Platelet C3G: a key player in vesicle exocytosis, spreading and clot retraction.

C3G is a Rap1 GEF that plays a pivotal role in platelet-mediated processes such as angiogenesis, tumor growth, and metastasis by modulating the platelet secretome. Here, we explore the mechanisms through which C3G governs platelet secretion. For this, we utilized animal models featuring either overexpression or deletion of C3G in platelets, as well as PC12 cell clones expressing C3G mutants. We found that C3G specifically regulates α-granule secretion via PKCδ, but it does not affect δ-granules or lysosomes. C3G activated RalA through a GEF-dependent mechanism, facilitating vesicle docking, while interfering with the formation of the trans-SNARE complex, thereby restricting vesicle fusion. Furthermore, C3G promotes the formation of lamellipodia during platelet spreading on specific substrates by enhancing actin polymerization via Src and Rac1-Arp2/3 pathways, but not Rap1. Consequently, C3G deletion in platelets favored kiss-and-run exocytosis. C3G also controlled granule secretion in PC12 cells, including pore formation. Additionally, C3G-deficient platelets exhibited reduced phosphatidylserine exposure, resulting in decreased thrombin generation, which along with defective actin polymerization and spreading, led to impaired clot retraction. In summary, platelet C3G plays a dual role by facilitating platelet spreading and clot retraction through the promotion of outside-in signaling while concurrently downregulating α-granule secretion by restricting granule fusion.

Single vesicle chemistry reveals partial release happens at the mechanical stress-induced exocytosis.

Neuronal activity can be modulated by mechanical stress in the central nervous system (CNS) in neurodegenerative diseases, for example Alzheimer's disease. However, the impact of mechanical stress on chemical signal transmission, especially the storage and release of neurotransmitter in neuron vesicles, has not been fully clarified. In this study, a nanotip conical carbon fiber microelectrode (CFME) and a disk CFME are placed in and on a cell, respectively. The nanotip conical CFME functions for both the mechanical stress and the quantification of transmitter storage in single vesicles, while the disk CFME is used to monitor the transmitter release during exocytosis induced by mechanical stress at the same cell. By comparing the vesicular transmitter storage with its release during mechanical stress-induced exocytosis at the same cell, we find the release ratio of transmitter in chromaffin cells varies from 27 % to 100 %, while for PC12 cells from 30 % to 100 %. Our results indicate that the exocytosis of cells responding to mechanical stress shows individual difference obviously, with a significant population exhibiting partial release mode. The variation of Ca2+ channels and mechanosensitive ion channels on cell membrane may both contribute to this variation. Our discovery not only shows mechanical stress can change the transmission of cellular chemical signals at the vesicle level, but also provides an important reference perspective for the study of nervous system regulation and nervous system diseases.

Rab26 restricts insulin secretion via sequestering Synaptotagmin-1.

Rab26 is known to regulate multiple membrane trafficking events, but its role in insulin secretion in pancreatic ß cells remains unclear despite it was first identified in the pancreas. In this study, we generated Rab26-/- mice through CRISPR/Cas9 technique. Surprisingly, insulin levels in the blood of the Rab26-/- mice do not decrease upon glucose stimulation but conversely increase. Deficiency of Rab26 promotes insulin secretion, which was independently verified by Rab26 knockdown in pancreatic insulinoma cells. Conversely, overexpression of Rab26 suppresses insulin secretion in both insulinoma cell lines and isolated mouse islets. Islets overexpressing Rab26, upon transplantation, also failed to restore glucose homeostasis in type 1 diabetic mice. Immunofluorescence microscopy revealed that overexpression of Rab26 results in clustering of insulin granules. GST-pulldown experiments reveal that Rab26 interacts with synaptotagmin-1 (Syt1) through directly binding to its C2A domain, which interfering with the interaction between Syt1 and SNAP25, and consequently inhibiting the exocytosis of newcomer insulin granules revealed by TIRF microscopy. Our results suggest that Rab26 serves as a negative regulator of insulin secretion, via suppressing insulin granule fusion with plasma membrane through sequestering Syt1.

Design and validation of neuronal exocytosis blocking peptides as potential novel antiperspirants.

Thermoregulation and heat dissipation by sweat production and evaporation are vital for human survival. However, hyperhidrosis or excessive perspiration might affect people's quality of life by causing discomfort and stress. The prolonged use of classical antiperspirants, anticholinergic medications or botulinum toxin injections for persistent hyperhidrosis might produce diverse side effects that limit their clinical use. Inspired by botox molecular mode of action, we used an in silico molecular modelling approach to design novel peptides to target neuronal acetylcholine exocytosis by interfering with the Snapin-SNARE complex formation. Our exhaustive design rendered the selection of 11 peptides that decreased calcium-dependent vesicle exocytosis in rat DRG neurons, reducing αCGRP release and TRPV1 inflammatory sensitization. The most potent peptides were palmitoylated peptides SPSR38-4.1 and SPSR98-9.1 that significantly suppressed acetylcholine release in vitro in human LAN-2 neuroblastoma cells. Noteworthy, local acute and chronic administration of SPSR38-4.1 peptide significantly decreased, in a dose-dependent manner, pilocarpine-induced sweating in an in vivo mouse model. Taken together, our in silico approach lead to the identification of active peptides able to attenuate excessive sweating by modulating neuronal acetylcholine exocytosis, and identified peptide SPSR38-4.1 as a promising new antihyperhidrosis candidate for clinical development.

Spatiotemporal organization and correlation of tip-focused exocytosis and endocytosis in regulating pollen tube tip growth.

Pollen tube polar growth is a key cellular process during plant fertilization and is regulated by tip-focused exocytosis and endocytosis. However, the spatiotemporal dynamics and localizations of apical exocytosis and endocytosis in the tip region are still a matter of debate. Here, we use a refined spinning-disk confocal microscope coupled with fluorescence recovery after photobleaching for sustained live imaging and quantitative analysis of rapid vesicular activities in growing pollen tube tips. We traced and analyzed the occurrence site of exocytic plasma membrane-targeting of Arabidopsis secretory carrier membrane protein 4 and its subsequent endocytosis in tobacco pollen tube tips. We demonstrated that the pollen tube apex is the site for both vesicle polar exocytic fusion and endocytosis to take place. In addition, we disrupted either tip-focused exocytosis or endocytosis and found that their dynamic activities are closely correlated with one another basing on the spatial organization of actin fringe. Collectively, our findings attempt to propose a new exocytosis and endocytosis-coordinated yin-yang working model underlying the apical membrane organization and dynamics during pollen tube tip growth.

Two distinct pathways regulate chromaffin cell exocytosis.

JGP study reveals how the neurotransmitter PACAP induces a secretory response in chromaffin cells that differs from the one induced by acetylcholine.

Functionally Collaborative Nanostructure for Direct Monitoring of Neurotransmitter Exocytosis in Living Cells.

Neurotransmitter exocytosis of living cells plays a vital role in neuroscience. However, the available amperometric technique with carbon fiber electrodes typically measures exocytotic events from one cell during one procedure, which requires professional operations and takes time to produce statistical results of multiple cells. Here, we develop a functionally collaborative nanostructure to directly measure the neurotransmitter dopamine (DA) exocytosis from living rat pheochromocytoma (PC12) cells. The functionally collaborative nanostructure is constructed of metal-organic framework (MOF)-on-nanowires-on-graphene oxide, which is highly sensitive to DA molecules and enables direct detection of neurotransmitter exocytosis. Using the microsensor, the exocytosis from PC12 cells pretreated with the desired drugs (e.g., anticoronavirus drug, antiflu drug, or anti-inflammatory drug) has been successfully measured. Our achievements demonstrate the feasibility of the functionally collaborative nanostructure in the real-time detection of exocytosis and the potential applicability in the highly efficient assessment of the modulation effects of medications on exocytosis.

Calcium-dependent subquantal peptide release from single docked lawn-resident vesicles of pituitary lactotrophs.

Regulated exocytosis consists of the fusion between vesicles and the plasma membranes, leading to the formation of a narrow fusion pore through which secretions exit the vesicle lumen into the extracellular space. An increase in the cytosolic concentration of free Ca2+ ([Ca2+]i) is considered the stimulus of this process. However, whether this mechanism can be preserved in a simplified system of membrane lawns with docked secretory vesicles, devoid of cellular components, is poorly understood. Here, we studied peptide discharge from individual secretory vesicles docked at the plasma membrane, prepared from primary endocrine pituitary cells (the lactotrophs), releasing hormone prolactin. To label secretory vesicles, we transfected lactotrophs to express the fluorescent atrial natriuretic peptide (ANP.emd), previously shown to be expressed in and released from prolactin-containing vesicles. We used stimulating solutions containing different [Ca2+] to evoke vesicle peptide discharge, which appeared similar in membrane lawns and in intact stimulated lactotrophs. All vesicles examined discharged peptides in a subquantal manner, either exhibiting a unitary or sequential time course. In the membrane lawns, the unitary vesicle peptide discharge was predominant and slightly slower than that recorded in intact cells, but with a shorter delay with respect to the stimulation onset. This study revealed directly that Ca2+ triggers peptide discharge from docked single vesicles in the membrane lawns with a half-maximal response of ∼8 µM [Ca2+], consistent with previous whole-cell patch-clamp studies in endocrine cells where the rapid component of exocytosis, interpreted to represent docked vesicles, was fully activated at <10 µM [Ca2+]. Interestingly, the sequential subquantal peptide vesicle discharge indicates that fluctuations between constricted and dilated fusion pore states are preserved in membrane lawns and that fusion pore regulation appears to be an autonomously controlled process.

The molecular chaperone cysteine string protein is required for monomeric SNARE proteins to assemble in trans-complexes during human sperm acrosomal exocytosis†.

Membrane fusion in sperm cells is crucial for acrosomal exocytosis and must be preserved to ensure fertilizing capacity. Evolutionarily conserved protein machinery regulates acrosomal exocytosis. Molecular chaperones play a vital role in spermatogenesis and post-testicular maturation. Cysteine string protein (CSP) is a member of the Hsp40 co-chaperones, and the participation of molecular chaperones in acrosomal exocytosis is poorly understood. In particular, the role of CSP in acrosomal exocytosis has not been reported so far. Using western blot and indirect immunofluorescence, we show that CSP is present in human sperm, is palmitoylated, and predominantly bound to membranes. Moreover, using functional assays and transmission electron microscopy, we report that blocking the function of CSP avoided the assembly of trans-complexes and inhibited exocytosis. In summary, here, we describe the presence of CSP in human sperm and show that this protein has an essential role in membrane fusion during acrosomal exocytosis mediating the trans-SNARE complex assembly between the outer acrosomal and plasma membranes. In general, understanding CSP's role is critical in identifying new biomarkers and generating new rational-based approaches to treat male infertility.

Methodologies for Detecting Quantal Exocytosis in Adrenal Chromaffin Cells Through Diamond-Based MEAs.

Diamond-based multiarray sensors are suitable to detect in real-time exocytosis and action potentials from cultured, spontaneously firing chromaffin cells, primary hippocampal neurons, and midbrain dopaminergic neurons. Here, we focus on how amperometric measurements of catecholamine release are performed on micrographitic diamond multiarrays (µG-D-MEAs) with high temporal and spatial resolution by 16 electrodes simultaneously.

Electrostatic regulation of the cis- and trans-membrane interactions of synaptotagmin-1.

Synaptotagmin-1 is a vesicular protein and Ca2+ sensor for Ca2+-dependent exocytosis. Ca2+ induces synaptotagmin-1 binding to its own vesicle membrane, called the cis-interaction, thus preventing the trans-interaction of synaptotagmin-1 to the plasma membrane. However, the electrostatic regulation of the cis- and trans-membrane interaction of synaptotagmin-1 was poorly understood in different Ca2+-buffering conditions. Here we provide an assay to monitor the cis- and trans-membrane interactions of synaptotagmin-1 by using native purified vesicles and the plasma membrane-mimicking liposomes (PM-liposomes). Both ATP and EGTA similarly reverse the cis-membrane interaction of synaptotagmin-1 in free [Ca2+] of 10-100 µM. High PIP2 concentrations in the PM-liposomes reduce the Hill coefficient of vesicle fusion and synaptotagmin-1 membrane binding; this observation suggests that local PIP2 concentrations control the Ca2+-cooperativity of synaptotagmin-1. Our data provide evidence that Ca2+ chelators, including EGTA and polyphosphate anions such as ATP, ADP, and AMP, electrostatically reverse the cis-interaction of synaptotagmin-1.

NGF-Induced Upregulation of CGRP in Orofacial Pain Induced by Tooth Movement Is Dependent on Atp6v0a1 and Vesicle Release.

The nerve growth factor (NGF) and calcitonin gene-related peptide (CGRP) play a crucial role in the regulation of orofacial pain. It has been demonstrated that CGRP increases orofacial pain induced by NGF. V-type proton ATPase subunit an isoform 1 (Atp6v0a1) is involved in the exocytosis pathway, especially in vesicular transport in neurons. The objective was to examine the role of Atp6v0a1 in NGF-induced upregulation of CGRP in orofacial pain induced by experimental tooth movement. Orofacial pain was elicited by ligating closed-coil springs between incisors and molars in Sprague-Dawley rats. Gene and protein expression levels were determined through real-time polymerase chain reaction, immunostaining, and fluorescence in situ hybridization. Lentivirus vectors carrying Atp6v0a1 shRNA were used to knockdown the expression of Atp6v0a1 in TG and SH-SY5Y neurons. The release of vesicles in SH-SY5Y neurons was observed by using fluorescence dye FM1-43, and the release of CGRP was detected by Enzyme-Linked Immunosorbent Assy. Orofacial pain was evaluated through the rat grimace scale. Our results revealed that intraganglionic administration of NGF and Atp6v0a1 shRNA upregulated and downregulated CGRP in trigeminal ganglia (TG) and trigeminal subnucleus caudalis (Vc), respectively, and the orofacial pain was also exacerbated and alleviated, respectively, following administration of NGF and Atp6v0a1 shRNA. Besides, intraganglionic administration of NGF simultaneously caused the downregulation of Atp6v0a1 in TG. Moreover, the release of vesicles and CGRP in SH-SY5Y neurons was interfered by NGF and Atp6v0a1 shRNA. In conclusion, in the orofacial pain induced by experimental tooth movement, NGF induced the upregulation of CGRP in TG and Vc, and this process is dependent on Atp6v0a1 and vesicle release, suggesting that they are involved in the transmission of nociceptive information in orofacial pain.

Activation of TRPV4 Induces Exocytosis and Ferroptosis in Human Melanoma Cells.

TRPV4 (transient receptor potential vanilloid 4), a calcium permeable TRP ion channel, is known to play a key role in endocytosis. However, whether it contributes to exocytosis remains unclear. Here, we report that activation of TRPV4 induced massive exocytosis in both melanoma A375 cell and heterologous expression systems. We show here that, upon application of TRPV4-specific agonists, prominent vesicle priming from endoplasmic reticulum (ER) was observed, followed by morphological changes of mitochondrial crista may lead to cell ferroptosis. We further identified interactions between TRPV4 and folding/vesicle trafficking proteins, which were triggered by calcium entry through activated TRPV4. This interplay, in turn, enhanced TRPV4-mediated activation of folding and vesicle trafficking proteins to promote exocytosis. Our study revealed a signaling mechanism underlying stimulus-triggered exocytosis in melanoma and highlighted the role of cellular sensor TRPV4 ion channel in mediating ferroptosis.

Glucose inhibits glucagon secretion by decreasing [Ca2+]c and by reducing the efficacy of Ca2+ on exocytosis via somatostatin-dependent and independent mechanisms.

OBJECTIVE: The mechanisms by which glucose stimulates insulin secretion from ß-cells are well established and involve inhibition of ATP-sensitive K+ (KATP) channels, followed by a rise in [Ca2+]c that triggers exocytosis. However, the mechanisms by which glucose controls glucagon release from α-cells are much less known. In particular, it is debated whether the sugar controls glucagon secretion by changing α-cell [Ca2+]c, and whether KATP channels or paracrine factors are involved. The present study addresses these issues. METHODS: We tested the effect of a decrease or an increase of glucose concentration (Gx, with x = concentration in mM) on α-cell [Ca2+]c and glucagon secretion. α-cell [Ca2+]c was monitored using GluCreGCaMP6f mice expressing the Ca2+-sensitive fluorescent protein, GCaMP6f, specifically in α-cells. [Ca2+]c was compared between dispersed α-cells and α-cells within islets to evaluate the potential contribution of an indirect effect of glucose. The same protocols were used for experiments of glucagon secretion from whole islets and [Ca2+]c measurements to test if changes in glucagon release mirror those in α-cell [Ca2+]c. RESULTS: Blockade of KATP channels by sulfonylureas (tolbutamide 100 µM or gliclazide 25 µM) strongly increased [Ca2+]c in both dispersed α-cells and α-cells within islets. By contrast, glucose had no effect on [Ca2+]c in dispersed α-cells, whereas it affected it in α-cells within islets. The effect of glucose was however different in islets expressing (Sst+/+) or not somatostatin (SST) (Sst-/-). Decreasing glucose concentration from G7 to G1 modestly increased α-cell [Ca2+]c, but to a slightly larger extent in Sst+/+ islets than in Sst-/- islets. This G1-induced [Ca2+]c rise was also observed in the continuous presence of sulfonylureas in both Sst+/+ and Sst-/- islets. Increasing glucose concentration from G7 to G20 did not affect α-cell [Ca2+]c in Sst+/+ islets which remained low, whereas it strongly increased it in Sst-/- islets. The observations that this increase was seen only in α-cells within islets but never in dispersed α-cells and that it was abrogated by the gap junction inhibitor, carbenoxolone, point to an indirect effect of G20 and suggest that, in Sst-/- islets, G20-stimulated ß-cells entrain α-cells whereas, in Sst+/+ islets, the concomitant release of SST keeps α-cell [Ca2+]c at low levels. The [Ca2+]c lowering effect of endogenous SST is also supported by the observation that SST receptor antagonists (SSTR2/3) increased [Ca2+]c in α-cells from Sst+/+ islets. All these [Ca2+]c changes induced parallel changes in glucagon release. To test if glucose also controls glucagon release independently of [Ca2+]c changes, additional experiments were performed in the continuous presence of 30 mM K+ and the KATP channel opener diazoxide (250 µM). In these conditions, α-cell [Ca2+]c within islets was elevated and its steady-state level was unaffected by glucose. However, decreasing the glucose concentration from G7 to G1 stimulated glucagon release whereas increasing it from G1 to G15 inhibited it. These effects were also evident in Sst-/- islets, and opposite to those on insulin secretion. CONCLUSIONS: We propose a model according to which glucose controls α-cell [Ca2+]c and glucagon secretion through multiple mechanisms. Increasing the glucose concentration modestly decreases [Ca2+]c in α-cells independently of their KATP channels and partly via SST. The involvement of SST increases with the glucose concentration, and one major effect of SST is to keep α-cell [Ca2+]c at low levels by counteracting the effect of an entrainment of α-cells by ß-cells when ß-cells become stimulated by glucose. All these [Ca2+]c changes induce parallel changes in glucagon release. Glucose also decreases the efficacy of Ca2+ on exocytosis by an attenuating pathway that is opposite to the well-established amplifying pathway controlling insulin release in ß-cells.

A two-subpopulation model that reflects heterogeneity of large dense core vesicles in exocytosis.

Exocytosis of large dense core vesicles is responsible for hormone secretion in neuroendocrine cells. The population of primed vesicles ready to release upon cell excitation demonstrates large heterogeneity. However, there are currently no models that clearly reflect such heterogeneity. Here, we develop a novel model based on single vesicle release events from amperometry recordings of PC12 cells using carbon fiber microelectrode. In this model, releasable vesicles can be grouped into two subpopulations, namely, SP1 and SP2. SP1 vesicles replenish quickly, with kinetics of ~0.0368 s-1, but likely undergo slow fusion pore expansion (amperometric signals rise at ~2.5 pA/ms), while SP2 vesicles demonstrate slow replenishment (kinetics of ~0.0048 s-1) but prefer fast dilation of fusion pore, with an amperometric signal rising rate of ~9.1 pA/ms. Phorbol ester enlarges the size of SP2 partially via activation of protein kinase C and conveys SP1 vesicles into SP2. Inhibition of Rho GTPase-dependent actin rearrangement almost completely depletes SP2. We also propose that the phorbol ester-sensitive vesicle subpopulation (SP2) is analogous to the subset of superprimed synaptic vesicles in neurons. This model provides a meticulous description of the architecture of the readily releasable vesicle pool and elucidates the heterogeneity of the vesicle priming mechanism.

Calcium-dependent ESCRT recruitment and lysosome exocytosis maintain epithelial integrity during Candida albicans invasion.

Candida albicans is both a commensal and an opportunistic fungal pathogen. Invading hyphae of C. albicans secrete candidalysin, a pore-forming peptide toxin. To prevent cell death, epithelial cells must protect themselves from direct damage induced by candidalysin and by the mechanical forces exerted by expanding hyphae. We identify two key Ca2+-dependent repair mechanisms employed by epithelial cells to withstand candidalysin-producing hyphae. Using camelid nanobodies, we demonstrate candidalysin secretion directly into the invasion pockets induced by elongating C. albicans hyphae. The toxin induces oscillatory increases in cytosolic [Ca2+], which cause hydrolysis of PtdIns(4,5)P2 and loss of cortical actin. Epithelial cells dispose of damaged membrane regions containing candidalysin by an Alg-2/Alix/ESCRT-III-dependent blebbing process. At later stages, plasmalemmal tears induced mechanically by invading hyphae are repaired by exocytic insertion of lysosomal membranes. These two repair mechanisms maintain epithelial integrity and prevent mucosal damage during both commensal growth and infection by C. albicans.

SNARE Proteins Mediate α-Synuclein Secretion via Multiple Vesicular Pathways.

The cell-to-cell transmission of pathological α-synuclein (α-syn) has been proposed to be a critical event in the development of synucleinopathies. Recent studies have begun to reveal the underlying molecular mechanism of α-syn propagation. As one of the central steps, α-syn secretion is reported to be Ca2+-dependent and mediated by unconventional exocytosis. However, the soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) requirement and vesicle identity of α-syn secretion remain elusive. Here we found that α-syn secretion is SNARE-dependent by systematically knocking down Q-SNAREs and R-SNAREs in exocytosis pathways. α-Syn secretion was mainly mediated by syntaxin 4 (STX4) and synaptosomal-associated protein 23 (SNAP23), but did not require STX1 and SNAP25, in differentiated SH-SY5Y cells. On the other hand, vesicle-associated membrane protein 3 (VAMP3), VAMP7, and VAMP8 were all involved in α-syn secretion, most likely in overlapping pathways. Application of super-resolution microscopy revealed localization of both endogenous and overexpressed α-syn in endosomes, lysosomes, and autophagosomes in rat primary cortical neurons. α-Syn co-localized with microtubule-associated protein 1 light chain 3 (LC3) most extensively, suggesting its tight association with the autophagy pathway. Consistently, α-syn secretion was regulated by the autophagy-lysosome pathway. Collectively, our data suggest that α-syn secretion is SNARE-dependent and is mediated by multiple vesicular pathways including exocytosis of recycling endosomes, multivesicular bodies, autophagosomes, and lysosomes.

Osteosarcoma exocytosis of soluble LGALS3BP mediates macrophages toward a tumoricidal phenotype.

This study aimed to elucidate the interactions between osteosarcoma (OS) and M1 macrophages infiltrated into the tumor microenvironment and to explore the underlying mechanisms whereby M1 macrophages influence the growth of OS, so that novel treatments of OS can be developed. A transwell co-culture system, an indirect conditioned medium culture system and two orthotopic bearing OS models were established to assess for the interplay between M1 macrophages and OS. We found that the co-culture of M1 macrophages with OS cells significantly inhibited the growth of the tumor cells by inducing apoptosis. Furthermore, HSPA1L secreted by M1 macrophages exerted this anti-tumor effect through the IRAK1 and IRAK4 pathways. LGALS3BP secreted by OS cells bound to the ligand LGALS3 on M1 macrophages and thereby induced the secretion of Hspa11 via Akt phosphorylation. In vivo experiments demonstrated that the culture supernatant of OS-stimulated M1 macrophages significantly inhibited the growth of OS, whereas silencing Lgals3bp promoted the progression of OS. In conclusion, OS modifies the phenotype of tumor-associated macrophages (TAMs) and thereby influences the apoptosis of OS cells through soluble factors. The modulation of TAMs may be a promising and effective therapeutic approach in OS.

Glycine Release Is Potentiated by cAMP via EPAC2 and Ca2+ Stores in a Retinal Interneuron.

Neuromodulation via the intracellular second messenger cAMP is ubiquitous at presynaptic nerve terminals. This modulation of synaptic transmission allows exocytosis to adapt to stimulus levels and reliably encode information. The AII amacrine cell (AII-AC) is a central hub for signal processing in the mammalian retina. The main apical dendrite of the AII-AC is connected to several lobular appendages that release glycine onto OFF cone bipolar cells and ganglion cells. However, the influence of cAMP on glycine release is not well understood. Using membrane capacitance measurements from mouse AII-ACs to directly measure exocytosis, we observe that intracellular dialysis of 1 mm cAMP enhances exocytosis without affecting the L-type Ca2+ current. Responses to depolarizing pulses of various durations show that the size of the readily releasable pool of vesicles nearly doubles with cAMP, while paired-pulse depression experiments suggest that release probability does not change. Specific agonists and antagonists for exchange protein activated by cAMP 2 (EPAC2) revealed that the cAMP-induced enhancement of exocytosis requires EPAC2 activation. Furthermore, intact Ca2+ stores were also necessary for the cAMP potentiation of exocytosis. Postsynaptic recordings from OFF cone bipolar cells showed that increasing cAMP with forskolin potentiated the frequency of glycinergic spontaneous IPSCs. We propose that cAMP elevations in the AII-AC lead to a robust enhancement of glycine release through an EPAC2 and Ca2+ store signaling pathway. Our results thus contribute to a better understanding of how AII-AC crossover inhibitory circuits adapt to changes in ambient luminance.SIGNIFICANCE STATEMENT The mammalian retina operates over a wide dynamic range of light intensities and contrast levels. To optimize the signal-to-noise ratio of processed visual information, both excitatory and inhibitory synapses within the retina must modulate their gain in synaptic transmission to adapt to different levels of ambient light. Here we show that increases of cAMP concentration within AII amacrine cells produce enhanced exocytosis from these glycinergic interneurons. Therefore, we propose that light-sensitive neuromodulators may change the output of glycine release from AII amacrine cells. This novel mechanism may fine-tune the amount of tonic and phasic synaptic inhibition received by bipolar cell terminals and, consequently, the spiking patterns that ganglion cells send to the upstream visual areas of the brain.

Glucagon-like peptide-1 receptor controls exocytosis in chromaffin cells by increasing full-fusion events.

Agonists for glucagon-like-peptide-1 receptor (GLP-1R) are currently used for the treatment of type 2 diabetes and obesity. Their benefits have been centered on pancreas and hypothalamus, but their roles in other organ systems are not well understood. We studied the action of GLP-1R on secretions of adrenal medulla. Exendin-4, a synthetic analog of GLP-1, increases the synthesis and the release of catecholamines (CAs) by increasing cyclic AMP (cAMP) production, without apparent participation of cAMP-regulated guanine nucleotide exchange factor (Epac). Exendin-4, when incubated for 24 h, increases CA synthesis by promoting the activation of tyrosine hydroxylase. Short incubation (20 min) increases the quantum size of exocytotic events by switching exocytosis from partial to full fusion. Our results give a strong support to the role of GLP-1 in the fine control of exocytosis.