Phospholipase Cη2 Activation Redirects Vesicle Trafficking by Regulating F-actin

PI(4,5)P₂ localizes to sites of dense core vesicle exocytosis in neuroendocrine cells and is required for Ca²⁺-triggered vesicle exocytosis, but the impact of local PI(4,5)P₂ hydrolysis on exocytosis is poorly understood. Previously, we reported that Ca²⁺-dependent activation of phospholipase Cη2 (PLCη2) catalyzes PI(4,5)P₂ hydrolysis, which affected vesicle exocytosis by regulating the activities of the lipid-dependent priming factors CAPS (also known as CADPS) and ubiquitous Munc13-2 in PC12 cells. Here we describe an additional role for PLCη2 in vesicle exocytosis as a Ca²⁺-dependent regulator of the actin cytoskeleton. Depolarization of neuroendocrine PC12 cells with 56 or 95 mm KCl buffers increased peak Ca²⁺ levels to ∼400 or ∼800 nm, respectively, but elicited similar numbers of vesicle exocytic events. However, 56 mm K⁺ preferentially elicited the exocytosis of plasma membrane-resident vesicles, whereas 95 mm K⁺ preferentially elicited the exocytosis of cytoplasmic vesicles arriving during stimulation. Depolarization with 95 mm K⁺ but not with 56 mm K⁺ activated PLCη2 to catalyze PI(4,5)P₂ hydrolysis. The decrease in PI(4,5)P₂ promoted F-actin disassembly, which increased exocytosis of newly arriving vesicles. Consistent with its role as a Ca²⁺-dependent regulator of the cortical actin cytoskeleton, PLCη2 localized with F-actin filaments. The results highlight the importance of PI(4,5)P₂ for coordinating cytoskeletal dynamics with vesicle exocytosis and reveal a new role for PLCη2 as a Ca²⁺-dependent regulator of F-actin dynamics and vesicle trafficking.     

Adenosine Triphosphate and the Late Steps in Calcium-dependent Exocytosis at a Ribbon Synapse

The ATP dependence of the kinetics of Ca²⁺-dependent exocytosis after flash photolysis of caged Ca²⁺ was studied by capacitance measurements with submillisecond resolution in single synaptic terminals of retinal bipolar neurons. After control experiments verified that this combination of techniques is valid for the study of exocytosis in synaptic terminals, a comparison was made between the Ca²⁺ dependence of the rate of exocytosis in synaptic terminals internally dialyzed with MgATP, MgATP-γ-S, or no added Mg²⁺ or nucleotide. The Ca²⁺ threshold for release, the maximum rate of release, and the overall relationship between the rate of synaptic vesicle fusion and [Ca²⁺]_i were found to be independent of MgATP. A decrease in the average rate at near-threshold [Ca²⁺]_i was observed in terminals with MgATP-γ-S, but due to the small sample size is of unclear significance. The Ca²⁺ dependence of the delay between the elevation of [Ca²⁺]_i and the beginning of the capacitance rise was also found to be independent of MgATP. In contrast, MgATP had a marked effect on the ability of terminals to respond to multiple stimuli. Terminals with MgATP typically exhibited a capacitance increase to a second stimulus that was >70% of the amplitude of the first response and to a third stimulus with a response amplitude that was >50% of the first, whereas terminals without MgATP responded to a second stimulus with a response <35% of the first and rarely responded to a third flash. These results suggest a major role for MgATP in preparing synaptic vesicles for fusion, but indicate that cytosolic MgATP may have little role in events downstream of calcium entry, provided that [Ca²⁺]_i near release sites is elevated above ≈30 μM.     

Calcium microdomains in regulated exocytosis

Katz and co-workers showed that Ca²⁺ triggers exocytosis. The existence of sub-micrometer domains of greater than 100 μM [Ca²⁺]_i was postulated on theoretical grounds. Using a modified, low-affinity aequorin, Llinas et al. were the first to demonstrate the existence of Ca²⁺ ‘microdomains’ in squid presynaptic terminals. Over the past several years, it has become clear that individual Ca²⁺ nano- and microdomains forming around the mouth of voltage-gated Ca²⁺ channels ascertain the tight coupling of fast synaptic vesicle release to membrane depolarization by action potentials. Recent work has established different geometric arrangements of vesicles and Ca²⁺ channels at different central synapses and pointed out the role of Ca²⁺ syntillas – localized, store operated Ca²⁺ signals – in facilitation and spontaneous release. The coupling between Ca²⁺ increase and evoked exocytosis is more sluggish in peripheral terminals and neuroendocrine cells, where channels are less clustered and Ca²⁺ comes from different sources, including Ca²⁺ influx via the plasma membrane and the mobilization of Ca²⁺ from intracellular stores. Finally, also non- (electrically) excitable cells display highly localized Ca²⁺ signaling domains. We discuss in particular the organization of structural microdomains of Bergmann glia, specialized astrocytes of the cerebellum that have only recently been considered as secretory cells. Glial microdomains are the spatial substrate for functionally segregated Ca²⁺ signals upon metabotropic activation. Our review emphasizes the large diversity of different geometric arrangements of vesicles and Ca²⁺ sources, leading to a wide spectrum of Ca²⁺ signals triggering release.     

Pathways that control cortical F-actin dynamics during secretion

Chromaffin cells possess a mesh of filamentous actin underneath the plasma membrane which acts as a barrier to the chromaffin vesicles access to exocytotic sites. Disassembly of cortical F-actin in response to stimulation allows the movement of vesicles from the reserve pool to the release-ready vesicle pool and, therefore, to exocytotic sites. The dynamics of cortical F-actin is controlled by two mechanisms: a) stimulation-induced Ca²⁺ entry and scinderin activation and b) protein kinase C (PKC) activation and MARCKS phosphorylation as demonstrated here by experiments with recombinant proteins, antisense olygodeoxynucleotides and vector mediated transient expressions. Under physiological conditions (i.e., cholinergic receptor stimulation followed by Ca²⁺ entry), mechanism (a) is the most important for the control of cortical F-actin network whereas when Ca²⁺ is released from intracellular stores (i.e., histamine stimulation) cortical F-actin is regulated mainly by mechanism b.     

How to make a synaptic ribbon: RIBEYE deletion abolishes ribbons in retinal synapses and disrupts neurotransmitter release

Synaptic ribbons are large proteinaceous scaffolds at the active zone of ribbon synapses that are specialized for rapid sustained synaptic vesicles exocytosis. A single ribbon‐specific protein is known, RIBEYE, suggesting that ribbons may be constructed from RIBEYE protein. RIBEYE knockdown in zebrafish, however, only reduced but did not eliminate ribbons, indicating a more ancillary role. Here, we show in mice that full deletion of RIBEYE abolishes all presynaptic ribbons in retina synapses. Using paired recordings in acute retina slices, we demonstrate that deletion of RIBEYE severely impaired fast and sustained neurotransmitter release at bipolar neuron/AII amacrine cell synapses and rendered spontaneous miniature release sensitive to the slow Ca²⁺‐buffer EGTA, suggesting that synaptic ribbons mediate nano‐domain coupling of Ca²⁺ channels to synaptic vesicle exocytosis. Our results show that RIBEYE is essential for synaptic ribbons as such, and may organize presynaptic nano‐domains that position release‐ready synaptic vesicles adjacent to Ca²⁺ channels.     

Synaptic Vesicle Exocytosis

Presynaptic nerve terminals release neurotransmitters by synaptic vesicle exocytosis. Membrane fusion mediating synaptic exocytosis and other intracellular membrane traffic is affected by a universal machinery that includes SNARE (for “soluble NSF-attachment protein receptor”) and SM (for “Sec1/Munc18-like”) proteins. During fusion, vesicular and target SNARE proteins assemble into an α-helical trans-SNARE complex that forces the two membranes tightly together, and SM proteins likely wrap around assembling trans-SNARE complexes to catalyze membrane fusion. After fusion, SNARE complexes are dissociated by the ATPase NSF (for “N-ethylmaleimide sensitive factor”). Fusion-competent conformations of SNARE proteins are maintained by chaperone complexes composed of CSPα, Hsc70, and SGT, and by nonenzymatically acting synuclein chaperones; dysfunction of these chaperones results in neurodegeneration. The synaptic membrane-fusion machinery is controlled by synaptotagmin, and additionally regulated by a presynaptic protein matrix (the “active zone”) that includes Munc13 and RIM proteins as central components.Synaptic vesicles are uniform organelles of ∼40 nm diameter that constitute the central organelle for neurotransmitter release. Each presynaptic nerve terminal contains hundreds of synaptic vesicles that are filled with neurotransmitters. When an action potential depolarizes the presynaptic plasma membrane, Ca²⁺-channels open, and Ca²⁺ flows into the nerve terminal to trigger the exocytosis of synaptic vesicles, thereby releasing their neurotransmitters into the synaptic cleft (Fig. 1). Ca²⁺ triggers exocytosis by binding to synaptotagmin; after exocytosis, vesicles are re-endocytosed, recycled, and refilled with neurotransmitters. Recycling can occur by multiple parallel pathways, either by fast recycling via local reuse of vesicles (“kiss-and-run” and “kiss-and-stay”), or by slower recycling via an endosomal intermediate (Fig. 1).Open in a separate windowFigure 1.The synaptic vesicle cycle. A presynaptic nerve terminal is depicted schematically as it contacts a postsynaptic neuron. The synaptic vesicle cycle consists of exocytosis (red arrows) followed by endocytosis and recycling (yellow arrows). Synaptic vesicles (green circles) are filled with neurotransmitters (NT; red dots) by active transport (neurotransmitter uptake) fueled by an electrochemical gradient established by a proton pump that acidifies the vesicle interior (vesicle acidification; green background). In preparation to synaptic exocytosis, synaptic vesicles are docked at the active zone, and primed by an ATP-dependent process that renders the vesicles competent to respond to a Ca²⁺-signal. When an action potential depolarizes the presynaptic membrane, Ca²⁺-channels open, causing a local increase in intracellular Ca²⁺ at the active zone that triggers completion of the fusion reaction. Released neurotransmitters then bind to receptors associated with the postsynaptic density (PSD). After fusion pore opening, synaptic vesicles probably recycle via three alternative pathways: local refilling with neurotransmitters without undocking (“kiss-and-stay”), local recycling with undocking (“kiss-and-run”), and full recycling of vesicles with passage through an endosomal intermediate. (Adapted from Südhof 2004.)Due to their small size, synaptic vesicles contain a limited complement of proteins that have been described in detail (Südhof 2004; Takamori et al. 2006). Although the functions of several vesicle components remain to be identified, most vesicle components participate in one of three processes: neurotransmitter uptake and storage, vesicle exocytosis, and vesicle endocytosis and recycling. In addition, it is likely that at least some vesicle proteins are involved in the biogenesis of synaptic vesicles and the maintenance of their exquisite uniformity and stability, but little is known about how vesicles are made, and what determines their size.     

Osthole and imperatorin, the active constituents of Cnidium monnieri (L.) Cusson, facilitate glutamate release from rat hippocampal nerve terminals

We examined the effects of osthole and imperatorin, two active compounds of Cnidium monnieri (L.) Cusson, on the release of glutamate from rat hippocampal synaptosomes and investigated the possible mechanism. The results showed that osthole or imperatorin significantly facilitated 4-aminopridine (4-AP)-evoked glutamate release in a concentration-dependent manner. The facilitatory action of osthole or imperatorin was blocked by the vesicular transporter inhibitor bafilomycin A1, not by the glutamate transporter inhibitor l-transpyrrolidine-2,4-dicarboxylic acid (l-trans-PDC), indicating that the release facilitation by osthole or imperatorin results from a enhancement of vesicular exocytosis and not from an increase of Ca²⁺-independent efflux via glutamate transporter. Examination of the effect of osthole and imperatorin on cytosolic [Ca²⁺] revealed that the facilitation of glutamate release could be attributed to an increase in voltage-dependent Ca²⁺ influx. Consistent with this, ω-conotoxin MVIIC, a wide-spectrum blocker of the N- and P/Q-type Ca²⁺ channels, significantly suppressed the osthole or imperatorin-mediated facilitation of glutamate release, but intracellular Ca²⁺ release inhibitor dantrolene had no effect. Osthole or imperatorin did not alter the resting synaptosomal membrane potential or 4-AP-mediated depolarization; thus, the facilitation of 4-AP-evoked Ca²⁺ influx and glutamate release produced by osthole or imperatorin was not due to it decreasing synaptosomal excitability. In addition, osthole or imperatorin-mediated inhibition of 4-AP-evoked release was prevented by protein kinase C (PKC) inhibitors. Furthermore, osthole or imperatorin increased 4-AP-induced phosphorylation of PKC. Together, these results suggest that osthole or imperatorin effects a facilitation of glutamate release from nerve terminals by positively modulating N-and P/Q-type Ca²⁺ channel activation through a signaling cascade involving PKC.     

A Highlights from MBoC Selection: Synaptobrevin-2 dependent regulation of single synaptic vesicle endocytosis

Evidence from multiple systems indicates that vesicle SNARE (soluble NSF attachment receptor) proteins are involved in synaptic vesicle endocytosis, although their exact action at the level of single vesicles is unknown. Here we interrogate the role of the main synaptic vesicle SNARE mediating fusion, synaptobrevin-2 (also called VAMP2), in modulation of single synaptic vesicle retrieval. We report that in the absence of synaptobrevin-2, fast and slow modes of single synaptic vesicle retrieval are impaired, indicating a role of the SNARE machinery in coupling exocytosis to endocytosis of single synaptic vesicles. Ultrafast endocytosis was impervious to changes in the levels of synaptobrevin-2, pointing to a separate molecular mechanism underlying this type of recycling. Taken together with earlier studies suggesting a role of synaptobrevin-2 in endocytosis, these results indicate that the machinery for fast synchronous release couples fusion to retrieval and regulates the kinetics of endocytosis in a Ca²⁺-dependent manner.     

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.     

Scinderin,a Ca2+-Dependent Actin Filament Severing Protein that Controls Cortical Actin Network Dynamics During Secretion

Secretory vesicles are localized in specific compartments within neurosecretory cells. These are different pools in which vesicles are in various states of releasability. The transit of vesicles between compartments is controlled and regulated by Ca²⁺, scinderin and the cortical F-actin network. Cortical F-actin disassembly is produced by the filament severing activity of scinderin. This Ca²⁺-dependent activity of scinderin together with its Ca²⁺-independent actin nucleating activity, control cortical F-actin dynamics during the secretory cycle. A good understanding of the interaction of actin with scinderin and of the role of this protein in secretion has been provided by the analysis of the molecular structure of scinderin together with the use of recombinant proteins corresponding to its different domains.     

Suppression of Ca2+ syntillas increases spontaneous exocytosis in mouse adrenal chromaffin cells

A central concept in the physiology of neurosecretion is that a rise in cytosolic [Ca²⁺] in the vicinity of plasmalemmal Ca²⁺ channels due to Ca²⁺ influx elicits exocytosis. Here, we examine the effect on spontaneous exocytosis of a rise in focal cytosolic [Ca²⁺] in the vicinity of ryanodine receptors (RYRs) due to release from internal stores in the form of Ca²⁺ syntillas. Ca²⁺ syntillas are focal cytosolic transients mediated by RYRs, which we first found in hypothalamic magnocellular neuronal terminals. (scintilla, Latin for spark; found in nerve terminals, normally synaptic structures.) We have also observed Ca²⁺ syntillas in mouse adrenal chromaffin cells. Here, we examine the effect of Ca²⁺ syntillas on exocytosis in chromaffin cells. In such a study on elicited exocytosis, there are two sources of Ca²⁺: one due to influx from the cell exterior through voltage-gated Ca²⁺ channels, and that due to release from intracellular stores. To eliminate complications arising from Ca²⁺ influx, we have examined spontaneous exocytosis where influx is not activated. We report here that decreasing syntillas leads to an increase in spontaneous exocytosis measured amperometrically. Two independent lines of experimentation each lead to this conclusion. In one case, release from stores was blocked by ryanodine; in another, stores were partially emptied using thapsigargin plus caffeine, after which syntillas were decreased. We conclude that Ca²⁺ syntillas act to inhibit spontaneous exocytosis, and we propose a simple model to account quantitatively for this action of syntillas.     

APP Is Cleaved by Bace1 in Pre-Synaptic Vesicles and Establishes a Pre-Synaptic Interactome,via Its Intracellular Domain,with Molecular Complexes that Regulate Pre-Synaptic Vesicles Functions

Amyloid Precursor Protein (APP) is a type I membrane protein that undergoes extensive processing by secretases, including BACE1. Although mutations in APP and genes that regulate processing of APP, such as PSENs and BRI2/ITM2B, cause dementias, the normal function of APP in synaptic transmission, synaptic plasticity and memory formation is poorly understood. To grasp the biochemical mechanisms underlying the function of APP in the central nervous system, it is important to first define the sub-cellular localization of APP in synapses and the synaptic interactome of APP. Using biochemical and electron microscopy approaches, we have found that APP is localized in pre-synaptic vesicles, where it is processed by Bace1. By means of a proteomic approach, we have characterized the synaptic interactome of the APP intracellular domain. We focused on this region of APP because in vivo data underline the central funtional and pathological role of the intracellular domain of APP. Consistent with the expression of APP in pre-synaptic vesicles, the synaptic APP intracellular domain interactome is predominantly constituted by pre-synaptic, rather than post-synaptic, proteins. This pre-synaptic interactome of the APP intracellular domain includes proteins expressed on pre-synaptic vesicles such as the vesicular SNARE Vamp2/Vamp1 and the Ca²⁺ sensors Synaptotagmin-1/Synaptotagmin-2, and non-vesicular pre-synaptic proteins that regulate exocytosis, endocytosis and recycling of pre-synaptic vesicles, such as target-membrane-SNAREs (Syntaxin-1b, Syntaxin-1a, Snap25 and Snap47), Munc-18, Nsf, α/β/γ-Snaps and complexin. These data are consistent with a functional role for APP, via its carboxyl-terminal domain, in exocytosis, endocytosis and/or recycling of pre-synaptic vesicles.     

Real-time cellular impedance measurements detect Ca channel-dependent oscillations of morphology in human H295R adrenoma cells

Endocrine cells, such as H295R have been widely used to study secretion of steroid and other hormones. Exocytosis-dependent hormone release is accompanied by an increase in plasma membrane surface area and a decrease in vesicle content. Recovery of vesicles and decrease in plasma membrane area is achieved by endocytotic processes. These changes in the extent of the surface area lead to morphological changes which can be determined by label-free real-time impedance measurements. Exo- and endocytosis have been described to be triggered by activation of L-type Ca²⁺ channels. The present study demonstrates that activation of L-type calcium channels induces prolonged oscillating changes in cellular impedance. The data support the hypothesis that a tight regulation of the intracellular Ca²⁺ concentration is a prerequisite for the observed cellular impedance oscillations. Furthermore evidence is presented for a mechanism in which the oscillations depend on a Ca²⁺-triggered calmodulin-dependent cascade involving myosin light chain kinase, nonmuscle myosin II and ultimately actin polymerization, a known determinant for cell shape changes and exocytosis in secretory cells. The described assay provides a method to determine continuously prolonged changes in cellular morphology such as exo/endocytosis cycles.     

Two synaptic vesicle pools,vesicle recruitment and replenishment of pools at the Drosophila neuromuscular junction

Drosophila neuromuscular junctions (DNMJs) are malleable and its synaptic strength changes with activities. Mobilization and recruitment of synaptic vesicles (SVs), and replenishment of SV pools in the presynaptic terminal are involved in control of synaptic efficacy. We have studied dynamics of SVs using a fluorescent styryl dye, FM1-43, which is loaded into SVs during endocytosis and released during exocytosis, and identified two SV pools. The exo/endo cycling pool (ECP) is loaded with FM1-43 during low frequency nerve stimulation and releases FM1-43 during exocytosis induced by high K⁺. The ECP locates close to release sites in the periphery of presynaptic boutons. The reserve pool (RP) is loaded and unloaded only during high frequency stimulation and resides primarily in the center of boutons. The size of ECP closely correlates with the efficacy of synaptic transmission during low frequency neuronal firing. An increase of cAMP facilitates SV movement from RP to ECP. Post-tetanic potentiation (PTP) correlates well with recruitment of SVs from RP. Neither PTP nor post-tetanic recruitment of SVs from RP occurs in memory mutants that have defects in the cAMP/PKA cascade. Cyotochalasin D slows mobilization of SVs from RP, suggesting involvement of actin filaments in SV movement. During repetitive nerve stimulation the ECP is replenished, while RP replenishment occurs after tetanic stimulation in the absence of external Ca²⁺. Mobilization of internal Ca²⁺ stores underlies RP replenishment. SV dynamics is involved in synaptic plasticity and DNMJs are suitable for further studies.     

Molecular cloning and functional expression of chromaffin cell scinderin indicates that it belongs to the family of Ca2+-dependent F-actin severing proteins

Scienderin is a Ca⁺-dependent actin filament severing protein present in chromaffin cells, platelets and a variety of secretory cells. It has been suggested that scinderin is involved in chromaffin cell F-actin dynamics and that this actin network controls the delivery of secretory vesicles to plasma membrane exocytotic sites. Moreover, scinderin redistribution and activity may be regulated by pH and Ca²⁺ in resting and stimulated cells. Here we describe the molecular cloning, the nucleotide sequence and the expression of bovine chromaffin cell scinderin cDNA. The fusion protein obtained cross-reacts with native scinderin antibodies and binds phosphatidylserine (PS), phosphatidylinositol 4,5-bisphosphate (PIP₂) and actin in a Ca⁺-dependent manner. Antibodies raised against the fusion protein produced the same cellular staining patterns for scinderin as anti-native scinderin. Nucleotide and amino acid sequence analysis indicate that scinderin has six domains each containing three internal sequence motifs, two actin and two PIP₂ binding sites and has 63 and 53% homology with gelsolin and villin. These data indicate that scinderin is a novel member of the family of Ca²⁺-dependent F-actin severing proteins which includes gelsolin and villin.Abbreviations PIP₂ phosphatidylinositol 4,5 bisphosphate - PKC protein kinase C - Sc scinderin - PS phosphatidyl serine - F-Sc scinderin fusion protein - PCR polymerase chain reaction     

Synaptotagmin IV regulates dense core vesicle (DCV) release in LbetaT2 cells

Synaptotagmins (Syts) are calcium-binding proteins which are conserved from nematodes to humans. Fifteen Syts have been identified in mammalian species. Syt I is recognized as a Ca²⁺ sensor for the synchronized release of synaptic vesicles in some types of neurons, but its role in the secretion of dense core vesicles (DCVs) remains unclear. The function of Syt IV is of particular interest because it is rapidly up-regulated by chronic depolarization and seizures. Using RNAi-mediated gene silencing, we have explored the role of Syt I and IV on secretion in a pituitary gonadotrope cell line. Downregulation of Syt IV clearly reduced Ca²⁺-triggered exocytosis of dense core vesicles (DCVs) in LβT2 cells. Syt I silencing, however, had no effect on vesicular release.     

Studies on the Role of B-50 (GAP-43) in the Mechanism of Ca2+-Induced Noradrenaline Release: Lack of Involvement of Protein Kinase C After the Ca2+ Trigger

Abstract: The involvement of B-50, protein kinase C (PKC), and PKC-mediated B-50 phosphorylation in the mechanism of Ca²⁺-induced noradrenaline (NA) release was studied in highly purified rat cerebrocortical synaptosomes permeated with streptolysin-O. Under optimal permeation conditions, 12% of the total NA content (8.9 pmol of NA/mg of synaptosomal protein) was released in a largely (>60%) ATP-dependent manner as a result of an elevation of the free Ca²⁺ concentration from 10^?8 to 10^?5M Ca²⁺ The Ca²⁺ sensitivity in the micromolar range is identical for [³H]NA and endogenous NA release, indicating that Ca²⁺-induced [³H]NA release originates from vesicular pools in noradrenergic synaptosomes. Ca²⁺-induced NA release was inhibited by either N- or C-terminal-directed anti-B-50 antibodies, confirming a role of B-50 in the process of exocytosis. In addition, both anti-B-50 antibodies inhibited PKC-mediated B-50 phosphorylation with a similar difference in inhibitory potency as observed for NA release. However, in a number of experiments, evidence was obtained challenging a direct role of PKC and PKC-mediated B-50 phosphorylation in Ca²⁺-induced NA release. PKC pseudosubstrate PKC_19-36, which inhibited B-50 phosphorylation (IC₅₀ value, 10^?5M), failed to inhibit Ca²⁺-induced NA release, even when added before the Ca²⁺ trigger. Similar results were obtained with PKC inhibitor H-7, whereas polymyxin B inhibited B-50 phosphorylation as well as Ca²⁺-induced NA release. Concerning the Ca²⁺ sensitivity, we demonstrate that PKC-mediated B-50 phosphorylation is initiated at a slightly higher Ca²⁺ concentration than NA release. Moreover, phorbol ester-induced PKC down-regulation was not paralleled by a decrease in Ca²⁺-induced NA release from streptolysin-O-permeated synaptosomes. Finally, the Ca²⁺- and phorbol ester-induced NA release was found to be additive, suggesting that they stimulate release through different mechanisms. In summary, we show that B-50 is involved in Ca²⁺-induced NA release from streptolysin-O-permeated synaptosomes. Evidence is presented challenging a role of PKC-mediated B-50 phosphorylation in the mechanism of NA exocytosis after Ca²⁺ influx. An involvement of PKC or PKC-mediated B-50 phosphorylation before the Ca²⁺ trigger is not ruled out. We suggest that the degree of B-50 phosphorylation, rather than its phosphorylation after PKC activation itself, is important in the molecular cascade after the Ca²⁺ influx resulting in exocytosis of NA.     

Osthole and imperatorin,the active constituents of Cnidium monnieri (L.) Cusson,facilitate glutamate release from rat hippocampal nerve terminals

We examined the effects of osthole and imperatorin, two active compounds of Cnidium monnieri (L.) Cusson, on the release of glutamate from rat hippocampal synaptosomes and investigated the possible mechanism. The results showed that osthole or imperatorin significantly facilitated 4-aminopridine (4-AP)-evoked glutamate release in a concentration-dependent manner. The facilitatory action of osthole or imperatorin was blocked by the vesicular transporter inhibitor bafilomycin A1, not by the glutamate transporter inhibitor l-transpyrrolidine-2,4-dicarboxylic acid (l-trans-PDC), indicating that the release facilitation by osthole or imperatorin results from a enhancement of vesicular exocytosis and not from an increase of Ca²⁺-independent efflux via glutamate transporter. Examination of the effect of osthole and imperatorin on cytosolic [Ca²⁺] revealed that the facilitation of glutamate release could be attributed to an increase in voltage-dependent Ca²⁺ influx. Consistent with this, ω-conotoxin MVIIC, a wide-spectrum blocker of the N- and P/Q-type Ca²⁺ channels, significantly suppressed the osthole or imperatorin-mediated facilitation of glutamate release, but intracellular Ca²⁺ release inhibitor dantrolene had no effect. Osthole or imperatorin did not alter the resting synaptosomal membrane potential or 4-AP-mediated depolarization; thus, the facilitation of 4-AP-evoked Ca²⁺ influx and glutamate release produced by osthole or imperatorin was not due to it decreasing synaptosomal excitability. In addition, osthole or imperatorin-mediated inhibition of 4-AP-evoked release was prevented by protein kinase C (PKC) inhibitors. Furthermore, osthole or imperatorin increased 4-AP-induced phosphorylation of PKC. Together, these results suggest that osthole or imperatorin effects a facilitation of glutamate release from nerve terminals by positively modulating N-and P/Q-type Ca²⁺ channel activation through a signaling cascade involving PKC.     

Ca2+–Calmodulin regulates SNARE assembly and spontaneous neurotransmitter release via v-ATPase subunit V0a1

Most chemical neurotransmission occurs through Ca²⁺-dependent evoked or spontaneous vesicle exocytosis. In both cases, Ca²⁺ sensing is thought to occur shortly before exocytosis. In this paper, we provide evidence that the Ca²⁺ dependence of spontaneous vesicle release may partly result from an earlier requirement of Ca²⁺ for the assembly of soluble N-ethylmaleimide–sensitive fusion attachment protein receptor (SNARE) complexes. We show that the neuronal vacuolar-type H⁺-adenosine triphosphatase V0 subunit a1 (V100) can regulate the formation of SNARE complexes in a Ca²⁺–Calmodulin (CaM)-dependent manner. Ca²⁺–CaM regulation of V100 is not required for vesicle acidification. Specific disruption of the Ca²⁺-dependent regulation of V100 by CaM led to a >90% loss of spontaneous release but only had a mild effect on evoked release at Drosophila melanogaster embryo neuromuscular junctions. Our data suggest that Ca²⁺–CaM regulation of V100 may control SNARE complex assembly for a subset of synaptic vesicles that sustain spontaneous release.     

Secretory Vesicle Pools and Rate and Kinetics of Single Vesicle Exocytosis in Neurosecretory Cells

Secretory vesicles are localized in specific compartments within neurosecretory cells. Morphometric, cytochemical and electrophysiological techniques have allowed the definition of secretory vesicle compartments. These are different pools in which vesicles are in various states of releasability. The transit of vesicles between compartments is not random, but an event controlled and regulated by Ca²⁺ and the cortical F-actin network. Cortical F-actin disassembly, a Ca²⁺-dependent event, controls the transit of secretory vesicles from the reserve compartment to the release-ready vesicle pool. Furthermore, the recent development of new technical approaches (patch-clamp membrane capacitance, electrochemical detection of amines with carbon-fibre microelectrodes) has now permitted us to understand the kinetics of single vesicle exocytosis.