) was equal at all investigated temperatures (23°C, 27°C, 32°C and 37°C). However, the augmentation of exocytosis induced by an elevation of the free cytosolic [Ca²⁺] to ~1.5 μM (9 mM Ca²⁺ + 10 mM EGTA) was potent at 32°C or 37°C but less distinct at 27°C and abolished at 23°C. Adiponectin secretion stimulated by 30 min incubations with the membrane permeable cAMP analogue 8-Br-cAMP (1 mM) or a combination of 10 μM forskolin and 200 μM IBMX was unaffected by a reduction of temperature from 32°C to 23°C. At 32°C, cAMP-stimulated secretion was 2-fold amplified by inclusion of the Ca²⁺ ionophore ionomycin (1μM), an effect that was not observed at 23°C. We suggest that cooling affects adipocyte exocytosis/adiponectin secretion at a Ca²⁺-dependent step, likely involving ATP-dependent processes, important for augmentation of cAMP-stimulated adiponectin release.     

Polarized TIRFM Reveals Changes in Plasma Membrane Topology Before and During Granule Fusion

We have recently developed a combination of polarization and total internal reflection fluorescence microscopy (pTIRFM) to monitor changes in plasma membrane topology occurring after fusion of chromaffin granules. In this report, pTIRFM is further exploited to reveal two major findings in regards to the secretory pathway in bovine chromaffin cells. First, we show that changes in membrane topology are sometimes detected even prior to fusion. This occurs with high probability in a small subset of granules that appear in the evanescent field during the experiment. On these occasions, the plasma membrane invaginates with the movement just preceding the appearance of a granule in the evanescent field. Such events may represent a direct interaction of the granule with the plasma membrane. Second, we show that the topological fate of the post-fusion, granule/plasma membrane intermediate is regulated by divalent cation. When Sr²⁺ is used instead of Ca²⁺ to trigger exocytosis, membrane topology in the exocytotic region is stabilized with significant curvature and indentation.     

Identifying the targets of the amplifying pathway for insulin secretion in pancreatic beta-cells by kinetic modeling of granule exocytosis

A kinetic model for insulin secretion in pancreatic β-cells is adapted from a model for fast exocytosis in chromaffin cells. The fusion of primed granules with the plasma membrane is assumed to occur only in the “microdomain” near voltage-sensitive L-type Ca²⁺-channels, where [Ca²⁺] can reach micromolar levels. In contrast, resupply and priming of granules are assumed to depend on the cytosolic [Ca²⁺]. Adding a two-compartment model to handle the temporal distribution of Ca²⁺ between the microdomain and the cytosol, we obtain a unified model that can generate both the fast granule fusion and the slow insulin secretion found experimentally in response to a step of membrane potential. The model can simulate the potentiation induced in islets by preincubation with glucose and the reduction in second-phase insulin secretion induced by blocking R-type Ca²⁺-channels (Ca_V2.3). The model indicates that increased second-phase insulin secretion induced by the amplifying signal is controlled by the “resupply” step of the exocytosis cascade. In contrast, enhancement of priming is a good candidate for amplification of first-phase secretion by glucose, cyclic adenosine 3′:5′-cyclic monophosphate, and protein kinase C. Finally, insulin secretion is enhanced when the amplifying signal oscillates in phase with the triggering Ca²⁺-signal.     

Synaptotagmin IV Acts as a Multi-Functional Regulator of Ca<Superscript>2+</Superscript>-Dependent Exocytosis

In response to stimuli, secretary cells secrete a variety of signaling molecules packed in vesicles (e.g., neurotransmitters and peptide hormones) into the extracellular space by exocytosis. The vesicle secretion is often triggered by calcium ion (Ca²⁺) entered into secretary cells and achieved by the fusion of secretory vesicles with the plasma membrane. Recent accumulating evidence has indicated that members of the synaptotagmin (Syt) family play a major role in Ca²⁺-dependent exocytosis, and Syt I, in particular, is now widely accepted as the major Ca²⁺-sensor for synchronous neurotransmitter release. Involvement of other Syt isoforms in Ca²⁺-dependent exocytotic events other than neurotransmitter release has also been reported, and the Syt IV isoform is of particular interest, because Syt IV has several unique features not found in Syt I (e.g., immediate early gene product induced by deporalization and postsynaptic localization). In this article, we summarize the literature on the multi-functional role of Syt IV in Ca²⁺-dependent exocytosis.