Prazosin Potentiates Mast Cell-Stabilizing Property of Adrenaline.

BACKGROUND/AIMS: Adrenaline quickly inhibits the release of histamine from mast cells. Besides ß2-adrenergic receptors, several in vitro studies also indicate the involvement of α-adrenergic receptors in the process of exocytosis. Since exocytosis in mast cells can be detected electrophysiologically by the changes in the membrane capacitance (Cm), its continuous monitoring in the presence of drugs would determine their mast cell-stabilizing properties. METHODS: Employing the whole-cell patch-clamp technique in rat peritoneal mast cells, we examined the effects of adrenaline on the degranulation of mast cells and the increase in the Cm during exocytosis. We also examined the degranulation of mast cells in the presence or absence of α-adrenergic receptor agonists or antagonists. RESULTS: Adrenaline dose-dependently suppressed the GTP-γ-S-induced increase in the Cm and inhibited the degranulation from mast cells, which was almost completely erased in the presence of butoxamine, a ß2-adrenergic receptor antagonist. Among α-adrenergic receptor agonists or antagonists, high dose prazosin, a selective α1-adrenergic receptor antagonist, significantly reduced the ratio of degranulating mast cells and suppressed the increase in the Cm. Additionally, prazosin augmented the inhibitory effects of adrenaline on the degranulation of mast cells. CONCLUSION: This study provided electrophysiological evidence for the first time that adrenaline dose-dependently inhibited the process of exocytosis, confirming its usefulness as a potent mast cell-stabilizer. The pharmacological blockade of α1-adrenergic receptor by prazosin synergistically potentiated such mast cell-stabilizing property of adrenaline, which is primarily mediated by ß2-adrenergic receptors.

Role of ESCCAL-1 in regulating exocytosis of AuNPs in human esophageal squamous carcinoma cells.

Exocytosis is a critical factor for designing efficient nanocarriers and determining cytotoxicity. However, the research on the exocytosis mechanism of nanoparticles, especially the role of long non-coding RNAs (lncRNAs), has not been reported. In this study, the exocytosis of AuNPs in the KYSE70 cells and the involved molecular pathways of exocytosis are analyzed. It demonstrates that nanoparticles underwent time-dependent release from the cells by exocytosis, and the release of ß-hexosaminidase confirms that AuNPs are excreted through lysosomes. Mechanistic studies reveal that lncRNA ESCCAL-1 plays a vital role in controlling the exocytosis of AuNPs through activation of the MAPK pathway, including the phosphorylation of ERK and JNK. The study implies that the ESCCAL-1-mediated pathway plays an important role in the exocytosis of AuNPs in KYSE70 cells. This finding has implications for the role of ESCCAL-1 on the drug resistance of esophagus cancer by controlling lysosome-mediated exocytosis.

Simultaneous TIRF imaging of subplasmalemmal Ca2+ dynamics and granule fusions in insulin-secreting INS-1 cells reveals coexistent synchronized and asynchronous release.

The basal and glucose-induced insulin secretion from pancreatic beta cells is a tightly regulated process that is triggered in a Ca2+-dependent fashion and further positively modulated by substances that raise intracellular levels of adenosine 3',5'-cyclic monophosphate (cAMP) or by certain antidiabetic drugs. In a previous study, we have temporally resolved the subplasmalemmal [Ca2+]i dynamics in beta cells that are characterized by trains of sharply delimited spikes, reaching peak values up to 5 µM. Applying total internal reflection fluorescence (TIRF) microscopy and synaptopHluorin to visualize fusion events of individual granules, we found that several fusion events can coincide within 50 to 150 ms. To test whether subplasmalemmal [Ca2+]i microdomains around single or clustered Ca2+ channels may cause a synchronized release of insulin-containing vesicles, we applied simultaneous dual-color TIRF microscopy and monitored Ca2+ fluctuations and exocytotic events in INS-1 cells at high frame rates. The results indicate that fusions can be triggered by subplasmalemmal Ca2+ spiking. This, however, does account for a minority of fusion events. About 90 %-95 % of fusion events either happen between Ca2+ spikes or incidentally overlap with subplasmalemmal Ca2+ spikes. We conclude that only a fraction of exocytotic events in glucose-induced and tolbutamide- or forskolin-enhanced insulin release from INS-1 cells is tightly coupled to Ca2+ microdomains around voltage-gated Ca2+ channels.

ShlA toxin of Serratia induces P2Y2- and α5ß1-dependent autophagy and bacterial clearance from host cells.

Serratia marcescens is an opportunistic human pathogen involved in antibiotic-resistant hospital acquired infections. Upon contact with the host epithelial cell and prior to internalization, Serratia induces an early autophagic response that is entirely dependent on the ShlA toxin. Once Serratia invades the eukaryotic cell and multiples inside an intracellular vacuole, ShlA expression also promotes an exocytic event that allows bacterial egress from the host cell without compromising its integrity. Several toxins, including ShlA, were shown to induce ATP efflux from eukaryotic cells. Here, we demonstrate that ShlA triggered a nonlytic release of ATP from Chinese hamster ovary (CHO) cells. Enzymatic removal of accumulated extracellular ATP (eATP) or pharmacological blockage of the eATP-P2Y2 purinergic receptor inhibited the ShlA-promoted autophagic response in CHO cells. Despite the intrinsic ecto-ATPase activity of CHO cells, the effective concentration and kinetic profile of eATP was consistent with the established affinity of the P2Y2 receptor and the known kinetics of autophagy induction. Moreover, eATP removal or P2Y2 receptor inhibition also suppressed the ShlA-induced exocytic expulsion of the bacteria from the host cell. Blocking α5ß1 integrin highly inhibited ShlA-dependent autophagy, a result consistent with α5ß1 transactivation by the P2Y2 receptor. In sum, eATP operates as the key signaling molecule that allows the eukaryotic cell to detect the challenge imposed by the contact with the ShlA toxin. Stimulation of P2Y2-dependent pathways evokes the activation of a defensive response to counteract cell damage and promotes the nonlytic clearance of the pathogen from the infected cell.

The Mood Stabilizer Lithium Slows Down Synaptic Vesicle Cycling at Glutamatergic Synapses.

Lithium is a mood stabilizer broadly used to prevent and treat symptoms of mania and depression in people with bipolar disorder (BD). Little is known, however, about its mode of action. Here, we analyzed the impact of lithium on synaptic vesicle (SV) cycling at presynaptic terminals releasing glutamate, a neurotransmitter previously implicated in BD and other neuropsychiatric conditions. We used the pHluorin-based synaptic tracer vGpH and a fully automated image processing pipeline to quantify the effect of lithium on both SV exocytosis and endocytosis in hippocampal neurons. We found that lithium selectively reduces SV exocytic rates during electrical stimulation, and markedly slows down SV recycling post-stimulation. Analysis of single-bouton responses revealed the existence of functionally distinct excitatory synapses with varying sensitivity to lithium-some terminals show responses similar to untreated cells, while others are markedly impaired in their ability to recycle SVs. While the cause of this heterogeneity is unclear, these data indicate that lithium interacts with the SV machinery and influences glutamate release in a large fraction of excitatory synapses. Together, our findings show that lithium down modulates SV cycling, an effect consistent with clinical reports indicating hyperactivation of glutamate neurotransmission in BD.

Adrenergic receptors control frequency-dependent switching of the exocytosis mode between "full-collapse" and "kiss-and-run" in murine motor nerve terminal.

AIMS: Neurotransmitter release from the synaptic vesicles can occur through two modes of exocytosis: "full-collapse" or "kiss-and-run". Here we investigated how increasing the nerve activity and pharmacological stimulation of adrenoceptors can influence the mode of exocytosis in the motor nerve terminal. METHODS: Recording of endplate potentials with intracellular microelectrodes was used to estimate acetylcholine release. A fluorescent dye FM1-43 and its quenching with sulforhodamine 101 were utilized to visualize synaptic vesicle recycling. KEY FINDINGS: An increase in the frequency of stimulation led to a decrease in the rate of FM1-43 unloading despite the higher number of quanta released. High frequency activity promoted neurotransmitter release via the kiss-and-run mechanism. This was confirmed by experiments utilizing (I) FM1-43 dye quencher, that is able to pass into the synaptic vesicle via fusion pore, and (II) loading of FM1-43 by compensatory endocytosis. Noradrenaline and specific α2-adrenoreceptors agonist, dexmedetomidine, controlled the mode of synaptic vesicle recycling at high frequency activity. Their applications favored neurotransmitter release via full-collapse exocytosis rather than the kiss-and-run pathway. SIGNIFICANCE: At the diaphragm neuromuscular junctions, neuronal commands are translated into contractions necessary for respiration. During stress, an increase in discharge rate of the phrenic nerve shifts the exocytosis from the full-collapse to the kiss-and-run mode. The stress-related molecule, noradrenaline, restricts neurotransmitter release in response to a high frequency activity, and prevents the shift in the mode of exocytosis through α2-adrenoceptor activation. This may be a component of the mechanism that limits overstimulation of the respiratory system during stress.

Vesicle Fusion as a Target Process for the Action of Sphingosine and Its Derived Drugs.

The fusion of membranes is a central part of the physiological processes involving the intracellular transport and maturation of vesicles and the final release of their contents, such as neurotransmitters and hormones, by exocytosis. Traditionally, in this process, proteins, such SNAREs have been considered the essential components of the fusion molecular machinery, while lipids have been seen as merely structural elements. Nevertheless, sphingosine, an intracellular signalling lipid, greatly increases the release of neurotransmitters in neuronal and neuroendocrine cells, affecting the exocytotic fusion mode through the direct interaction with SNAREs. Moreover, recent studies suggest that FTY-720 (Fingolimod), a sphingosine structural analogue used in the treatment of multiple sclerosis, simulates sphingosine in the promotion of exocytosis. Furthermore, this drug also induces the intracellular fusion of organelles such as dense vesicles and mitochondria causing cell death in neuroendocrine cells. Therefore, the effect of sphingosine and synthetic derivatives on the heterologous and homologous fusion of organelles can be considered as a new mechanism of action of sphingolipids influencing important physiological processes, which could underlie therapeutic uses of sphingosine derived lipids in the treatment of neurodegenerative disorders and cancers of neuronal origin such neuroblastoma.

Identification of a Potent Cytotoxic Pyrazole with Anti-Breast Cancer Activity That Alters Multiple Pathways.

In this study, we identified a novel pyrazole-based derivative (P3C) that displayed potent cytotoxicity against 27 human cancer cell lines derived from different tissue origins with 50% cytotoxic concentrations (CC50) in the low micromolar and nanomolar range, particularly in two triple-negative breast cancer (TNBC) cell lines (from 0.25 to 0.49 µM). In vitro assays revealed that P3C induces reactive oxygen species (ROS) accumulation leading to mitochondrial depolarization and caspase-3/7 and -8 activation, suggesting the participation of both the intrinsic and extrinsic apoptotic pathways. P3C caused microtubule disruption, phosphatidylserine externalization, PARP cleavage, DNA fragmentation, and cell cycle arrest on TNBC cells. In addition, P3C triggered dephosphorylation of CREB, p38, ERK, STAT3, and Fyn, and hyperphosphorylation of JNK and NF-kB in TNBC cells, indicating the inactivation of both p38MAPK/STAT3 and ERK1/2/CREB signaling pathways. In support of our in vitro assays, transcriptome analyses of two distinct TNBC cell lines (MDA-MB-231 and MDA-MB-468 cells) treated with P3C revealed 28 genes similarly affected by the treatment implicated in apoptosis, oxidative stress, protein kinase modulation, and microtubule stability.

NGF Enhances CGRP Release Evoked by Capsaicin from Rat Trigeminal Neurons: Differential Inhibition by SNAP-25-Cleaving Proteases.

Nerve growth factor (NGF) is known to intensify pain in various ways, so perturbing pertinent effects without negating its essential influences on neuronal functions could help the search for much-needed analgesics. Towards this goal, cultured neurons from neonatal rat trigeminal ganglia-a locus for craniofacial sensory nerves-were used to examine how NGF affects the Ca2+-dependent release of a pain mediator, calcitonin gene-related peptide (CGRP), that is triggered by activating a key signal transducer, transient receptor potential vanilloid 1 (TRPV1) with capsaicin (CAP). Measurements utilised neurons fed with or deprived of NGF for 2 days. Acute re-introduction of NGF induced Ca2+-dependent CGRP exocytosis that was inhibited by botulinum neurotoxin type A (BoNT/A) or a chimera of/E and/A (/EA), which truncated SNAP-25 (synaptosomal-associated protein with Mr = 25 k) at distinct sites. NGF additionally caused a Ca2+-independent enhancement of the neuropeptide release evoked by low concentrations (<100 nM) of CAP, but only marginally increased the peak response to ≥100 nM. Notably, BoNT/A inhibited CGRP exocytosis evoked by low but not high CAP concentrations, whereas/EA effectively reduced responses up to 1 µM CAP and inhibited to a greater extent its enhancement by NGF. In addition to establishing that sensitisation of sensory neurons to CAP by NGF is dependent on SNARE-mediated membrane fusion, insights were gleaned into the differential ability of two regions in the C-terminus of SNAP-25 (181-197 and 198-206) to support CAP-evoked Ca2+-dependent exocytosis at different intensities of stimulation.

Vitamin D Inhibits the Early Aggregation of α-Synuclein and Modulates Exocytosis Revealed by Electrochemical Measurements.

Alpha-synuclein (α-Syn) localizes at presynaptic terminal and modulates synaptic functions. Increasing evidence demonstrate that α-Syn oligomers, forming at the early of aggregation, are cytotoxic and is thus related to brain neurodegenerative diseases. Herein, we find that vitamin D (VD) can reduce neurocytotoxicity. The reduced neurocytotoxicity might be attributed to the less amount of large-sized α-Syn oligomers inhibited by VD, measured by electrochemical collision at single particle level, which are not observable with traditionally ensembled method. Single-cell amperometry (SCA) results show that VD can recover the amount of neurotransmitter release during exocytosis induced by α-Syn oligomers, further verifying the neuroprotection of VD. Our study reveals the neuroprotective role of VD through inhibiting α-Syn aggregation, which is envisioned to be of great importance in treatment and prevention of the neurodegenerative diseases.

1α,25-(OH)2 vitamin D3 prevents insulin resistance and regulates coordinated exocytosis and insulin secretion.

Vitamin D3 is associated with improvements in insulin resistance and glycemia. In this study, we investigated the short-term effect of 1α,25(OH)2 Vitamin D3 (1,25-D3) and cholecalciferol (vitamin D3) on the glycemia and insulin sensitivity of control and dexamethasone-induced insulin-resistance rats. 45Ca2+ influx responses to 1,25-D3 and its role in insulin secretion were investigated in isolated pancreatic islets from control rats. In vivo, 5 d treatment with 1,25-D3 (i.p.) prevented insulin resistance in dexamethasone-treated rats. Treatment with 1,25-D3 improved the activities of hepatic enzymes, serum lipids and calcium concentrations in insulin-resistant rats. 25-D3 (o.g.) does not affect insulin resistance. In pancreatic islets, 1,25-D3 increased insulin secretion and stimulated rapid response 45Ca2+ influx. The stimulatory effect of 1,25-D3 on 45Ca2+ influx was decreased by diazoxide, apamine, thapsigargin, dantrolene, 2-APB, nifedipine, TEA, PKA, PKC, and cytoskeleton inhibitor, while it was increased by glibenclamide and N-ethylmaleimide. The stimulatory effect of 1,25-D3 on 45Ca2+ influx involves the activation of L-type VDCC, K+-ATP, K+-Ca2+, and Kv channels, which augment cytosolic calcium. These ionic changes mobilize calcium from stores and downstream activation of PKC, PKA tethering vesicle traffic and fusion at the plasma membrane for insulin secretion. This is the first study highlighting the unprecedented role of 1,25-D3 (short-term effect) in the regulation of glucose homeostasis and on prevention of insulin resistance. Furthermore, this study shows the intracellular ß-cell signal transduction of 1,25-D3 through the modulation of pivotal ionic channels and proteins exhibiting a coordinated exocytosis of vesicles for insulin secretion.

Targeting the insulin granule for modulation of insulin exocytosis.

The pancreatic ß-cells control insulin secretion in the body to regulate glucose homeostasis, and ß-cell stress and dysfunction is characteristic of Type 2 Diabetes. Pharmacological targeting of the ß-cell to increase insulin secretion is typically utilised, however, extended use of common drugs such as sulfonylureas are known to result in secondary failure. Moreover, there is evidence they may induce ß-cell failure in the long term. Within ß-cells, insulin secretory granules (ISG) serve as compartments to store, process and traffic insulin for exocytosis. There is now growing evidence that ISG exist in multiple populations, distinct in their protein composition, motility, age, and capacity for secretion. In this review, we discuss the implications of a heterogenous ISG population in ß-cells and highlight the need for more understanding into how unique ISG populations may be targeted in anti-diabetic therapies.

Nanobody-mediated macromolecular crowding induces membrane fission and remodeling in the African trypanosome.

The dense variant surface glycoprotein (VSG) coat of African trypanosomes represents the primary host-pathogen interface. Antigenic variation prevents clearing of the pathogen by employing a large repertoire of antigenically distinct VSG genes, thus neutralizing the host's antibody response. To explore the epitope space of VSGs, we generate anti-VSG nanobodies and combine high-resolution structural analysis of VSG-nanobody complexes with binding assays on living cells, revealing that these camelid antibodies bind deeply inside the coat. One nanobody causes rapid loss of cellular motility, possibly due to blockage of VSG mobility on the coat, whose rapid endocytosis and exocytosis are mechanistically linked to Trypanosoma brucei propulsion and whose density is required for survival. Electron microscopy studies demonstrate that this loss of motility is accompanied by rapid formation and shedding of nanovesicles and nanotubes, suggesting that increased protein crowding on the dense membrane can be a driving force for membrane fission in living cells.

Selective inhibition of gamma aminobutyric acid release from mouse hippocampal interneurone subtypes by the volatile anaesthetic isoflurane.

BACKGROUND: The cellular and molecular mechanisms by which general anaesthesia occurs is poorly understood. Hippocampal interneurone subpopulations, which are critical regulators of cognitive function, have diverse neurophysiological and synaptic properties, but their responses to anaesthetics are unclear. METHODS: We used live-cell imaging of fluorescent biosensors expressed in mouse hippocampal neurones to delineate interneurone subtype-specific effects of isoflurane on synaptic vesicle exocytosis. The role of voltage-gated sodium channel (Nav) subtype expression in determining isoflurane sensitivity was probed by overexpression or knockdown of specific Nav subtypes in identified interneurones. RESULTS: Clinically relevant concentrations of isoflurane differentially inhibited synaptic vesicle exocytosis: to 83.1% (11.7%) of control in parvalbumin-expressing interneurones, and to 58.6% (13.3%) and 64.5% (8.5%) of control in somatostatin-expressing interneurones and glutamatergic neurones, respectively. The relative expression of Nav1.1 (associated with lower sensitivity) and Nav1.6 (associated with higher sensitivity) determined the sensitivity of exocytosis to isoflurane. CONCLUSIONS: Isoflurane inhibits synaptic vesicle exocytosis from hippocampal glutamatergic neurones and GABAergic interneurones in a cell-type-specific manner depending on their expression of voltage-gated sodium channel subtypes.

Nano-analysis Reveals High Fraction of Serotonin Release during Exocytosis from a Gut Epithelium Model Cell.

Electrochemical methods were used to explore the exocytotic nature of serotonin (5-HT) release in human carcinoid BON cells, an in vitro human enterochromaffin cell model, to understand the mechanisms operating the release of gut-derived 5-HT in the intestinal mucosal epithelium. We show that the fractional vesicular 5-HT release in BON cells is 80 % compared to previous work in pancreatic beta cells (34 %). The fractional release increased from 80 % in control BON cells to 87 % with 5-HT preincubation and nearly 100 % with the combination of 5-HT and the 5-HT4 autoreceptor agonist, cisapride. Thus, partial release is the primary mechanism of exocytosis in BON cells, resulting in a variable amount of the vesicular content being released. Factors that control secretion of 5-HT from enterochromaffin cells or BON cells are important as partial release provides a mechanism for development of effective therapeutic strategies to treat gastrointestinal diseases.

DD04107-Derived neuronal exocytosis inhibitor peptides: Evidences for synaptotagmin-1 as a putative target.

The analgesic peptide DD04107 (Pal-EEMQRR-NH2) and its acetylated analogue inhibit α-calcitonin gene-related peptide (α-CGRP) exocytotic release from primary sensory neurons. Examining the crystal structure of the SNARE-Synaptotagmin-1(Syt1) complex, we hypothesized that these peptides could inhibit neuronal exocytosis by binding to Syt1, hampering at least partially its interaction with the SNARE complex. To address this hypothesis, we first interrogate the role of individual side-chains on the inhibition of α-CGRP release, finding that E1, M3, Q4 and R6 residues were crucial for activity. CD and NMR conformational analysis showed that linear peptides have tendency to adopt α-helical conformations, but the results with cyclic analogues indicated that this secondary structure is not needed for activity. Isothermal titration calorimetry (ITC) measurements demonstrate a direct interaction of some of these peptides with Syt1-C2B domain, but not with Syt7-C2B region, indicating selectivity. As expected for a compound able to inhibit α-CGRP release, cyclic peptide derivative Pal-E-cyclo[EMQK]R-NH2 showed potent in vivo analgesic activity, in a model of inflammatory pain. Molecular dynamics simulations provided a model consistent with KD values for the interaction of peptides with Syt1-C2B domain, and with their biological activity. Altogether, these results identify Syt1 as a potential new analgesic target.

Amyloid-ß induced membrane damage instigates tunneling nanotube-like conduits by p21-activated kinase dependent actin remodulation.

Alzheimer's disease (AD) pathology progresses gradually via anatomically connected brain regions. Direct transfer of amyloid-ß1-42 oligomers (oAß) between connected neurons has been shown, however, the mechanism is not fully revealed. We observed formation of oAß induced tunneling nanotubes (TNTs)-like nanoscaled f-actin containing membrane conduits, in differentially differentiated SH-SY5Y neuronal models. Time-lapse images showed that oAß propagate from one cell to another via TNT-like structures. Preceding the formation of TNT-like conduits, we detected oAß-induced plasma membrane (PM) damage and calcium-dependent repair through lysosomal-exocytosis, followed by massive endocytosis to re-establish the PM. Massive endocytosis was monitored by an influx of the membrane-staining dye TMA-DPH and PM damage was quantified by propidium iodide influx in the absence of Ca2+. The massive endocytosis eventually caused accumulation of internalized oAß in Lamp1 positive multivesicular bodies/lysosomes via the actin cytoskeleton remodulating p21-activated kinase1 (PAK1) dependent endocytic pathway. Three-dimensional quantitative confocal imaging, structured illumination superresolution microscopy, and flowcytometry quantifications revealed that oAß induces activation of phospho-PAK1, which modulates the formation of long stretched f-actin extensions between cells. Moreover, the formation of TNT-like conduits was inhibited by preventing PAK1-dependent internalization of oAß using the small-molecule inhibitor IPA-3, a highly selective cell-permeable auto-regulatory inhibitor of PAK1. The present study reveals that the TNT-like conduits are probably instigated as a consequence of oAß induced PM damage and repair process, followed by PAK1 dependent endocytosis and actin remodeling, probably to maintain cell surface expansion and/or membrane tension in equilibrium.

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.

Morphological profiling of human T and NK lymphocytes by high-content cell imaging.

The immunological synapse is a complex structure that decodes stimulatory signals into adapted lymphocyte responses. It is a unique window to monitor lymphocyte activity because of development of systematic quantitative approaches. Here we demonstrate the applicability of high-content imaging to human T and natural killer (NK) cells and develop a pipeline for unbiased analysis of high-definition morphological profiles. Our approach reveals how distinct facets of actin cytoskeleton remodeling shape immunological synapse architecture and affect lytic granule positioning. Morphological profiling of CD8+ T cells from immunodeficient individuals allows discrimination of the roles of the ARP2/3 subunit ARPC1B and the ARP2/3 activator Wiskott-Aldrich syndrome protein (WASP) in immunological synapse assembly. Single-cell analysis further identifies uncoupling of lytic granules and F-actin radial distribution in ARPC1B-deficient lymphocytes. Our study provides a foundation for development of morphological profiling as a scalable approach to monitor primary lymphocyte responsiveness and to identify complex aspects of lymphocyte micro-architecture.

Therapeutic concentrations of varenicline increases exocytotic release of catecholamines from human and rat adrenal chromaffin cells in the presence of nicotine.

Cardiovascular side effects of varenicline and a case report of a hypertensive crisis in a varenicline-prescribed patient with pheochromocytoma have been reported. The goal of the present study was to determine whether such side effects might derive, in part, from increased exocytosis of secretory vesicles and subsequent catecholamine release triggered by varenicline in human chromaffin cells of the adrenal gland. In this study, we performed electrophysiological plasma membrane capacitance and carbon fiber amperometry experiments to evaluate the effect of varenicline on exocytosis and catecholamine release, respectively, at concentrations reached during varenicline therapy (100 nM). Experiments were conducted in the absence or presence of nicotine, at plasma concentrations achieved right after smoking (250 nM) or steady-state concentrations (110 nM), in chromaffin cells of the adrenal gland obtained from human organ donors. Cells were stimulated with short pulses (10 ms) of acetylcholine (ACh; 300 µM) applied at 0.2 Hz, in order to closer mimic the physiological situation at the splanchnic nerve-chromaffin cell synapse. In addition, rat chromaffin cells were used to compare the effects obtained in cells from a more readily available species. Varenicline increased the exocytosis of secretory vesicles in human and rat chromaffin cells in the presence of nicotine, effects that were not due to an increase of plasma membrane capacitance or currents triggered by the nicotinic agonists alone. These results should be considered in nicotine addiction therapies when varenicline is used.